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Sample records for protein storage vacuoles

  1. Isolation of Protein Storage Vacuoles and Their Membranes.

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    Shimada, Tomoo; Hara-Nishimura, Ikuko

    2017-01-01

    Protein-storage vacuoles (PSVs) are specialized vacuoles that sequester large amounts of storage proteins. During seed development, PSVs are formed de novo and/or from preexisting lytic vacuoles. Seed PSVs can be subdivided into four distinct compartments: membrane, globoid, matrix, and crystalloid. In this chapter, we introduce easy methods for isolation of PSVs and their membranes from pumpkin seeds. These methods facilitate the identification and characterization of PSV components.

  2. Live Cell Imaging During Germination Reveals Dynamic Tubular Structures Derived from Protein Storage Vacuoles of Barley Aleurone Cells

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    Verena Ibl

    2014-09-01

    Full Text Available The germination of cereal seeds is a rapid developmental process in which the endomembrane system undergoes a series of dynamic morphological changes to mobilize storage compounds. The changing ultrastructure of protein storage vacuoles (PSVs in the cells of the aleurone layer has been investigated in the past, but generally this involved inferences drawn from static pictures representing different developmental stages. We used live cell imaging in transgenic barley plants expressing a TIP3-GFP fusion protein as a fluorescent PSV marker to follow in real time the spatially and temporally regulated remodeling and reshaping of PSVs during germination. During late-stage germination, we observed thin, tubular structures extending from PSVs in an actin-dependent manner. No extensions were detected following the disruption of actin microfilaments, while microtubules did not appear to be involved in the process. The previously-undetected tubular PSV structures were characterized by complex movements, fusion events and a dynamic morphology. Their function during germination remains unknown, but might be related to the transport of solutes and metabolites.

  3. Rapid degradation of abnormal proteins in vacuoles from Acer pseudoplatanus L. cells

    International Nuclear Information System (INIS)

    Canut, H.; Alibert, G.; Carrasco, A.; Boudet, A.M.

    1986-01-01

    In Acer pseudoplatanus cells, the proteins synthesized in the presence of an amino acid analog ([ 14 C]p-fluorophenylalanine), were degraded more rapidly than normal ones ([ 14 C]phenylalanine as precursor). The degradation of an important part of these abnormal proteins occurred inside the vacuoles. The degradation process was not apparently associated to a specific proteolytic system but was related to a preferential transfer of these aberrant proteins from the cytoplasm to the vacuole

  4. Plant storage proteins – the main nourisching products – from biosynthesis to cellular storage depots

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    Agnieszka Chmielnicka

    2017-06-01

    Full Text Available Storage proteins of legumes are one of the main components of the human and animal diet. The substances collected in their seeds have the pro-health values, supporting the prevention of many civilization diseases. However, there are still many uncertainties about the mechanisms leading to the production of nutritious seeds. It is also difficult to identify which of their constituents and in what final form are responsible for the observed protective effects in vivo. In this work, on the background of different types of storage proteins, these deposited mainly in legumes were in the focus of interest. They were characterized on the example of pea (Pisum sativum proteins. Mechanisms associated with their biosynthesis and transport to specific cellular compartments was presented. Ways of their post-translational processing, segregation and storage in the specific vacuoles were also discussed. Therefore, the paper presents the state-of-the-art knowledge concerning the processes making the accumulated protein deposits ready to use by plants, animals and humans.

  5. A novel PNPLA2 mutation causes neutral lipid storage disease with myopathy (NLSDM) presenting muscular dystrophic features with lipid storage and rimmed vacuoles.

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    Chen, J; Hong, D; Wang, Z; Yuan, Y

    2010-01-01

    Neutral lipid storage disease with myopathy (NLSDM) is a type of lipid storage myopathy arising due to a mutation in the PNPLA2 gene encoding an adipose triglyceride lipase responsible for the degradation of intracellular triglycerides. Herein, we report the cases of two siblings manifesting slowly progressive proximal and distal limb weakness in adulthood. One of the patients had dilated cardiomyopathy, hearing loss and short stature. Muscle specimens of the 2 patients revealed muscular dystrophic features with massive lipid droplets and numerous rimmed vacuoles in the fibers. A novel homozygous mutation IVS2+1G > A in the PNPLA2 gene was identified in the 2 cases, but not in the healthy familial individuals. The presence of massive lipid droplets with muscular dystrophic changes and rimmed vacuoles in muscle fibers might be one of the characteristic pathological changes of NLSDM.

  6. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors required during Trypanosoma cruzi parasitophorous vacuole development.

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    Cueto, Juan Agustín; Vanrell, María Cristina; Salassa, Betiana Nebaí; Nola, Sébastien; Galli, Thierry; Colombo, María Isabel; Romano, Patricia Silvia

    2017-06-01

    Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular parasite that exploits different host vesicular pathways to invade the target cells. Vesicular and target soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are key proteins of the intracellular membrane fusion machinery. During the early times of T. cruzi infection, several vesicles are attracted to the parasite contact sites in the plasma membrane. Fusion of these vesicles promotes the formation of the parasitic vacuole and parasite entry. In this work, we study the requirement and the nature of SNAREs involved in the fusion events that take place during T. cruzi infection. Our results show that inhibition of N-ethylmaleimide-sensitive factor protein, a protein required for SNARE complex disassembly, impairs T. cruzi infection. Both TI-VAMP/VAMP7 and cellubrevin/VAMP3, two v-SNAREs of the endocytic and exocytic pathways, are specifically recruited to the parasitophorous vacuole membrane in a synchronized manner but, although VAMP3 is acquired earlier than VAMP7, impairment of VAMP3 by tetanus neurotoxin fails to reduce T. cruzi infection. In contrast, reduction of VAMP7 activity by expression of VAMP7's longin domain, depletion by small interfering RNA or knockout, significantly decreases T. cruzi infection susceptibility as a result of a minor acquisition of lysosomal components to the parasitic vacuole. In addition, overexpression of the VAMP7 partner Vti1b increases the infection, whereas expression of a KIF5 kinesin mutant reduces VAMP7 recruitment to vacuole and, concomitantly, T. cruzi infection. Altogether, these data support a key role of TI-VAMP/VAMP7 in the fusion events that culminate in the T. cruzi parasitophorous vacuole development. © 2016 John Wiley & Sons Ltd.

  7. Differential compartmentation of sucrose and gentianose in the cytosol and vacuoles of storage root protoplasts from Gentiana Lutea L.

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    Keller, F; Wiemken, A

    1982-12-01

    The storage roots of perennial Gentiana lutea L.plants contain several sugars. The predominant carbohydrate reserve is gentianose (β-D-glucopyranosyl-(1 → 6)-α-D-glucopyranosyl-(1 ↔ 2)-β-D-fructofuranoside). Vacuoles were isolated from root protoplasts and purified through a betaine density gradient. The yield was about 75%. Gentianose and gentiobiose were localized to 100% in the vacuoles, fructose and glucose to about 80%, and sucrose to only about 50%. Taking the volumes of the vacuolar and extravacuolar (cytosolic) compartments into account it is inferred that gentianose is located exclusively in the vacuoles, whilst sucrose is much more concentrated in the cytosol where it may play a role as a cryoprotectant. The concentration of fructose and glucose appeared to be similar on both sides of the tonoplast.

  8. Cell Vacuolation Caused by Vibrio cholerae Hemolysin

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    Figueroa-Arredondo, Paula; Heuser, John E.; Akopyants, Natalia S.; Morisaki, J. Hiroshi; Giono-Cerezo, Silvia; Enríquez-Rincón, Fernando; Berg, Douglas E.

    2001-01-01

    Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (∼1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (∼16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses. PMID:11179335

  9. Cell vacuolation caused by Vibrio cholerae hemolysin.

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    Figueroa-Arredondo, P; Heuser, J E; Akopyants, N S; Morisaki, J H; Giono-Cerezo, S; Enríquez-Rincón, F; Berg, D E

    2001-03-01

    Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemic V. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin of Aeromonas; it involved primarily the endoplasmic reticulum at early times (approximately 1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (approximately 16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. cholerae vacuolating activity was cloned and was found to correspond to hlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. cholerae strains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

  10. A Legionella pneumophila effector protein encoded in a region of genomic plasticity binds to Dot/Icm-modified vacuoles.

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    Shira Ninio

    2009-01-01

    Full Text Available Legionella pneumophila is an opportunistic pathogen that can cause a severe pneumonia called Legionnaires' disease. In the environment, L. pneumophila is found in fresh water reservoirs in a large spectrum of environmental conditions, where the bacteria are able to replicate within a variety of protozoan hosts. To survive within eukaryotic cells, L. pneumophila require a type IV secretion system, designated Dot/Icm, that delivers bacterial effector proteins into the host cell cytoplasm. In recent years, a number of Dot/Icm substrate proteins have been identified; however, the function of most of these proteins remains unknown, and it is unclear why the bacterium maintains such a large repertoire of effectors to promote its survival. Here we investigate a region of the L. pneumophila chromosome that displays a high degree of plasticity among four sequenced L. pneumophila strains. Analysis of GC content suggests that several genes encoded in this region were acquired through horizontal gene transfer. Protein translocation studies establish that this region of genomic plasticity encodes for multiple Dot/Icm effectors. Ectopic expression studies in mammalian cells indicate that one of these substrates, a protein called PieA, has unique effector activities. PieA is an effector that can alter lysosome morphology and associates specifically with vacuoles that support L. pneumophila replication. It was determined that the association of PieA with vacuoles containing L. pneumophila requires modifications to the vacuole mediated by other Dot/Icm effectors. Thus, the localization properties of PieA reveal that the Dot/Icm system has the ability to spatially and temporally control the association of an effector with vacuoles containing L. pneumophila through activities mediated by other effector proteins.

  11. Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation

    Czech Academy of Sciences Publication Activity Database

    Liu, Q.; Vain, T.; Viotti, C.; Doyle, S. M.; Tarkowská, Danuše; Novák, Ondřej; Zipfel, C.; Sitbon, F.; Robert, S.; Hofius, D.

    2018-01-01

    Roč. 11, č. 4 (2018), s. 553-567 ISSN 1674-2052 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Arabidopsis * brassinosteroid signaling * DUF300 proteins * tonoplast * vacuole integrity Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Plant sciences, botany Impact factor: 8.827, year: 2016

  12. Characteristics of weak base-induced vacuoles formed around individual acidic organelles.

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    Hiruma, Hiromi; Kawakami, Tadashi

    2011-01-01

    We have previously found that the weak base 4-aminopyridine induces Brownian motion of acidic organelles around which vacuoles are formed, causing organelle traffic disorder in neurons. Our present study investigated the characteristics of vacuoles induced by weak bases (NH(4)Cl, aminopyridines, and chloroquine) using mouse cells. Individual vacuoles included acidic organelles identified by fluorescent protein expression. Mitochondria and actin filaments were extruded outside the vacuoles, composing the vacuole rim. Staining with amine-reactive fluorescence showed no protein/amino acid content in vacuoles. Thus, serous vacuolar contents are probably partitioned by viscous cytosol, other organelles, and cytoskeletons, but not membrane. The weak base (chloroquine) was immunochemically detected in intravacuolar organelles, but not in vacuoles. Early vacuolization was reversible, but long-term vacuolization caused cell death. The vacuolization and cell death were blocked by the vacuolar H(+)-ATPase inhibitor and Cl--free medium. Staining with LysoTracker or LysoSensor indicated that intravacuolar organelles were strongly acidic and vacuoles were slightly acidic. This suggests that vacuolization is caused by accumulation of weak base and H(+) in acidic organelles, driven by vacuolar H(+)-ATPase associated with Cl(-) entering, and probably by subsequent extrusion of H(+) and water from organelles to the surrounding cytoplasm.

  13. Rimmed vacuoles in Becker muscular dystrophy have similar features with inclusion myopathies.

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    Momma, Kazunari; Noguchi, Satoru; Malicdan, May Christine V; Hayashi, Yukiko K; Minami, Narihiro; Kamakura, Keiko; Nonaka, Ikuya; Nishino, Ichizo

    2012-01-01

    Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

  14. Rimmed vacuoles in Becker muscular dystrophy have similar features with inclusion myopathies.

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    Kazunari Momma

    Full Text Available Rimmed vacuoles in myofibers are thought to be due to the accumulation of autophagic vacuoles, and can be characteristic in certain myopathies with protein inclusions in myofibers. In this study, we performed a detailed clinical, molecular, and pathological characterization of Becker muscular dystrophy patients who have rimmed vacuoles in muscles. Among 65 Becker muscular dystrophy patients, we identified 12 patients who have rimmed vacuoles and 11 patients who have deletions in exons 45-48 in DMD gene. All patients having rimmed vacuoles showed milder clinical features compared to those without rimmed vacuoles. Interestingly, the rimmed vacuoles in Becker muscular dystrophy muscles seem to represent autophagic vacuoles and are also associated with polyubiquitinated protein aggregates. These findings support the notion that rimmed vacuoles can appear in Becker muscular dystrophy, and may be related to the chronic changes in muscle pathology induced by certain mutations in the DMD gene.

  15. Effector Protein Cig2 Decreases Host Tolerance of Infection by Directing Constitutive Fusion of Autophagosomes with the Coxiella-Containing Vacuole

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    Lara J. Kohler

    2016-07-01

    Full Text Available Coxiella burnetii replicates in an acidified lysosome-derived vacuole. Biogenesis of the Coxiella-containing vacuole (CCV requires bacterial effector proteins delivered into host cells by the Dot/Icm secretion system. Genetic and cell biological analysis revealed that an effector protein called Cig2 promotes constitutive fusion of autophagosomes with the CCV to maintain this compartment in an autolysosomal stage of maturation. This distinguishes the CCV from other pathogen-containing vacuoles that are targeted by the host autophagy pathway, which typically confers host resistance to infection by delivering the pathogen to a toxic lysosomal environment. By maintaining the CCV in an autolysosomal stage of maturation, Cig2 enabled CCV homotypic fusion and enhanced bacterial virulence in the Galleria mellonella (wax moth model of infection by a mechanism that decreases host tolerance. Thus, C. burnetii residence in an autolysosomal organelle alters host tolerance of infection, which indicates that Cig2-dependent manipulation of a lysosome-derived vacuole influences the host response to infection.

  16. Autophagy-Related Direct Membrane Import from ER/Cytoplasm into the Vacuole or Apoplast: A Hidden Gateway also for Secondary Metabolites and Phytohormones?

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    Ivan Kulich

    2014-04-01

    Full Text Available Transportation of low molecular weight cargoes into the plant vacuole represents an essential plant cell function. Several lines of evidence indicate that autophagy-related direct endoplasmic reticulum (ER to vacuole (and also, apoplast transport plays here a more general role than expected. This route is regulated by autophagy proteins, including recently discovered involvement of the exocyst subcomplex. Traffic from ER into the vacuole bypassing Golgi apparatus (GA acts not only in stress-related cytoplasm recycling or detoxification, but also in developmentally-regulated biopolymer and secondary metabolite import into the vacuole (or apoplast, exemplified by storage proteins and anthocyanins. We propose that this pathway is relevant also for some phytohormones’ (e.g., auxin, abscisic acid (ABA and salicylic acid (SA degradation. We hypothesize that SA is not only an autophagy inducer, but also a cargo for autophagy-related ER to vacuole membrane container delivery and catabolism. ER membrane localized enzymes will potentially enhance the area of biosynthetic reactive surfaces, and also, abundant ER localized membrane importers (e.g., ABC transporters will internalize specific molecular species into the autophagosome biogenesis domain of ER. Such active ER domains may create tubular invaginations of tonoplast into the vacuoles as import intermediates. Packaging of cargos into the ER-derived autophagosome-like containers might be an important mechanism of vacuole and exosome biogenesis and cytoplasm protection against toxic metabolites. A new perspective on metabolic transformations intimately linked to membrane trafficking in plants is emerging.

  17. pH measurement of tubular vacuoles of an arbuscular mycorrhizal fungus, Gigaspora margarita.

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    Funamoto, Rintaro; Saito, Katsuharu; Oyaizu, Hiroshi; Aono, Toshihiro; Saito, Masanori

    2015-01-01

    Arbuscular mycorrhizal fungi play an important role in phosphate supply to the host plants. The fungal hyphae contain tubular vacuoles where phosphate compounds such as polyphosphate are accumulated. Despite their importance for the phosphate storage, little is known about the physiological properties of the tubular vacuoles in arbuscular mycorrhizal fungi. As an indicator of the physiological state in vacuoles, we measured pH of tubular vacuoles in living hyphae of arbuscular mycorrhizal fungus Gigaspora margarita using ratio image analysis with pH-dependent fluorescent probe, 6-carboxyfluorescein. Fluorescent images of the fine tubular vacuoles were obtained using a laser scanning confocal microscope, which enabled calculation of vacuolar pH with high spatial resolution. The tubular vacuoles showed mean pH of 5.6 and a pH range of 5.1-6.3. These results suggest that the tubular vacuoles of arbuscular mycorrhizal fungi have a mildly acidic pH just like vacuoles of other fungal species including yeast and ectomycorrhizal fungi.

  18. Delivering of Proteins to the Plant Vacuole—An Update

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    Cláudia Pereira

    2014-05-01

    Full Text Available Trafficking of soluble cargo to the vacuole is far from being a closed issue as it can occur by different routes and involve different intermediates. The textbook view of proteins being sorted at the post-Golgi level to the lytic vacuole via the pre-vacuole or to the protein storage vacuole mediated by dense vesicles is now challenged as novel routes are being disclosed and vacuoles with intermediate characteristics described. The identification of Vacuolar Sorting Determinants is a key signature to understand protein trafficking to the vacuole. Despite the long established vacuolar signals, some others have been described in the last few years, with different properties that can be specific for some cells or some types of vacuoles. There are also reports of proteins having two different vacuolar signals and their significance is questionable: a way to increase the efficiency of the sorting or different sorting depending on the protein roles in a specific context? Along came the idea of differential vacuolar sorting, suggesting a possible specialization of the trafficking pathways according to the type of cell and specific needs. In this review, we show the recent advances in the field and focus on different aspects of protein trafficking to the vacuoles.

  19. Formation of the food vacuole in Plasmodium falciparum: a potential role for the 19 kDa fragment of merozoite surface protein 1 (MSP1(19.

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    Anton R Dluzewski

    2008-08-01

    Full Text Available Plasmodium falciparum Merozoite Surface Protein 1 (MSP1 is synthesized during schizogony as a 195-kDa precursor that is processed into four fragments on the parasite surface. Following a second proteolytic cleavage during merozoite invasion of the red blood cell, most of the protein is shed from the surface except for the C-terminal 19-kDa fragment (MSP1(19, which is still attached to the merozoite via its GPI-anchor. We have examined the fate of MSP1(19 during the parasite's subsequent intracellular development using immunochemical analysis of metabolically labeled MSP1(19, fluorescence imaging, and immuno-electronmicroscopy. Our data show that MSP1(19 remains intact and persists to the end of the intracellular cycle. This protein is the first marker for the biogenesis of the food vacuole; it is rapidly endocytosed into small vacuoles in the ring stage, which coalesce to form the single food vacuole containing hemozoin, and persists into the discarded residual body. The food vacuole is marked by the presence of both MSP1(19 and the chloroquine resistance transporter (CRT as components of the vacuolar membrane. Newly synthesized MSP1 is excluded from the vacuole. This behavior indicates that MSP1(19 does not simply follow a classical lysosome-like clearance pathway, instead, it may play a significant role in the biogenesis and function of the food vacuole throughout the intra-erythrocytic phase.

  20. The vacuole within

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    Ellis, Kathryn; Hoffman, Brenton D.; Bagnat, Michel

    2013-01-01

    The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuoles results in a shortened embryo along the anterior-posterior axis. Interestingly, we discovered that notochord vacuoles are also essential for proper spine morphogenesis and that vacuole defects lead to scoliosis of the spine. Here we discuss the cellular organization of the notochord and how key features of its architecture allow the notochord to function in embryonic axis elongation and spine formation. PMID:23887209

  1. A Rab-centric perspective of bacterial pathogen-occupied vacuoles.

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    Sherwood, Racquel Kim; Roy, Craig R

    2013-09-11

    The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

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    Coreen Johnson

    Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

  3. Identification of genes affecting vacuole membrane fragmentation in Saccharomyces cerevisiae.

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    Lydie Michaillat

    Full Text Available The equilibrium of membrane fusion and fission influences the volume and copy number of organelles. Fusion of yeast vacuoles has been well characterized but their fission and the mechanisms determining vacuole size and abundance remain poorly understood. We therefore attempted to systematically characterize factors necessary for vacuole fission. Here, we present results of an in vivo screening for deficiencies in vacuolar fragmentation activity of an ordered collection deletion mutants, representing 4881 non-essential genes of the yeast Saccharomyces cerevisiae. The screen identified 133 mutants with strong defects in vacuole fragmentation. These comprise numerous known fragmentation factors, such as the Fab1p complex, Tor1p, Sit4p and the V-ATPase, thus validating the approach. The screen identified many novel factors promoting vacuole fragmentation. Among those are 22 open reading frames of unknown function and three conspicuous clusters of proteins with known function. The clusters concern the ESCRT machinery, adaptins, and lipases, which influence the production of diacylglycerol and phosphatidic acid. A common feature of these factors of known function is their capacity to change membrane curvature, suggesting that they might promote vacuole fragmentation via this property.

  4. A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells

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    Marianna Faraco

    2017-06-01

    Full Text Available It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

  5. Pleiotropic Actions of Helicobacter pylori Vacuolating Cytotoxin, VacA

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    Isomoto, Hajime; Moss, Joel; Hirayama, Toshiya

    2010-01-01

    Helicobacter pylori produces a vacuolating cytotoxin, VacA, and most virulent H. pylori strains secrete VacA. VacA binds to two types of receptor-like protein tyrosine phosphatase (RPTP), RPTPα and RPTPβ, on the surface of host cells. VacA bound to RPTPβ, relocates and concentrates in lipid rafts in the plasma membrane. VacA causes vacuolization, membrane anion-selective channel and pore formation, and disruption of endosomal and lysosomal activity in host cells. Secreted VacA is processed in...

  6. Determination of Glutathione and Its Redox Status in Isolated Vacuoles of Red Beetroot Cells

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    E.V. Pradedova

    2016-02-01

    Full Text Available The glutathione of the red beetroot vacuoles (Beta vulgaris L. was measured using three well-known methods: the spectrofluorimetric method with orthophthalic aldehyde (OPT; the spectrophotometric method with 5.5'-dithiobis-2-nitrobenzoic acid (DTNB; the high-performance liquid chromatography (HPLC. The content of reduced (GSH and oxidized glutathione (GSSG differed depending on the research method. With OPT the concentration of glutathione was: GSH – 0.059 µmol /mg protein; GSSG – 0.019 µmol/mg protein and total glutathione (GSHtotal – 0.097 µmol/mg protein. In the case of determining with DTNB the concentration of glutathione was: GSH – 0.091 µmol/mg protein; GSSG – 0.031 µmol/mg protein; GSHtotal – 0.153 µmol/mg protein. HPLC-defined concentration of glutathione was lower: GSH – 0.039 µmol/mg protein; GSSG – 0.007 µmol/mg protein; GSHtotal – 0.053 µmol/mg protein. Redox ratio of GSH/GSSG was also dependent on the method of determination: with OPT – 3.11; with DTNB – 2.96 and HPLC – 5.57. Redox ratio of glutathione in vacuoles was much lower than the tissue extracts of red beetroot, which, depending on the method of determination, was: 7.23, 7.16 and 9.22. The results showed the vacuoles of red beetroot parenchyma cells contain glutathione. Despite the low value of the redox ratio GSH/GSSG, in vacuoles the pool of reduced glutathione prevailed over the pool of oxidized glutathione.

  7. Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

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    Hayley J Newton

    Full Text Available The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into

  8. Mucolipin co-deficiency causes accelerated endolysosomal vacuolation of enterocytes and failure-to-thrive from birth to weaning.

    Directory of Open Access Journals (Sweden)

    Natalie N Remis

    2014-12-01

    Full Text Available During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3-/-;Trpml1-/- vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3-/-;Trpml1-/- mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns

  9. Mucolipin Co-deficiency Causes Accelerated Endolysosomal Vacuolation of Enterocytes and Failure-to-Thrive from Birth to Weaning

    Science.gov (United States)

    Castiglioni, Andrew J.; Flores, Emma N.; Cantú, Jorge A.; García-Añoveros, Jaime

    2014-01-01

    During the suckling period, intestinal enterocytes are richly endowed with endosomes and lysosomes, which they presumably utilize for the uptake and intracellular digestion of milk proteins. By weaning, mature intestinal enterocytes replace those rich in lysosomes. We found that mouse enterocytes before weaning express high levels of two endolysosomal cation channels, mucolipins 3 and 1 -products of Trpml3 and Trpml1 genes; moreover neonatal enterocytes of mice lacking both mucolipins (Trpml3−/−;Trpml1−/−) vacuolated pathologically within hours of birth and remained so until weaning. Ultrastructurally and chemically these fast-forming vacuoles resembled those that systemically appear in epithelial cells of mucolipidosis type IV (MLIV) patients, which bear mutations in Trpml1. Hence, lack of both mucolipins 1 and 3 causes an accelerated MLIV-type of vacuolation in enterocytes. The vacuoles were aberrant hybrid organelles with both endosomal and lysosomal components, and were not generated by alterations in endocytosis or exocytosis, but likely by an imbalance between fusion of lysosomes and endosomes and their subsequent scission. However, upon extensive vacuolation enterocytes displayed reduced endocytosis from the intestinal lumen, a defect expected to compromise nutrient uptake. Mice lacking both mucolipins suffered a growth delay that began after birth and continued through the suckling period but recovered after weaning, coinciding with the developmental period of enterocyte vacuolation. Our results demonstrate genetic redundancy between lysosomal mucolipins 3 and 1 in neonatal enterocytes. Furthermore, our Trpml3−/−;Trpml1−/− mice represent a polygenic animal model of the poorly-understood, and often intractable, neonatal failure-to-thrive with intestinal pathology. Our results implicate lysosomes in neonatal intestinal pathologies, a major cause of infant mortality worldwide, and suggest transient intestinal dysfunction might affect newborns

  10. Tuber storage proteins.

    Science.gov (United States)

    Shewry, Peter R

    2003-06-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits activity as an acylhydrolase and esterase, sporamin from sweet potato is an inhibitor of trypsin, and dioscorin from yam is a carbonic anhydrase. Both sporamin and dioscorin also exhibit antioxidant and radical scavenging activity. Taro differs from the other three crops in that it contains two major types of storage protein: a trypsin inhibitor related to sporamin and a mannose-binding lectin. These characteristics indicate that tuber storage proteins have evolved independently in different species, which contrasts with the highly conserved families of storage proteins present in seeds. Furthermore, all exhibit biological activities which could contribute to resistance to pests, pathogens or abiotic stresses, indicating that they may have dual roles in the tubers.

  11. Neuronal vacuolation and spinocerebellar degeneration associated with altered neurotransmission

    Directory of Open Access Journals (Sweden)

    Aggeliki Giannakopoulou

    2017-06-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species, such as the domestic dog (Canis lupus familiaris, and many are caused by mutations in the same genes as corresponding human conditions. In the present study, we report an inherited neurodegenerative condition, termed ‘neuronal vacuolation and spinocerebellar degeneration’ (NVSD which affects neonatal or young dogs, mainly Rottweilers, which recently has been linked with the homozygosity for the RAB3GAP1:c.743delC allele. Mutations in human RAB3GAP1 cause Warburg micro syndrome (WARBM, a severe developmental disorder characterized predominantly by abnormalities of the nervous system including axonal peripheral neuropathy. RAB3GAP1 encodes the catalytic subunit of a GTPase activator protein and guanine exchange factor for Rab3 and Rab18 proteins, respectively. Rab proteins are involved in membrane trafficking in the endoplasmic reticulum, autophagy, axonal transport and synaptic transmission. The present study attempts to carry out a detailed histopathological examination of NVSD disease, extending from peripheral nerves to lower brain structures focusing on the neurotransmitter alterations noted in the cerebellum, the major structure affected. NVSD dogs presented with progressive cerebellar ataxia and some clinical manifestations that recapitulate the WARBM phenotype. Neuropathological examination revealed dystrophic axons, neurodegeneration and intracellular vacuolization in specific nuclei. In the cerebellum, severe vacuolation of cerebellar nuclei neurons, atrophy of Purkinje cells, and diminishing of GABAergic and glutamatergic fibres constitute the most striking lesions. The balance of evidence suggests that the neuropathological lesions are a reaction to the altered neurotransmission. The canine phenotype could serve as a model to delineate the disease-causing pathological mechanisms in RAB3GAP1 mutation.

  12. THE TONOPLAST TRANSPORT SYSTEMS OF PLANT VACUOLES AND THEIR POTENTIAL APPLICATION IN BIOTECHNOLOGY

    Directory of Open Access Journals (Sweden)

    S. V. Isayenkov

    2013-06-01

    Full Text Available The pivotal role of plant vacuoles in plant survival was discussed in the review. Particularly, the providing of cellular turgor, accumulation of inorganic osmolytes and nutrients are the primary tasks of these cellular organelles. The main mechanisms of tonoplast transport systems were described. The known transport pathways of minerals, heavy metals, vitamins and other organic compounds were classified and outlined. The main systems of membrane vacuolar transport were reviewed. The outline of the physiological functions and features of vacuolar membrane transport proteins were performed. The physiological role of transport of minerals, nutrients and other compounds into vacuoles were discussed. This article reviews the main types of plant vacuoles and their functional role in plant cell. Current state and progress in vacuolar transport research was outlined. The examples of application for rinciples and mechanisms of vacuolar membrane transport in plant biotechnology were iven. The perspectives and approaches in plant and food biotechnology concerning transport and physiology of vacuoles are discussed.

  13. Thioploca spp: filamentous sulfur bacteria with nitrate vacuoles

    DEFF Research Database (Denmark)

    Jørgensen, BB; Gallardo, VA

    1999-01-01

    communities of large Thioploca species live along the Pacific coast of South America and in other upwelling areas of high organic matter sedimentation with bottom waters poor in oxygen and rich in nitrate. Each cell of these thioplocas harbors a large liquid vacuole which is used as a storage for nitrate...... with a concentration of lip to 506 mM. The nitrate is used as an electron acceptor for sulfide oxidation and the bacteria may grow autotrophically or mixotrophically using acetate or other organic molecules as carbon source. The filaments stretch up into the overlying seawater, from which they take up nitrate...

  14. The trafficking pathway of a wheat storage protein in transgenic rice endosperm.

    Science.gov (United States)

    Oszvald, Maria; Tamas, Laszlo; Shewry, Peter R; Tosi, Paola

    2014-04-01

    The trafficking of proteins in the endoplasmic reticulum (ER) of plant cells is a topic of considerable interest since this organelle serves as an entry point for proteins destined for other organelles, as well as for the ER itself. In the current work, transgenic rice was used to study the pattern and pathway of deposition of the wheat high molecular weight (HMW) glutenin sub-unit (GS) 1Dx5 within the rice endosperm using specific antibodies to determine whether it is deposited in the same or different protein bodies from the rice storage proteins, and whether it is located in the same or separate phases within these. The protein distribution and the expression pattern of HMW sub-unit 1Dx5 in transgenic rice endosperm at different stages of development were determined using light and electron microscopy after labelling with antibodies. The use of HMW-GS-specific antibodies showed that sub-unit 1Dx5 was expressed mainly in the sub-aleurone cells of the endosperm and that it was deposited in both types of protein body present in the rice endosperm: derived from the ER and containing prolamins, and derived from the vacuole and containing glutelins. In addition, new types of protein bodies were also formed within the endosperm cells. The results suggest that the HMW 1Dx5 protein could be trafficked by either the ER or vacuolar pathway, possibly depending on the stage of development, and that its accumulation in the rice endosperm could compromise the structural integrity of protein bodies and their segregation into two distinct populations in the mature endosperm.

  15. Proglobulin processing enzyme in vacuoles isolated from developing pumpkin cotyledons

    International Nuclear Information System (INIS)

    Hara-Nishimura, I.; Nishimura, M.

    1987-01-01

    The enzymic conversion of proglobulin to globulin catalyzed by the extracts of vacuoles isolated from developing pumpkin (Cucurbita sp. cv Kurokawa Amakuri Nankin) cotyledons was investigated. The endoplasmic reticulum fraction isolated from the developing cotyledons pulse-labeled with [ 35 S]methionine was shown to contain mainly the radiolabeled proglobulin, which was used as a substrate for assaying the proteolytic processing in vitro. The vacuolar extracts catalyzed the proteolytic processing of the proglobulin molecule to produce globulin containing two kinds of polypeptide chains, γ and δ. The pH optimum for the vacuole-mediated conversion was at pH 5.0. The proteolytic processing of proglobulin by the vacuolar extracts was inhibited in the presence of various thiol reagents, e.g. p-chloromercuribenzoate, N-ethylmaleimide, iodoacetic acid, Hg 2+ , and Cu 2+ , but not phenylmethylsulfonyl fluoride, EDTA, o-phenanthroline, leupeptin, antipain, pepstatin, chymostatin, or pumpkin trypsin inhibitor, and was activated in the presence of dithiothreitol and cysteine, indicating that the processing enzyme is a thiol protease. The suborganellar fractionation of the vacuoles showed that the processing activity was localized in the matrix fraction, but not in the membrane or crystalloid fractions. During the seed development, the enzyme was shown to increase, exhibiting the maximal activity at the late developmental stage. The matrix fraction of the protein bodies isolated from the dry castor bean (Ricinus communis) exhibited the processing activity toward the pumpkin proglobulin molecules in the same manner as that by the matrix fraction of pumpkin vacuoles

  16. Shigella subverts the host recycling compartment to rupture its vacuole.

    Science.gov (United States)

    Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

    2014-10-08

    Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. In vivo inhibition of cysteine proteases provides evidence for the involvement of 'senescence-associated vacuoles' in chloroplast protein degradation during dark-induced senescence of tobacco leaves.

    Science.gov (United States)

    Carrión, Cristian A; Costa, María Lorenza; Martínez, Dana E; Mohr, Christina; Humbeck, Klaus; Guiamet, Juan J

    2013-11-01

    Breakdown of leaf proteins, particularly chloroplast proteins, is a massive process in senescing leaves. In spite of its importance in internal N recycling, the mechanism(s) and the enzymes involved are largely unknown. Senescence-associated vacuoles (SAVs) are small, acidic vacuoles with high cysteine peptidase activity. Chloroplast-targeted proteins re-localize to SAVs during senescence, suggesting that SAVs might be involved in chloroplast protein degradation. SAVs were undetectable in mature, non-senescent tobacco leaves. Their abundance, visualized either with the acidotropic marker Lysotracker Red or by green fluorescent protein (GFP) fluorescence in a line expressing the senescence-associated cysteine protease SAG12 fused to GFP, increased during senescence induction in darkness, and peaked after 2-4 d, when chloroplast dismantling was most intense. Increased abundance of SAVs correlated with higher levels of SAG12 mRNA. Activity labelling with a biotinylated derivative of the cysteine protease inhibitor E-64 was used to detect active cysteine proteases. The two apparently most abundant cysteine proteases of senescing leaves, of 40kDa and 33kDa were detected in isolated SAVs. Rubisco degradation in isolated SAVs was completely blocked by E-64. Treatment of leaf disks with E-64 in vivo substantially reduced degradation of Rubisco and leaf proteins. Overall, these results indicate that SAVs contain most of the cysteine protease activity of senescing cells, and that SAV cysteine proteases are at least partly responsible for the degradation of stromal proteins of the chloroplast.

  18. Initiation and elimination of vacuoles in microencapsulated shells

    International Nuclear Information System (INIS)

    Du Kai; You Dan

    2000-01-01

    Two mechanisms of vacuole formation in microencapsulated micro-shells wall are introduced. It is verified that phase separation of trace amount of water in the organic solvent is the most possible course of vacuole formation

  19. Cytoplasmic vacuolation in cultured rat astrocytes induced by an organophosphorus agent requires extracellular signal-regulated kinase activation

    International Nuclear Information System (INIS)

    Isobe, Ichiro; Maeno, Yoshitaka; Nagao, Masataka; Iwasa, Mineo; Koyama, Hiroyoshi; Seko-Nakamura, Yoshimi; Monma-Ohtaki, Jun

    2003-01-01

    There are various toxic chemicals that cause cell death. However, in certain cases deleterious agents elicit various cellular responses prior to cell death. To determine the cellular mechanisms by which such cellular responses are induced is important, but sufficient attention has not been paid to this issue to date. In this study, we showed the characteristic effects of an organophosphorus (OP) agent, bis(pinacolyl methyl)phosphonate (BPMP), which we synthesized for the study of OP nerve agents, on cultured rat astrocytes. Morphologically, BPMP induced cytoplasmic vacuolation and stellation in the rat astrocytes. Cytoplasmic vacuolation is a cell pathological change observed, for example, in vacuolar degeneration, and stellation has been reported in astrocytic reactions against various stimuli. By pretreatment with cycloheximide, a protein synthesis inhibitor, stellation was inhibited, although vacuolation was not. Cell staining with a mitochondrion-selective dye indicated that the vacuolation probably occurs in the mitochondria that are swollen and vacuolatred in the center. Interestingly, the extracellular signal-regulated kinase (ERK) cascade inhibitor inhibited vacuolation and, to some extent, stellation. These results suggest that the ERK signaling cascade is important for the induction of mitochondrial vacuolation. We expect that a detailed study of these astrocytic reactions will provide us new perspectives regarding the variation and pathological significance of cell morphological changes, such as vacuolar degeneration, and also the mechanisms underlying various neurological disorders

  20. The vacuole within: How cellular organization dictates notochord function

    OpenAIRE

    Ellis, Kathryn; Hoffman, Brenton D.; Bagnat, Michel

    2013-01-01

    The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuole...

  1. The relationship between vacuolation and initiation of PCD in rice (Oryza sativa) aleurone cells

    Science.gov (United States)

    Zheng, Yan; Zhang, Heting; Deng, Xiaojiang; Liu, Jing; Chen, Huiping

    2017-01-01

    Vacuole fusion is a necessary process for the establishment of a large central vacuole, which is the central location of various hydrolytic enzymes and other factors involved in death at the beginning of plant programmed cell death (PCD). In our report, the fusion of vacuoles has been presented in two ways: i) small vacuoles coalesce to form larger vacuoles through membrane fusion, and ii) larger vacuoles combine with small vacuoles when small vacuoles embed into larger vacuoles. Regardless of how fusion occurs, a large central vacuole is formed in rice (Oryza sativa) aleurone cells. Along with the development of vacuolation, the rupture of the large central vacuole leads to the loss of the intact plasma membrane and the degradation of the nucleus, resulting in cell death. Stabilizing or disrupting the structure of actin filaments (AFs) inhibits or promotes the fusion of vacuoles, which delays or induces PCD. In addition, the inhibitors of the vacuolar processing enzyme (VPE) and cathepsin B (CathB) block the occurrence of the large central vacuole and delay the progression of PCD in rice aleurone layers. Overall, our findings provide further evidence for the rupture of the large central vacuole triggering the PCD in aleruone layers.

  2. Pumping up the volume - vacuole biogenesis in Arabidopsis thaliana.

    Science.gov (United States)

    Krüger, Falco; Schumacher, Karin

    2017-07-08

    Plant architecture follows the need to collect CO 2, solar energy, water and mineral nutrients via large surface areas. It is by the presence of a central vacuole that fills much of the cell volume that plants manage to grow at low metabolic cost. In addition vacuoles buffer the fluctuating supply of essential nutrients and help to detoxify the cytosol when plants are challenged by harmful molecules. Despite their large size and multiple important functions, our knowledge of vacuole biogenesis and the machinery underlying their amazing dynamics is still fragmentary. In this review, we try to reconcile past and present models for vacuole biogenesis with the current knowledge of multiple parallel vacuolar trafficking pathways and the molecular machineries driving membrane fusion and organelle shape. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Molecular markers for granulovacuolar degeneration are present in rimmed vacuoles.

    Directory of Open Access Journals (Sweden)

    Masahiro Nakamori

    Full Text Available BACKGROUND: Rimmed vacuoles (RVs are round-oval cytoplasmic inclusions, detected in muscle cells of patients with myopathies, such as inclusion body myositis (IBM and distal myopathy with RVs (DMRV. Granulovacuolar degeneration (GVD bodies are spherical vacuoles containing argentophilic and hematoxyphilic granules, and are one of the pathological hallmarks commonly found in hippocampal pyramidal neurons of patients with aging-related neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. These diseases are common in the elderly and share some pathological features. Therefore, we hypothesized that mechanisms of vacuolar formation in RVs and GVD bodies are common despite their role in two differing pathologies. We explored the components of RVs by immunohistochemistry, using antibodies for GVD markers. METHODS: Subjects included one AD case, eight cases of sporadic IBM, and three cases of DMRV. We compared immunoreactivity and staining patterns for GVD markers. These markers included: (1 tau-modifying proteins (caspase 3, cyclin-dependent kinase 5 [CDK5], casein kinase 1δ [CK1δ], and c-jun N-terminal kinase [JNK], (2 lipid raft-associated materials (annexin 2, leucine-rich repeat kinase 2 [LRRK2], and flotillin-1, and (3 other markers (charged multi-vesicular body protein 2B [CHMP2B] and phosphorylated transactive response DNA binding protein-43 [pTDP43] in both GVD bodies and RVs. Furthermore, we performed double staining of each GVD marker with pTDP43 to verify the co-localization. RESULTS: GVD markers, including lipid raft-associated proteins and tau kinases, were detected in RVs. CHMP2B, pTDP43, caspase 3, LRRK2, annexin 2 and flotillin-1 were detected on the rim and were diffusely distributed in the cytoplasm of RV-positive fibers. CDK5, CK1δ and JNK were detected only on the rim. In double staining experiments, all GVD markers colocalized with pTDP43 in RVs. CONCLUSIONS: These results suggest that RVs of muscle

  4. Essential domain of receptor tyrosine phosphatase beta (RPTPbeta) for interaction with Helicobacter pylori vacuolating cytotoxin

    DEFF Research Database (Denmark)

    Yahiro, Kinnosuke; Wada, Akihiro; Yamasaki, Eiki

    2004-01-01

    Helicobacter pylori produces a potent exotoxin, VacA, which causes progressive vacuolation as well as gastric injury. Although VacA was able to interact with two receptor-like protein tyrosine phosphatases, RPTPbeta and RPTPalpha, RPTPbeta was found to be responsible for gastric damage caused...

  5. The rapid isolation of vacuoles from leaves of crassulacean Acid metabolism plants.

    Science.gov (United States)

    Kringstad, R; Kenyon, W H; Black, C C

    1980-09-01

    A technique is presented for the isolation of vacuoles from Sedum telephium L. leaves. Leaf material is digested enzymically to produce protoplasts rapidly which are partially lysed by gentle osmotic shock and the inclusion of 5 millimolar ethyleneglycol-bis (beta-aminoethyl ether)N,N'-tetraacetic acid in the wash medium. Vacuoles are isolated from the partially lysed protoplasts by brief centrifugation on a three-step Ficoll-400 gradient consisting of 5, 10, and 15% (w/v) Ficoll-400. A majority of the vacuoles accumulate at the 5 to 10% Ficoll interface, whereas a smaller proportion sediments at the 10 to 15% Ficoll-400 interface. The total time required for vacuole isolation is 2 to 2.5 hours, beginning from leaf harvest.The yield of vacuoles is approximately 44%. The major vacuole layer is 15 hours when left in Ficoll; however, dispersion into media of various osmotic concentrations resulted in decreased stability. Addition of mercaptobenzothiazole, CaCl(2), MgCl(2), bovine serum albumin, ethylenediaminetetraacetic acid, polyethylene glycol 600, and KH(2)PO(4) to the vacuole isolation media did not increase the stability of the isolated vacuoles.THIS TECHNIQUE WITH ONLY SLIGHT MODIFICATIONS HAS BEEN USED TO ISOLATE LEAF CELL VACUOLES FROM THE FOLLOWING CRASSULACEAN ACID METABOLISM PLANTS: pineapple, Kalanchoë fedtschenkoi, and Echeveria elegans. Spinach leaves also were used successfully.

  6. Hydrolytic enzymes in the central vacuole of plant cells.

    Science.gov (United States)

    Boller, T; Kende, H

    1979-06-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. (a) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. (b) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. (c) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated.THE INTRACELLULAR ACTIVITIES OF THE FOLLOWING ACID HYDROLASES WERE PRIMARILY LOCALIZED IN THE VACUOLE OF TOBACCO CELLS: alpha-mannosidase, beta-N-acetylglucosaminidase, beta-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar

  7. Increasing cyclic electron flow is related to Na+ sequestration into vacuoles for salt tolerance in soybean.

    Science.gov (United States)

    He, Yi; Fu, Junliang; Yu, Chenliang; Wang, Xiaoman; Jiang, Qinsu; Hong, Jian; Lu, Kaixing; Xue, Gangping; Yan, Chengqi; James, Andrew; Xu, Ligen; Chen, Jianping; Jiang, Dean

    2015-11-01

    In land plants, the NAD(P)H dehydrogenase (NDH) complex reduces plastoquinones and drives cyclic electron flow (CEF) around PSI. It also produces extra ATP for photosynthesis and improves plant fitness under conditions of abiotic environmental stress. To elucidate the role of CEF in salt tolerance of the photosynthetic apparatus, Na(+) concentration, chlorophyll fluorescence, and expression of NDH B and H subunits, as well as of genes related to cellular and vacuolar Na(+) transport, were monitored. The salt-tolerant Glycine max (soybean) variety S111-9 exhibited much higher CEF activity and ATP accumulation in light than did the salt-sensitive variety Melrose, but similar leaf Na(+) concentrations under salt stress. In S111-9 plants, ndhB and ndhH were highly up-regulated under salt stress and their corresponding proteins were maintained at high levels or increased significantly. Under salt stress, S111-9 plants accumulated Na(+) in the vacuole, but Melrose plants accumulated Na(+) in the chloroplast. Compared with Melrose, S111-9 plants also showed higher expression of some genes associated with Na(+) transport into the vacuole and/or cell, such as genes encoding components of the CBL10 (calcineurin B-like protein 10)-CIPK24 (CBL-interacting protein kinase 24)-NHX (Na(+)/H(+) antiporter) and CBL4 (calcineurin B-like protein 4)-CIPK24-SOS1 (salt overly sensitive 1) complexes. Based on the findings, it is proposed that enhanced NDH-dependent CEF supplies extra ATP used to sequester Na(+) in the vacuole. This reveals an important mechanism for salt tolerance in soybean and provides new insights into plant resistance to salt stress. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  8. Hydrolytic enzymes in the central vacuole of plant cells

    International Nuclear Information System (INIS)

    Boller, T.; Kende, H.

    1979-01-01

    The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast: (a) purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation; (b) hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation; and (c) vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β-N-acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. None of the vacuolar enzymes investigated ws found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter

  9. Formation, function, and exhaustion of notochordal cytoplasmic vacuoles within intervertebral disc: current understanding and speculation

    Science.gov (United States)

    Sinkemani, Arjun; Xie, Zhi-Yang; Shi, Rui; Wei, Ji-Nan; Wu, Xiao-Tao

    2017-01-01

    Notochord nucleus pulposus cells are characteristic of containing abundant and giant cytoplasmic vacuoles. This review explores the embryonic formation, biological function, and postnatal exhaustion of notochord vacuoles, aiming to characterize the signal network transforming the vacuolated nucleus pulposus cells into the vacuole-less chondrocytic cells. Embryonically, the cytoplasmic vacuoles within vertebrate notochord originate from an evolutionarily conserved vacuolation process during neurulation, which may continue to provide mechanical and signal support in constructing a mammalian intervertebral disc. For full vacuolation, a vacuolating specification from dorsal organizer cells, synchronized convergent extension, well-structured notochord sheath, and sufficient post-Golgi trafficking in notochord cells are required. Postnatally, age-related and species-specific exhaustion of vacuolated nucleus pulposus cells could be potentiated by Fas- and Fas ligand-induced apoptosis, intolerance to mechanical stress and nutrient deficiency, vacuole-mediated proliferation check, and gradual de-vacuolation within the avascular and compression-loaded intervertebral disc. These results suggest that the notochord vacuoles are active and versatile organelles for both embryonic notochord and postnatal nucleus pulposus, and may provide novel information on intervertebral disc degeneration to guide cell-based regeneration. PMID:28915712

  10. Protein construct storage: Bayesian variable selection and prediction with mixtures.

    Science.gov (United States)

    Clyde, M A; Parmigiani, G

    1998-07-01

    Determining optimal conditions for protein storage while maintaining a high level of protein activity is an important question in pharmaceutical research. A designed experiment based on a space-filling design was conducted to understand the effects of factors affecting protein storage and to establish optimal storage conditions. Different model-selection strategies to identify important factors may lead to very different answers about optimal conditions. Uncertainty about which factors are important, or model uncertainty, can be a critical issue in decision-making. We use Bayesian variable selection methods for linear models to identify important variables in the protein storage data, while accounting for model uncertainty. We also use the Bayesian framework to build predictions based on a large family of models, rather than an individual model, and to evaluate the probability that certain candidate storage conditions are optimal.

  11. Tuber Storage Proteins

    OpenAIRE

    SHEWRY, PETER R.

    2003-01-01

    A wide range of plants are grown for their edible tubers, but five species together account for almost 90 % of the total world production. These are potato (Solanum tuberosum), cassava (Manihot esculenta), sweet potato (Ipomoea batatus), yams (Dioscorea spp.) and taro (Colocasia, Cyrtosperma and Xanthosoma spp.). All of these, except cassava, contain groups of storage proteins, but these differ in the biological properties and evolutionary relationships. Thus, patatin from potato exhibits act...

  12. A genome-wide immunodetection screen in S. cerevisiae uncovers novel genes involved in lysosomal vacuole function and morphology.

    Directory of Open Access Journals (Sweden)

    Florante Ricarte

    Full Text Available Vacuoles of yeast Saccharomyces cerevisiae are functionally analogous to mammalian lysosomes. Both are cellular organelles responsible for macromolecular degradation, ion/pH homeostasis, and stress survival. We hypothesized that undefined gene functions remain at post-endosomal stage of vacuolar events and performed a genome-wide screen directed at such functions at the late endosome and vacuole interface - ENV genes. The immunodetection screen was designed to identify mutants that internally accumulate precursor form of the vacuolar hydrolase carboxypeptidase Y (CPY. Here, we report the uncovering and initial characterizations of twelve ENV genes. The small size of the collection and the lack of genes previously identified with vacuolar events are suggestive of the intended exclusive functional interface of the screen. Most notably, the collection includes four novel genes ENV7, ENV9, ENV10, and ENV11, and three genes previously linked to mitochondrial processes - MAM3, PCP1, PPE1. In all env mutants, vesicular trafficking stages were undisturbed in live cells as assessed by invertase and active α-factor secretion, as well as by localization of the endocytic fluorescent marker FM4-64 to the vacuole. Several mutants exhibit defects in stress survival functions associated with vacuoles. Confocal fluorescence microscopy revealed the collection to be significantly enriched in vacuolar morphologies suggestive of fusion and fission defects. These include the unique phenotype of lumenal vesicles within vacuoles in the novel env9Δ mutant and severely fragmented vacuoles upon deletion of GET4, a gene recently implicated in tail anchored membrane protein insertion. Thus, our results establish new gene functions in vacuolar function and morphology, and suggest a link between vacuolar and mitochondrial events.

  13. The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

    Science.gov (United States)

    Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

    2008-12-01

    Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

  14. New insights into roles of acidocalcisomes and contractile vacuole complex in osmoregulation in protists.

    Science.gov (United States)

    Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

    2013-01-01

    While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. © 2013, Elsevier Inc. All Rights Reserved.

  15. Nano rare-earth oxides induced size-dependent vacuolization: an independent pathway from autophagy.

    Science.gov (United States)

    Zhang, Ying; Yu, Chenguang; Huang, Guanyi; Wang, Changli; Wen, Longping

    2010-09-07

    Four rare earth oxides have been shown to induce autophagy. Interestingly, we often noticed plentiful vacuolization, which was not always involved in this autophagic process. In this study, we investigated three other rare-earth elements, including Yttrium (Y), Ytterbium (Yb), and Lanthanum (La). Autophagic effect could be induced by all of them but only Y(2)O(3) and Yb(2)O(3) could cause massive vacuolization. Y(2)O(3) and Yb(2)O(3) treated by sonication or centrifugation to reduce particle size were used to test vacuolization level in HeLa cell lines. The results showed that rare earth oxides-induced vacuolization is size-dependent and differs from autophagic pathway. To further clarify the characteristics of this autophagic process, we used MEF Atg-5 (autophagy associated gene 5) knockout cell line, and the result showed that the autophagic process induced by rare earth oxides is Atg-5-dependent and the observed vacuolization was independent from autophagy. Similar results could also be observed in our tests on 3-methyladenine(3-MA), a well-known autophagy inhibitor. In conclusion, for the first time, we clarified the relationship between massive vacuolization and autophagic process induced by rare earth oxides and pointed out the size effect of rare earth oxides on the formation of vacuoles, which give clues to further investigation on the mechanisms underlying their biological effects.

  16. Identification and characterisation of seed storage protein transcripts from Lupinus angustifolius

    Directory of Open Access Journals (Sweden)

    Goggin Danica E

    2011-04-01

    Full Text Available Abstract Background In legumes, seed storage proteins are important for the developing seedling and are an important source of protein for humans and animals. Lupinus angustifolius (L., also known as narrow-leaf lupin (NLL is a grain legume crop that is gaining recognition as a potential human health food as the grain is high in protein and dietary fibre, gluten-free and low in fat and starch. Results Genes encoding the seed storage proteins of NLL were characterised by sequencing cDNA clones derived from developing seeds. Four families of seed storage proteins were identified and comprised three unique α, seven β, two γ and four δ conglutins. This study added eleven new expressed storage protein genes for the species. A comparison of the deduced amino acid sequences of NLL conglutins with those available for the storage proteins of Lupinus albus (L., Pisum sativum (L., Medicago truncatula (L., Arachis hypogaea (L. and Glycine max (L. permitted the analysis of a phylogenetic relationships between proteins and demonstrated, in general, that the strongest conservation occurred within species. In the case of 7S globulin (β conglutins and 2S sulphur-rich albumin (δ conglutins, the analysis suggests that gene duplication occurred after legume speciation. This contrasted with 11S globulin (α conglutin and basic 7S (γ conglutin sequences where some of these sequences appear to have diverged prior to speciation. The most abundant NLL conglutin family was β (56%, followed by α (24%, δ (15% and γ (6% and the transcript levels of these genes increased 103 to 106 fold during seed development. We used the 16 NLL conglutin sequences identified here to determine that for individuals specifically allergic to lupin, all seven members of the β conglutin family were potential allergens. Conclusion This study has characterised 16 seed storage protein genes in NLL including 11 newly-identified members. It has helped lay the foundation for efforts to use

  17. Macroautophagy and microautophagy in relation to vacuole formation in mesophyll cells of Dendrobium tepals.

    Science.gov (United States)

    van Doorn, Wouter G; Kirasak, Kanjana; Ketsa, Saichol

    2015-04-01

    Prior to flower opening, mesophyll cells at the vascular bundles of Dendrobium tepals showed a large increase in vacuolar volume, partially at the expense of the cytoplasm. Electron micrographs indicated that this increase in vacuolar volume was mainly due to vacuole fusion. Macroautophagous structures typical of plant cells were observed. Only a small part of the decrease in cytoplasmic volume seemed due to macroautophagy. The vacuoles contained vesicles of various types, including multilamellar bodies. It was not clear if these vacuolar inclusions were due to macroautophagy or microautophagy. Only a single structure was observed of a protruding vacuole, indicating microautophagy. It is concluded that macroautophagy occurs in these cells but its role in vacuole formation seems small, while a possible role of microautophagy in vacuole formation might be hypothesized. Careful labeling of organelle membranes seems required to advance our insight in plant macro- and microautophagy and their roles in vacuole formation. Copyright © 2015 Elsevier GmbH. All rights reserved.

  18. Characterization of the anion sensitive ATPase in intact vacuoles of Kalanchoe diagremontiana

    Energy Technology Data Exchange (ETDEWEB)

    Kobza, J.; Uribe, E.G.

    1986-04-01

    A method for the isolation of intact vacuoles from K. daigremontiana was developed which produced high yields of relatively pure vacuoles as determined by marker enzyme contamination. Upon isolation, the vacuoles were stabilized by the inclusion of 5% (w/v) ficoll. Enzyme activity was insensitive to vanadate and azide but was strongly inhibited by DCCD. Enzyme activity was strictly dependent on the inclusion of Mg/sup 2 +/ and was stimulated by anions as depicted by the series, NO/sub 3//sup -/ < Br/sup -/ < SO/sub 4//sup -/ < HCO/sub 3//sup -/ < Cl/sup -/. It was found that in intact vacuoles the ATPase activity was stimulated by phosphate to a level equivalent to that found with the chloride. The enzyme exhibited Michaelis-Menten kinetics with a Km for Mg-ATP complex of 0.51 mM.

  19. Calcium controls the formation of vacuoles from mitochondria to regulate microspore development in wheat.

    Science.gov (United States)

    Li, Dong Xiao; Hu, Hai Yan; Li, Gan; Ru, Zhen Gang; Tian, Hui Qiao

    2017-09-01

    Potassium antimonite was used to investigate the localisation of calcium in developing wheat anthers to examine the relationship between Ca 2+ and pollen development. During anther development, calcium precipitate formation increased in anther wall cells prior to microspore mother cell meiosis and appeared in microspores, suggesting the presence of a calcium influx from anther wall cells into the locule. Initially, the precipitates in microspore cytoplasm primarily accumulated in the mitochondria and destroyed their inner membranes (cisterns) to become small vacuoles, which expanded and fused, ultimately becoming a large vacuole during microspore vacuolisation. After microspore division and large vacuole decomposition, many calcium precipitates again accumulated in the small vacuoles, indicating that calcium from the large vacuole moved back into the cytoplasm of bicellular pollen.

  20. Vacuolar biogenesis and aquaporin expression at early germination of broad bean seeds.

    Science.gov (United States)

    Novikova, Galina V; Tournaire-Roux, Colette; Sinkevich, Irina A; Lityagina, Snejana V; Maurel, Christophe; Obroucheva, Natalie

    2014-09-01

    A key event in seed germination is water uptake-mediated growth initiation in embryonic axes. Vicia faba var. minor (broad bean) seeds were used for studying cell growth, vacuolar biogenesis, expression and function of tonoplast water channel proteins (aquaporins) in embryonic axes during seed imbibition, radicle emergence and growth. Hypocotyl and radicle basal cells showed vacuole restoration from protein storage vacuoles, whereas de novo vacuole formation from provacuoles was observed in cells newly produced by root meristem. cDNA fragments of seven novel aquaporin isoforms including five Tonoplast Intrinsic Proteins (TIP) from three sub-types were amplified by PCR. The expression was probed using q-RT-PCR and when possible with isoform-specific antibodies. Decreased expression of TIP3s was associated to the transformation of protein storage vacuoles to vacuoles, whereas enhanced expression of a TIP2 homologue was closely linked to the fast cell elongation. Water channel functioning checked by inhibitory test with mercuric chloride showed closed water channels prior to growth initiation and active water transport into elongating cells. The data point to a crucial role of tonoplast aquaporins during germination, especially during growth of embryonic axes, due to accelerated water uptake and vacuole enlargement resulting in rapid cell elongation. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  1. New Insights into the Roles of Acidocalcisomes and the Contractile Vacuole Complex in Osmoregulation in Protists

    Science.gov (United States)

    Docampo, Roberto; Jimenez, Veronica; Lander, Noelia; Li, Zhu-Hong; Niyogi, Sayantanee

    2013-01-01

    While free-living protists are usually subjected to hyposmotic environments, parasitic protists are also in contact with hyperosmotic habitats. Recent work in one of these parasites, Trypanosoma cruzi, has revealed that its contractile vacuole complex, which usually collects and expels excess water as a mechanism of regulatory volume decrease after hyposmotic stress, has also a role in cell shrinking when the cells are submitted to hyperosmotic stress. Trypanosomes also have an acidic calcium store rich in polyphosphate (polyP), named the acidocalcisome, which is involved in their response to osmotic stress. Here, we review newly emerging insights on the role of acidocalcisomes and the contractile vacuole complex in the cellular response to hyposmotic and hyperosmotic stresses. We also review the current state of knowledge on the composition of these organelles and their other roles in calcium homeostasis and protein trafficking. PMID:23890380

  2. Notochord vacuoles are lysosome-related organelles that function in axis and spine morphogenesis.

    Science.gov (United States)

    Ellis, Kathryn; Bagwell, Jennifer; Bagnat, Michel

    2013-03-04

    The notochord plays critical structural and signaling roles during vertebrate development. At the center of the vertebrate notochord is a large fluid-filled organelle, the notochord vacuole. Although these highly conserved intracellular structures have been described for decades, little is known about the molecular mechanisms involved in their biogenesis and maintenance. Here we show that zebrafish notochord vacuoles are specialized lysosome-related organelles whose formation and maintenance requires late endosomal trafficking regulated by the vacuole-specific Rab32a and H(+)-ATPase-dependent acidification. We establish that notochord vacuoles are required for body axis elongation during embryonic development and identify a novel role in spine morphogenesis. Thus, the vertebrate notochord plays important structural roles beyond early development.

  3. Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

    Science.gov (United States)

    Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

    2016-01-01

    High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

  4. Seed storage protein components are associated with curled ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-11-16

    Nov 16, 2009 ... analysis suggests that the two increased protein spots in mutants were ... The main objective of this work was to gain further understanding of the influence of curled cotyledon on the seed storage protein components in soybean by com- .... cotyledon formation during Arabidopsis embryogenesis: interaction.

  5. Raman Microspectroscopy of the Yeast Vacuoles

    Czech Academy of Sciences Publication Activity Database

    Bednárová, Lucie; Palacký, J.; Bauerová, Václava; Hrušková-Heidingsfeldová, Olga; Pichová, Iva; Mojzeš, P.

    2012-01-01

    Roč. 27, 5-6 (2012), s. 503-507 ISSN 0712-4813 R&D Projects: GA ČR GAP208/10/0376; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Raman microspectroscopy * living cell * yeast * vacuole * chemical composition * polyphospate * Candida albicans Subject RIV: CE - Biochemistry Impact factor: 0.530, year: 2012

  6. Effects of Long-Term Storage Time and Original Sampling Month on Biobank Plasma Protein Concentrations

    Directory of Open Access Journals (Sweden)

    Stefan Enroth

    2016-10-01

    Full Text Available The quality of clinical biobank samples is crucial to their value for life sciences research. A number of factors related to the collection and storage of samples may affect the biomolecular composition. We have studied the effect of long-time freezer storage, chronological age at sampling, season and month of the year and on the abundance levels of 108 proteins in 380 plasma samples collected from 106 Swedish women. Storage time affected 18 proteins and explained 4.8–34.9% of the observed variance. Chronological age at sample collection after adjustment for storage-time affected 70 proteins and explained 1.1–33.5% of the variance. Seasonal variation had an effect on 15 proteins and month (number of sun hours affected 36 proteins and explained up to 4.5% of the variance after adjustment for storage-time and age. The results show that freezer storage time and collection date (month and season exerted similar effect sizes as age on the protein abundance levels. This implies that information on the sample handling history, in particular storage time, should be regarded as equally prominent covariates as age or gender and need to be included in epidemiological studies involving protein levels.

  7. Biomimetic materials for protein storage and transport

    Science.gov (United States)

    Firestone, Millicent A [Elmhurst, IL; Laible, Philip D [Villa Park, IL

    2012-05-01

    The invention provides a method for the insertion of protein in storage vehicles and the recovery of the proteins from the vehicles, the method comprising supplying isolated protein; mixing the isolated protein with a fluid so as to form a mixture, the fluid comprising saturated phospholipids, lipopolymers, and a surfactant; cycling the mixture between a first temperature and a second temperature; maintaining the mixture as a solid for an indefinite period of time; diluting the mixture in detergent buffer so as to disrupt the composition of the mixture, and diluting to disrupt the fluid in its low viscosity state for removal of the guest molecules by, for example, dialysis, filtering or chromatography dialyzing/filtering the emulsified solid.

  8. Behavior of whey protein concentrates under extreme storage conditions

    Science.gov (United States)

    The overseas demand for whey protein concentrates (WPC) has increased steadily in recent years. Emergency aid foods often include WPC, but shelf-life studies of whey proteins under different shipment and storage conditions have not been conducted in the last 50 yr. Microbial quality, compound form...

  9. Dynamic trafficking of wheat γ-gliadin and of its structural domains in tobacco cells, studied with fluorescent protein fusions

    Science.gov (United States)

    Francin-Allami, Mathilde; Saumonneau, Amélie; Lavenant, Laurence; Bouder, Axelle; Sparkes, Imogen; Hawes, Chris; Popineau, Yves

    2011-01-01

    Prolamins, the main storage proteins of wheat seeds, are synthesized and retained in the endoplasmic reticulum (ER) of the endosperm cells, where they accumulate in protein bodies (PBs) and are then exported to the storage vacuole. The mechanisms leading to these events are unresolved. To investigate this unconventional trafficking pathway, wheat γ-gliadin and its isolated repeated N-terminal and cysteine-rich C-terminal domains were fused to fluorescent proteins and expressed in tobacco leaf epidermal cells. The results indicated that γ-gliadin and both isolated domains were able to be retained and accumulated as protein body-like structures (PBLS) in the ER, suggesting that tandem repeats are not the only sequence involved in γ-gliadin ER retention and PBLS formation. The high actin-dependent mobility of γ-gliadin PBLS is also reported, and it is demonstrated that most of them do not co-localize with Golgi body or pre-vacuolar compartment markers. Both γ-gliadin domains are found in the same PBLS when co-expressed, which is most probably due to their ability to interact with each other, as indicated by the yeast two-hybrid and FRET-FLIM experiments. Moreover, when stably expressed in BY-2 cells, green fluorescent protein (GFP) fusions to γ-gliadin and its isolated domains were retained in the ER for several days before being exported to the vacuole in a Golgi-dependent manner, and degraded, leading to the release of the GFP ‘core’. Taken together, the results show that tobacco cells are a convenient model to study the atypical wheat prolamin trafficking with fluorescent protein fusions. PMID:21617248

  10. Variant Exported Blood-Stage Proteins Encoded by Plasmodium Multigene Families Are Expressed in Liver Stages Where They Are Exported into the Parasitophorous Vacuole.

    Directory of Open Access Journals (Sweden)

    Aurélie Fougère

    2016-11-01

    Full Text Available Many variant proteins encoded by Plasmodium-specific multigene families are exported into red blood cells (RBC. P. falciparum-specific variant proteins encoded by the var, stevor and rifin multigene families are exported onto the surface of infected red blood cells (iRBC and mediate interactions between iRBC and host cells resulting in tissue sequestration and rosetting. However, the precise function of most other Plasmodium multigene families encoding exported proteins is unknown. To understand the role of RBC-exported proteins of rodent malaria parasites (RMP we analysed the expression and cellular location by fluorescent-tagging of members of the pir, fam-a and fam-b multigene families. Furthermore, we performed phylogenetic analyses of the fam-a and fam-b multigene families, which indicate that both families have a history of functional differentiation unique to RMP. We demonstrate for all three families that expression of family members in iRBC is not mutually exclusive. Most tagged proteins were transported into the iRBC cytoplasm but not onto the iRBC plasma membrane, indicating that they are unlikely to play a direct role in iRBC-host cell interactions. Unexpectedly, most family members are also expressed during the liver stage, where they are transported into the parasitophorous vacuole. This suggests that these protein families promote parasite development in both the liver and blood, either by supporting parasite development within hepatocytes and erythrocytes and/or by manipulating the host immune response. Indeed, in the case of Fam-A, which have a steroidogenic acute regulatory-related lipid transfer (START domain, we found that several family members can transfer phosphatidylcholine in vitro. These observations indicate that these proteins may transport (host phosphatidylcholine for membrane synthesis. This is the first demonstration of a biological function of any exported variant protein family of rodent malaria parasites.

  11. The Metalloprotease Mpl Supports Listeria monocytogenes Dissemination through Resolution of Membrane Protrusions into Vacuoles.

    Science.gov (United States)

    Alvarez, Diego E; Agaisse, Hervé

    2016-06-01

    Listeria monocytogenes is an intracellular pathogen that disseminates within the intestinal epithelium through acquisition of actin-based motility and formation of plasma membrane protrusions that project into adjacent cells. The resolution of membrane protrusions into vacuoles from which the pathogen escapes results in bacterial spread from cell to cell. This dissemination process relies on the mlp-actA-plcB operon, which encodes ActA, a bacterial nucleation-promoting factor that mediates actin-based motility, and PlcB, a phospholipase that mediates vacuole escape. Here we investigated the role of the metalloprotease Mpl in the dissemination process. In agreement with previous findings showing that Mpl is required for PlcB activation, infection of epithelial cells with the ΔplcB or Δmpl strains resulted in the formation of small infection foci. As expected, the ΔplcB strain displayed a strong defect in vacuole escape. However, the Δmpl strain showed an unexpected defect in the resolution of protrusions into vacuoles, in addition to the expected but mild defect in vacuole escape. The Δmpl strain displayed increased levels of ActA on the bacterial surface in protrusions. We mapped an Mpl-dependent processing site in ActA between amino acid residues 207 to 238. Similar to the Δmpl strain, the ΔactA207-238 strain displayed increased levels of ActA on the bacterial surface in protrusions. Although the ΔactA207-238 strain displayed wild-type actin-based motility, it formed small infection foci and failed to resolve protrusions into vacuoles. We propose that, in addition to its role in PlcB processing and vacuole escape, the metalloprotease Mpl is required for ActA processing and protrusion resolution. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  12. Morphological changes in the sperm storage tubules of the ...

    African Journals Online (AJOL)

    TEM results showed pyknosis, swollen mitochondria, vacuolation and increased number of lysosomes in degenerating SST. The observed morphological changes indicate the ability of carbendazim to disrupt structural integrity of SST as well as its storage capacity. This poses a great threat to the fertility of exposed birds ...

  13. Mature forms of the major seed storage albumins in sunflower: A mass spectrometric approach.

    Science.gov (United States)

    Franke, Bastian; Colgrave, Michelle L; Mylne, Joshua S; Rosengren, K Johan

    2016-09-16

    Seed storage albumins are abundant, water-soluble proteins that are degraded to provide critical nutrients for the germinating seedling. It has been established that the sunflower albumins encoded by SEED STORAGE ALBUMIN 2 (SESA2), SESA20 and SESA3 are the major components of the albumin-rich fraction of the common sunflower Helianthus annuus. To determine the structure of sunflowers most important albumins we performed a detailed chromatographic and mass spectrometric characterization to assess what post-translational processing they receive prior to deposition in the protein storage vacuole. We found that SESA2 and SESA20 each encode two albumins. The first of the two SESA2 albumins (SESA2-1) exists as a monomer of 116 or 117 residues, differing by a threonine at the C-terminus. The second of the two SESA2 albumins (SESA2-2) is a monomer of 128 residues. SESA20 encodes the albumin SESA20-2, which is a 127-residue monomer, whereas SESA20-1 was not abundant enough to be structurally described. SESA3, which has been partly characterized previously, was found in several forms with methylation of its asparagine residues. In contrast to other dicot albumins, which are generally matured into a heterodimer, all the dominant mature sunflower albumins SESA2, SESA20-2, SESA3 and its post-translationally modified analogue SESA3-a are monomeric. Sunflower plants have been bred to thrive in various climate zones making them favored crops to meet the growing worldwide demand by humans for protein. The abundance of seed storage proteins makes them an important source of protein for animal and human nutrition. This study explores the structures of the dominant sunflower napin-type seed storage albumins to understand what structures evolution has favored in the most abundant proteins in sunflower seed. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

  14. Irradiation and Post-Irradiation Storage of Chicken: Effects on Fat and Proteins

    International Nuclear Information System (INIS)

    Abou-Tarboush, H.M.; Al-Kahtani, H.A.; Abou-Arab, A.A.; Atia, M.; Bajaber, A.S.; Ahmed, M.A.; El-Mojaddidi, M.A.

    1997-01-01

    Chicken were subjected to gamma irradiation doses of 2.5, 5.0, 7.5 and 10.0 KGy and post-irradiation storage of 21 days at 4±2º. The effects on fat and protein of chicken were studied. Rate of formation of total volatile basic-nitrogen was less in irradiated samples particularly in samples treated with 5.0KGy during the entire storage. Fatty acid profiles of chicken lipids were not significantly (P≤ 0.05) affected by irradiation especially at doses of 5.0 KGy. However, irradiation caused a large increase in thiobarbituric acid (TBA) values which continued gradually during storage. Changes in amino acids were minimal. Irradiated and unirradiated samples showed the appearance of protein subunits with molecular weights in the range of 10.0 to 88.0 and 10.0 to 67.0 KD, respectively. No changes were observed in the sarcoplasmic protein but the intensity of bands in all irradiated samples decreased after 21 days of storage

  15. Dorfman-Chanarin syndrome: a rare neutral lipid storage disease.

    Science.gov (United States)

    Mitra, Souvik; Samanta, Moumita; Sarkar, Mihir; Chatterjee, Sukanta

    2010-01-01

    Dorfman-Chanarin syndrome is a rare neutral lipid storage disorder characterized by ichthyosis, lipid vacuolations in peripheral leucocytes, and multisystem involvement. It is an autosomal recessive disorder caused by mutations in the CGI-58 gene. A total of 42 cases have been reported worldwide till February 2009 out of which 4 have been previously reported from India. We report a case of a 20-month-old male with congenital ichthyosis, organomegaly, and bilateral cryptorchidism. Examination of the peripheral smear revealed lipid vacuoles in the leucocytes consistent with Jordan's anomaly, which was confirmed by transmission electron microscopy. Liver biopsy revealed micronodular cirrhosis with macrovesicular steatosis while skin biopsy showed ichthyosis vulgaris. Dorfman-Chanarin syndrome was diagnosed on the basis of clinical and laboratory criteria with certain unreported manifestations. Dietary modifications were instituted and followed up after 1 year with promising results. This emphasizes the importance of neonatal screening for lipid vacuolations in peripheral blood in all cases of congenital ichthyosis.

  16. Dorfman-Chanarin syndrome: A rare neutral lipid storage disease

    Directory of Open Access Journals (Sweden)

    Mitra Souvik

    2010-10-01

    Full Text Available Dorfman-Chanarin syndrome is a rare neutral lipid storage disorder characterized by ichthyosis, lipid vacuolations in peripheral leucocytes, and multisystem involvement. It is an autosomal recessive disorder caused by mutations in the CGI-58 gene. A total of 42 cases have been reported worldwide till February 2009 out of which 4 have been previously reported from India. We report a case of a 20-month-old male with congenital ichthyosis, organomegaly, and bilateral cryptorchidism. Examination of the peripheral smear revealed lipid vacuoles in the leucocytes consistent with Jordan′s anomaly, which was confirmed by transmission electron microscopy. Liver biopsy revealed micronodular cirrhosis with macrovesicular steatosis while skin biopsy showed ichthyosis vulgaris. Dorfman-Chanarin syndrome was diagnosed on the basis of clinical and laboratory criteria with certain unreported manifestations. Dietary modifications were instituted and followed up after 1 year with promising results. This emphasizes the importance of neonatal screening for lipid vacuolations in peripheral blood in all cases of congenital ichthyosis.

  17. Intense pseudotransport of a cationic drug mediated by vacuolar ATPase: Procainamide-induced autophagic cell vacuolization

    International Nuclear Information System (INIS)

    Morissette, Guillaume; Lodge, Robert; Marceau, Francois

    2008-01-01

    Cationic drugs frequently exhibit large apparent volumes of distribution, consistent with various forms of cellular sequestration. The contributions of organelles and metabolic processes that may mimic drug transport were defined in human vascular smooth muscle cells. We hypothesized that procainamide-induced vacuolar cytopathology is driven by intense pseudotransport mediated by the vacuolar (V)-ATPase and pursued the characterization of vesicular trafficking alterations in this model. Large amounts of procainamide were taken up by intact cells (maximal in 2 h, reversible upon washout, apparent K M 4.69 mM; fluorometric determination of cell-associated drug). Procainamide uptake was extensively prevented or reversed by pharmacological inhibition of the V-ATPase with bafilomycin A1 or FR 167356, decreased at low extracellular pH and preceded vacuolar cell morphology. However, the uptake of procainamide was unaffected by mitochondrial poisons that reduced the uptake of rhodamine 6G. Large vacuoles induced by millimolar procainamide were labeled with the late endosome/lysosome markers Rab7 and CD63 and the autophagy effector LC3; their osmotic formation (but not procainamide uptake) was reduced by extracellular mannitol and parallel to LC3 II formation. Procainamide-induced vacuolization is associated with defective endocytosis of fluorophore-labeled bovine serum albumin, but not with induction of the unfolded protein response. The contents of a vacuole subset slowly (≥ 24 h) become positive for Nile red staining (phospholipidosis-like response). V-ATPase-driven ion trapping is a form of intense cation pseudotransport that concerns the uncharged form of the drugs, and is associated with a vacuolar, autophagic and evolutive cytopathology and profound effects on vesicular trafficking

  18. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    International Nuclear Information System (INIS)

    Polatnick, J.; Wool, S.H.

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated [ 3 H] uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity. (Author)

  19. Localization of foot-and-mouth disease - RNA synthesis on newly formed cellular smooth membranous vacuoles

    Energy Technology Data Exchange (ETDEWEB)

    Polatnick, J.; Wool, S.H. (United States Department of Agriculture, Science and Education, Greenport, New York (USA). Agricultural Research, Plum Island Animal Disease Center)

    1982-01-01

    Viral RNA synthesis in foot-and-mouth disease infected bovine kidney cell cultures was associated throughout the infectious period with newly formed smooth membranous vacuoles. Membrane formation was measured by choline uptake. The site of RNA synthesis was determined by electron microscopic examination of autoradiograms of incorporated (/sup 3/H) uridine. Both membrane formation and RNA synthesis became signifcant at 2.5 hours postinfection, but membrane formation increased steadily to 4.5 hours while RNA synthesis peaked at 3.5 hours. Percent density distributions of developed silver grains on autoradiograms showed that almost all RNA synthesis was concentrated on the smooth vacuoles of infected cells. Histogram analysis of grain density distributions established that the site of RNA synthesis was the vacuolar membrane. The newly formed smooth membrane-bound vacuoles were not seen to coalesce into the large vacuolated areas typical of poliovirus cytopathogenicity.

  20. Plasmodium berghei Δp52&p36 parasites develop independent of a parasitophorous vacuole membrane in Huh-7 liver cells.

    Science.gov (United States)

    Ploemen, Ivo H J; Croes, Huib J; van Gemert, Geert-Jan J; Wijers-Rouw, Mietske; Hermsen, Cornelus C; Sauerwein, Robert W

    2012-01-01

    The proteins P52 and P36 are expressed in the sporozoite stage of the murine malaria parasite Plasmodium berghei. Δp52&p36 sporozoites lacking expression of both proteins are severely compromised in their capability to develop into liver stage parasites and abort development soon after invasion; presumably due to the absence of a parasitophorous vacuole membrane (PVM). However, a small proportion of P. berghei Δp52&p36 parasites is capable to fully mature in hepatocytes causing breakthrough blood stage infections. We have studied the maturation of replicating Δp52&p36 parasites in cultured Huh-7 hepatocytes. Approximately 50% of Δp52&p36 parasites developed inside the nucleus of the hepatocyte but did not complete maturation and failed to produce merosomes. In contrast cytosolic Δp52&p36 parasites were able to fully mature and produced infectious merozoites. These Δp52&p36 parasites developed into mature schizonts in the absence of an apparent parasitophorous vacuole membrane as shown by immunofluorescence and electron microscopy. Merozoites derived from these maturing Δp52&p36 liver stages were infectious for C57BL/6 mice.

  1. Identification of a Peptide-Pheromone that Enhances Listeria monocytogenes Escape from Host Cell Vacuoles

    Science.gov (United States)

    Xayarath, Bobbi; Alonzo, Francis; Freitag, Nancy E.

    2015-01-01

    Listeria monocytogenes is a Gram-positive facultative intracellular bacterial pathogen that invades mammalian cells and escapes from membrane-bound vacuoles to replicate within the host cell cytosol. Gene products required for intracellular bacterial growth and bacterial spread to adjacent cells are regulated by a transcriptional activator known as PrfA. PrfA becomes activated following L. monocytogenes entry into host cells, however the signal that stimulates PrfA activation has not yet been defined. Here we provide evidence for L. monocytogenes secretion of a small peptide pheromone, pPplA, which enhances the escape of L. monocytogenes from host cell vacuoles and may facilitate PrfA activation. The pPplA pheromone is generated via the proteolytic processing of the PplA lipoprotein secretion signal peptide. While the PplA lipoprotein is dispensable for pathogenesis, bacteria lacking the pPplA pheromone are significantly attenuated for virulence in mice and have a reduced efficiency of bacterial escape from the vacuoles of nonprofessional phagocytic cells. Mutational activation of PrfA restores virulence and eliminates the need for pPplA-dependent signaling. Experimental evidence suggests that the pPplA peptide may help signal to L. monocytogenes its presence within the confines of the host cell vacuole, stimulating the expression of gene products that contribute to vacuole escape and facilitating PrfA activation to promote bacterial growth within the cytosol. PMID:25822753

  2. Genotypic variability and mutant identification in cicer arietinum L. by seed storage protein profiling

    International Nuclear Information System (INIS)

    Hameed, A.; Iqbal, N.; Shah, T.M.

    2012-01-01

    A collection of thirty-four chickpea genotypes, including five kabuli and twenty-nine desi, were analyzed by SDS-PAGE for seed storage protein profiling. Total soluble seed proteins were resolved on 12% gels. A low level of variability was observed in desi as compared to kabuli genotypes. Dendrogram based on electrophoretic data clustered the thirty-four genotypes in four major groups. As large number of desi genotypes illustrated identical profiles, therefore could not be differentiated on the basis of seed storage protein profiles. One kabuli genotype ILC-195 found to be the most divergent showing 86% similarity with all other genotypes. ILC-195 can be distinguished from its mutant i.e., CM-2000 and other kabuli genotypes on the basis of three peptides i.e. SSP-66, SSP-43 and SSP-39. Some proteins peptides were found to be genotype specific like SSP-26 for ICCV-92311. Uniprot and NCBI protein databases were searched for already reported and characterized seed storage proteins in chickpea. Among 33 observed peptides, only six seed storages proteins from chickpea source were available in databases. On the basis of molecular weight similarity, identified peptides were SSP-64 as Serine/Threonine dehydratase, SSP-56 as Alpha-amylase inhibitor, SSP-50 as Provicillin, SSP-39 as seed imbibition protein, SSP-35 as Isoflavane reductase and SSP-19 as lipid transport protein. Highest variability was observed in vicillin subunits and beta subunits of legumins and its polymorphic forms. In conclusion, seed storage profiling can be economically used to asses the genetic variation, phylogenetic relationship and as markers to differentiate mutants from their parents. (author)

  3. Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles

    Science.gov (United States)

    Cna'ani, Alon; Shavit, Reut; Ravid, Jasmin; Aravena-Calvo, Javiera; Skaliter, Oded; Masci, Tania; Vainstein, Alexander

    2017-01-01

    Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography–mass spectrometry (GC–MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger β-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC–MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites. PMID:29163617

  4. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

    Directory of Open Access Journals (Sweden)

    María Milagros López de Armentia

    2016-03-01

    Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

  5. Baking quality parameters of wheat in relation to endosperm storage proteins

    Directory of Open Access Journals (Sweden)

    Daniela Horvat

    2012-01-01

    Full Text Available Wheat storage proteins of twelve winter wheat cultivars grown at the experimental field of the Agricultural Institute Osijek in 2009 were studied for their contribution to the baking quality. Composition of high molecular weight glutenin subunits (HMW-GS was analyzed by SDS-PAGE method, while the proportions of endosperm storage proteins were determined by RP-HPLC method. Regarding the proportion of storage proteins, results of the linear correlation (p<0.05 showed that protein (P and wet gluten (WG content were highly negatively correlated with albumins and globulins (AG and positively with α- gliadins (GLI. A strong negative correlation between AG and water absorption (WA capacity of flour was found, while α- GLI had positive influence on this property. Dough development time (DDT was positively significantly correlated with HMW-GS and negatively with AG. Degree of dough softening (DS was strongly positively affected by γ- GLI and gliadins to glutenins ratio (GLI/GLU and negatively by total GLU and HMW-GS. Dough energy (E and maximum resistance (RMAX were significantly positively affected by Glu-1 score and negatively by GLI/GLU ratio. Resistance to extensibility ratio (R/EXT was significantly negatively correlated with total GLI. Bread volume was significantly negatively influenced by AG.

  6. Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB-1 from rice

    NARCIS (Netherlands)

    Chen, Y.; Sun, A.; Wang, M.; Zhu, Z.; Ouwerkerk, P.B.F.

    2014-01-01

    Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we

  7. Culture supernatants from V. cholerae O1 El Tor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity.

    Science.gov (United States)

    Vidal, Jorge E; Enríquez-Rincón, Fernando; Giono-Cerezo, Silvia; Ribas-Aparicio, Rosa María; Figueroa-Arredondo, Paula

    2009-01-01

    To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

  8. Purification and Initial Functions of Sex-Specific Storage Protein 2 in Bombyx mori.

    Science.gov (United States)

    Chen, Jianqing; Shu, Tejun; Chen, Jian; Ye, Man; Lv, Zhengbing; Nie, Zuoming; Gai, Qijing; Yu, Wei; Zhang, Yaozhou

    2015-08-01

    In this study, we identified a heat-resistant protein from the chrysalis stage of the silkworm which we named sex-specific storage protein 2 (SSP2). This protein was stable even at 80 °C, and has an amino acid sequence that is 90.65 % homologous to SP2. We utilized the heat-resistant characteristics of SSP2 to purify the protein and maintain its biological activity. In addition, using flow cytometry and the MTT assay, we found that SSP2 had anti-apoptotic effects on BmN cells, and that SSP2 could also inhibit cell apoptosis induced by chemical factors. These results suggest that SSP2 has a cell-protective function, and provides a basis for future work on the function of storage proteins in silkworm.

  9. Whey protein concentrate storage at elevated temperature and humidity

    Science.gov (United States)

    Dairy processors are finding new export markets for whey protein concentrate (WPC), a byproduct of cheesemaking, but they need to know if full-sized bags of this powder will withstand high temperature and relative humidity (RH) levels during unrefrigerated storage under tropical conditions. To answ...

  10. Examining the impact of grazing on iron remineralization: effect of prey type on digestive vacuole pH

    Science.gov (United States)

    Pritchard, K. R.; Nuester, J.; Twining, B.

    2012-12-01

    Most of the iron available to phytoplankton in high-nutrient, low-chlorophyll areas is regenerated by zooplankton grazers. The extent to which the bioavailability of this regenerated iron is a function of prey-type and the chemical conditions within digestive systems of zooplankton is unknown. The chemical composition of the prey, including silica frustules of diatoms and calcium carbonate coccoliths of cocolithophores, might buffer the acidity within a digestive vacuole and thereby influencing the resulting speciation and bioavailability of regenerated iron. In order to test the effect of prey-type on the chemical condition in the digestive vacuole of the heterotrophic dinoflagellate Oxyrrhis marina, we used the ratiometric fluorescent dye Lysosensor Yellow/Blue DND-160 in conjunction with confocal microscopy to measure and compare digestive vacuole acidity after feeding O. marina with either the diatom Thalassiosira pseudonana, the coccolithophore Emiliana huxleyi, or the chlorophyte Dunaliella tertiolecta. After feeding and loading O. marina with the Lysosensor dye, we recorded the total fluorescence (f) of the wavelength regions λ1=500-555 nm and λ2=410-490 nm using an excitation wavelength of 405 nm, and calculated the Lysosensor fluorescence ratio r=f(λ1)/f(λ2). External calibration curves show that this ratio (r) is inversely related to pH. In addition, we also measured the emission of chlorophyll fluorescence above 640 nm in order to identify prey within the grazers and study the timing chlorophyll degradation in conjunction with vacuole pH. After the initial addition of either prey, O. marina consumed 10 times and 2 times more D. tertiolecta cells than E. huxleyi and T. pseudonana cells, respectively. The clearance of the digestive vacuole measured as the disappearance of chlorophyll fluorescence is ca. twice as long for O. marina feeding on D. tertiolecta than on E. huxleyi or T. pseudonana. Initial r was inversely proportional to prey preference

  11. The effects of frozen tissue storage conditions on the integrity of RNA and protein.

    Science.gov (United States)

    Auer, H; Mobley, J A; Ayers, L W; Bowen, J; Chuaqui, R F; Johnson, L A; Livolsi, V A; Lubensky, I A; McGarvey, D; Monovich, L C; Moskaluk, C A; Rumpel, C A; Sexton, K C; Washington, M K; Wiles, K R; Grizzle, W E; Ramirez, N C

    2014-10-01

    Unfixed tissue specimens most frequently are stored for long term research uses at either -80° C or in vapor phase liquid nitrogen (VPLN). There is little information concerning the effects such long term storage on tissue RNA or protein available for extraction. Aliquots of 49 specimens were stored for 5-12 years at -80° C or in VPLN. Twelve additional paired specimens were stored for 1 year under identical conditions. RNA was isolated from all tissues and assessed for RNA yield, total RNA integrity and mRNA integrity. Protein stability was analyzed by surface-enhanced or matrix-assisted laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS, MALDI-TOF-MS) and nano-liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RNA yield and total RNA integrity showed significantly better results for -80° C storage compared to VPLN storage; the transcripts that were preferentially degraded during VPLN storage were these involved in antigen presentation and processing. No consistent differences were found in the SELDI-TOF-MS, MALDI-TOF-MS or nLC-ESI-MS/MS analyses of specimens stored for more than 8 years at -80° C compared to those stored in VPLN. Long term storage of human research tissues at -80° C provides at least the same quality of RNA and protein as storage in VPLN.

  12. Conservation of polypyrimidine tract binding proteins and their putative target RNAs in several storage root crops.

    Science.gov (United States)

    Kondhare, Kirtikumar R; Kumar, Amit; Hannapel, David J; Banerjee, Anjan K

    2018-02-07

    Polypyrimidine-tract binding proteins (PTBs) are ubiquitous RNA-binding proteins in plants and animals that play diverse role in RNA metabolic processes. PTB proteins bind to target RNAs through motifs rich in cytosine/uracil residues to fine-tune transcript metabolism. Among tuber and root crops, potato has been widely studied to understand the mobile signals that activate tuber development. Potato PTBs, designated as StPTB1 and StPTB6, function in a long-distance transport system by binding to specific mRNAs (StBEL5 and POTH1) to stabilize them and facilitate their movement from leaf to stolon, the site of tuber induction, where they activate tuber and root growth. Storage tubers and root crops are important sustenance food crops grown throughout the world. Despite the availability of genome sequence for sweet potato, cassava, carrot and sugar beet, the molecular mechanism of root-derived storage organ development remains completely unexplored. Considering the pivotal role of PTBs and their target RNAs in potato storage organ development, we propose that a similar mechanism may be prevalent in storage root crops as well. Through a bioinformatics survey utilizing available genome databases, we identify the orthologues of potato PTB proteins and two phloem-mobile RNAs, StBEL5 and POTH1, in five storage root crops - sweet potato, cassava, carrot, radish and sugar beet. Like potato, PTB1/6 type proteins from these storage root crops contain four conserved RNA Recognition Motifs (characteristic of RNA-binding PTBs) in their protein sequences. Further, 3´ UTR (untranslated region) analysis of BEL5 and POTH1 orthologues revealed the presence of several cytosine/uracil motifs, similar to those present in potato StBEL5 and POTH1 RNAs. Using RT-qPCR assays, we verified the presence of these related transcripts in leaf and root tissues of these five storage root crops. Similar to potato, BEL5-, PTB1/6- and POTH1-like orthologue RNAs from the aforementioned storage root

  13. Differential Induction of Cytoplasmic Vacuolization and Methuosis by Novel 2-Indolyl-Substituted Pyridinylpropenones.

    Science.gov (United States)

    Trabbic, Christopher J; Dietsch, Heather M; Alexander, Evan M; Nagy, Peter I; Robinson, Michael W; Overmeyer, Jean H; Maltese, William A; Erhardt, Paul W

    2014-01-09

    Because many cancers harbor mutations that confer resistance to apoptosis, there is a need for therapeutic agents that can trigger alternative forms of cell death. Methuosis is a novel form of non-apoptotic cell death characterized by accumulation of vacuoles derived from macropinosomes and endosomes. Previous studies identified an indole-based chalcone, 3-(5-methoxy-2-methylindol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), that induces methuosis in human cancer cells. Herein, we describe the synthesis of related 2-indolyl substituted pyridinylpropenones and their effects on U251 glioblastoma cells. Increasing the size of the 2-indolyl substituent substantially reduces growth inhibitory activity and cytotoxicity, but does not prevent cell vacuolization. Computational models suggest that the results are not due to steric-driven conformational effects. The unexpected uncoupling of vacuolization and cell death implies that the relationship between endosomal perturbations and methuotic cell death is more complex than previously realized. The new series of compounds will be useful in further defining the molecular and cellular mechanisms underlying methuosis.

  14. Semiautomated Segmentation and Measurement of Cytoplasmic Vacuoles in a Neutrophil With General-Purpose Image Analysis Software.

    Science.gov (United States)

    Mizukami, Maki; Yamada, Misaki; Fukui, Sayaka; Fujimoto, Nao; Yoshida, Shigeru; Kaga, Sanae; Obata, Keiko; Jin, Shigeki; Miwa, Keiko; Masauzi, Nobuo

    2016-11-01

    Morphological observation of blood or marrow film is still described nonquantitatively. We developed a semiautomatic method for segmenting vacuoles from the cytoplasm using Photoshop (PS) and Image-J (IJ), called PS-IJ, and measured the relative entire cell area (rECA) and relative areas of vacuoles (rAV) in the cytoplasm of neutrophil with PS-IJ. Whole-blood samples were stored at 4°C with ethylenediaminetetraacetate and in two different preserving manners (P1 and P2). Color-tone intensity levels of neutrophil images were semiautomatically compensated using PS, and then vacuole portions were automatically segmented by IJ. The rAV and rECA were measured by counting pixels by IJ. For evaluating the accuracy in segmentations of vacuoles with PS-IJ, the rAV/rECA ratios calculated with results from PS-IJ were compared with those calculated with human eye and IJ (HE-IJ). The rECA and rAV/ in P1 significantly (P < 0.05, P < 0.05) were enlarged and increased, but did not significantly (P = 0.46, P = 0.21) change in P2. The rAV/rECA ratios by PS-IJ were significantly correlated (r = 0.90, P < 0.01) with those by HE-IJ. PS-IJ method can successfully segment vacuoles and measure the rAV and rECA, becoming a useful tool for quantitative description of morphological observation of blood and marrow film. © 2016 Wiley Periodicals, Inc.

  15. Changes in cod muscle proteins during frozen storage revealed by proteome analysis and multivariate data analysis

    DEFF Research Database (Denmark)

    Kjærsgård, Inger Vibeke Holst; Nørrelykke, M.R.; Jessen, Flemming

    2006-01-01

    Multivariate data analysis has been combined with proteomics to enhance the recovery of information from 2-DE of cod muscle proteins during different storage conditions. Proteins were extracted according to 11 different storage conditions and samples were resolved by 2-DE. Data generated by 2-DE...... was subjected to principal component analysis (PCA) and discriminant partial least squares regression (DPLSR). Applying PCA to 2-DE data revealed the samples to form groups according to frozen storage time, whereas differences due to different storage temperatures or chilled storage in modified atmosphere...... light chain 1, 2 and 3, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase A and two ?-actin fragments, and a nuclease diphosphate kinase B fragment to change in concentration, during frozen storage. Application of proteomics, multivariate data analysis and MS/MS to analyse...

  16. Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis.

    Science.gov (United States)

    Puchkov, Evgeny O

    2010-06-01

    In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate-labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two-dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein-Smoluchowski equation using the obtained data, were found to be 2.16 +/- 0.60, 2.52 +/- 0.63, 3.32 +/- 0.9 and 11.3 +/- 1.7 cP. The first three viscosity values correspond to 30-40% glycerol solutions. The viscosity value of 11.3 +/- 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells.

  17. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Pramod Kumar Singh

    2016-01-01

    Full Text Available Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE across a broad range 3.0–10.0 immobilized pH gradient (IPG strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected in these accessions. In-gel protein expression patterns revealed three protein spots as upregulated and three other as downregulated. Using trypsin in-gel digestion, these differentially expressed proteins were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS which showed 45% amino acid homology of chickpea seed storage proteins with Arabidopsis thaliana.

  18. Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

    Directory of Open Access Journals (Sweden)

    Erhui Xiong

    Full Text Available The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1 is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize and dicot (soybean and pea seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

  19. Effects of water deficit on breadmaking quality and storage protein compositions in bread wheat (Triticum aestivum L.).

    Science.gov (United States)

    Zhou, Jiaxing; Liu, Dongmiao; Deng, Xiong; Zhen, Shoumin; Wang, Zhimin; Yan, Yueming

    2018-03-12

    Water deficiency affects grain proteome dynamics and storage protein compositions, resulting in changes in gluten viscoelasticity. In this study, the effects of field water deficit on wheat breadmaking quality and grain storage proteins were investigated. Water deficiency produced a shorter grain-filling period, a decrease in grain number, grain weight and grain yield, a reduced starch granule size and increased protein content and glutenin macropolymer contents, resulting in superior dough properties and breadmaking quality. Reverse phase ultra-performance liquid chromatography analysis showed that the total gliadin and glutenin content and the accumulation of individual components were significantly increased by water deficiency. Two-dimensional gel electrophoresis detected 144 individual storage protein spots with significant accumulation changes in developing grains under water deficit. Comparative proteomic analysis revealed that water deficiency resulted in significant upregulation of 12 gliadins, 12 high-molecular-weight glutenin subunits and 46 low-molecular-weight glutenin subunits. Quantitative real-time polymerase chain reaction analysis revealed that the expression of storage protein biosynthesis-related transcription factors Dof and Spa was upregulated by water deficiency. The present results illustrated that water deficiency leads to increased accumulation of storage protein components and upregulated expression of Dof and Spa, resulting in an improvement in glutenin strength and breadmaking quality. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

  20. Moessbauer spectroscopic studies of iron-storage proteins

    Energy Technology Data Exchange (ETDEWEB)

    St. Pierre, T.G.

    1986-01-01

    /sup 57/Fe Moessbauer spectroscopy was used to study iron storage proteins. Various cryostats and a superconducting magnet were used to obtain sample environment temperatures from 1.3 to 200K and applied magnetic fields of up to 10T. The Moessbauer spectra of ferritins isolated from iron-overloaded human spleen, limpet (Patella vulgata), giant limpet (Patella laticostata) and chiton (Clavarizona hirtosa) hemolymph, and bacterial (Pseudomonas aeruginosa) cells are used to gain information on the magnetic ordering- and superparamagnetic transition temperatures of the microcrystalline cores of the proteins. Investigations were made about the cause of the difference in the magnetic anisotropy constants of the cores of iron-overloaded human spleen ferritin and hemosiderin. Livers taken from an iron-overloaded hornbill and artificially iron-loaded rats showed no component with a superparamagnetic transition temperature approaching that of the human spleen hemosiderin.

  1. Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT) Protein 2

    Science.gov (United States)

    Gross, David A.; Snapp, Erik L.; Silver, David L.

    2010-01-01

    Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation. PMID:20520733

  2. Structural insights into triglyceride storage mediated by fat storage-inducing transmembrane (FIT protein 2.

    Directory of Open Access Journals (Sweden)

    David A Gross

    2010-05-01

    Full Text Available Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2 belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9AAA in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

  3. Synergy of cell-cell repulsion and vacuolation in a computational model of lumen formation.

    Science.gov (United States)

    Boas, Sonja E M; Merks, Roeland M H

    2014-03-06

    A key step in blood vessel development (angiogenesis) is lumen formation: the hollowing of vessels for blood perfusion. Two alternative lumen formation mechanisms are suggested to function in different types of blood vessels. The vacuolation mechanism is suggested for lumen formation in small vessels by coalescence of intracellular vacuoles, a view that was extended to extracellular lumen formation by exocytosis of vacuoles. The cell-cell repulsion mechanism is suggested to initiate extracellular lumen formation in large vessels by active repulsion of adjacent cells, and active cell shape changes extend the lumen. We used an agent-based computer model, based on the cellular Potts model, to compare and study both mechanisms separately and combined. An extensive sensitivity analysis shows that each of the mechanisms on its own can produce lumens in a narrow region of parameter space. However, combining both mechanisms makes lumen formation much more robust to the values of the parameters, suggesting that the mechanisms may work synergistically and operate in parallel, rather than in different vessel types.

  4. Synergy of cell–cell repulsion and vacuolation in a computational model of lumen formation

    Science.gov (United States)

    Boas, Sonja E. M.; Merks, Roeland M. H.

    2014-01-01

    A key step in blood vessel development (angiogenesis) is lumen formation: the hollowing of vessels for blood perfusion. Two alternative lumen formation mechanisms are suggested to function in different types of blood vessels. The vacuolation mechanism is suggested for lumen formation in small vessels by coalescence of intracellular vacuoles, a view that was extended to extracellular lumen formation by exocytosis of vacuoles. The cell–cell repulsion mechanism is suggested to initiate extracellular lumen formation in large vessels by active repulsion of adjacent cells, and active cell shape changes extend the lumen. We used an agent-based computer model, based on the cellular Potts model, to compare and study both mechanisms separately and combined. An extensive sensitivity analysis shows that each of the mechanisms on its own can produce lumens in a narrow region of parameter space. However, combining both mechanisms makes lumen formation much more robust to the values of the parameters, suggesting that the mechanisms may work synergistically and operate in parallel, rather than in different vessel types. PMID:24430123

  5. Seed storage protein polymorphism in ten elite rice (Oryza sativa L ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... for several economical traits by conserving landrace genotypes and ... plasm, seed storage protein analysis represents a valid alternative ... of each variety was taken and ground into fine powder using pestle and mortal and ...

  6. Dorfman-Chanarin syndrome: A rare neutral lipid storage disease

    OpenAIRE

    Mitra Souvik; Samanta Moumita; Sarkar Mihir; Chatterjee Sukanta

    2010-01-01

    Dorfman-Chanarin syndrome is a rare neutral lipid storage disorder characterized by ichthyosis, lipid vacuolations in peripheral leucocytes, and multisystem involvement. It is an autosomal recessive disorder caused by mutations in the CGI-58 gene. A total of 42 cases have been reported worldwide till February 2009 out of which 4 have been previously reported from India. We report a case of a 20-month-old male with congenital ichthyosis, organomegaly, and bilateral cryptorchidism. Examination ...

  7. Cell survival under nutrient stress is dependent on metabolic conditions regulated by Akt and not by autophagic vacuoles.

    Science.gov (United States)

    Bruno, P; Calastretti, A; Priulla, M; Asnaghi, L; Scarlatti, F; Nicolin, A; Canti, G

    2007-10-01

    Akt activation assists tumor cell survival and promotes resistance to chemotherapy. Here we show that constitutively active Akt (CA-Akt) cells are highly sensitized to cell death induced by nutrient and growth factor deprivation, whereas dominant-negative Akt (DN-Akt) cells have a high rate of survival. The content of autophagosomes in starved CA-Akt cells was high, while DN-Akt cells expressed autophagic vacuoles constitutively, independently of nutrition conditions. Thus Akt down-regulation and downstream events can induce autophagosomes which were not directly determinants of cell death. Biochemical analysis in Akt-mutated cells show that (i) Akt and mTOR proteins were degraded more rapidly than the housekeeping proteins, (ii) mTOR phosphorylation at position Thr(2446) was relatively high in DN-Akt and low in CA-Akt cells, induced by starvation in mock cells only, which suggests reduced autoregulation of these pathways in Akt-mutated cells, (iii) both protein synthesis and protein degradation were significantly higher in starved CA-Akt cells than in starved DN-Akt cells or mock cells. In conclusion, constitutively active Akt, unable to control synthesis and wasting of proteins, accelerates the death of starved cells.

  8. The vacuolar V1/V0-ATPase is involved in the release of the HOPS subunit Vps41 from vacuoles, vacuole fragmentation and fusion

    DEFF Research Database (Denmark)

    Takeda, Kozue; Cabrera, Margarita; Rohde, Jan

    2008-01-01

    At yeast vacuoles, phosphorylation of the HOPS subunit Vps41 depends on the Yck3 kinase. In a screen for mutants that mimic the yck3Delta phenotype, in which Vps41 accumulates in vacuolar dots, we observed that mutants in the V0-part of the V0/V1-ATPase, in particular in vma16Delta, also accumulate...

  9. Vacuolating Cytotoxin of Helicobacter pylori Plays a Role during Colonization in a Mouse Model of Infection

    OpenAIRE

    Salama, Nina R.; Otto, Glen; Tompkins, Lucy; Falkow, Stanley

    2001-01-01

    Helicobacter pylori, the causative agent of gastritis and ulcer disease in humans, secretes a toxin called VacA (vacuolating cytotoxin) into culture supernatants. VacA was initially characterized and purified on the basis of its ability to induce the formation of intracellular vacuoles in tissue culture cells. H. pylori strains possessing different alleles of vacA differ in their ability to express active toxin. Those strains expressing higher toxin levels are correlated with more severe gast...

  10. Purification and proteomics of pathogen-modified vacuoles and membranes

    Directory of Open Access Journals (Sweden)

    Jo-Ana eHerweg

    2015-06-01

    Full Text Available Certain pathogenic bacteria adopt an intracellular lifestyle and proliferate in eukaryotic host cells. The intracellular niche protects the bacteria from cellular and humoral components of the mammalian immune system, and at the same time, allows the bacteria to gain access to otherwise restricted nutrient sources. Yet, intracellular protection and access to nutrients comes with a price, i.e. the bacteria need to overcome cell-autonomous defense mechanisms, such as the bactericidal endocytic pathway. While a few bacteria rupture the early phagosome and escape into the host cytoplasm, most intracellular pathogens form a distinct, degradation-resistant and replication-permissive membranous compartment. Intracellular bacteria that form unique pathogen vacuoles include Legionella, Mycobacterium, Chlamydia, Simkania and Salmonella species. In order to understand the formation of these pathogen niches on a global scale and in a comprehensive and quantitative manner, an inventory of compartment-associated host factors is required. To this end, the intact pathogen compartments need to be isolated, purified and biochemically characterized. Here, we review recent progress on the isolation and purification of pathogen-modified vacuoles and membranes, as well as their proteomic characterization by mass spectrometry and different validation approaches. These studies provide the basis for further investigations on the specific mechanisms of pathogen-driven compartment formation.

  11. Dynamic changes in proteins during apple (Malus x domestica) fruit ripening and storage

    OpenAIRE

    Shi, Yun; Jiang, Li; Zhang, Li; Kang, Ruoyi; Yu, Zhifang

    2014-01-01

    A proteomic study, using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight, was conducted in apple fruit (cv. ‘Golden Delicious’) starting at 10 days prior to harvest through 50 days in storage. Total protein was extracted using a phenol/sodium dodecyl sulfate protocol. More than 400 protein spots were detected in each gel and 55 differentially expressed proteins (p

  12. Maternal exposure to hexachlorophene targets intermediate-stage progenitor cells of the hippocampal neurogenesis in rat offspring via dysfunction of cholinergic inputs by myelin vacuolation

    International Nuclear Information System (INIS)

    Itahashi, Megu; Abe, Hajime; Tanaka, Takeshi; Mizukami, Sayaka; Kimura, Masayuki; Yoshida, Toshinori; Shibutani, Makoto

    2015-01-01

    Highlights: • The effect of maternal exposure to HCP on rat hippocampal neurogenesis was examined. • HCP induces myelin vacuolation of nerve tracts in the septal–hippocampal pathway. • Myelin changes suppress Chrnb2-mediated cholinergic inputs to the dentate gyrus. • SGZ apoptosis occurs via the mitochondrial pathway and targets type-2b cells. • Dysfunction of cholinergic inputs is related to type-2b SGZ cell apoptosis. - Abstract: Hexachlorophene (HCP) is known to induce myelin vacuolation corresponding to intramyelinic edema of nerve fibers in the central and peripheral nervous system in animals. This study investigated the effect of maternal exposure to HCP on hippocampal neurogenesis in rat offspring using pregnant rats supplemented with 0 (controls), 100, or 300 ppm HCP in the diet from gestational day 6 to day 21 after delivery. On postnatal day (PND) 21, the numbers of T box brain 2 + progenitor cells and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling + apoptotic cells in the hippocampal subgranular zone (SGZ) decreased in female offspring at 300 ppm, which was accompanied by myelin vacuolation and punctate tubulin beta-3 chain staining of nerve fibers in the hippocampal fimbria. In addition, transcript levels of the cholinergic receptor, nicotinic beta 2 (Chrnb2) and B-cell CLL/lymphoma 2 (Bcl2) decreased in the dentate gyrus. HCP-exposure did not alter the numbers of SGZ proliferating cells and reelin- or calcium-binding protein-expressing γ-aminobutyric acid (GABA)-ergic interneuron subpopulations in the dentate hilus on PND 21 and PND 77. Although some myelin vacuolation remained, all other changes observed in HCP-exposed offspring on PND 21 disappeared on PND 77. These results suggest that maternal HCP exposure reversibly decreases type-2b intermediate-stage progenitor cells via the mitochondrial apoptotic pathway in offspring hippocampal neurogenesis at 300 ppm HCP. Neurogenesis may be affected by dysfunction

  13. Translocation of an 89-kDa periplasmic protein is associated with Holospora infection

    International Nuclear Information System (INIS)

    Iwatani, Koichi; Dohra, Hideo; Lang, B. Franz; Burger, Gertraud; Hori, Manabu; Fujishima, Masahiro

    2005-01-01

    The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus

  14. Integrated transcriptomics and proteomics analysis of storage protein composition in developing barley grain to improve nutritional profile

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Dionisio, Giuseppe; Renaut, Jenny

    2012-01-01

    The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe......-regimes caused significant differences in both quantity and quality of the storage proteins transcripts. Principal Component Analysis of the amino acid (AA) profiles also indicated dissimilarity in individual AA percentages, correlated to hordein content. The abundance values of proteins of interest confirmed...

  15. Biochemical characterization of amandin, the major storage protein in almond (Prunus dulcis L.).

    Science.gov (United States)

    Sathe, Shridhar K; Wolf, Walter J; Roux, Kenneth H; Teuber, Suzanne S; Venkatachalam, Mahesh; Sze-Tao, Kar Wai Clara

    2002-07-17

    The almond major storage protein, amandin, was prepared by column chromatography (amandin-1), cryoprecipitation (amandin-2), and isoelectric precipitation (amandin-3) methods. Amandin is a legumin type protein characterized by a sedimentation value of 14S. Amandin is composed of two major types of polypeptides with estimated molecular weights of 42-46 and 20-22 kDa linked via disulfide bonds. Several additional minor polypeptides were also present in amandin. Amandin is a storage protein with an estimated molecular weight of 427,300 +/- 47,600 Da (n = 7) and a Stokes radius of 65.88 +/- 3.21 A (n = 7). Amandin is not a glycoprotein. Amandin-1, amandin-2, and amandin-3 are antigenically related and have similar biochemical properties. Amandin-3 is more negatively charged than either amandin-1 or amandin-2. Methionine is the first essential limiting amino acid in amandin followed by lysine and threonine.

  16. Influence of dietary lipid and protein sources on the sensory quality of organic rainbow trout (Oncorhynchus mykiss) after ice storage

    DEFF Research Database (Denmark)

    Green-Petersen, Ditte; Hyldig, Grethe; Jacobsen, Charlotte

    2014-01-01

    The influence of dietary protein and lipid sources on the quality of organic rainbow trout (Oncorhynchus mykiss) was studied. The protein and oil sources were fishmeal, fish oil, and organic vegetable protein and oils. Sensory profiling was performed during 3 to 14 days of ice storage along...... with lipid analyses of the fillet. Overall, the results showed that the sensory characteristics of the trout were affected in different ways during ice storage. The source of lipid seemed to affect the sensory quality at the beginning of the storage period, while the protein source seemed to have a more...

  17. Nutritional value and digestion rate of rhea meat proteins in association with storage and cooking processes.

    Science.gov (United States)

    Filgueras, Renata S; Gatellier, Philippe; Ferreira, Claude; Zambiazi, Rui C; Santé-Lhoutellier, Véronique

    2011-09-01

    The nutritional value of proteins was investigated after the storage and cooking of rhea M. Gastrocnemius pars interna. Oxidation of basic and aromatic amino acids, surface hydrophobicity and aggregation state of proteins, were determined in raw and cooked meat. In addition, myofibrillar proteins were exposed in vitro to proteases of the digestive tract. Cooking markedly affected the protein surface hydrophobicity. The BBP bound content was three times greater in cooked than in fresh rhea meat. A small increment in tryptophan content after cooking was observed. Storage influenced Schiff bases formation indicating the presence of protein-aldehyde adducts after cooking. High content of Schiff bases was found after cooking of samples stored for 5 days, demonstrating a probable implication of free amino groups, most likely from lysine. Cooking decreased the myofibrillar protein susceptibility to pepsin activity. After cooking, the proteolysis rate by pancreatic enzymes increased. Our findings support the importance of protein aggregation in the nutritional value of meat proteins. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. Vitellogenin-RNAi and ovariectomy each increase lifespan, increase protein storage, and decrease feeding, but are not additive in grasshoppers.

    Science.gov (United States)

    Tetlak, Alicia G; Burnett, Jacob B; Hahn, Daniel A; Hatle, John D

    2015-12-01

    Reduced reproduction has been shown to increase lifespan in many animals, yet the mechanisms behind this trade-off are unclear. We addressed this question by combining two distinct, direct means of life-extension via reduced reproduction, to test whether they were additive. In the lubber grasshopper, Romalea microptera, ovariectomized (OVX) individuals had a ~20% increase in lifespan and a doubling of storage relative to controls (Sham operated). Similarly, young female grasshoppers treated with RNAi against vitellogenin (the precursor to egg yolk protein) had increased fat body mass and halted ovarian growth. In this study, we compared VgRNAi to two control groups that do not reduce reproduction, namely buffer injection (Buffer) and injection with RNAi against a hexameric storage protein (Hex90RNAi). Each injection treatment was tested with and without ovariectomy. Hence, we tested feeding, storage, and lifespans in six groups: OVX and Buffer, OVX and Hex90RNAi, OVX and VgRNAi, Sham and Buffer, Sham and Hex90RNAi, and Sham and VgRNAi. Ovariectomized grasshoppers and VgRNAi grasshoppers each had similar reductions in feeding (~40%), increases in protein storage in the hemolymph (150-300%), and extensions in lifespan (13-21%). Ovariectomized grasshoppers had higher vitellogenin protein levels than did VgRNAi grasshoppers. Last but not least, when ovariectomy and VgRNAi were applied together, there was no greater effect on feeding, protein storage, or longevity. Hence, feeding regulation, and protein storage in insects, may be conserved components of life-extension via reduced reproduction.

  19. Adjustment of host cells for accommodation of symbiotic bacteria: vacuole defunctionalization, HOPS suppression, and TIP1g retargeting in Medicago

    NARCIS (Netherlands)

    Gavrin, A.Y.; Kaiser, B.N.; Geiger, D.; Tyerman, S.D.; Wen, Z.; Bisseling, T.; Fedorova, E.E.

    2014-01-01

    In legume–rhizobia symbioses, the bacteria in infected cells are enclosed in a plant membrane, forming organelle-like compartments called symbiosomes. Symbiosomes remain as individual units and avoid fusion with lytic vacuoles of host cells. We observed changes in the vacuole volume of infected

  20. Physicochemical properties and storage stability of soybean protein nanoemulsions prepared by ultra-high pressure homogenization.

    Science.gov (United States)

    Xu, Jing; Mukherjee, Dipaloke; Chang, Sam K C

    2018-02-01

    This study investigated the effects of the ultrahigh pressure homogenization (pressure, protein concentration, oil phase fraction, pH, temperature, and ionic strength) and storage on the properties of nanoemulsions (100-500nm range), which were stabilized by laboratory-prepared soybean protein isolate (SPI), β-conglycinin (7S) and glycinin (11S). The nanoemulsions made with SPI, 7S and 11S proteins exhibited considerable stability over various ionic strengths (0-500mM NaCl), pH (7), thermal treatments (30-60°C) and storage (0-45days). The far-UV spectra of SPI, 7S, 11S dispersions, and SPI-, 7S-, 11S protein-stabilized nanoemulsions were analyzed for the protein structural changes following lipid removal. The ultra-high pressure homogenization changed the secondary structure of SPI, 7S, 11S proteins in the nanoemulsions, and enhanced their stability. This study demonstrated that SPI, 7S, and 11S proteins can be used as effective emulsifiers in nanoemulsions prepared by ultra-high pressure homogenization. Copyright © 2017. Published by Elsevier Ltd.

  1. Yersinia pestis Targets the Host Endosome Recycling Pathway during the Biogenesis of the Yersinia-Containing Vacuole To Avoid Killing by Macrophages

    Science.gov (United States)

    Connor, Michael G.; Pulsifer, Amanda R.; Ceresa, Brian K.

    2018-01-01

    ABSTRACT Yersinia pestis has evolved many strategies to evade the innate immune system. One of these strategies is the ability to survive within macrophages. Upon phagocytosis, Y. pestis prevents phagolysosome maturation and establishes a modified compartment termed the Yersinia-containing vacuole (YCV). Y. pestis actively inhibits the acidification of this compartment, and eventually, the YCV transitions from a tight-fitting vacuole into a spacious replicative vacuole. The mechanisms to generate the YCV have not been defined. However, we hypothesized that YCV biogenesis requires Y. pestis interactions with specific host factors to subvert normal vesicular trafficking. In order to identify these factors, we performed a genome-wide RNA interference (RNAi) screen to identify host factors required for Y. pestis survival in macrophages. This screen revealed that 71 host proteins are required for intracellular survival of Y. pestis. Of particular interest was the enrichment for genes involved in endosome recycling. Moreover, we demonstrated that Y. pestis actively recruits Rab4a and Rab11b to the YCV in a type three secretion system-independent manner, indicating remodeling of the YCV by Y. pestis to resemble a recycling endosome. While recruitment of Rab4a was necessary to inhibit YCV acidification and lysosomal fusion early during infection, Rab11b appeared to contribute to later stages of YCV biogenesis. We also discovered that Y. pestis disrupts global host endocytic recycling in macrophages, possibly through sequestration of Rab11b, and this process is required for bacterial replication. These data provide the first evidence that Y. pestis targets the host endocytic recycling pathway to avoid phagolysosomal maturation and generate the YCV. PMID:29463656

  2. Storage stability of cauliflower soup powder: The effect of lipid oxidation and protein degradation reactions.

    Science.gov (United States)

    Raitio, Riikka; Orlien, Vibeke; Skibsted, Leif H

    2011-09-15

    Soups based on cauliflower soup powders, prepared by dry mixing of ingredients and rapeseed oil, showed a decrease in quality, as evaluated by a sensory panel, during the storage of the soup powder in the dark for up to 12weeks under mildly accelerated conditions of 40°C and 75% relative humidity. Antioxidant, shown to be effective in protecting the rapeseed bulk oil, used for the powder preparation, had no effect on storage stability of the soup powder. The freshly prepared soup powder had a relatively high concentration of free radicals, as measured by electron spin resonance spectroscopy, which decreased during storage, and most remarkably during the first two weeks of storage, with only marginal increase in lipid hydroperoxides as primary lipid oxidation products, and without any increase in secondary lipid oxidation products. Analyses of volatiles by SPME-GC-MS revealed a significant increase in concentrations of 2-methyl- and 3-methyl butanals, related to Maillard reactions, together with an increase in 2-acetylpyrrole concentration. The soup powders became more brown during storage, as indicated by a decreasing Hunter L-value, in accord with non-enzymatic browning reactions. A significant increase in the concentrations of dimethyl disulfide in soup powder headspace indicated free radical-initiated protein oxidation. Protein degradation, including Maillard reactions and protein oxidation, is concluded to be more important than lipid oxidation in determining the shelf-life of dry cauliflower soup powder. Copyright © 2011 Elsevier Ltd. All rights reserved.

  3. Late-Onset Glycogen Storage Disease Type II (Pompe's Disease) with a Novel Mutation: A Malaysian Experience.

    Science.gov (United States)

    Fu Liong, Hiew; Abdul Wahab, Siti Aishah; Yakob, Yusnita; Lock Hock, Ngu; Thong, Wong Kum; Viswanathan, Shanthi

    2014-01-01

    Pompe's disease (acid maltase deficiency, glycogen storage disease type II) is an autosomal recessive disorder caused by a deficiency of lysosomal acid α-1,4-glucosidase, resulting in excessive accumulation of glycogen in the lysosomes and cytoplasm of all tissues, most notably in skeletal muscles. We present a case of adult-onset Pompe's disease with progressive proximal muscles weakness over 5 years and respiratory failure on admission, requiring prolonged mechanical ventilation. Electromyography showed evidence of myopathic process with small amplitudes, polyphasic motor unit action potentials, and presence of pseudomyotonic discharges. Muscle biopsy showed glycogen-containing vacuoles in the muscle fibers consistent with glycogen storage disease. Genetic analysis revealed two compound heterozygous mutations at c.444C>G (p.Tyr148∗) in exon 2 and c.2238G>C (p.Trp746Cys) in exon 16, with the former being a novel mutation. This mutation has not been reported before, to our knowledge. The patient was treated with high protein diet during the admission and subsequently showed good clinical response to enzyme replacement therapy with survival now to the eighth year. Conclusion. In patients with late-onset adult Pompe's disease, careful evaluation and early identification of the disease and its treatment with high protein diet and enzyme replacement therapy improve muscle function and have beneficial impact on long term survival.

  4. Liver-specific Aquaporin 11 knockout mice show rapid vacuolization of the rough endoplasmic reticulum in periportal hepatocytes after amino acid feeding

    DEFF Research Database (Denmark)

    Rojek, Aleksandra; Füchtbauer, Ernst-Martin; Füchtbauer, Annette C.

    2013-01-01

    -specific Aqp11 KO mice, allowing us to study the role of AQP11 protein in liver of mice with normal kidney function. The unchallenged liver-specific Aqp11 KO mice have normal longevity, their livers appeared normal, and the plasma biochemistries revealed only a minor defect in lipid handling. Fasting......Aquaporin 11 (AQP11) is a protein channel expressed intracellularly in multiple organs, yet its physiological function is unclear. Aqp11 knockout (KO) mice die early due to malfunction of the kidney, a result of hydropic degeneration of proximal tubule cells. Here we report the generation of liver...... protein or larger doses of various amino acids. The fasting/refeeding challenge is associated with increased expression of markers of ER stress Grp78 and GADD153 and decreased glutathione levels, suggesting that ER stress may play role in the development of vacuoles in the AQP11-deficient hepatocytes. NMR...

  5. Full-thickness human skin explants for testing the toxicity of topically applied chemicals

    International Nuclear Information System (INIS)

    Nakamura, M.; Rikimaru, T.; Yano, T.; Moore, K.G.; Pula, P.J.; Schofield, B.H.; Dannenberg, A.M. Jr.

    1990-01-01

    This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components

  6. Full-thickness human skin explants for testing the toxicity of topically applied chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Nakamura, M.; Rikimaru, T.; Yano, T.; Moore, K.G.; Pula, P.J.; Schofield, B.H.; Dannenberg, A.M. Jr. (Johns Hopkins Univ., Baltimore, MD (USA))

    1990-09-01

    This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin. In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically. The human skin specimens were discards from a variety of surgical procedures. They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant. The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C. In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis. Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants. Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells. The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium. During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity. Incubation (for 4 or 24 h at 36{degrees}C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage. This reduction was probably caused by autolysis of many of the vacuolated cells. Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type. The storage type seemed to be caused by autolysis of cell components.

  7. Characterization of seed storage protein patterns of Heliotropium digynum.

    Science.gov (United States)

    Alwhibi, Mona Soliman

    2017-09-01

    Heliotropium digynum , is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8) software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir'iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

  8. Using a two-layered sphere model to investigate the impact of gas vacuoles on the inherent optical properties of Microcystis aeruginosa

    CSIR Research Space (South Africa)

    Matthews, MW

    2013-01-01

    Full Text Available A two-layered sphere model is used to investigate the impact of gas vacuoles on the inherent optical properties (IOPs) of the cyanophyte Microcystis aeruginosa. Enclosing a vacuole-like particle within a chromatoplasm shell layer significantly...

  9. Proteome analysis of potato juice and tuber vacuoles from cv. Kuras

    DEFF Research Database (Denmark)

    Jørgensen, Malene

    %), Kunitz protease inhibitors (KPI, 30 %) and lipoxygenases (10 %). The large amount of lipoxygenase indicates that lipoxygenase also function as a storage protein. The tetraploid nature of most potatoes seems to give rise to a huge complexity of the proteome. The KPI superfamily was found to be represented...

  10. Influence of storage and heating on protein glycation levels of processed lactose-free and regular bovine milk products.

    Science.gov (United States)

    Milkovska-Stamenova, Sanja; Hoffmann, Ralf

    2017-04-15

    Thermal treatment preserves the microbiological safety of milk, but also induces Maillard reactions modifying for example proteins. The purpose of this study was evaluating the influence of consumer behaviors (storage and heating) on protein glycation degrees in bovine milk products. Lactosylation and hexosylation sites were identified in ultra-high temperature (UHT), lactose-free pasteurized, and lactose-free UHT milk (ULF) and infant formula (IF) using tandem mass spectrometry (electron transfer dissociation). Overall, 303 lactosylated and 199 hexosylated peptides were identified corresponding to 170 lactosylation (31 proteins) and 117 hexosylation sites (25 proteins). In quantitative terms, storage increased lactosylation up to fourfold in UHT and IF and hexosylation up to elevenfold in ULF and threefold in IF. These levels increased additionally twofold when the stored samples were heated (40°C). In conclusion, storage and heating appear to influence protein glycation levels in milk at similar or even higher degrees than industrial processing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Salvage and storage of infectious disease protein targets in the SSGCID high-throughput crystallization pathway using microfluidics

    International Nuclear Information System (INIS)

    Christensen, Jeff; Gerdts, Cory J.; Clifton, Mathew C.; Stewart, Lance

    2011-01-01

    SSGCID protein crystals were salvaged and stored using the MPCS Plug Maker and CrystalCards when high-throughput traditional sitting-drop vapor diffusion initially failed. The MPCS Plug Maker is a microcapillary-based protein-crystallization system for generating diffraction-ready crystals from nanovolumes of protein. Crystallization screening using the Plug Maker was used as a salvage pathway for proteins that failed to crystallize during the initial observation period using the traditional sitting-drop vapor-diffusion method. Furthermore, the CrystalCards used to store the crystallization experiments set up by the Plug Maker are shown be a viable container for long-term storage of protein crystals without a discernable loss of diffraction quality with time. Use of the Plug Maker with SSGCID proteins is demonstrated to be an effective crystal-salvage and storage method

  12. Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H(+)-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8.

    Science.gov (United States)

    Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

    2015-01-01

    Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H(+)-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H(+)-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation.

  13. Dissection of autophagy in tobacco BY-2 cells under sucrose starvation conditions using the vacuolar H+-ATPase inhibitor concanamycin A and the autophagy-related protein Atg8

    Science.gov (United States)

    Yano, Kanako; Yanagisawa, Takahiro; Mukae, Kyosuke; Niwa, Yasuo; Inoue, Yuko; Moriyasu, Yuji

    2015-01-01

    Tobacco BY-2 cells undergo autophagy in sucrose-free culture medium, which is the process mostly responsible for intracellular protein degradation under these conditions. Autophagy was inhibited by the vacuolar H+-ATPase inhibitors concanamycin A and bafilomycin A1, which caused the accumulation of autophagic bodies in the central vacuoles. Such accumulation did not occur in the presence of the autophagy inhibitor 3-methyladenine, and concanamycin in turn inhibited the accumulation of autolysosomes in the presence of the cysteine protease inhibitor E-64c. Electron microscopy revealed not only that the autophagic bodies were accumulated in the central vacuole, but also that autophagosome-like structures were more frequently observed in the cytoplasm in treatments with concanamycin, suggesting that concanamycin affects the morphology of autophagosomes in addition to raising the pH of the central vacuole. Using BY-2 cells that constitutively express a fusion protein of autophagosome marker protein Atg8 and green fluorescent protein (GFP), we observed the appearance of autophagosomes by fluorescence microscopy, which is a reliable morphological marker of autophagy, and the processing of the fusion protein to GFP, which is a biochemical marker of autophagy. Together, these results suggest the involvement of vacuole type H+-ATPase in the maturation step of autophagosomes to autolysosomes in the autophagic process of BY-2 cells. The accumulation of autophagic bodies in the central vacuole by concanamycin is a marker of the occurrence of autophagy; however, it does not necessarily mean that the central vacuole is the site of cytoplasm degradation. PMID:26368310

  14. Late-Onset Glycogen Storage Disease Type II (Pompe’s Disease with a Novel Mutation: A Malaysian Experience

    Directory of Open Access Journals (Sweden)

    Hiew Fu Liong

    2014-01-01

    Full Text Available Pompe’s disease (acid maltase deficiency, glycogen storage disease type II is an autosomal recessive disorder caused by a deficiency of lysosomal acid α-1,4-glucosidase, resulting in excessive accumulation of glycogen in the lysosomes and cytoplasm of all tissues, most notably in skeletal muscles. We present a case of adult-onset Pompe’s disease with progressive proximal muscles weakness over 5 years and respiratory failure on admission, requiring prolonged mechanical ventilation. Electromyography showed evidence of myopathic process with small amplitudes, polyphasic motor unit action potentials, and presence of pseudomyotonic discharges. Muscle biopsy showed glycogen-containing vacuoles in the muscle fibers consistent with glycogen storage disease. Genetic analysis revealed two compound heterozygous mutations at c.444C>G (p.Tyr148* in exon 2 and c.2238G>C (p.Trp746Cys in exon 16, with the former being a novel mutation. This mutation has not been reported before, to our knowledge. The patient was treated with high protein diet during the admission and subsequently showed good clinical response to enzyme replacement therapy with survival now to the eighth year. Conclusion. In patients with late-onset adult Pompe’s disease, careful evaluation and early identification of the disease and its treatment with high protein diet and enzyme replacement therapy improve muscle function and have beneficial impact on long term survival.

  15. Transcriptome study of storage protein genes of field-grown barley in response to inorganic nitrogen fertilizers

    DEFF Research Database (Denmark)

    Hansen, Michael; Bowra, Steve; Lange, Mette

    2010-01-01

    The storage proteins of barley, in terms of both amino acid profile and quantity, are traits strongly influenced by the amount of nitrogen applied. Given this, we performed a developmental expression analysis of the genes from barley grains grown under field conditions to further our understanding...... profile under different N regimes. Reviewing the expression of the storage protein homologues within the families revealed markedly different temporal profiles; for example, some alleles were expressed very early in development. Furthermore, the differential temporal expression of the homologues suggested...

  16. PsVPS1, a dynamin-related protein, is involved in cyst germination and soybean infection of Phytophthora sojae.

    Directory of Open Access Journals (Sweden)

    Delong Li

    Full Text Available Plant pathogens secrete effector proteins to suppress plant immunity. However, the mechanism by which oomycete pathogens deliver effector proteins during plant infection remains unknown. In this report, we characterized a Phytophthora sojae vps1 gene. This gene encodes a homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene vps1 that mediates budding of clathrin-coated vesicles from the late Golgi, which are diverted from the general secretory pathway to the vacuole. PsVPS1-silenced mutants were generated using polyethylene glycol-mediated protoplast stable transformation and were viable but had reduced extracellular protein activity. The PsVPS1-silenced mutants showed impaired hyphal growth, and the shapes of the vacuoles were highly fragmented. Silencing of PsVPS1 affected cyst germination as well as the polarized growth of germinated cysts. Silenced mutants showed impaired invasion of susceptible soybean plants regardless of wounding. These results suggest that PsVPS1 is involved in vacuole morphology and cyst development. Moreover, it is essential for the virulence of P. sojae and extracellular protein secretion.

  17. Antioxidant activity of pomegranate peel extract on lipid and protein oxidation in beef meatballs during frozen storage.

    Science.gov (United States)

    Turgut, Sebahattin Serhat; Işıkçı, Fatma; Soyer, Ayla

    2017-07-01

    Antioxidant effect of pomegranate peel extract (PE) to retard lipid and protein oxidation in beef meatballs was investigated during frozen storage at -18±1°C. Concentrated and freeze dried aqueous extract of pomegranate peel was incorporated into freshly prepared meatball mix at 0.5% and 1.0% concentrations, and compared with 0.01% butylated hydroxytoluene (BHT) and control (without any antioxidant). In PE treated samples, particularly in high PE concentration, peroxide, malondialdehyde and carbonyl formation, loss of total protein solubility and sulfhydryl groups were significantly lower than control after 6months of storage. A diminution of both myofibrillar (MP) and sarcoplasmic (SP) proteins of high molecular weight was detected after 6months of the storage according to gel electrophoresis patterns. The 1.0% PE led to maintain colour intensity (C) and hue (h°) value. The results from sensory analyses revealed that PE addition to meatballs was effective on preventing rancid odour formation. Addition of both 0.5 and 1% PE in meatballs reduced lipid and protein oxidation and improved sensory scores. These results indicated that PE was effective on retarding lipid and protein oxidations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Influence of yogurt fermentation and refrigerated storage on the stability of protein toxin contaminants.

    Science.gov (United States)

    Jackson, Lauren S; Triplett, Odbert A; Tolleson, William H

    2015-06-01

    Dairy products sold in a ready-to-eat form present the risk that adulterants persisting through manufacturing, storage, and distribution would reach consumers. Pathogenic microbes, including shigatoxigenic strains of Escherichia coli and the toxins they produce, are common food safety hazards associated with dairy products. Ricin and abrin are plant-derived ribosome-inactivating protein toxins related to the shiga-like toxins produced by E. coli. Limited information exists on the effects of manufacturing processes on the stabilities of these heat-resistant ribosome-inactivating proteins in the presence of foods. The goal of this study was to determine how typical yogurt manufacturing and storage processes influence ribosome-inactivating protein toxins. Ricin and abrin were added to skim or whole milk and batch pasteurized. Complete inactivation of both toxins was observed after 30 minutes at 85 °C. If the toxins were added after pasteurization, the levels of ricin and abrin in yogurt and their cytotoxic activities did not change significantly during fermentation or refrigerated storage for 4 weeks. The activities of ricin and abrin were inhibited by skim milk, nonfat yogurt, whole milk, and whole milk yogurt. The results showed minimal effects of the toxins on yogurt pH and %titratable acidity but inhibitory effects of yogurt on toxin activity. Published by Elsevier Ltd.

  19. Characterization of seed storage protein patterns of Heliotropium digynum

    Directory of Open Access Journals (Sweden)

    Mona Soliman Alwhibi

    2017-09-01

    Full Text Available Heliotropium digynum, is a shrub that has ecological importance. The height of the plant differs from one population to another and the difference in length of the inflorescence can be attributed to environmental factors, such as rainfall or type of soil and temperature. To date, no study has shed light on estimation in seed samples of H. digynum in Saudi Arabia. So, the aim is to evaluate and characterize the protein patterns of seed storage proteins of H. digynum to be used as fingerprint of this plant in Saudi Arabia. It is collected from different locations in the central region of Saudi Arabia and total protein extraction from plant was compared in SDS-PAGE. The genetic relationships among all cultivars were analyzed using UPGMA and NJ using Total Lab TL and in the same way using Jaccard Similarity Coefficient dendrogram using STATISTICA (ver.8 software. Results, our data show that amounts of protein are different, although they are of the same type or from the same geographical region. Amounts ranged between 22 and 1.5 mg/g of dry weight. Less amount of protein was obtained from the group of samples collected from Dir’iyah area, and the highest amount of protein was from the group of samples collected from Dyrab area in general.

  20. Pep3p/Pep5p complex: a putative docking factor at multiple steps of vesicular transport to the vacuole of Saccharomyces cerevisiae.

    OpenAIRE

    Srivastava, A; Woolford, C A; Jones, E W

    2000-01-01

    Pep3p and Pep5p are known to be necessary for trafficking of hydrolase precursors to the vacuole and for vacuolar biogenesis. These proteins are present in a hetero-oligomeric complex that mediates transport at the vacuolar membrane. PEP5 interacts genetically with VPS8, implicating Pep5p in the earlier Golgi to endosome step and/or in recycling from the endosome to the Golgi. To understand further the cellular roles of Pep3p and Pep5p, we isolated and characterized a set of pep3 conditional ...

  1. The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

    Science.gov (United States)

    Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

    2015-01-01

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. PMID:25713145

  2. Proteomic Analysis of Differentially Accumulated Proteins in Cucumber (Cucumis sativus Fruit Peel in Response to Pre-storage Cold Acclimation

    Directory of Open Access Journals (Sweden)

    Bin Wang

    2018-01-01

    Full Text Available Harvested fruits are still living organs and respond to environmental stimuli. Low temperature storage is effective in extending life of harvested fruit, but it may also cause chilling injury. Cold acclimation has been shown to induce chilling tolerance in plants, but what proteomic changes caused by cold acclimation are related to defense against chilling stress remains largely unclear. Here, 3 d of pre-storage cold acclimation (PsCA at 10°C reduced chilling injury and secondary disease severity in cucumber stored at 5°C by 51 and 94%, respectively, compared with the control which was directly stored at 5°C. Proteomic analysis of cucumber peel identified 21 significant differentially-accumulated proteins (SDAPs right after PsCA treatment and 23 after the following cold storage (PsCA+CS. These proteins are mainly related to stress response and defense (SRD, energy metabolism, protein metabolism, signal transduction, primary metabolism, and transcription. The SRD proteins, which made up 37% of the 21 and 47% of the 23, respectively, represented the largest class of SDAPs, and all but one protein were up-regulated, suggesting accumulation of proteins involved in defense response is central feature of proteomic profile changes brought about by PsCA. In fruit just after PsCA treatment, the identified SDAPs are related to responses to various stresses, including chilling, salt stress, dehydration, fungi, bacteria, insects, and DNA damage. However, after prolonged cold storage, the targeted proteins in acclimated fruit were narrowed down in scope to those involved in defense against chilling and pathogens. The change patterns at the transcription level of the majority of the up-regulated differentially-accumulated proteins were highly consistent with those at protein level. Taken all, the results suggest that the short-time cold acclimation initiated comprehensive defense responses in cucumber fruit at first, while the long term storage thereafter

  3. Proteomic Analysis of Differentially Accumulated Proteins in Cucumber (Cucumis sativus) Fruit Peel in Response to Pre-storage Cold Acclimation.

    Science.gov (United States)

    Wang, Bin; Shen, Fei; Zhu, Shijiang

    2017-01-01

    Harvested fruits are still living organs and respond to environmental stimuli. Low temperature storage is effective in extending life of harvested fruit, but it may also cause chilling injury. Cold acclimation has been shown to induce chilling tolerance in plants, but what proteomic changes caused by cold acclimation are related to defense against chilling stress remains largely unclear. Here, 3 d of pre-storage cold acclimation (PsCA) at 10°C reduced chilling injury and secondary disease severity in cucumber stored at 5°C by 51 and 94%, respectively, compared with the control which was directly stored at 5°C. Proteomic analysis of cucumber peel identified 21 significant differentially-accumulated proteins (SDAPs) right after PsCA treatment and 23 after the following cold storage (PsCA+CS). These proteins are mainly related to stress response and defense (SRD), energy metabolism, protein metabolism, signal transduction, primary metabolism, and transcription. The SRD proteins, which made up 37% of the 21 and 47% of the 23, respectively, represented the largest class of SDAPs, and all but one protein were up-regulated, suggesting accumulation of proteins involved in defense response is central feature of proteomic profile changes brought about by PsCA. In fruit just after PsCA treatment, the identified SDAPs are related to responses to various stresses, including chilling, salt stress, dehydration, fungi, bacteria, insects, and DNA damage. However, after prolonged cold storage, the targeted proteins in acclimated fruit were narrowed down in scope to those involved in defense against chilling and pathogens. The change patterns at the transcription level of the majority of the up-regulated differentially-accumulated proteins were highly consistent with those at protein level. Taken all, the results suggest that the short-time cold acclimation initiated comprehensive defense responses in cucumber fruit at first, while the long term storage thereafter altered the

  4. Protein mobilities and P-selectin storage in Weibel-Palade bodies.

    Science.gov (United States)

    Kiskin, Nikolai I; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J; Carter, Tom

    2010-09-01

    Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

  5. Characterization of Seed Storage Proteins from Chickpea Using 2D Electrophoresis Coupled with Mass Spectrometry

    OpenAIRE

    Singh, Pramod Kumar; Shrivastava, Nidhi; Chaturvedi, Krishna; Sharma, Bechan; Bhagyawant, Sameer S.

    2016-01-01

    Proteomic analysis was employed to map the seed storage protein network in landrace and cultivated chickpea accessions. Protein extracts were separated by two-dimensional gel electrophoresis (2D-GE) across a broad range 3.0–10.0 immobilized pH gradient (IPG) strips. Comparative elucidation of differentially expressed proteins between two diverse geographically originated chickpea accessions was carried out using 2D-GE coupled with mass spectrometry. A total of 600 protein spots were detected ...

  6. Fat storage-inducing transmembrane protein 2 is required for normal fat storage in adipose tissue.

    Science.gov (United States)

    Miranda, Diego A; Kim, Ji-Hyun; Nguyen, Long N; Cheng, Wang; Tan, Bryan C; Goh, Vera J; Tan, Jolene S Y; Yaligar, Jadegoud; Kn, Bhanu Prakash; Velan, S Sendhil; Wang, Hongyan; Silver, David L

    2014-04-04

    Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.

  7. The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

    Science.gov (United States)

    Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

    2015-04-10

    Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Effect of pomegranate peel extract on lipid and protein oxidation in beef meatballs during refrigerated storage.

    Science.gov (United States)

    Turgut, Sebahattin Serhat; Soyer, Ayla; Işıkçı, Fatma

    2016-06-01

    Antioxidant effect of pomegranate peel extract (PE) to retard lipid and protein oxidation was investigated in meatballs during refrigerated storage at 4±1°C. Concentrated lyophilised water extract of pomegranate peel was incorporated into freshly minced beef meat at 0.5% and 1% concentrations and compared with 0.01% butylated hydroxytoluene (BHT) as a reference and control (without any antioxidant). PE showed high phenolic content and antioxidant activity. In PE added samples, thiobarbituric acid reactive substances (TBARS) value, peroxide formation, loss of sulfhydryl groups and formation of protein carbonyls were lower than control (Pmeatballs prolonged the refrigerated storage up to 8 days. Addition of both 0.5 and 1% PE in meatballs reduced lipid and protein oxidation and improved sensory scores. These results indicated that PE was effective on retarding lipid and protein oxidation. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Isolation and characterisation of cDNA clones representing the genes encoding the major tuber storage protein (dioscorin) of yam (Dioscorea cayenensis Lam.).

    Science.gov (United States)

    Conlan, R S; Griffiths, L A; Napier, J A; Shewry, P R; Mantell, S; Ainsworth, C

    1995-06-01

    cDNA clones encoding dioscorins, the major tuber storage proteins (M(r) 32,000) of yam (Dioscorea cayenesis) have been isolated. Two classes of clone (A and B, based on hybrid release translation product sizes and nucleotide sequence differences) which are 84.1% similar in their protein coding regions, were identified. The protein encoded by the open reading frame of the class A cDNA insert is of M(r) 30,015. The difference in observed and calculated molecular mass might be attributed to glycosylation. Nucleotide sequencing and in vitro transcription/translation suggest that the class A dioscorin proteins are synthesised with signal peptides of 18 amino acid residues which are cleaved from the mature peptide. The class A and class B proteins are 69.6% similar with respect to each other, but show no sequence identity with other plant proteins or with the major tuber storage proteins of potato (patatin) or sweet potato (sporamin). Storage protein gene expression was restricted to developing tubers and was not induced by growth conditions known to induce expression of tuber storage protein genes in other plant species. The codon usage of the dioscorin genes suggests that the Dioscoreaceae are more closely related to dicotyledonous than to monocotyledonous plants.

  10. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis.

    Science.gov (United States)

    Conn, Simon J; Gilliham, Matthew; Athman, Asmini; Schreiber, Andreas W; Baumann, Ute; Moller, Isabel; Cheng, Ning-Hui; Stancombe, Matthew A; Hirschi, Kendal D; Webb, Alex A R; Burton, Rachel; Kaiser, Brent N; Tyerman, Stephen D; Leigh, Roger A

    2011-01-01

    The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from Arabidopsis thaliana leaf cells differing in calcium concentration ([Ca], epidermis 60 mM) were compared using a microarray screen and single-cell quantitative PCR. Three tonoplast-localized Ca(2+) transporters, CAX1 (Ca(2+)/H(+)-antiporter), ACA4, and ACA11 (Ca(2+)-ATPases), were identified as preferentially expressed in Ca-rich mesophyll. Analysis of respective loss-of-function mutants demonstrated that only a mutant that lacked expression of both CAX1 and CAX3, a gene ectopically expressed in leaves upon knockout of CAX1, had reduced mesophyll [Ca]. Reduced capacity for mesophyll Ca accumulation resulted in reduced cell wall extensibility, stomatal aperture, transpiration, CO(2) assimilation, and leaf growth rate; increased transcript abundance of other Ca(2+) transporter genes; altered expression of cell wall-modifying proteins, including members of the pectinmethylesterase, expansin, cellulose synthase, and polygalacturonase families; and higher pectin concentrations and thicker cell walls. We demonstrate that these phenotypes result from altered apoplastic free [Ca(2+)], which is threefold greater in cax1/cax3 than in wild-type plants. We establish CAX1 as a key regulator of apoplastic [Ca(2+)] through compartmentation into mesophyll vacuoles, a mechanism essential for optimal plant function and productivity.

  11. Acidification of the parasitophorous vacuole containing Toxoplasma gondii in the presence of hydroxyurea

    Directory of Open Access Journals (Sweden)

    Cristiane S. Carvalho

    2006-09-01

    Full Text Available Toxoplasma gondii multiplies within parasitophorous vacuole that is not recognized by the primary no oxidative defense of host cells, mainly represented by the fusion with acidic organelles. Recent studies have already shown that hydroxyurea arrested the intracellular parasites leading to its destruction. In the present work we investigated the cellular mechanism involved in the destruction of intracellular Toxoplasma gondii. Fluorescent vital stains were used in order to observe possible acidification of parasitophorous vacuole-containing Toxoplasma gondii in presence of hydroxyurea. Vero cells infected with tachyzoites were treated with hydroxyurea for 12, 24 or 48 hours. Fluorescence, indicative of acidification, was observed in the parasitophorous vacuole when the cultures were incubated in presence of acridine orange. LysoTracker red was used in order to determine whether lysosomes were involved in the acidification process. An intense fluorescence was observed after 12 and 24 hours of incubation with hydroxyurea, achieving it is highly intensity after 48 hours of treatment. Ultrastructural cytochemistry for localization of the acid phosphatase lysosomal enzyme was realized. Treated infected cultures showed reaction product in vesicles fusing with vacuole or associated with intravacuolar parasites. These results suggest that fusion with lysosomes and acidification of parasitophorous vacuole leads to parasites destruction in the presence pf hydroxyurea.Toxoplasma gondii se multiplica dentro do vacúolo parasitóforo que não é reconhecido pela defesa primária não oxidativa de células hospedeiras: a fusão com organelas ácidas. Estudos anteriores mostraram que hidroxiuréia interrompeu a multiplicação dos parasitos intracelulares causando sua eliminação. No presente trabalho nós investigamos o mecanismo celular envolvido na destruição do Toxoplasma gondii intracelular. Marcadores vitais fluorescentes foram usados para observar a

  12. Calcium Signals from the Vacuole

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    Gerald Schönknecht

    2013-10-01

    Full Text Available The vacuole is by far the largest intracellular Ca2+ store in most plant cells. Here, the current knowledge about the molecular mechanisms of vacuolar Ca2+ release and Ca2+ uptake is summarized, and how different vacuolar Ca2+ channels and Ca2+ pumps may contribute to Ca2+ signaling in plant cells is discussed. To provide a phylogenetic perspective, the distribution of potential vacuolar Ca2+ transporters is compared for different clades of photosynthetic eukaryotes. There are several candidates for vacuolar Ca2+ channels that could elicit cytosolic [Ca2+] transients. Typical second messengers, such as InsP3 and cADPR, seem to trigger vacuolar Ca2+ release, but the molecular mechanism of this Ca2+ release still awaits elucidation. Some vacuolar Ca2+ channels have been identified on a molecular level, the voltage-dependent SV/TPC1 channel, and recently two cyclic-nucleotide-gated cation channels. However, their function in Ca2+ signaling still has to be demonstrated. Ca2+ pumps in addition to establishing long-term Ca2+ homeostasis can shape cytosolic [Ca2+] transients by limiting their amplitude and duration, and may thus affect Ca2+ signaling.

  13. Arabidopsis senescence-associated protein DMP1 is involved in membrane remodeling of the ER and tonoplast

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    Kasaras Alexis

    2012-04-01

    Full Text Available Abstract Background Arabidopsis DMP1 was discovered in a genome-wide screen for senescence-associated membrane proteins. DMP1 is a member of a novel plant-specific membrane protein family of unknown function. In rosette leaves DMP1 expression increases from very low background level several 100fold during senescence progression. Results Expression of AtDMP1 fused to eGFP in Nicotiana benthamiana triggers a complex process of succeeding membrane remodeling events affecting the structure of the endoplasmic reticulum (ER and the vacuole. Induction of spherical structures (“bulbs”, changes in the architecture of the ER from tubular to cisternal elements, expansion of smooth ER, formation of crystalloid ER, and emergence of vacuolar membrane sheets and foamy membrane structures inside the vacuole are proceeding in this order. In some cells it can be observed that the process culminates in cell death after breakdown of the entire ER network and the vacuole. The integrity of the plasma membrane, nucleus and Golgi vesicles are retained until this stage. In Arabidopsis thaliana plants expressing AtDMP1-eGFP by the 35S promoter massive ER and vacuole vesiculation is observed during the latest steps of leaf senescence, whereas earlier in development ER and vacuole morphology are not perturbed. Expression by the native DMP1 promoter visualizes formation of aggregates termed “boluses” in the ER membranes and vesiculation of the entire ER network, which precedes disintegration of the central vacuole during the latest stage of senescence in siliques, rosette and cauline leaves and in darkened rosette leaves. In roots tips, DMP1 is strongly expressed in the cortex undergoing vacuole biogenesis. Conclusions Our data suggest that DMP1 is directly or indirectly involved in membrane fission during breakdown of the ER and the tonoplast during leaf senescence and in membrane fusion during vacuole biogenesis in roots. We propose that these properties of DMP1

  14. Ocatin. A novel tuber storage protein from the andean tuber crop oca with antibacterial and antifungal activities.

    Science.gov (United States)

    Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E

    2002-04-01

    The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens.

  15. Engineering of soybean seed storage proteins

    International Nuclear Information System (INIS)

    Dickinson, C.D.; Floener, L.A.; Evans, R.P.; Nielsen, N.C.

    1987-01-01

    Protein engineering is one approach to the improvement of seed quality. With this in mind, a rapid in vitro system has been developed to assay the effect structural modifications have on the assembly of glycinin and β-conglycinin subunit complexes. Transcription plasmids were constructed for production of synthetic glycinin and β-conglycinin mRNAs by SP6 RNA-polymerase. Radiolabeled translation products from these messages were tested for their ability to form complexes. Gy4 and Gy5 proglycinins (group-2 subunits) and the a-subunit of β-conglycinin self-assembled into trimers. Proglycinin Gy2 (group-1 subunit) did not self-assemble, but assembled into mixed trimers in combination with Gy4 proglycinin. No assembly was observed for preproglycinins Gyl and Gy4, or for a Gy4 proglycinin which lacked 27 amino acids in a highly conserved internal sequence. Insertion of alternating MET-ARG residues in predicted turn regions of a hypervariable sequence in Gy4 proglycinin were tolerated when the string was short but inhibited trimer assembly as it became longer. The response to several different long deletions in this hypervariable region have also been tested. Different levels of trimer assembly were obtained and may depend on the secondary structures of the regions being joined in the engineered subunits. This system will be useful to study the assembly of storage protein complexes and to screen against modifications that interfere with subunit assembly

  16. Changes of microbial spoilage, lipid-protein oxidation and physicochemical properties during post mortem refrigerated storage of goat meat.

    Science.gov (United States)

    Sabow, Azad Behnan; Sazili, Awis Qurni; Aghwan, Zeiad Amjad; Zulkifli, Idrus; Goh, Yong Meng; Ab Kadir, Mohd Zainal Abidin; Nakyinsige, Khadijah; Kaka, Ubedullah; Adeyemi, Kazeem Dauda

    2016-06-01

    Examined was the effect of post mortem refrigerated storage on microbial spoilage, lipid-protein oxidation and physicochemical traits of goat meat. Seven Boer bucks were slaughtered, eviscerated and aged for 24 h. The Longissimus lumborum (LL) and Semitendinosus (ST) muscles were excised and subjected to 13 days post mortem refrigerated storage. The pH, lipid and protein oxidation, tenderness, color and drip loss were determined in LL while microbiological analysis was performed on ST. Bacterial counts generally increased with increasing aging time and the limit for fresh meat was reached at day 14 post mortem. Significant differences were observed in malondialdehyde (MDA) content at day 7 of storage. The thiol concentration significantly reduced as aging time increased. The band intensities of myosin heavy chain (MHC) and troponin-T significantly decreased as storage progressed, while actin remained relatively stable. After 14 days of aging, tenderness showed significant improvement while muscle pH and drip loss reduced with increase in storage time. Samples aged for 14 days had higher lightness (P goat meat. © 2016 Japanese Society of Animal Science.

  17. Prolonging hypothermic storage (4 C) of bovine embryos with fish antifreeze protein.

    Science.gov (United States)

    Ideta, Atsushi; Aoyagi, Yoshito; Tsuchiya, Kanami; Nakamura, Yuuki; Hayama, Kou; Shirasawa, Atsushi; Sakaguchi, Kenichiro; Tominaga, Naomi; Nishimiya, Yoshiyuki; Tsuda, Sakae

    2015-01-01

    Embryos obtained via superovulation are necessary for mammalian artificial reproduction, and viability is a key determinant of success. Nonfreezing storage at 4 C is possible, but currently used storage solutions can maintain embryo viability for only 24-48 h. Here we found that 10 mg/ml antifreeze protein (AFP) dissolved in culture medium 199 with 20% (v/v) fetal bovine serum and 25 mM HEPES could keep bovine embryos alive for 10 days at 4 C. We used a recombinant AFP isolated from the notched-fin eelpout (Zoarces elongatus Kner). Photomicroscopy indicated that the AFP-embryo interaction was enhanced at 37 C. Embryos pre-warmed with the AFP solution at 37 C for 60 min maintained high viability, whereas those that were not pre-warmed could live no longer than 7 days. Thus, short-term storage of bovine embryos was achieved by a combination of AFP-containing medium and controlled pre-warming.

  18. Biomarkers in the diagnosis of lysosomal storage disorders: proteins, lipids, and inhibodies.

    Science.gov (United States)

    Aerts, Johannes M F G; Kallemeijn, Wouter W; Wegdam, Wouter; Joao Ferraz, Maria; van Breemen, Marielle J; Dekker, Nick; Kramer, Gertjan; Poorthuis, Ben J; Groener, Johanna E M; Cox-Brinkman, Josanne; Rombach, Saskia M; Hollak, Carla E M; Linthorst, Gabor E; Witte, Martin D; Gold, Henrik; van der Marel, Gijs A; Overkleeft, Herman S; Boot, Rolf G

    2011-06-01

    A biomarker is an analyte indicating the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. In the case of lysosomal storage disorders (LSDs), primary and secondary accumulating metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Clinical applications of biomarkers are found in improved diagnosis, monitoring disease progression, and assessing therapeutic correction. These are illustrated by reviewing the discovery and use of biomarkers for Gaucher disease and Fabry disease. In addition, recently developed chemical tools allowing specific visualization of enzymatically active lysosomal glucocerebrosidase are described. Such probes, coined inhibodies, offer entirely new possibilities for more sophisticated molecular diagnosis, enzyme replacement therapy monitoring, and fundamental research.

  19. Vacúolos de gás e flutuação em Difflugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea Gas vacuoles and flotation in Diffugia mitriformis Wallich (Protista, Rhizopoda, Testaceolobosea

    Directory of Open Access Journals (Sweden)

    Vladimir Stolzenberg Torres

    1996-01-01

    Full Text Available The natural formation of gas vacuoles as a method of locomotion is described for Difflugia mitriformis Wallich, 1984. These vacuoles may contain different compositions of gases, basicly carbodioxyde or oxigen, with a membranous limitation similar or identical to other types of vacuoles. Those vacuoles are utilised by the organism as a mode of dislocation frorn the bottom to the water surface by flotation permiting better conditions for the survival of the individual, with the consequence of the perpetuance of the taxon.

  20. Oculopharyngeal Weakness, Hypophrenia, Deafness, and Impaired Vision: A Novel Autosomal Dominant Myopathy with Rimmed Vacuoles

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    Ting Chen

    2016-01-01

    Conclusions: We reported a novel autosomal dominant myopathy with rimmed vacuoles characterized by dysarthria, dysphagia, external ophthalmoplegia, limb weakness, hypophrenia, deafness, and impaired vision, but the causative gene has not been found and needs further study.

  1. A new look on protein-polyphenol complexation during honey storage: is this a random or organized event with the help of dirigent-like proteins?

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    Katrina Brudzynski

    Full Text Available Honey storage initiates melanoidin formation that involves a cascade of seemingly unguided redox reactions between amino acids/proteins, reducing sugars and polyphenols. In the process, high molecular weight protein-polyphenol complexes are formed, but the mechanism involved remains unknown. The objective of this study was twofold: to determine quantitative and qualitative changes in proteins in honeys stored for prolonged times and in different temperatures and to relate these changes to the formation of protein-polyphenol complexes. Six -month storage decreased the protein content by 46.7% in all tested honeys (t-test, p<0.002 with the rapid reduction occurring during the first three month. The changes in protein levels coincided with alterations in molecular size and net charge of proteins on SDS -PAGE. Electro-blotted proteins reacted with a quinone-specific nitro blue tetrazolium (NBT on nitrocellulose membranes indicating that quinones derived from oxidized polyphenols formed covalent bonds with proteins. Protein-polyphenol complexes isolated by size-exclusion chromatography differed in size and stoichiometry and fall into two categories: (a high molecular weight complexes (230-180 kDa enriched in proteins but possessing a limited reducing activity toward the NBT and (b lower molecular size complexes (110-85 kDa enriched in polyphenols but strongly reducing the dye. The variable stoichiometry suggest that the large, "protein-type" complexes were formed by protein cross-linking, while in the smaller, "polyphenol-type" complexes polyphenols were first polymerized prior to protein binding. Quinones preferentially bound a 31 kDa protein which, by the electrospray quadrupole time of flight mass spectrometry (ESI-Qtof-MS analysis, showed homology to dirigent-like proteins known for assisting in radical coupling and polymerization of phenolic compounds. These findings provide a new look on protein-polyphenol interaction in honey where the

  2. Effects of sterilization, packaging, and storage on vitamin C degradation, protein denaturation, and glycation in fortified milks.

    Science.gov (United States)

    Gliguem, H; Birlouez-Aragon, I

    2005-03-01

    Monitoring the nutritional quality of dietetic milk throughout its shelf life is particularly important due to the high susceptibility of some vitamins to oxidation, and the continuous development of the Maillard reaction during storage. The objective of this paper was to evaluate the vitamin C content and protein modification by denaturation and glycation on fortified milk samples (growth milks) destined for 1- to 3-yr-old children. The influences of the sterilization process, formulation, packaging, and storage duration at ambient temperature in the dark were studied. Vitamin C degradation was particularly influenced by type of packaging. The use of a 3-layered opaque bottle was associated with complete oxidation of vitamin C after 1 mo of storage, whereas in the 6-layered opaque bottle, which has an oxygen barrier, the vitamin C content slowly decreased to reach 25% of the initial concentration after 4 mo of storage. However, no significant effect of vitamin C degradation during storage could be observed in terms of Maillard reaction, despite the fact that a probable impact occurred during sterilization. Furosine content and the FAST (fluorescence of advanced Maillard products and soluble tryptophan) index-indicators of the early and advanced Maillard reaction, respectively-were significantly higher in the in-bottle sterilized milk samples compared with UHT samples, and in fortified milk samples compared with cow milk. However, after 1 mo, the impact of storage was predominant, increasing the furosine level and the FAST index at similar levels for the differently processed samples. The early Maillard reaction developed continuously throughout the storage period.In conclusion, only packaging comprising an oxygen and light barrier is compatible with vitamin C fortification of milk. Furthermore, short storage time or low storage temperature is needed to retard vitamin C degradation, protein denaturation, and development of the Maillard reaction.

  3. Effects of Storage and Granary Weevil Infestation on Gel Electrophoresis and Protein Solubility Properties of Hard and Soft Wheat Flours.

    Science.gov (United States)

    Keskin, Sule; Yalçin, Erkan; Özkaya, Hazim

    2018-02-24

    The objective of this study was to investigate the effects of storage and granary weevil, Sitophilus granarius (L.; Coleoptera: Curculionidae), infestation on pH, protein solubility (PS) and gel electrophoresis properties of meal and roller-milled flours of hard (Ceyhan-99 cv.) and soft (Eser cv.) wheat cultivars, respectively, after 6 mo of storage. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) technique was applied for studying the electrophoretic properties. Hard and soft wheats were infested with non-sexed S. granarius at a rate of two adults/ kg, and stored for 6 mo at 30 ± 1°C and 70 ± 5% RH. The pest-free wheat samples were used as control. The infested and its control samples were collected monthly, and after cleaning the granary weevils, they were hammer-milled or roller-milled in order to get meal flours and roller-milled flours, respectively. The effect of infestation on the storage proteins was more obvious in meal flours than that of the roller-milled flours. Granary weevil feeding resulted secreting of hydrolyzing enzymes and increased the acidity of flours; subsequently the breaking and releasing of some storage proteins generally caused a decrease in pH and an increase in PS values of the meal flours of wheat cultivars. SDS-PAGE results generally indicated that towards the end of storage, the insect population, that greatly increased, caused minor protein depletions resulting decreasing protein band intensities between 113 and 58 kDa of hard wheat meal flour and 101 and 40 kDa of soft wheat roller-milled flour. Consequently, the potential effect of changes probably occurred in high molecular weight glutenin subunits of both wheat cultivars.

  4. Ocatin. A Novel Tuber Storage Protein from the Andean Tuber Crop Oca with Antibacterial and Antifungal Activities1

    Science.gov (United States)

    Flores, Teresita; Alape-Girón, Alberto; Flores-Díaz, Marietta; Flores, Hector E.

    2002-01-01

    The most abundant soluble tuber protein from the Andean crop oca (Oxalis tuberosa Mol.), named ocatin, has been purified and characterized. Ocatin accounts for 40% to 60% of the total soluble oca tuber proteins, has an apparent molecular mass of 18 kD and an isoelectric point of 4.8. This protein appears to be found only in tubers and is accumulated only within the cells of the pith and peridermis layers (peel) of the tuber as it develops. Ocatin inhibits the growth of several phytopathogenic bacteria (Agrobacterium tumefaciens, Agrobacterium radiobacter, Serratia marcescens, and Pseudomonas aureofaciens) and fungi (Phytophthora cinnamomi, Fusarium oxysporum, Rhizoctonia solani, and Nectria hematococcus). Ocatin displays substantial amino acid sequence similarity with a widely distributed group of intracellular pathogenesis-related proteins with a hitherto unknown biological function. Our results showed that ocatin serves as a storage protein, has antimicrobial properties, and belongs to the Betv 1/PR-10/MLP protein family. Our findings suggest that an ancient scaffolding protein was recruited in the oca tuber to serve a storage function and that proteins from the Betv 1/PR-10/MLP family might play a role in natural resistance to pathogens. PMID:11950978

  5. Proteomics for exploiting diversity of lupin seed storage proteins and their use as nutraceuticals for health and welfare.

    Science.gov (United States)

    Cabello-Hurtado, Francisco; Keller, Jean; Ley, José; Sanchez-Lucas, Rosa; Jorrín-Novo, Jesús V; Aïnouche, Abdelkader

    2016-06-30

    Lupins have a variety of both traditional and modern uses. In the last decade, reports assessing the benefits of lupin seed proteins have proliferated and, nowadays, the pharmaceutical industry is interested in lupin proteins for human health. Modern genomics and proteomics have hugely contributed to describing the diversity of lupin storage genes and, above all, proteins. Most of these studies have been centered on few edible lupin species. However, Lupinus genus comprises hundreds of species spread throughout the Old and New Worlds, and these resources have been scarcely explored and exploited. We present here a detailed review of the literature on the potential of lupin seed proteins as nutraceuticals, and the use of -omic tools to analyze seed storage polypeptides in main edible lupins and their diversity at the Lupinus inter- and intra-species level. In this sense, proteomics, more than any other, has been a key approach. Proteomics has shown that lupin seed protein diversity, where post-translational modifications yield a large number of peptide variants with a potential concern in bioactivity, goes far beyond gene diversity. The future extended use of second and third generation proteomics should definitely help to go deeper into coverage and characterization of lupin seed proteome. Some important topics concerning storage proteins from lupin seeds are presented and analyzed in an integrated way in this review. Proteomic approaches have been essential in characterizing lupin seed protein diversity, which goes far beyond gene diversity since the protein level adds to the latter differential proteolytic cleavage of conglutin pro-proteins and a diverse array of glycosylation forms and sites. Proteomics has also proved helpful for screening and studying Lupinus germplasm with the future aim of exploiting and improving food production, quality, and nutritional values. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Effects of storage conditions on results for quantitative and qualitative evaluation of proteins in canine urine.

    Science.gov (United States)

    Théron, Marie-Laure; Piane, Laetitia; Lucarelli, Laetitia; Henrion, Rémi; Layssol-Lamour, Catherine; Palanché, Florence; Concordet, Didier; Braun, Jean-Pierre D; Trumel, Catherine; Lavoué, Rachel

    2017-08-01

    OBJECTIVE To investigate effects of storage conditions on the canine urine protein-to-creatinine ratio (UPC) and on SDS-agarose gel electrophoresis (AGE) of urinary proteins. SAMPLE Urine specimens from 20 proteinuric (UPC > 0.5) and 20 nonproteinuric (UPC ≤ 0.2) dogs. PROCEDURES UPC and SDS-AGE were performed on urine specimens stored at room temperature (20°C) and 4°C for up to 5 days and at -20° and -80°C for up to 360 days; some specimens were subjected to 3 freeze-thaw cycles. Results were compared with those obtained for fresh urine specimens. RESULTS UPC was not affected by storage at room temperature or by freezing. A decrease in UPC was observed for specimens from nonproteinuric dogs after 5 days at 4°C (10%) and from both groups after 90 days at -20° and -80°C (≤ 20% and ≤ 15%, respectively). The SDS-AGE profiles revealed no visual changes regardless of duration of storage for specimens stored at room temperature, 4°C, and -80°C, except for 1 profile after 360 days at -80°C. Repeated freeze-thaw cycles did not affect SDS-AGE profiles. Appearance or strengthening of high-molecular-weight bands that could alter interpretation was evident in SDS-AGE profiles after storage at -20°C for ≥ 15 days (31/40 dogs). CONCLUSIONS AND CLINICAL RELEVANCE Storage of urine at -20° or -80°C for up to 1 year influenced the UPC without affecting clinical interpretation. Storage of urine specimens at -20°C impaired visual analysis of SDS-AGE. When SDS-AGE cannot be performed on fresh or recently refrigerated urine specimens, storage at -80°C is recommended.

  7. Organization of the cytoplasmic reticulum in the central vacuole of parenchyma cells in Allium cepa L.

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    Tomasz J. Wodzicki

    2015-01-01

    Full Text Available An elaborate and complex cytoplasmic reticulum composed of fine filaments and lamellae ranging from 0.1 to 4 microns in size is revealed by viewing the central vacuole of onion bulb parenchyma cells with the scanning election microscope. The larger cytoplasmic strands, visible with the light microscope, are composed of numerous smaller filaments (some tubular which might explain the observed bidirectional movement of particles in these larger strands. The finely divided cytoplasmic network of filaments is continuous with the parietal cytoplasm inclosing the vacuolar sap. In these highly vacuolated cells the mass of the protoplast is in the form of an intravacuolar reticulum immersed in the cell sap. The probable significance of the vacuolar sap in relation to physiological processes of the cell is discussed.

  8. Convergent evolution of plant and animal embryo defences by hyperstable non-digestible storage proteins.

    Science.gov (United States)

    Pasquevich, María Yanina; Dreon, Marcos Sebastián; Qiu, Jian-Wen; Mu, Huawei; Heras, Horacio

    2017-11-20

    Plants have evolved sophisticated embryo defences by kinetically-stable non-digestible storage proteins that lower the nutritional value of seeds, a strategy that have not been reported in animals. To further understand antinutritive defences in animals, we analysed PmPV1, massively accumulated in the eggs of the gastropod Pomacea maculata, focusing on how its structure and structural stability features affected its capacity to withstand passage through predator guts. The native protein withstands >50 min boiling and resists the denaturing detergent sodium dodecyl sulphate (SDS), indicating an unusually high structural stability (i.e., kinetic stability). PmPV1 is highly resistant to in vitro proteinase digestion and displays structural stability between pH 2.0-12.0 and 25-85 °C. Furthermore, PmPV1 withstands in vitro and mice digestion and is recovered unchanged in faeces, supporting an antinutritive defensive function. Subunit sequence similarities suggest a common origin and tolerance to mutations. This is the first known animal genus that, like plant seeds, lowers the nutritional value of eggs by kinetically-stable non-digestible storage proteins that survive the gut of predators unaffected. The selective pressure of the harsh gastrointestinal environment would have favoured their appearance, extending by convergent evolution the presence of plant-like hyperstable antinutritive proteins to unattended reproductive stages in animals.

  9. Protein changes in the albedo of citrus fruits on postharvesting storage.

    Science.gov (United States)

    Lliso, Ignacio; Tadeo, Francisco R; Phinney, Brett S; Wilkerson, Curtis G; Talón, Manuel

    2007-10-31

    In this work, major protein changes in the albedo of the fruit peel of Murcott tangor (tangerine x sweet orange) during postharvest ageing were studied through 2D PAGE. Protein content in matured on-tree fruits and in fruits stored in nonstressing [99% relative humidity (RH) and 25 degrees C], cold (99% RH and 4 degrees C), and drought (60% RH and 25 degrees C) conditions was initially determined. Protein identification through MS/MS determinations revealed in all samples analyzed the occurrence of manganese superoxide dismutase (Mn SOD), actin, ATP synthase beta subunit (ATPase), citrus salt-stress associated protein (CitSap), ascorbate peroxidase (APX), translationally controlled tumor protein (TCTP), and a cysteine proteinase (CP) of the papain family. The latter protein was identified in two different gel spots, with different molecular mass, suggesting the simultaneous presence of the proteinase precursor and its active form. While Mn SOD, actin, ATPase, and CitSap were unchanged in the assayed conditions, TCTP and APX were downregulated during the postharvest ageing process. Ageing-induced APX repression was also reversed by drought. CP contents in albedo, which were similar in on- and off-tree fruits, were strongly dependent upon cold storage. The active/total CP protein ratio significantly increased after cold exposure. This proteomic survey indicates that major changes in protein content in the albedo of the peel of postharvest stored citrus fruits are apparently related to the activation of programmed cell death (PCD).

  10. Biophysical evaluation of aminoclay as an effective protectant for protein stabilization during freeze-drying and storage

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    Song JG

    2016-12-01

    Full Text Available Jae Geun Song, Sang Hoon Lee, Hyo-Kyung Han College of Pharmacy, Dongguk University, Goyang, South Korea Abstract: This study aimed to evaluate aminoclay (3-aminopropyl-functionalized magnesium phyllosilicate as an effective protectant for the stabilization of protein formulation in freeze-drying. Bovine serum albumin (BSA, as a model protein, was freeze-dried with aminoclay at various concentrations, and the effects of aminoclay on the structural stability of proteins were compared with those of the conventional stabilizers. The structural characteristics of the protein were determined by size exclusion chromatography (SEC, circular dichroism (CD, and Fourier transform infrared (FTIR spectroscopy. Furthermore, physicochemical and morphological characteristics were examined by X-ray powder diffraction (XRPD, differential scanning calorimetry (DSC, and scanning electron microscopy (SEM. XRPD and DSC patterns indicated that the glass transition temperature (Tg of the amorphous formulation of aminoclay mixed with proteins was gradually elevated as the concentration of aminoclay increased. FTIR and CD spectral analysis suggested that the protein structure was well maintained with aminoclay during the freeze-drying process and 3 months of storage at 4°C and 40°C. Furthermore, aminoclay conferred the greatest protection against aggregation and retained the monomer content of BSA even at a high temperature. The morphological characteristics of lyophilized proteins were also well conserved during the storage with aminoclay. These results suggested that aminoclay may be useful as an alternative stabilizer for maintaining the structural stability of protein formulations. Keywords: aminoclay, cryoprotectant, lyoprotectant, freeze-drying, protein, stability

  11. Molecular structure, dynamics and hydration studies of soybean storage proteins and model systems by nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Kakalis, L.T.

    1989-01-01

    The potential of high-resolution 13 C NMR for the characterization of soybean storage proteins was explored. The spectra of a commercial soy protein isolate as well as those of alkali-denatured 7S and 11S soybean globulins were well resolved and tentatively assigned. Relaxation measurements indicated fast motion for several side chains and the protein backbone. Protein fractions (11S and 7S) were also investigated at various states of molecular association. The large size of the multisubunit soybean storage proteins affected adversely both the resolution and the sensitivity of their 13 C NMR spectra. A comparison of 17 O and 2 H NMR relaxation rates of water in solutions of lysozyme (a model system) as a function of concentration, pH and magnetic field suggested that only 17 O monitors directly the hydration of lysozyme. Analysis of 17 O NMR lysozyme hydration data in terms of a two-state, fast-exchange, anisotropic model resulted in hydration parameters which are consistent with the protein's physico-chemical properties. The same model was applied to the calculation of the amount and mobility of bound water in soy protein dispersions by means of 17 O NMR relaxation measurements as a function of protein concentration. The protein concentration dependences of 1 H transverse NMR relaxation measurements at various pH and ionic strength values were fitted by a viral expansion. The interpretation of the data was based on the effects of protein aggregation, salt binding and protein group ionization on the NMR measurements. In all cases, relaxation rates showed a linear dependence on protein activity

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

    Science.gov (United States)

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

    2015-10-23

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

  13. The use of CaCl2 and other salts to improve surface finish and eliminate vacuoles in ICF microencapsulated shells

    International Nuclear Information System (INIS)

    McQuillan, B.W.; Elsner, F.H.; Stephens, R.B.; Brown, L.C.

    1999-01-01

    Polystyrene and poly(α-methylstyrene) (PAMS) shells made by microencapsulation are prone to having vacuoles in the walls and a concomitant surface roughness. These defects can be detrimental to the implosion required for ICF shots. The authors have found that adding sufficient salt (typically CaCl 2 or NH 4 Cl) to the exterior polyvinylalcohol (PVA) solution during the drying phase inhibits the formation of vacuoles and decreases the surface roughness of the shells. The use of such salts does affect other shell specifications, for which other process variables must be adjusted

  14. Specific binding of Ulex europaeus agglutinin I lectin to sarcolemma of distal myopathy with rimmed vacuole formation.

    Science.gov (United States)

    Yatabe, K; Kawai, M

    1997-08-01

    Ulex europaeus agglutinin I (UEA I) binding was studied in 83 patients with various neuromuscular disorders. UEA I labelled endomysial capillaries and endothelial cells of perimysial blood vessels in all the examined muscles. There was no UEA I binding to muscle fibres except for all (9) cases of distal myopathy with rimmed vacuole formation (DMRV), 1 of 5 cases of inclusion body myositis and 1 of 36 cases of inflammatory myopathies. The UEA I binding was completely eliminated by preincubation of UEA I solution with L-fucose. Using electron microscopy, the UEA I binding was localized to sarcolemma and intrasarco-plasmic membranous organelles other than mitochondria. Myosatellite cells were not labelled. These findings revealed the existence of fucosylated proteins or lipids in a subset of skeletal muscles suffering from DMRV. Biochemical identification of the fucosylated substance and further detailed study on subcellular localization of UEA I binding may yield important clues to the unknown pathogenesis of DMRV.

  15. Efficacy of various protein-based coating on enhancing the shelf life of fresh eggs during storage.

    Science.gov (United States)

    Caner, Cengiz; Yüceer, Muhammed

    2015-07-01

    The effectiveness of various coatings (whey protein isolate [WPI], whey protein concentrate [WPC], zein, and shellac) on functional properties, interior quality, and eggshell breaking strength of fresh eggs were evaluated during storage at 24 °: C for 6 weeks. Coatings and storage time had significant effects on Haugh unit, yolk index, albumen pH, dry matter (DMA), relative whipping capacity (RWC), and albumen viscosity. Uncoated eggs had higher albumen pH (9.56) and weight loss, and lower albumen viscosity (5.73), Haugh unit (HU), and yolk index (YI) during storage. Among the coated eggs, the shellac and zein coated eggs had the highest value of albumen viscosity (27.26 to 26.90), HU (74.10 to 73.61), and YI (44.84 to 44.63) after storage. Shellac (1.44%) was more effective in preventing weight loss than WPC (4.59%), WPI (4.60%), and zein (2.13%) coatings. Uncoated eggs had the higest value (6.71%) of weight lost. All coatings increased shell strength (5.18 to 5.73 for top and 3.58 to 4.71 for bottom) significantly (P eggs (4.70 for top and 3.15 for bottom). The functional properties such as albumen DMA (14.50 to 16.66 and 18.97 for uncoated) and albumen RWC (841 to 891 and 475 for uncoated) of fresh eggs can be preserved during storage when they are coated. The shellac and zein coatings were more effective for maintaining the internal quality of fresh eggs during storage. Fourier transform near infrared (FT-NIR) in the 800 to 2500 nm reflection spectra were used to quantify the contents of the fresh eggs at the end of storage. Eggs coated with shellac or zein displayed a higher absorbance at 970 and 1,197 nm respectively (OH vibration of water) compared with those coated with WPI or WPC and the uncoated group at the end of storage. The coatings improved functional properties and also shell strength and could be a viable alternative technology for maintaining the internal quality of eggs during long-term storage. This study highlights the promising use of

  16. Potential costs of bacterial infection on storage protein gene expression and reproduction in queenless Apis mellifera worker bees on distinct dietary regimes.

    Science.gov (United States)

    Lourenço, Anete Pedro; Martins, Juliana Ramos; Guidugli-Lazzarini, Karina Rosa; Macedo, Liliane Maria Fróes; Bitondi, Márcia Maria Gentile; Simões, Zilá Luz Paulino

    2012-09-01

    Insects are able to combat infection by initiating an efficient immune response that involves synthesizing antimicrobial peptides and a range of other defense molecules. These responses may be costly to the organism, resulting in it exploiting endogenous resources to maintain homeostasis or support defense to the detriment of other physiological needs. We used queenless worker bees on distinct dietary regimes that may alter hemolymph protein storage and ovary activation to investigate the physiological costs of infection with Serratia marcescens. The expression of the genes encoding the storage proteins vitellogenin and hexamerin 70a, the vitellogenin receptor, and vasa (which has a putative role in reproduction), was impaired in the infected bees. This impairment was mainly evident in the bees fed beebread, which caused significantly higher expression of these genes than did royal jelly or syrup, and this was confirmed at the vitellogenin and hexamerin 70a protein levels. Beebread was also the only diet that promoted ovary activation in the queenless bees, but this activation was significantly impaired by the infection. The expression of the genes encoding the storage proteins apolipophorins-I and -III and the lipophorin receptor was not altered by infection regardless the diet provided to the bees. Similarly, the storage of apolipophorin-I in the hemolymph was only slightly impaired by the infection, independently of the supplied diet. Taken together these results indicate that, infection demands a physiological cost from the transcription of specific protein storage-related genes and from the reproductive capacity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  17. Integrin Subunit CD18 Is the T-Lymphocyte Receptor for the Helicobacter pylori Vacuolating Cytotoxin

    Czech Academy of Sciences Publication Activity Database

    Sewald, X.; Gebert-Vogl, B.; Prassl, S.; Barwig, I.; Weiss, E.; Fabbri, M.; Osička, Radim; Schiemann, M.; Busch, D. H.; Semmrich, M.; Holzmann, B.; Šebo, Peter; Haas, R.

    2008-01-01

    Roč. 3, č. 1 (2008), s. 20-29 ISSN 1931-3128 R&D Projects: GA ČR GP204/07/P105 Institutional research plan: CEZ:AV0Z50200510 Keywords : helicobacter pylori * vacuolating cytotoxin * adenocarcinoma Subject RIV: EE - Microbiology, Virology Impact factor: 7.436, year: 2008

  18. Genetic mapping and validation of the loci controlling 7S α' and 11S A-type storage protein subunits in soybean [Glycine max (L.) Merr.].

    Science.gov (United States)

    Boehm, Jeffrey D; Nguyen, Vi; Tashiro, Rebecca M; Anderson, Dale; Shi, Chun; Wu, Xiaoguang; Woodrow, Lorna; Yu, Kangfu; Cui, Yuhai; Li, Zenglu

    2018-03-01

    Four soybean storage protein subunit QTLs were mapped using bulked segregant analysis and an F 2 population, which were validated with an F 5 RIL population. The storage protein globulins β-conglycinin (7S subunit) and glycinin (11S subunits) can affect the quantity and quality of proteins found in soybean seeds and account for more than 70% of the total soybean protein. Manipulating the storage protein subunits to enhance soymeal nutrition and for desirable tofu manufacturing characteristics are two end-use quality goals in soybean breeding programs. To aid in developing soybean cultivars with desired seed composition, an F 2 mapping population (n = 448) and an F 5 RIL population (n = 180) were developed by crossing high protein cultivar 'Harovinton' with the breeding line SQ97-0263_3-1a, which lacks the 7S α', 11S A 1 , 11S A 2 , 11S A 3 and 11S A 4 subunits. The storage protein composition of each individual in the F 2 and F 5 populations were profiled using SDS-PAGE. Based on the presence/absence of the subunits, genomic DNA bulks were formed among the F 2 plants to identify genomic regions controlling the 7S α' and 11S protein subunits. By utilizing polymorphic SNPs between the bulks characterized with Illumina SoySNP50K iSelect BeadChips at targeted genomic regions, KASP assays were designed and used to map QTLs causing the loss of the subunits. Soybean storage protein QTLs were identified on Chromosome 3 (11S A 1 ), Chromosome 10 (7S α' and 11S A 4 ), and Chromosome 13 (11S A 3 ), which were also validated in the F 5 RIL population. The results of this research could allow for the deployment of marker-assisted selection for desired storage protein subunits by screening breeding populations using the SNPs linked with the subunits of interest.

  19. Effects of storage structures and moisture contents on seed quality attributes of quality protein maize

    Directory of Open Access Journals (Sweden)

    Gopal Bhandari

    2017-12-01

    Full Text Available The study was aimed to examine the effects of various storage structures and moisture contents on seed quality attributes of quality protein maize seed. The quality protein maize (QPM-1 seed was tested in conventional seed storage containers (Fertilizer sack and earthen pot and the improved hermetic ones (Metal bin, Super grain bag, and Purdue Improved Crop Storage (PICS bag at Seed Science and Technology Division, Khumaltar, Nepal during February, 2015 to January 2016. Ten treatments comprising 5 storage devices in two moisture regimes (11% and 9% replicated thrice and laid out in Completely Randomized Design (CRD. Data on temperature, relative humidity (RH, germination, electrical conductivity (EC, seed moisture content (MC were collected bimonthly. The conventional containers were found liable to the external environmental condition whereas the hermetic structures observed with controlled RH level below 40% in all combinations. Electrical conductivity (EC for seed vigor showed that hermetic containers provide higher seed vigor than the conventional ones. Up to 4 months all treatments were found statistically at par for germination. A significant difference was observed in each treatment after 4 months where PICS bag & Super grain bag showed best germination followed by metal bin while fertilizer bag & earthen-pot showed poorer and poorest germination respectively till one year. Almost all treatments with lower MC showed better results than the treatments with higher MC. A negative correlation (R2=69.7% was found between EC and Germination. All six figures from 2 to 12 months on MC showed statistically different where hermetic plastic bags were found maintaining MC as initial whereas MC of fertilizer bags and earthen pot was spiked than the basal figure. The finding evidenced that the hermetic containers and low MC are the seed storage approaches for retaining the quality of seed even in an ambient environmental condition for more than a year.

  20. Effect of irradiation and soaking in BHT and sodium pyrophosphate on meat proteins and lipids during cold storage

    International Nuclear Information System (INIS)

    Hassan, I.M.; Emam, O.A.

    1988-01-01

    The effect of irradiation treatments up to 10 KGy, soaking in a solution containing 0.5% Na-pyrophosphate and 250 ppm butylated hydroxy toluene (BHT) and a combination of both treatments on the nitrogen content and solubility, protein fractions and lipids stability in beef steaks during cold storage at 4 ± 1°C was followed until the samples were rejected by sensory evaluation. The least effective radiation doses for soluble protein nitrogen (SPN), total soluble nitrogen (TSN) and total nitrogen (TN) were 2, 5 and 10 KGy, respectively. Such effects were proportionally related to the applied dose. The loss in nitrogen compounds and/or their solubility which occurred upon irradiation appeared to be retarded by soaking treatment. Irradiation treatments induced additional protein fraction which seems to be originated from the sarcoplasmic proteins. After the resolution of rigor mortis, the incremental rate of nitrogen extractability was inversely related to the irradiation dose. Another protein fraction was detected only in the 10 KGy irradiated samples after 14 days of cold storage which might be originating from fibrillar proteins as a result of its interaction with some lipid oxidation products. However, soaking treatment itself caused extensive changes in protein fractions, in contrast, protection from radiation and radiation after-effects were observed

  1. COMPARATIVE DYNAMICS OF PROTEIN DESTRUCTION IN CANNED FOODS IN SAUCE AT DIFFERENT THERMAL TREATMENT REGIMES AND SUBSEQUENT STORAGE

    OpenAIRE

    V. B. Krylova; T. V. Gustova

    2017-01-01

    In the course of investigations, the structural changes in proteins were established, which were associated with the preliminary treatment of meat ingredients, a pH level of the system and parameters of thermal treatment.The pasteurization regimes allowed retaining a protein nitrogen proportion up to 94% by the end of canned food storage duration. Upon sterilization, the losses in protein nitrogen were two times higher. A negative effect of more acidic sauce on preservation of the protein nitr...

  2. Fructan active enzymes (FAZY) activities and biosynthesis of fructooligosaccharides in the vacuoles of Agave tequilana Weber Blue variety plants of different age.

    Science.gov (United States)

    Mellado-Mojica, Erika; González de la Vara, Luis E; López, Mercedes G

    2017-02-01

    Biosynthesis of agave fructans occurs in mesontle vacuoles which showed fluctuations in FAZY activities and synthesized a diverse spectrum of fructooligosaccharide isomers. Agave tequilana Weber Blue variety is an important agronomic crop in Mexico. Fructan metabolism in A. tequilana exhibits changes in fructan content, type, degree of polymerization (DP), and molecular structure. Specific activities of vacuolar fructan active enzymes (FAZY) in A. tequilana plants of different age and the biosynthesis of fructooligosaccharides (FOSs) were analyzed in this work. Vacuoles from mesontle (stem) protoplasts were isolated and collected from 2- to 7-year-old plants. For the first time, agave fructans were identified in the vacuolar content by HPAEC-PAD. Several FAZY activities (1-SST, 6-SFT, 6G-FFT, 1-FFT, and FEH) with fluctuations according to the plant age were found in protein vacuolar extracts. Among vacuolar FAZY, 1-SST activities appeared in all plant developmental stages, as well as 1-FFT and FEH activities. The enzymes 6G-FFT and 6-SST showed only minimal activities. Lowest and highest FAZY activities were found in 2- and 6-year-old plants, respectively. Synthesized products (FOS) were analyzed by TLC and HPAEC-PAD. Vacuolar FAZYs yielded large FOS isomers diversity, being 7-year-old plants the ones that synthesized a greater variety of fructans with different DP, linkages, and molecular structures. Based on the above, we are proposing a model for the FAZY activities constituting the FOS biosynthetic pathways in Agave tequilana Weber Blue variety.

  3. LAMP-2 deficiency leads to hippocampal dysfunction but normal clearance of neuronal substrates of chaperone-mediated autophagy in a mouse model for Danon disease

    OpenAIRE

    Rothaug, Michelle; Stroobants, Stijn; Schweizer, Michaela; Peters, Judith; Zunke, Friederike; Allerding, Mirka; D?Hooge, Rudi; Saftig, Paul; Blanz, Judith

    2015-01-01

    The Lysosomal Associated Membrane Protein type-2 (LAMP-2) is an abundant lysosomal membrane protein with an important role in immunity, macroautophagy (MA) and chaperone-mediated autophagy (CMA). Mutations within the Lamp2 gene cause Danon disease, an X-linked lysosomal storage disorder characterized by (cardio)myopathy and intellectual dysfunction. The pathological hallmark of this disease is an accumulation of glycogen and autophagic vacuoles in cardiac and skeletal muscle that, along with ...

  4. An inhibition of p38 mitogen activated protein kinase delays the platelet storage lesion.

    Directory of Open Access Journals (Sweden)

    Andrey Skripchenko

    Full Text Available BACKGROUND AND OBJECTIVES: Platelets during storage undergo diverse alterations collectively known as the platelet storage lesion, including metabolic, morphological, functional and structural changes. Some changes correlate with activation of p38 mitogen activated protein kinase (p38 MAPK. Another MAPK, extracellular signal-related kinase (ERK, is involved in PLT activation. The aim of this study was to compare the properties of platelets stored in plasma in the presence or absence of p38 and ERK MAPK inhibitors. MATERIALS AND METHODS: A single Trima apheresis platelet unit (n = 12 was aliquoted into five CLX storage bags. Two aliquots were continuously agitated with or without MAPK inhibitors. Two aliquots were subjected to 48 hours of interruption of agitation with or without MAPK inhibitors. One aliquot contained the same amount of solvent vehicle used to deliver the inhibitor. Platelets were stored at 20-24°C for 7 days and sampled on Days 1, 4, and 7 for 18 in vitro parameters. RESULTS: Inhibition of p38 MAPK by VX-702 leads to better maintenance of all platelet in vitro storage parameters including platelet mitochondrial function. Accelerated by interruption of agitation, the platelet storage lesion of units stored with VX-702 was diminished to that of platelets stored with continuous agitation. Inhibition of ERK MAPK did not ameliorate decrements in any in vitro platelet properties. CONCLUSION: Signaling through p38 MAPK, but not ERK, is associated with platelet deterioration during storage.

  5. Vacuolization of mucolipidosis type II mouse exocrine gland cells represents accumulation of autolysosomes.

    Science.gov (United States)

    Boonen, Marielle; van Meel, Eline; Oorschot, Viola; Klumperman, Judith; Kornfeld, Stuart

    2011-04-15

    We previously reported that mice deficient in UDP-GlcNAc:lysosomal enzyme GlcNAc-1-phosphotransferase (mucolipidosis type II or Gnptab -/- mice), the enzyme that initiates the addition of the mannose 6-phosphate lysosomal sorting signal on acid hydrolases, exhibited extensive vacuolization of their exocrine gland cells, while the liver, brain, and muscle appeared grossly unaffected. Similar pathological findings were observed in several exocrine glands of patients with mucolipidosis II. To understand the basis for this cell type-specific abnormality, we analyzed these tissues in Gnptab -/- mice using a combined immunoelectron microscopy and biochemical approach. We demonstrate that the vacuoles in the exocrine glands are enlarged autolysosomes containing undigested cytoplasmic material that accumulate secondary to deficient lysosomal function. Surprisingly, the acid hydrolase levels in these tissues ranged from normal to modestly decreased, in contrast to skin fibroblasts, which accumulate enlarged lysosomes and/or autolysosomes also but exhibit very low levels of acid hydrolases. We propose that the lysosomal defect in the exocrine cells is caused by the combination of increased secretion of the acid hydrolases via the constitutive pathway along with their entrapment in secretory granules. Taken together, our results provide new insights into the mechanisms of the tissue-specific abnormalities seen in mucolipidosis type II.

  6. The purification and characterization of a third storage protein (convicilin) from the seeds of pea (Pisum sativum L.).

    OpenAIRE

    Croy, R R; Gatehouse, J A; Tyler, M; Boulter, D

    1980-01-01

    A third storage protein, distinct from legumin and vicilin, has been purified from the seeds of pea (Pisum sativum L.). This protein has been named 'convicilin' and is present in protein bodies isolated from pea seeds. Convicilin has a subunit mol.wt. of 71 000 and a mol.wt. in its native form of 290 000. Convicilin is antigenically dissimilar to legumin, but gives a reaction of identity with vicilin when tested against antibodies raised against both proteins. However, convicilin contains no ...

  7. The Proteome of the Isolated Chlamydia trachomatis Containing Vacuole Reveals a Complex Trafficking Platform Enriched for Retromer Components.

    Directory of Open Access Journals (Sweden)

    Lukas Aeberhard

    2015-06-01

    Full Text Available Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

  8. Effect of pasteurization on the protein composition and oxidative stability of beer during storage.

    Science.gov (United States)

    Lund, Marianne N; Hoff, Signe; Berner, Torben S; Lametsch, René; Andersen, Mogens L

    2012-12-19

    The impacts of pasteurization of a lager beer on protein composition and the oxidative stability were studied during storage at 22 °C for 426 days in the dark. Pasteurization clearly improved the oxidative stability of beer determined by ESR spectroscopy, whereas it had a minor negative effect on the volatile profile by increasing volatile compounds that is generally associated with heat treatment and a loss of fruity ester aroma. A faster rate of radical formation in unpasteurized beer was consistent with a faster consumption of sulfite. Beer proteins in the unpasteurized beer were more degraded, most likely due to proteolytic enzyme activity of yeast remnants and more precipitation of proteins was also observed. The differences in soluble protein content and composition are suggested to result in differences in the contents of prooxidative metals as a consequence of the proteins ability to bind metals. This also contributes to the differences in oxidative stabilities of the beers.

  9. Viability and growth promotion of starter and probiotic bacteria in yogurt supplemented with whey protein hydrolysate during refrigerated storage

    Directory of Open Access Journals (Sweden)

    Anna Dąbrowska

    2017-11-01

    Full Text Available The effect of whey protein hydrolysate (WPH addition on growth of standard yoghurt cultures and Bifidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. WPH was obtained with the use of serine protease from Y. lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either whey protein concentrate, skim milk powder (SMP, WPH-SMP (ratio 1:1, WPH. The hydrolysate increased the yoghurt culture counts at the initial stage of fermentation and significantly inhibited the decrease in population viability throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of WPH. The hydrolysate did not increase the Bifidobacterium adolescentis counts at the initial stage. However, the WPH significantly improved its viability. After 21 days of storage, in the yogurts supplemented with WPH, the population of these bacteria oscillated around 3.04 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.

  10. Viability and growth promotion of starter and probiotic bacteria in yogurt supplemented with whey protein hydrolysate during refrigerated storage.

    Science.gov (United States)

    Dąbrowska, Anna; Babij, Konrad; Szołtysik, Marek; Chrzanowska, Józefa

    2017-11-22

    The effect of whey protein hydrolysate (WPH) addition on growth of standard yoghurt cultures and Bifidobacterium adolescentis during co-fermentation and its viability during storage at 4ºC in yoghurts has been evaluated. WPH was obtained with the use of serine protease from Y. lipolytica yeast. Stirred probiotic yoghurts were prepared by using whole milk standardized to 16% of dry matter with the addition of either whey protein concentrate, skim milk powder (SMP), WPH-SMP (ratio 1:1), WPH. The hydrolysate increased the yoghurt culture counts at the initial stage of fermentation and significantly inhibited the decrease in population viability throughout the storage at 4ºC in comparison to the control. The post-fermentation acidification was also retarded by the addition of WPH. The hydrolysate did not increase the Bifidobacterium adolescentis counts at the initial stage. However, the WPH significantly improved its viability. After 21 days of storage, in the yogurts supplemented with WPH, the population of these bacteria oscillated around 3.04 log10 CFU/g, while in samples where SMP or whey protein concentrate was used, the bacteria were no longer detected.

  11. A functional genomics approach to understand the control and regulation of storage protein biosynthesis in barley grain

    DEFF Research Database (Denmark)

    Vincze, É; Hansen, M; Bowra, S

    2008-01-01

    assembled in our laboratory. To identify coregulated genes, a distance matrix was constructed and we identified three clusters corresponding to the early, middle and late seed development. The gene expression pattern associated with the clusters was investigated using pathway specific analysis with specific......The aim of the study was to obtain an insight into amino acid and storage protein metabolism in the developing barley grain at the molecular level. Our strategy was to analyse the transcriptome of relevant pathways in developing grains of field grown barley using a grain specific microarray...... pathways in the barley grain. The study described here could provide a strong complement to existing knowledge assisting further  understanding of seed development and thereby provide a foundation for plant breeding towards storage protein with improved nutritional quality....

  12. Effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on protein oxidation and textural properties of fish mince (Pagrosomus major) during frozen storage.

    Science.gov (United States)

    Wang, Tiantian; Li, Zhenxing; Yuan, Fangzhou; Lin, Hong; Pavase, Tushar Ramesh

    2017-03-01

    Frozen storage of minced fish is currently one of the most important techniques to maintain its functional properties. However, some deterioration does occur during frozen storage and cause quality loss. The effects of brown seaweed polyphenols, α-tocopherol, and ascorbic acid on lipid and protein oxidation and textural properties of red sea bream (Pagrosomus major) during 90 days of frozen storage at -18 °C were investigated. All added antioxidants at 1 g kg -1 resulted in significantly lower thiobarbituric acid-reactive substances (TBARS) compared to the control during the 45 days of frozen storage. The antioxidants were also effective in retarding protein oxidation concerning to total sulfhydryl content and protein carbonyl content. Brown seaweed polyphenols and α-tocopherol significantly retarded the inactivation of Ca 2+ -ATPase activity during the first 45 days, whereas ascorbic acid had no such effect. The antioxidant activity showed either an invariable or decrease trend after 45 days storage. Adding antioxidants had a significant effect on the breaking force of the gels during the frozen storage period. These results indicate that brown seaweed polyphenols and α-tocopherol can be used to prevent oxidative reactions and thus maintain the structure of the gel formed by fish mince during frozen storage. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  13. Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

    Science.gov (United States)

    Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

    2015-04-01

    Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  14. Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments.

    Science.gov (United States)

    Maltese, William A; Overmeyer, Jean H

    2014-06-01

    Apoptosis is the most widely recognized form of physiological programmed cell death. During the past three decades, various nonapoptotic forms of cell death have gained increasing attention, largely because of their potential importance in pathological processes, toxicology, and cancer therapy. A recent addition to the panoply of cell death phenotypes is methuosis. The neologism is derived from the Greek methuo (to drink to intoxication) because the hallmark of this form of cell death is displacement of the cytoplasm by large fluid-filled vacuoles derived from macropinosomes. The demise of the cell resembles many forms of necrosis, insofar as there is a loss of metabolic capacity and plasma membrane integrity, without the cell shrinkage and nuclear fragmentation associated with apoptosis. Methuosis was initially defined in glioblastoma cells after ectopic expression of activated Ras, but recent reports have described small molecules that can induce the features of methuosis in a broad spectrum of cancer cells, including those that are resistant to conventional apoptosis-inducing drugs. This review summarizes the available information about the distinguishing morphological characteristics and underlying mechanisms of methuosis. We compare and contrast methuosis with other cytopathological conditions in which accumulation of clear cytoplasmic vacuoles is a prominent feature. Finally, we highlight key questions that need to be answered to determine whether methuosis truly represents a unique form of regulated cell death. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  16. Juliprosopine and juliprosine from prosopis juliflora leaves induce mitochondrial damage and cytoplasmic vacuolation on cocultured glial cells and neurons.

    Science.gov (United States)

    Silva, Victor Diogenes A; Pitanga, Bruno P S; Nascimento, Ravena P; Souza, Cleide S; Coelho, Paulo Lucas C; Menezes-Filho, Noélio; Silva, André Mário M; Costa, Maria de Fátima D; El-Bachá, Ramon S; Velozo, Eudes S; Costa, Silvia L

    2013-12-16

    Prosopis juliflora is a shrub largely used for animal and human consumption. However, ingestion has been shown to induce intoxication in animals, which is characterized by neuromuscular alterations induced by mechanisms that are not yet well understood. In this study, we investigated the cytotoxicity of a total alkaloid extract (TAE) and one alkaloid fraction (F32) obtained from P. juliflora leaves to rat cortical neurons and glial cells. Nuclear magnetic resonance characterization of F32 showed that this fraction is composed of a mixture of two piperidine alkaloids, juliprosopine (majority constituent) and juliprosine. TAE and F32 at concentrations between 0.3 and 45 μg/mL were tested for 24 h on neuron/glial cell primary cocultures. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed that TAE and F32 were cytotoxic to cocultures, and their IC50 values were 31.07 and 7.362 μg/mL, respectively. Exposure to a subtoxic concentration of TAE or F32 (0.3-3 μg/mL) induced vacuolation and disruption of the astrocyte monolayer and neurite network, ultrastructural changes, characterized by formation of double-membrane vacuoles, and mitochondrial damage, associated with changes in β-tubulin III and glial fibrillary acidic protein expression. Microglial proliferation was also observed in cultures exposed to TAE or F32, with increasing levels of OX-42-positive cells. Considering that F32 was more cytotoxic than TAE and that F32 reproduced in vitro the main morphologic and ultrastructural changes of "cara torta" disease, we can also suggest that piperidine alkaloids juliprosopine and juliprosine are primarily responsible for the neurotoxic damage observed in animals after they have consumed the plant.

  17. Characteristics of the digestive vacuole membrane of the alga-bearing ciliate Paramecium bursaria.

    Science.gov (United States)

    Kodama, Yuuki; Fujishima, Masahiro

    2012-07-01

    Cells of the ciliate Paramecium bursaria harbor symbiotic Chlorella spp. in their cytoplasm. To establish endosymbiosis with alga-free P. bursaria, symbiotic algae must leave the digestive vacuole (DV) to appear in the cytoplasm by budding of the DV membrane. This budding was induced not only by intact algae but also by boiled or fixed algae. However, this budding was not induced when food bacteria or India ink were ingested into the DVs. These results raise the possibility that P. bursaria can recognize sizes of the contents in the DVs. To elucidate this possibility, microbeads with various diameters were mixed with alga-free P. bursaria and traced their fate. Microbeads with 0.20μm diameter did not induce budding of the DVs. Microbeads with 0.80μm diameter produced DVs of 5-10μm diameter at 3min after mixing; then the DVs fragmented and became vacuoles of 2-5μm diameter until 3h after mixing. Each microbead with a diameter larger than 3.00μm induced budding similarly to symbiotic Chlorella. These observations reveal that induction of DV budding depends on the size of the contents in the DVs. Dynasore, a dynamin inhibitor, greatly inhibited DV budding, suggesting that dynamin might be involved in DV budding. Copyright © 2011 Elsevier GmbH. All rights reserved.

  18. COMPARATIVE DYNAMICS OF PROTEIN DESTRUCTION IN CANNED FOODS IN SAUCE AT DIFFERENT THERMAL TREATMENT REGIMES AND SUBSEQUENT STORAGE

    Directory of Open Access Journals (Sweden)

    V. B. Krylova

    2017-01-01

    Full Text Available In the course of investigations, the structural changes in proteins were established, which were associated with the preliminary treatment of meat ingredients, a pH level of the system and parameters of thermal treatment.The pasteurization regimes allowed retaining a protein nitrogen proportion up to 94% by the end of canned food storage duration. Upon sterilization, the losses in protein nitrogen were two times higher. A negative effect of more acidic sauce on preservation of the protein nitrogen fraction in canned foods was established.An accumulation of the peptide nitrogen fraction in the canned foods in tomato sauce aſter pasteurization was two times more intensive. In the sterilized canned foods, the processes of accumulation of the low molecular weight nitrogenous compounds were more intensive, which suggests a depth of destruction of the protein and peptide nitrogen fraction. It was shown that an accumulation of amino-ammonia nitrogen during canned food storage was on average 12.4% irrespective of the pH value in the used sauces and the type of thermal treatment.A shiſt in the pH value of the canned foods toward the acid side upon pasteurization was noticed. With that, a degree of the shiſt in the canned foods in tomato sauce was 2.5 times higher than the pH value of the canned foods in sour cream sauce. When sterilizing canned foods, another dynamics of the pH values was observed: a pH value declined by 0.39 units in the canned foods in tomato sauce and grew by 0.22 units in the canned foods in sour cream sauce. During storage, the tendency of more intense pH decline was revealed for the canned foods in tomato sauce aſter pasteurization compared to the canned foods aſter sterilization. Another character of the pH value dynamics was found in the canned foods in sour cream sauce: an insignificant increase (by 0.7% of the pH value in the pasteurized canned foods and a significant decrease (by 8.4% in the sterilized canned foods

  19. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Science.gov (United States)

    Fauteux, François; Strömvik, Martina V

    2009-01-01

    Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

  20. Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

    Directory of Open Access Journals (Sweden)

    Fauteux François

    2009-10-01

    Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

  1. Diel pattern of circadian clock and storage protein gene expression in leaves and during seed filling in cowpea (Vigna unguiculata).

    Science.gov (United States)

    Weiss, Julia; Terry, Marta I; Martos-Fuentes, Marina; Letourneux, Lisa; Ruiz-Hernández, Victoria; Fernández, Juan A; Egea-Cortines, Marcos

    2018-02-14

    Cowpea (Vigna unguiculata) is an important source of protein supply for animal and human nutrition. The major storage globulins VICILIN and LEGUMIN (LEG) are synthesized from several genes including LEGA, LEGB, LEGJ and CVC (CONVICILIN). The current hypothesis is that the plant circadian core clock genes are conserved in a wide array of species and that primary metabolism is to a large extent controlled by the plant circadian clock. Our aim was to investigate a possible link between gene expression of storage proteins and the circadian clock. We identified cowpea orthologues of the core clock genes VunLHY, VunTOC1, VunGI and VunELF3, the protein storage genes VunLEG, VunLEGJ, and VunCVC as well as nine candidate reference genes used in RT-PCR. ELONGATION FACTOR 1-A (ELF1A) resulted the most suitable reference gene. The clock genes VunELF3, VunGI, VunTOC1 and VunLHY showed a rhythmic expression profile in leaves with a typical evening/night and morning/midday phased expression. The diel patterns were not completely robust and only VungGI and VungELF3 retained a rhythmic pattern under free running conditions of darkness. Under field conditions, rhythmicity and phasing apparently faded during early pod and seed development and was regained in ripening pods for VunTOC1 and VunLHY. Mature seeds showed a rhythmic expression of VunGI resembling leaf tissue under controlled growth chamber conditions. Comparing time windows during developmental stages we found that VunCVC and VunLEG were significantly down regulated during the night in mature pods as compared to intermediate ripe pods, while changes in seeds were non-significant due to high variance. The rhythmic expression under field conditions was lost under growth chamber conditions. The core clock gene network is conserved in cowpea leaves showing a robust diel expression pattern except VunELF3 under growth chamber conditions. There appears to be a clock transcriptional reprogramming in pods and seeds compared to

  2. A High-Content Phenotypic Screen Reveals the Disruptive Potency of Quinacrine and 3′,4′-Dichlorobenzamil on the Digestive Vacuole of Plasmodium falciparum

    OpenAIRE

    Lee, Yan Quan; Goh, Amanda S. P.; Ch'ng, Jun Hong; Nosten, François H.; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S. W.

    2014-01-01

    Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of in...

  3. Properties and regulation of biosynthesis of cottonseed storage proteins. Comprehensive progress report, December 1, 1976 to September 1, 1979

    Energy Technology Data Exchange (ETDEWEB)

    Dure, III, L S

    1979-01-01

    The regulation of gene expression in cotton seed embryogenesis was studied by attempting to define what gene products are likely to be highly regulated during this developmental progression. The flow of nitrogen into the free amino acids pools of the developing cotyledons, and into the principal nitrogen nutritional reserve of the seed, the storage proteins was measured. This was continued by following the flow of nitrogen from the storage proteins to the principal exported amino acid asparagine that occurs during the first several days of germination. In this fashion the rise and fall of certain enzymes of amino acid intermediary metabolism could be postulated, and in some cases, verified. The subsets of abundant mRNAs whose appearance and disappearance coincided with developmental events in cotyledon embryogenesis/germination with the short range goal of identifying proteins/enzyme activities were delineated as well as their mRNAs that represent specific developmental stages and the long range goal of using these representatives as probes for studying the mechanisms controlling the rise and fall of these mRNAs and their protein products.

  4. The second report of a new hypomyelinating disease due to a defect in the VPS11 gene discloses a massive lysosomal involvement.

    Science.gov (United States)

    Hörtnagel, Konstanze; Krägeloh-Mann, Inge; Bornemann, Antje; Döcker, Miriam; Biskup, Saskia; Mayrhofer, Heidi; Battke, Florian; du Bois, Gabriele; Harzer, Klaus

    2016-11-01

    Vesicular protein sorting-associated proteins (VPS, including VPS11) are indispensable in the endocytic network, in particular the endosome-lysosome biogenesis. Exome sequencing revealed the homozygous variant p.Leu387_ Gly395del in the VPS11 gene in two siblings. On immunoblotting, the mutant VPS11 protein showed a distinctly reduced immunostaining intensity. The children presented with primary and severe developmental delay associated with myoclonic seizures, spastic tetraplegia, trunk and neck hypotonia, blindness, hearing loss, and microcephaly. Neuro-imaging showed severe hypomyelination affecting cerebral and cerebellar white matter and corpus callosum, in the absence of a peripheral neuropathy. Electron microscopy of a skin biopsy revealed clusters of membranous cytoplasmic bodies in dermal unmyelinated nerve axons, and numbers of vacuoles in eccrine sweat glands, similar to what is seen in a classic lysosomal storage disease (LSD). Bone marrow cytology showed a high number of storage macrophages with a micro-vacuolated cytoplasm. Biochemically, changes in urinary glycosphingolipids were reminiscent of those in prosaposin deficiency (another LSD). The clinical and neuro-imaged features in our patients were almost identical to those in some recently reported patients with another variant in the VPS11 gene, p.Cys846Gly; underlining the presumed pathogenic potential of VPS11 defects. A new feature was the morphological evidence for lysosomal storage in VPS11 deficiency: This newly characterised disease can be viewed as belonging to the complex field of LSD.

  5. Major Proteins of the Amyloplast of Agar and Soil - Grown Potato Tubers

    DEFF Research Database (Denmark)

    Hald, Simon; Blennow, Andreas; Stensballe, Allan

    and total tuber extracts by SDS-PAGE and the specific activities of marker enzymes for amyloplast, cytosol, mitochondria and the vacuole. SDS-PAGE separated amyloplast and starch granule proteins were in-gel digested with trypsin, analyzed by mass spectrometry, and identified by searches against presently...

  6. Lack of Globulin Synthesis during Seed Development Alters Accumulation of Seed Storage Proteins in Rice

    Directory of Open Access Journals (Sweden)

    Hye-Jung Lee

    2015-06-01

    Full Text Available The major seed storage proteins (SSPs in rice seeds have been classified into three types, glutelins, prolamins, and globulin, and the proportion of each SSP varies. It has been shown in rice mutants that when either glutelins or prolamins are defective, the expression of another type of SSP is promoted to counterbalance the deficit. However, we observed reduced abundances of glutelins and prolamins in dry seeds of a globulin-deficient rice mutant (Glb-RNAi, which was generated with RNA interference (RNAi-induced suppression of globulin expression. The expression of the prolamin and glutelin subfamily genes was reduced in the immature seeds of Glb-RNAi lines compared with those in wild type. A proteomic analysis of Glb-RNAi seeds showed that the reductions in glutelin and prolamin were conserved at the protein level. The decreased pattern in glutelin was also significant in the presence of a reductant, suggesting that the polymerization of the glutelin proteins via intramolecular disulfide bonds could be interrupted in Glb-RNAi seeds. We also observed aberrant and loosely packed structures in the storage organelles of Glb-RNAi seeds, which may be attributable to the reductions in SSPs. In this study, we evaluated the role of rice globulin in seed development, showing that a deficiency in globulin could comprehensively reduce the expression of other SSPs.

  7. A cytosolic copper storage protein provides a second level of copper tolerance in Streptomyces lividans.

    Science.gov (United States)

    Straw, Megan L; Chaplin, Amanda K; Hough, Michael A; Paps, Jordi; Bavro, Vassiliy N; Wilson, Michael T; Vijgenboom, Erik; Worrall, Jonathan A R

    2018-01-24

    Streptomyces lividans has a distinct dependence on the bioavailability of copper for its morphological development. A cytosolic copper resistance system is operative in S. lividans that serves to preclude deleterious copper levels. This system comprises of several CopZ-like copper chaperones and P 1 -type ATPases, predominantly under the transcriptional control of a metalloregulator from the copper sensitive operon repressor (CsoR) family. In the present study, we discover a new layer of cytosolic copper resistance in S. lividans that involves a protein belonging to the newly discovered family of copper storage proteins, which we have named Ccsp (cytosolic copper storage protein). From an evolutionary perspective, we find Ccsp homologues to be widespread in Bacteria and extend through into Archaea and Eukaryota. Under copper stress Ccsp is upregulated and consists of a homotetramer assembly capable of binding up to 80 cuprous ions (20 per protomer). X-ray crystallography reveals 18 cysteines, 3 histidines and 1 aspartate are involved in cuprous ion coordination. Loading of cuprous ions to Ccsp is a cooperative process with a Hill coefficient of 1.9 and a CopZ-like copper chaperone can transfer copper to Ccsp. A Δccsp mutant strain indicates that Ccsp is not required under initial copper stress in S. lividans, but as the CsoR/CopZ/ATPase efflux system becomes saturated, Ccsp facilitates a second level of copper tolerance.

  8. Integrated transcriptomics and proteomics analysis of storage protein composition in developing barley grain to improve nutritional profile

    DEFF Research Database (Denmark)

    Kaczmarczyk, Agnieszka Ewa; Dionisio, Giuseppe; Renaut, Jenny

    2012-01-01

    The aim of the study was to understand the molecular and biochemical mechanisms underpinning the effect of nitrogen (N) on barley (Hordeum vulgare) storage protein production (hordeins) during grain filling. Using a combination of advanced biochemistry methods, we could comprehensively describe c...

  9. The guanine nucleotide exchange factor RIC8 regulates conidial germination through Gα proteins in Neurospora crassa.

    Directory of Open Access Journals (Sweden)

    Carla J Eaton

    Full Text Available Heterotrimeric G protein signaling is essential for normal hyphal growth in the filamentous fungus Neurospora crassa. We have previously demonstrated that the non-receptor guanine nucleotide exchange factor RIC8 acts upstream of the Gα proteins GNA-1 and GNA-3 to regulate hyphal extension. Here we demonstrate that regulation of hyphal extension results at least in part, from an important role in control of asexual spore (conidia germination. Loss of GNA-3 leads to a drastic reduction in conidial germination, which is exacerbated in the absence of GNA-1. Mutation of RIC8 leads to a reduction in germination similar to that in the Δgna-1, Δgna-3 double mutant, suggesting that RIC8 regulates conidial germination through both GNA-1 and GNA-3. Support for a more significant role for GNA-3 is indicated by the observation that expression of a GTPase-deficient, constitutively active gna-3 allele in the Δric8 mutant leads to a significant increase in conidial germination. Localization of the three Gα proteins during conidial germination was probed through analysis of cells expressing fluorescently tagged proteins. Functional TagRFP fusions of each of the three Gα subunits were constructed through insertion of TagRFP in a conserved loop region of the Gα subunits. The results demonstrated that GNA-1 localizes to the plasma membrane and vacuoles, and also to septa throughout conidial germination. GNA-2 and GNA-3 localize to both the plasma membrane and vacuoles during early germination, but are then found in intracellular vacuoles later during hyphal outgrowth.

  10. The pupylation machinery is involved in iron homeostasis by targeting the iron storage protein ferritin.

    Science.gov (United States)

    Küberl, Andreas; Polen, Tino; Bott, Michael

    2016-04-26

    The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.

  11. N-terminomics reveals control of Arabidopsis seed storage proteins and proteases by the Arg/N-end rule pathway.

    Science.gov (United States)

    Zhang, Hongtao; Gannon, Lucy; Hassall, Kirsty L; Deery, Michael J; Gibbs, Daniel J; Holdsworth, Michael J; van der Hoorn, Renier A L; Lilley, Kathryn S; Theodoulou, Frederica L

    2018-05-01

    The N-end rule pathway of targeted protein degradation is an important regulator of diverse processes in plants but detailed knowledge regarding its influence on the proteome is lacking. To investigate the impact of the Arg/N-end rule pathway on the proteome of etiolated seedlings, we used terminal amine isotopic labelling of substrates with tandem mass tags (TMT-TAILS) for relative quantification of N-terminal peptides in prt6, an Arabidopsis thaliana N-end rule mutant lacking the E3 ligase PROTEOLYSIS6 (PRT6). TMT-TAILS identified over 4000 unique N-terminal peptides representing c. 2000 protein groups. Forty-five protein groups exhibited significantly increased N-terminal peptide abundance in prt6 seedlings, including cruciferins, major seed storage proteins, which were regulated by Group VII Ethylene Response Factor (ERFVII) transcription factors, known substrates of PRT6. Mobilisation of endosperm α-cruciferin was delayed in prt6 seedlings. N-termini of several proteases were downregulated in prt6, including RD21A. RD21A transcript, protein and activity levels were downregulated in a largely ERFVII-dependent manner. By contrast, cathepsin B3 protein and activity were upregulated by ERFVIIs independent of transcript. We propose that the PRT6 branch of the pathway regulates protease activities in a complex manner and optimises storage reserve mobilisation in the transition from seed to seedling via control of ERFVII action. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  12. Amino acid and protein changes in tilapia and spanish mackerel after irradiation and storage

    Energy Technology Data Exchange (ETDEWEB)

    Al-Kahtani, Hassan A.; Abu-Tarboush, Hamza M.; Atia, Mohamed; Bajaber, Adnan S.; Ahmed, Mohamed A.; El-Mojaddidi, Mohamed A

    1998-01-01

    Some amino acids in tilapia decreased while some others increased when subjected to doses up to 10.0 kGy. However, 10 kGy contributed to a significant reduction in all amino acids of Spanish mackerel. Variations in amino acid contents continued during post-irradiation storage with no consistent trend of increase or decrease. SDS-PAGE of protein from both fish showed 27 bands of subunits with MW < 14.0-94.0 KD. Isoelectric focusing patterns of sarcoplasmic protein of unirradiated and irradiated fish showed no charge in the number of bands, while some changes were observed in the intensities of the anodic and cathodic bands depending on isoelectric points (pIs)

  13. Lipid storage myopathy with clinical markers of Marfan syndrome: A rare association

    Directory of Open Access Journals (Sweden)

    Subasree Ramakrishnan

    2012-01-01

    Full Text Available Disorders of lipid metabolism can cause variable clinical presentations, often involving skeletal muscle, alone or together with other tissues. A 19-year-old boy presented with a 2-year history of muscle pain, cramps, exercise intolerance and progressive weakness of proximal lower limbs. Examination revealed skeletal markers of Marfan syndrome in the form of increased arm span compared with height, Kyphoscoliois, moderate pectus excavatum, high arched palate and wrist sign. He also had mild neck flexor weakness and proximal lower limb weakness with areflexia. Pathologic findings revealed lipid-laden fine vacuoles in the muscle fibers. Possibility of carnitine deficiency myopathy was considered and the patient was started on carnitine and Co Q. The patient made remarkable clinical improvement over the next 2 months. This case is reported for rarity of the association of clinical markers of Marfan syndrome and lipid storage myopathy and sparse literature on lipid storage myopathy in the Indian context.

  14. Storage stability of biodegradable polyethylene glycol microspheres

    Science.gov (United States)

    Jain, Era; Sheth, Saahil; Polito, Kristen; Sell, Scott A.; Zustiak, Silviya P.

    2017-10-01

    Degradable hydrogel microspheres are popular choices for multiple biomedical applications, including drug, protein, or cell carriers for minimally invasive delivery. Clinical transitioning of such new, sensitive pharmaceutical preparations requires investigation of storage methods that retain key properties for extended time. In this study, we sought to determine the influence of seven common storage conditions on the physical and mechanical properties of degradable polyethylene glycol (PEG) hydrogel microspheres: 25 °C, 4 °C, -80 °C, lyophilization/-20 °C, dimethyl sulfoxide/-80 °C, dimethyl sulfoxide/lyophilization/-20 °C, vacuum/-20 °C. We have outlined the storage conditions in detail and explained their effect on swelling ratio, stiffness and degradation rate post-storage. Additionally, we have implemented protein-loaded hydrogels to evaluate the effect of storage conditions on diffusivity as well as protein stability post-storage. We found that hydrogels could be stored short-term (1-4 d) under moist conditions (i.e. storage without drying) without a substantial loss of properties. For extended storage (7-28 d), they could be stored either at  -80 °C (moist condition) or vacuum drying (dry condition).

  15. Phospholipase C-related catalytically inactive protein participates in the autophagic elimination of Staphylococcus aureus infecting mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Kae Harada-Hada

    Full Text Available Autophagy is an intrinsic host defense system that recognizes and eliminates invading bacterial pathogens. We have identified microtubule-associated protein 1 light chain 3 (LC3, a hallmark of autophagy, as a binding partner of phospholipase C-related catalytically inactive protein (PRIP that was originally identified as an inositol trisphosphate-binding protein. Here, we investigated the involvement of PRIP in the autophagic elimination of Staphylococcus aureus in infected mouse embryonic fibroblasts (MEFs. We observed significantly more LC3-positive autophagosome-like vacuoles enclosing an increased number of S. aureus cells in PRIP-deficient MEFs than control MEFs, 3 h and 4.5 h post infection, suggesting that S. aureus proliferates in LC3-positive autophagosome-like vacuoles in PRIP-deficient MEFs. We performed autophagic flux analysis using an mRFP-GFP-tagged LC3 plasmid and found that autophagosome maturation is significantly inhibited in PRIP-deficient MEFs. Furthermore, acidification of autophagosomes was significantly inhibited in PRIP-deficient MEFs compared to the wild-type MEFs, as determined by LysoTracker staining and time-lapse image analysis performed using mRFP-GFP-tagged LC3. Taken together, our data show that PRIP is required for the fusion of S. aureus-containing autophagosome-like vacuoles with lysosomes, indicating that PRIP is a novel modulator in the regulation of the innate immune system in non-professional phagocytic host cells.

  16. A high-content phenotypic screen reveals the disruptive potency of quinacrine and 3',4'-dichlorobenzamil on the digestive vacuole of Plasmodium falciparum.

    Science.gov (United States)

    Lee, Yan Quan; Goh, Amanda S P; Ch'ng, Jun Hong; Nosten, François H; Preiser, Peter Rainer; Pervaiz, Shazib; Yadav, Sanjiv Kumar; Tan, Kevin S W

    2014-01-01

    Plasmodium falciparum is the etiological agent of malignant malaria and has been shown to exhibit features resembling programmed cell death. This is triggered upon treatment with low micromolar doses of chloroquine or other lysosomotrophic compounds and is associated with leakage of the digestive vacuole contents. In order to exploit this cell death pathway, we developed a high-content screening method to select compounds that can disrupt the parasite vacuole, as measured by the leakage of intravacuolar Ca(2+). This assay uses the ImageStream 100, an imaging-capable flow cytometer, to assess the distribution of the fluorescent calcium probe Fluo-4. We obtained two hits from a small library of 25 test compounds, quinacrine and 3',4'-dichlorobenzamil. The ability of these compounds to permeabilize the digestive vacuole in laboratory strains and clinical isolates was validated by confocal microscopy. The hits could induce programmed cell death features in both chloroquine-sensitive and -resistant laboratory strains. Quinacrine was effective at inhibiting field isolates in a 48-h reinvasion assay regardless of artemisinin clearance status. We therefore present as proof of concept a phenotypic screening method with the potential to provide mechanistic insights to the activity of antimalarial drugs.

  17. Investigation of total seed storage proteins of pakistani and japanese maize (zea mays l.) through sds-page markers

    International Nuclear Information System (INIS)

    Shinwari, Z.K.

    2014-01-01

    The assessment of genetic diversity among the members of a species is of vital importance for successful breeding and adaptability. In the present study 83 genotypes of maize of Pakistani and Japanese origin were evaluated for the total seed storage proteins using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) through vertical slab unit. The total protein subunits were separated on 12% polyacrylamide gel using standard protocols. A total of 18 protein subunits were noted out of which 7 (39%) were monomorphic and 11 (61%) were polymorphic, with molecular weight ranging from 10 to 122 kDa. Coefficients of similarity among the accessions ranged between 0.89 and 1.00. The dendrogram obtained through UPGMA clustering method showed two main clusters: 1 and 2. First cluster comprised of 9 genotypes including Sahiwal-2002, while second cluster contained 74 genotypes including Aaiti-2002 and Sadaf. Over all a low level of polymorphism was observed in total seed storage protein patterns of maize genotypes from Pakistan as well as Japan. It is inferred from the present study that more genotypes of maize could be brought under study and more advanced biochemical techniques with more reliable results could be followed to bring assessment of genetic diversity of maize for planning breeding programs. (author)

  18. Ammonium accumulation in commercially available embryo culture media and protein supplements during storage at 2-8°C and during incubation at 37°C.

    Science.gov (United States)

    Kleijkers, Sander H M; van Montfoort, Aafke P A; Bekers, Otto; Coonen, Edith; Derhaag, Josien G; Evers, Johannes L H; Dumoulin, John C M

    2016-06-01

    Does ammonium accumulate in commercially available culture media and protein supplements used for in vitro development of human pre-implantation embryos during storage and incubation? Ammonium accumulates in ready-to-use in vitro fertilization (IVF) culture media during storage at 2-8°C and in ready-to-use IVF culture media and protein supplements during incubation at 37°C. Both animal and human studies have shown that the presence of ammonium in culture medium has detrimental effects on embryonic development and pregnancy rate. It is, therefore, important to assess the amount of ammonium accumulation in ready-to-use IVF culture media under conditions that are common in daily practice. Ammonium accumulation was investigated in 15 ready-to-use media, 11 protein-free media and 8 protein supplements. Ammonium was measured by the use of an enzymatic method with glutamate dehydrogenase. To simulate the storage and incubation conditions during IVF treatments, ammonium concentrations were measured at different time-points during storage at 2-8°C for 6 weeks and during incubation at 37°C for 4 days. All ready-to-use, i.e. protein supplemented, culture media showed ammonium accumulation during storage for 6 weeks (ranging from 9.2 to 99.8 µM) and during incubation for 4 days (ranging from 8.4 to 138.6 µM), resulting in levels that might affect embryo development. The protein supplements also showed ammonium accumulation, while the culture media without protein supplementation did not. The main sources of ammonium buildup in ready-to-use culture media were unstable glutamine and the protein supplements. No additional ammonium buildup was found during incubation when using an oil overlay or with the presence of an embryo in the culture droplet. In addition to the unstable glutamine and the protein supplements, other free amino acids might contribute to the ammonium buildup. We did not investigate the deterioration of other components in the media. Break-down of

  19. Effects of diet, packaging, and irradiation on protein oxidation, lipid oxidation, and color of raw broiler thigh meat during refrigerated storage.

    Science.gov (United States)

    Xiao, S; Zhang, W G; Lee, E J; Ma, C W; Ahn, D U

    2011-06-01

    This study was designed to evaluate the effects of dietary treatment, packaging, and irradiation singly or in combination on the oxidative stability of broiler chicken thigh meat. A total of 120 four-week-old chickens were divided into 12 pens (10 birds/pen), and 4 pens of broilers were randomly assigned to a control oxidized diet (5% oxidized oil) or an antioxidant-added diet [500 IU of vitamin E + 200 mg/kg of butylated hydroxyanisole (BHA)] and fed for 2 wk. After slaughter, thigh meats were separated, ground, packaged in either oxygen-permeable or oxygen-impermeable vacuum bags, and irradiated at 0 or 3 kGy. Lipid oxidation (TBA-reactive substances), protein oxidation (carbonyl), and color of the meat were measured at 1, 4, and 7 d of refrigerated storage. The lipid and protein oxidation of thigh meats from birds fed the diet supplemented with antioxidants (vitamin E + BHA) was significantly lower than the lipid and protein oxidation of birds fed the control diet, whereas the lipid and protein oxidation of broilers fed the oxidized oil diet was higher than that of birds fed the control diet. Vacuum packaging slowed, but irradiation accelerated, the lipid and protein oxidation of thigh meat during storage. Dietary antioxidants (vitamin E + BHA) and irradiation treatments showed a stronger effect on lipid oxidation than on protein oxidation. A significant correlation between lipid and protein oxidation in meat was found during storage. Dietary supplementation of vitamin E + BHA and the irradiation treatment increased the lightness and redness of thigh meat, respectively. It is suggested that appropriate use of dietary antioxidants in combination with packaging could be effective in minimizing oxidative changes in irradiated raw chicken thigh meat.

  20. Fabrication of Flexible, Fully Organic, Degradable Energy Storage Devices Using Silk Proteins.

    Science.gov (United States)

    Pal, Ramendra K; Kundu, Subhas C; Yadavalli, Vamsi K

    2018-03-21

    Flexible and thin-film devices are of great interest in epidermal and implantable bioelectronics. The integration of energy storage and delivery devices such as supercapacitors (SCs) with properties such as flexibility, miniaturization, biocompatibility, and degradability are sought for such systems. Reducing e-waste and using sustainable materials and processes are additional desirable qualities. Herein, a silk protein-based biocompatible and degradable thin-film microSC (μSC) is reported. A protein carrier with the conducting polymer poly(3,4-ethylenedioxythiophene) polystyrene sulfonate and reduced graphene oxide dopant is used as a photopatternable biocomposite ink. Active electrodes are fabricated using photolithography under benign conditions, using only water as the solvent. These electrodes are printed on flexible protein sheets to form degradable, organic devices with a benign agarose-NaCl gel electrolyte. High capacitance, power density, cycling stability over 500 cycles, and the ability to power a light-emitting diode are shown. The device is flexible, can sustain cyclic mechanical stresses over 450 cycles, and retain capacitive properties over several days in liquid. Significantly, the μSCs are cytocompatible and completely degraded over the period of ∼1 month. By precise control of the device configuration, these silk protein-based, all-polymer organic devices can be designed to be tunably transient and provide viable alternatives for powering flexible and implantable bioelectronics.

  1. New views of the Toxoplasma gondii parasitophorous vacuole as revealed by Helium Ion Microscopy (HIM).

    Science.gov (United States)

    de Souza, Wanderley; Attias, Marcia

    2015-07-01

    The Helium Ion Microscope (HIM) is a new technology that uses a highly focused helium ion beam to scan and interact with the sample, which is not coated. The images have resolution and depth of field superior to field emission scanning electron microscopes. In this paper, we used HIM to study LLC-MK2 cells infected with Toxoplasma gondii. These samples were chemically fixed and, after critical point drying, were scraped with adhesive tape to expose the inner structure of the cell and parasitophorous vacuoles. We confirmed some of the previous findings made by field emission-scanning electron microscopy and showed that the surface of the parasite is rich in structures suggestive of secretion, that the nanotubules of the intravacuolar network (IVN) are not always straight, and that bifurcations are less frequent than previously thought. Fusion of the tubules with the parasite membrane or the parasitophorous vacuole membrane (PVM) was also infrequent. Tiny adhesive links were observed for the first time connecting the IVN tubules. The PVM showed openings of various sizes that even allowed the observation of endoplasmic reticulum membranes in the cytoplasm of the host cell. These findings are discussed in relation to current knowledge on the cell biology of T. gondii. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Protein degradation and protein synthesis in long-term memory formation

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    2014-06-01

    Full Text Available Long-term memory (LTM formation requires transient changes in the activity of intracellular signaling cascades that are thought to regulate new gene transcription and de novo protein synthesis in the brain. Consistent with this, protein synthesis inhibitors impair LTM for a variety of behavioral tasks when infused into the brain around the time of training or following memory retrieval, suggesting that protein synthesis is a critical step in LTM storage in the brain. However, evidence suggests that protein degradation mediated by the ubiquitin-proteasome system may also be a critical regulator of LTM formation and stability following retrieval. This requirement for increased protein degradation has been shown in the same brain regions in which protein synthesis is required for LTM storage. Additionally, increases in the phosphorylation of proteins involved in translational control parallel increases in protein polyubiquitination and the increased demand for protein degradation is regulated by intracellular signaling molecules thought to regulate protein synthesis during LTM formation. In some cases inhibiting proteasome activity can rescue memory impairments that result from pharmacological blockade of protein synthesis, suggesting that protein degradation may control the requirement for protein synthesis during the memory storage process. Results such as these suggest that protein degradation and synthesis are both critical for LTM formation and may interact to properly consolidate and store memories in the brain. Here, we review the evidence implicating protein synthesis and degradation in LTM storage and highlight the areas of overlap between these two opposing processes. We also discuss evidence suggesting these two processes may interact to properly form and store memories. LTM storage likely requires a coordinated regulation between protein degradation and synthesis at multiple sites in the mammalian brain.

  3. Targeted modification of storage protein content resulting in improved amino acid composition of barley grain

    DEFF Research Database (Denmark)

    Sikdar, Md. Shafiqul Islam; Bowra, S; Schmidt, Daiana

    2016-01-01

    family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C......C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi...... silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7 % reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS...

  4. Sperm with large nuclear vacuoles and semen quality in the evaluation of male infertility.

    Science.gov (United States)

    Komiya, Akira; Watanabe, Akihiko; Kawauchi, Yoko; Fuse, Hideki

    2013-02-01

    This study compared the sperm nuclear vacuoles and semen quality in the evaluation of male infertility. One hundred and forty-two semen samples were obtained from patients who visited the Male Infertility Clinic at Toyama University Hospital. Semen samples were evaluated by conventional semen analyses and the Sperm Motility Analysis System (SMAS). In addition, spermatozoa were analyzed at 3,700-6,150x magnification on an inverted microscope equipped with DIC/Nomarski differential interference contrast optics. A large nuclear vacuole (LNV) was defined as one or more vacuoles with the maximum diameter showing > 50% width of the sperm head. The percentage of spermatozoa with LNV (% LNV) was calculated for each sample. Correlations between the % LNV and parameters in SMAS and conventional semen analyses were analyzed. Processed motile spermatozoa from each sample were evaluated. The mean age of patients was 35 years old. Semen volume was 2.9 ± 1.6mL (0.1-11.0; mean ± standard deviation, minimum-maximum), sperm count was 39.3 ± 54.9 (x10(6)/mL, 0.01-262.0), sperm motility was 25.1 ± 17.8% (0-76.0), and normal sperm morphology was 10.3 ± 10.1% (0-49.0). After motile spermatozoa selection, we could evaluate % LNV in 125 ejaculates (88.0%) and at least one spermatozoon with LNV was observed in 118 ejaculates (94.4%). The percentage of spermatozoa with LNV was 28.0 ± 22.4% (0-100) and % LNV increased significantly when semen quality decreased. The correlation between the % LNV and the semen parameters was weak to moderate; correlation coefficients were -0.3577 in sperm count (p sperm motility (p = 0.0084), -0.2769 in motile sperm count (p = 0.019), -0.2419 in total motile sperm count (p = 0.0070), and -0.1676 in normal sperm morphology (p = 0.0639). The % LNV did not show a significant correlation with the SMAS parameters except for weak correlation to beat/cross frequency (r = -0.2414, p = 0.0071). The percentage of

  5. A study of different indicators of Maillard reaction with whey proteins and different carbohydrates under adverse storage conditions.

    Science.gov (United States)

    Leiva, Graciela E; Naranjo, Gabriela B; Malec, Laura S

    2017-01-15

    This study examined different indicators of each stage of Maillard reaction under adverse storage conditions in a system with whey proteins and lactose or glucose. The analysis of lysine loss by the o-phthaldialdehyde method can be considered a good indicator of the early stage, showing considerable differences in reactivity when systems with mono and disaccharides were analyzed. Capillary electrophoresis proved to be a sensitive method for evaluating the extent of glycosylation of the native proteins, providing valuable information when the loss of lysine was not significant. The estimation of the Amadori compound from the determination of total 5-hydroxymethyl-2-furfuraldehyde would have correlate well with reactive lysine content if the advanced stages of the reaction had not been reached. For assessing the occurrence of the intermediate and final stages, the measurement of free 5-hydroxymethyl-2-furfuraldehyde and color, proved not to be suitable for storage conditions. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. [Study of the reineta protein modifications (Brama australis), put under freezing and storage to -18 degrees C and -30 degrees C].

    Science.gov (United States)

    Abugoch, Lilian; Quitral, Vilma; Larraín, M Angélica; Vinagre, Julia; Kriukov, Andrei; Chávez, Gloria

    2006-12-01

    The objective of the present work was to study functional and thermal properties of reineta (Brama australis) frozen meat, analysed by water retention capacity (WRC), gel forming capacity (GFC), texture, emulsifying capacity and differential scanning calorimetry (DSC). For this study, reineta fillets were obtained and extracted by the same conditions, and cutted, packaged, frozen and stored at -18 degrees C and -30 degrees C for 7 months. The results obtained, showed that there were no signifficant differences in the responses to thermal treatment for all the specimens. For samples frozen at -18 degrees C and -30 degrees C, the protein contents were 23.5 + 0.0 and 25.4 + 1.0%, respectively. The WRC values were 0.45 + 0.1 and 1.59 +/- 0.0 g water/g protein, respectively. The gel forming capacity was only present in the fresh samples, whereas the frozen stored ones only form protein aggregates. The emulsifying capacity was between 960 and 1400 g oil / g protein, and the storage time increased this value. The miosin denaturation temperature (Td) and denaturation enthalpy (?H), obtained by DSC, fluctuated between 39.2 +/- 0.5 to 44.8 +/- 0.8 degrees C and 1.12 +/- 0.3 to 0.52 +/- 0.2 J/g, respectively. The actina values were between 71.0 +/- 0.6 to 75.3 +/- 0.5 degrees C and between 0.5 +/- 0.1 to 0.7 +/- 0.1 J/g. Cooperativity decreased as the storage time increased. This is showing a certain degree of protein displacement. The values found by thermal analyses showed a direct relationship with the functional properties, both decreasing with storage time.

  7. Yip1A, a Novel Host Factor for the Activation of the IRE1 Pathway of the Unfolded Protein Response during Brucella Infection

    Science.gov (United States)

    Taguchi, Yuki; Imaoka, Koichi; Kataoka, Michiyo; Uda, Akihiko; Nakatsu, Daiki; Horii-Okazaki, Sakuya; Kunishige, Rina; Kano, Fumi; Murata, Masayuki

    2015-01-01

    Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments.  On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation. PMID:25742138

  8. Multinodular and Vacuolating Neuronal Tumor of the Cerebrum: A New "Leave Me Alone" Lesion with a Characteristic Imaging Pattern.

    Science.gov (United States)

    Nunes, R H; Hsu, C C; da Rocha, A J; do Amaral, L L F; Godoy, L F S; Watkins, T W; Marussi, V H; Warmuth-Metz, M; Alves, H C; Goncalves, F G; Kleinschmidt-DeMasters, B K; Osborn, A G

    2017-10-01

    Multinodular and vacuolating neuronal tumor of the cerebrum is a recently reported benign, mixed glial neuronal lesion that is included in the 2016 updated World Health Organization classification of brain neoplasms as a unique cytoarchitectural pattern of gangliocytoma. We report 33 cases of presumed multinodular and vacuolating neuronal tumor of the cerebrum that exhibit a remarkably similar pattern of imaging findings consisting of a subcortical cluster of nodular lesions located on the inner surface of an otherwise normal-appearing cortex, principally within the deep cortical ribbon and superficial subcortical white matter, which is hyperintense on FLAIR. Only 4 of our cases are biopsy-proven because most were asymptomatic and incidentally discovered. The remaining were followed for a minimum of 24 months (mean, 3 years) without interval change. We demonstrate that these are benign, nonaggressive lesions that do not require biopsy in asymptomatic patients and behave more like a malformative process than a true neoplasm. © 2017 by American Journal of Neuroradiology.

  9. Canola/rapeseed protein-functionality and nutrition

    Directory of Open Access Journals (Sweden)

    Wanasundara Janitha P.D.

    2016-07-01

    Full Text Available Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S and napin (2S dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed.

  10. Survival of salmonella transformed to express green fluorescent protein on Italian parsley as affected by processing and storage.

    Science.gov (United States)

    Duffy, E A; Cisneros-Zevallos, L; Castillo, A; Pillai, S D; Ricke, S C; Acuff, G R

    2005-04-01

    To study the effect of processing and storage parameters on the survival of Salmonella on fresh Italian parsley, parsley bunches were dipped for 3 or 15 min in suspensions that were preequilibrated to 5, 25, or 35 degrees C and inoculated with Salmonella transformed to express enhanced green fluorescent protein. Loosely attached and/or associated, strongly attached and/or associated, and internalized and/or entrapped Salmonella cells were enumerated over 0, 1, and 7 days of storage at 25 degrees C and over 0, 1, 7, 14, and 30 days of storage at 4 degrees C using surface-plating procedures. Leaf sections obtained from samples after 0, 1, and 7 days of storage were examined using confocal scanning laser microscopy. Temperature of the dip suspension had little effect on the attachment and survival of Salmonella cells on parsley. Regardless of the temperature or duration of dip, Salmonella was internalized. Immersion for longer times resulted in higher numbers of attached and internalized cells. Microscopic observations supported these results and revealed Salmonella cells near the stomata and within cracks in the cuticle. Storage temperature had the greatest impact on the survival of Salmonella cells on parsley. When stored at 25 degrees C, parsley had a shelf life of 7 days, and Salmonella populations significantly increased over the 7 days of storage. For parsley stored at 4 degrees C, numbers of Salmonella cells decreased over days 0, 1, and 7. After 7 days of storage, there were no viable internalized Salmonella cells detected. Storage temperature represents an important control point for the safety of fresh parsley.

  11. Transfer of phagocytosed particles to the parasitophorous vacuole of Leishmania mexicana is a transient phenomenon preceding the acquisition of annexin I by the phagosome.

    Science.gov (United States)

    Collins, H L; Schaible, U E; Ernst, J D; Russell, D G

    1997-01-01

    The eukaryotic intracellular pathogen Leishmania mexicana resides inside macrophages contained within a membrane bound parasitophorous vacuole which, as it matures, acquires the characteristics of a late endosomal compartment. This study reports the selectivity of fusion of this compartment with other particle containing vacuoles. Phagosomes containing zymosan or live Listeria monocytogenes rapidly fused with L. mexicana parasitophorous vacuoles, while those containing latex beads or heat killed L. monocytogenes failed to do so. Fusigenicity of phagosomes was not primarily dependent on the receptor utilized for ingestion, as opsonization with defined ligands could not overcome the exclusion of either latex beads or heat killed organisms. However modulation of intracellular pH by pharmacological agents such as chloroquine and ammonium chloride increased delivery of live Listeria and also induced transfer of previously excluded particles. The absence of fusion correlated with the acquisition of annexin I, a putative lysosomal targeting, molecule, on the phagosome membrane. We propose that the acquisition of cellular membrane constituents such as annexin I during phagosome maturation can ultimately direct the fusion pathway of the vesicles formed and have described a model system to further document changes in vesicle fusigenicity within cells.

  12. Gold nanoparticles induced cloudy swelling to hydropic degeneration, cytoplasmic hyaline vacuolation, polymorphism, binucleation, karyopyknosis, karyolysis, karyorrhexis and necrosis in the liver

    Directory of Open Access Journals (Sweden)

    Jarrar Bashir M

    2011-09-01

    Full Text Available Abstract Background Nanoparticles (NPs can potentially cause adverse effects on organ, tissue, cellular, subcellular and protein levels due to their unusual physicochemical properties. Advances in nanotechnology have identified promising candidates for many biological and biomedical applications. The aim of the present study was to investigate the particle-size, dose and exposure duration effects of gold nanoparticles (GNPs on the hepatic tissue in an attempt to cover and understand the toxicity and their potential therapeutic and diagnostic use. Methods A total of 70 healthy male Wistar-Kyoto rats were exposed to GNPs received 50 or 100 ul of GNPs infusion of size (10, 20 and 50 nm for 3 or 7 days to investigate particle-size, dose and exposure duration effects of GNPs on the hepatic tissue. Results In comparison with respective control rats, exposure to GNPs doses has produced alterations in the hepatocytes, portal triads and the sinusoids. The alterations in the hepatocytes were mainly vacuolar to hydropic degeneration, cytopasmic hyaline vacuolation, polymorphism, binucleation, karyopyknosis, karyolysis, karyorrhexis and necrosis. Conclusions The hepatocytes swelling might be exhibited as a result of disturbances of membranes function that lead to massive influx of water and Na+ due to GNPs effects accompanied by leakage of lysosomal hydrolytic enzymes that lead to cytoplasmic degeneration and macromolecular crowding. Hydropic degeneration is a result of ion and fluid homestasis that lead to an increase of intracellular water. The vacuolated swelling of the cytoplasm of the hepatocytes of the GNPs treated rats might indicate acute and subacute liver injury induced by the GNPs. Binucleation represents a consequence of cell injury and is a sort of chromosomes hyperplasia which is usually seen in regenerating cells. The induced histological alterations might be an indication of injured hepatocytes due to GNPs toxicity that became unable to deal

  13. Dioscorin, the major tuber storage protein of yam (Dioscorea batatas decne) with carbonic anhydrase and trypsin inhibitor activities.

    Science.gov (United States)

    Hou, W C; Liu, J S; Chen, H J; Chen, T E; Chang, C F; Lin, Y H

    1999-05-01

    Dioscorin, the tuber storage protein of yam (Dioscorea batatas Decne), was purified successively by ammonium sulfate fractionation, DE-52 ion exchange chromatography, and Sephadex G-75 column. Two protein bands (82 and 28 kDa) were found under nonreducing conditions after SDS-PAGE; but only one band (32 kDa) was detected under reducing conditions. The first 21 amino acids in the N-terminal region of the 28 kDa form were VEDEFSYIEGNPNGPENWGNL, which was highly homologous to deductive sequence of dioscorin from cDNA of another yam species (Dioscoreacayenensis Lam) reported by Conlan et al. (Plant Mol. Biol. 1995, 28, 369-380). Hewett-Emmett and Tashian (Mol. Phylogenet. Evol. 1996, 5, 50 -77) mentioned that, according to DNA alignments, dioscorin from yam (D. cayenensis) was alpha-carbonic anhydrase (alpha-CA) related. In this report, we found that the purified dioscorin showed both CA dehydration activity using sodium bicarbonate as a substrate and CA activity staining after SDS-PAGE. A polyclonal antibody, which was raised against trypsin inhibitor (TI), a storage protein of sweet potato (Ipomoea batatas [L.] Lam var. Tainong 57), cross-reacted with dioscorin, which also showed TI activity determined by both activity staining after SDS-PAGE and trypsin inhibition determination.

  14. Actin polymerization in the endosomal pathway, but not on the Coxiella-containing vacuole, is essential for pathogen growth.

    Directory of Open Access Journals (Sweden)

    Heather E Miller

    2018-04-01

    Full Text Available Coxiella burnetii is an intracellular bacterium that replicates within an expansive phagolysosome-like vacuole. Fusion between the Coxiella-containing vacuole (CCV and late endosomes/multivesicular bodies requires Rab7, the HOPS tethering complex, and SNARE proteins, with actin also speculated to play a role. Here, we investigated the importance of actin in CCV fusion. Filamentous actin patches formed around the CCV membrane that were preferred sites of vesicular fusion. Accordingly, the mediators of endolysosomal fusion Rab7, VAMP7, and syntaxin 8 were concentrated in CCV actin patches. Generation of actin patches required C. burnetii type 4B secretion and host retromer function. Patches decorated with VPS29 and VPS35, components of the retromer, FAM21 and WASH, members of the WASH complex that engage the retromer, and Arp3, a component of the Arp2/3 complex that generates branched actin filaments. Depletion by siRNA of VPS35 or VPS29 reduced CCV actin patches and caused Rab7 to uniformly distribute in the CCV membrane. C. burnetii grew normally in VPS35 or VPS29 depleted cells, as well as WASH-knockout mouse embryo fibroblasts, where CCVs are devoid of actin patches. Endosome recycling to the plasma membrane and trans-Golgi of glucose transporter 1 (GLUT1 and cationic-independent mannose-6-phosphate receptor (CI-M6PR, respectively, was normal in infected cells. However, siRNA knockdown of retromer resulted in aberrant trafficking of GLUT1, but not CI-M6PR, suggesting canonical retrograde trafficking is unaffected by retromer disruption. Treatment with the specific Arp2/3 inhibitor CK-666 strongly inhibited CCV formation, an effect associated with altered endosomal trafficking of transferrin receptor. Collectively, our results show that CCV actin patches generated by retromer, WASH, and Arp2/3 are dispensable for CCV biogenesis and stability. However, Arp2/3-mediated production of actin filaments required for cargo transport within the

  15. Free Fatty Acid Storage in Human Visceral and Subcutaneous Adipose Tissue

    Science.gov (United States)

    Ali, Asem H.; Koutsari, Christina; Mundi, Manpreet; Stegall, Mark D.; Heimbach, Julie K.; Taler, Sandra J.; Nygren, Jonas; Thorell, Anders; Bogachus, Lindsey D.; Turcotte, Lorraine P.; Bernlohr, David; Jensen, Michael D.

    2011-01-01

    OBJECTIVE Because direct adipose tissue free fatty acid (FFA) storage may contribute to body fat distribution, we measured FFA (palmitate) storage rates and fatty acid (FA) storage enzymes/proteins in omental and abdominal subcutaneous fat. RESEARCH DESIGN AND METHODS Elective surgery patients received a bolus of [1-14C]palmitate followed by omental and abdominal subcutaneous fat biopsies to measure direct FFA storage. Long chain acyl-CoA synthetase (ACS) and diacylglycerol acyltransferase activities, CD36, fatty acid-binding protein, and fatty acid transport protein 1 were measured. RESULTS Palmitate tracer storage (dpm/g adipose lipid) and calculated palmitate storage rates were greater in omental than abdominal subcutaneous fat in women (1.2 ± 0.8 vs. 0.7 ± 0.4 μmol ⋅ kg adipose lipid−1 ⋅ min−1, P = 0.005) and men (0.7 ± 0.2 vs. 0.2 ± 0.1, P < 0.001), and both were greater in women than men (P < 0.0001). Abdominal subcutaneous adipose tissue palmitate storage rates correlated with ACS activity (women: r = 0.66, P = 0.001; men: r = 0.70, P = 0.007); in men, CD36 was also independently related to palmitate storage rates. The content/activity of FA storage enzymes/proteins in omental fat was dramatically lower in those with more visceral fat. In women, only omental palmitate storage rates were correlated (r = 0.54, P = 0.03) with ACS activity. CONCLUSIONS Some adipocyte FA storage factors correlate with direct FFA storage, but sex differences in this process in visceral fat do not account for sex differences in visceral fatness. The reduced storage proteins in those with greater visceral fat suggest that the storage factors we measured are not a predominant cause of visceral adipose tissue accumulation. PMID:21810594

  16. Vacuolating encephalitis in mice infected by human coronavirus OC43

    International Nuclear Information System (INIS)

    Jacomy, Helene; Talbot, Pierre J.

    2003-01-01

    Involvement of viruses in human neurodegenerative diseases and the underlying pathologic mechanisms remain generally unclear. Human respiratory coronaviruses (HCoV) can infect neural cells, persist in human brain, and activate myelin-reactive T cells. As a means of understanding the human infection, we characterized in vivo the neurotropic and neuroinvasive properties of HCoV-OC43 through the development of an experimental animal model. Virus inoculation of 21-day postnatal C57BL/6 and BALB/c mice led to a generalized infection of the whole CNS, demonstrating HCoV-OC43 neuroinvasiveness and neurovirulence. This acute infection targeted neurons, which underwent vacuolation and degeneration while infected regions presented strong microglial reactivity and inflammatory reactions. Damage to the CNS was not immunologically mediated and microglial reactivity was instead a consequence of direct virus-mediated neuronal injury. Although this acute encephalitis appears generally similar to that induced by murine coronaviruses, an important difference rests in the prominent spongiform-like degeneration that could trigger neuropathology in surviving animals

  17. Wherever I may roam: protein and membrane trafficking in P. falciparum-infected red blood cells.

    Science.gov (United States)

    Deponte, Marcel; Hoppe, Heinrich C; Lee, Marcus C S; Maier, Alexander G; Richard, Dave; Rug, Melanie; Spielmann, Tobias; Przyborski, Jude M

    2012-12-01

    Quite aside from its immense importance as a human pathogen, studies in recent years have brought to light the fact that the malaria parasite Plasmodium falciparum is an interesting eukaryotic model system to study protein trafficking. Studying parasite cell biology often reveals an overrepresentation of atypical cell biological features, possibly driven by the parasites' need to survive in an unusual biological niche. Malaria parasites possess uncommon cellular compartments to which protein traffic must be directed, including secretory organelles such as rhoptries and micronemes, a lysosome-like compartment referred to as the digestive vacuole and a complex (four membrane-bound) plastid, the apicoplast. In addition, the parasite must provide proteins to extracellular compartments and structures including the parasitophorous vacuole, the parasitophorous vacuolar membrane, the Maurer's clefts and both cytosol and plasma membrane of the host cell, the mature human red blood cell. Although some of these unusual destinations are possessed by other cell types, only Plasmodium parasites contain them all within one cell. Here we review what is known about protein and membrane transport in the P. falciparum-infected cell, highlighting novel features of these processes. A growing body of evidence suggests that this parasite is a real "box of tricks" with regards to protein traffic. Possibly, these tricks may be turned against the parasite by exploiting them as novel therapeutic targets. Copyright © 2012 Elsevier B.V. All rights reserved.

  18. Effects of abscisic acid and high osmoticum on storage protein gene expression in microspore embryos of Brassica napus

    International Nuclear Information System (INIS)

    Wilen, R.W.; Mandel, R.M.; Pharis, R.P.; Moloney, M.M.; Holbrook, L.A.

    1990-01-01

    Storage protein gene expression, characteristic of mid- to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using [ 2 H 6 ]ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos

  19. Ceramide-mediated macroautophagy involves inhibition of protein kinase B and up-regulation of beclin 1.

    Science.gov (United States)

    Scarlatti, Francesca; Bauvy, Chantal; Ventruti, Annamaria; Sala, Giusy; Cluzeaud, Françoise; Vandewalle, Alain; Ghidoni, Riccardo; Codogno, Patrice

    2004-04-30

    The sphingolipid ceramide is involved in the cellular stress response. Here we demonstrate that ceramide controls macroautophagy, a major lysosomal catabolic pathway. Exogenous C(2)-ceramide stimulates macroautophagy (proteolysis and accumulation of autophagic vacuoles) in the human colon cancer HT-29 cells by increasing the endogenous pool of long chain ceramides as demonstrated by the use of the ceramide synthase inhibitor fumonisin B(1). Ceramide reverted the interleukin 13-dependent inhibition of macroautophagy by interfering with the activation of protein kinase B. In addition, C(2)-ceramide stimulated the expression of the autophagy gene product beclin 1. Ceramide is also the mediator of the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells. Monodansylcadaverine staining and electron microscopy showed that this accumulation was abrogated by myriocin, an inhibitor of de novo synthesis ceramide. The tamoxifen-dependent accumulation of vacuoles was mimicked by 1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of glucosylceramide synthase. 1-Phenyl-2-decanoylamino-3-morpholino-1-propanol, tamoxifen, and C(2)-ceramide stimulated the expression of beclin 1, whereas myriocin antagonized the tamoxifen-dependent up-regulation. Tamoxifen and C(2)-ceramide interfere with the activation of protein kinase B, whereas myriocin relieved the inhibitory effect of tamoxifen. In conclusion, the control of macroautophagy by ceramide provides a novel function for this lipid mediator in a cell process with major biological outcomes.

  20. Expression of Bax in yeast affects not only the mitochondria but also vacuolar integrity and intracellular protein traffic

    DEFF Research Database (Denmark)

    Dimitrova, Irina; Toby, Garabet G; Tili, Esmerina

    2004-01-01

    -transferase (BI-GST) leads to aggregation, but not fusion of the mitochondria. In addition, Bax affects the integrity of yeast vacuoles, resulting in the disintegration and eventual loss of the organelles, and the disruption of intracellular protein traffic. While Bcl-2 coexpression only partially corrects...

  1. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

    NARCIS (Netherlands)

    Korstanje, Ron; Desai, Jigar; Lazar, Gloria; King, Benjamin; Rollins, Jarod; Spurr, Melissa; Joseph, Jamie; Kadambi, Sindhuja; Li, Yang; Cherry, Allison; Matteson, Paul G.; Paigen, Beverly; Millonig, James H.

    Korstanje R, Desai J, Lazar G, King B, Rollins J, Spurr M, Joseph J, Kadambi S, Li Y, Cherry A, Matteson PG, Paigen B, Millonig JH. Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects. Physiol Genomics 35:

  2. Effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage.

    Science.gov (United States)

    Kim, Hyun-Wook; Kim, Ji-Han; Yan, Feifei; Cheng, Heng-Wei; Brad Kim, Yuan H

    2017-12-01

    The present study aimed to evaluate the effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg during display storage. Two hundred and forty, 1-day-old male chicks (5 birds per pen) were subjected to four treatments in a 2 (thermoneutral condition at 21 °C and cyclic heat stress at 32-21-32 °C for 10 h day -1 ) × 2 (regular diet with 0 or 0.25 g kg -1 Bacillus subtilis) factorial design. Chickens were harvested at day 46, and pairs of whole legs were collected at 1 day postmortem. The chicken legs were deboned, ground, tray-packaged with oxygen-permeable film, and displayed for 3 days. Heat stress and probiotic supplementation had no impact on pH, water-holding capacity, color, protein functionality, lipid lipolysis and lipid/protein oxidation stability (P > 0.05). Display storage increased the pH and lipid oxidation of ground chicken legs (P chicken leg meat. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  3. Phenotypic Changes in Transgenic Tobacco Plants Overexpressing Vacuole-Targeted Thermotoga maritima BglB Related to Elevated Levels of Liberated Hormones

    Science.gov (United States)

    Nguyen, Quynh Anh; Lee, Dae-Seok; Jung, Jakyun; Bae, Hyeun-Jong

    2015-01-01

    The hyperthermostable β-glucosidase BglB of Thermotoga maritima was modified by adding a short C-terminal tetrapeptide (AFVY, which transports phaseolin to the vacuole, to its C-terminal sequence). The modified β-glucosidase BglB was transformed into tobacco (Nicotiana tabacum L.) plants. We observed a range of significant phenotypic changes in the transgenic plants compared to the wild-type (WT) plants. The transgenic plants had faster stem growth, earlier flowering, enhanced root systems development, an increased biomass biosynthesis rate, and higher salt stress tolerance in young plants compared to WT. In addition, programed cell death was enhanced in mature plants. Furthermore, the C-terminal AFVY tetrapeptide efficiently sorted T. maritima BglB into the vacuole, which was maintained in an active form and could perform its glycoside hydrolysis function on hormone conjugates, leading to elevated hormone [abscisic acid (ABA), indole 3-acetic acid (IAA), and cytokinin] levels that likely contributed to the phenotypic changes in the transgenic plants. The elevation of cytokinin led to upregulation of the transcription factor WUSCHELL, a homeodomain factor that regulates the development, division, and reproduction of stem cells in the shoot apical meristems. Elevation of IAA led to enhanced root development, and the elevation of ABA contributed to enhanced tolerance to salt stress and programed cell death. These results suggest that overexpressing vacuole-targeted T. maritima BglB may have several advantages for molecular farming technology to improve multiple targets, including enhanced production of the β-glucosidase BglB, increased biomass, and shortened developmental stages, that could play pivotal roles in bioenergy and biofuel production. PMID:26618153

  4. Influence of pre-cooking protein paste gelation conditions and post-cooking gel storage conditions on gel texture.

    Science.gov (United States)

    Paker, Ilgin; Matak, Kristen E

    2016-01-15

    Gelation conditions affect the setting of myofibrillar fish protein gels. Therefore the impact of widely applied pre-cooking gelation time/temperature strategies and post-cooking period on the texture and color of final protein gels was determined. Four pre-cooking gelation strategies (no setting time, 30 min at 25 °C, 1 h at 40 °C or 24 h at 4 °C) were applied to protein pastes (fish protein concentrate and standard functional additives). After cooking, texture and color were analyzed either directly or after 24 h at 4 °C on gels adjusted to 25 °C. No-set gels were harder, gummier and chewier (P cooking. Gel-setting conditions had a greater (P cooking stored gels in texture and color, depending on the pre-cooking gelation strategy. Pre-cooking gelation conditions will affect final protein gel texture and color, with gel stability benefiting from a gel-setting period. However, post-cooking storage may have a greater impact on final gels, with textural attributes becoming more consistent between all samples. © 2015 Society of Chemical Industry.

  5. The effect of melanin-free extract from Sepia esculenta ink on lipid peroxidation, protein oxidation and water-holding capacity of tilapia fillet during cold storage.

    Science.gov (United States)

    Duan, Zhen-Hua; Liu, Hua-Zhong; Luo, Ping; Gu, Yi-Peng; Li, Yan-Qun

    2018-03-14

    Preservative effect of melanin-free extract of Sepia esculenta ink (MFESI) on Sparus latus fillet has been verified in our previous work. This study aims to further approach the mechanism of MFESI for extending the shelf-life of fish fillet during cold storage. Tilapia fillets were treated with different dosage of MFESI (0, 15, 25 and 35 mg/ml) and packed with preservative film for succedent cold-storage at 4 °C for scheduled time. Contents of total volatile basic nitrogen and sulfydryl and carbanyl groups were measured for evaluating protein oxidation. Malondialdehyde contents were measured for estimating lipid peroxidation and loss of water was used to determine water-holding capacity of fillet. The data indicated that MFESI not only possessed certain degree of antioxidant capacity in vitro, also lengthened shelf-life of tilapia fillet in cold-storage condition. Apart from 15 mg/ml, both 25 and 35 mg/ml of MFESI obviously prevented lipid and protein from oxidation and reduced loss of water from tilapia fillets, and the latter was more effective than the former. MFESI can repress lipid peroxidation and protein oxidation and reduce water loss, maintain the tilapia fillets quality and, thus, it could be an effective and natural preservative for extending the shelf-life of tilapia fillets during cold storage.

  6. Zinc deficiency-induced iron accumulation, a consequence of alterations in iron regulatory protein-binding activity, iron transporters, and iron storage proteins.

    Science.gov (United States)

    Niles, Brad J; Clegg, Michael S; Hanna, Lynn A; Chou, Susan S; Momma, Tony Y; Hong, Heeok; Keen, Carl L

    2008-02-22

    One consequence of zinc deficiency is an elevation in cell and tissue iron concentrations. To examine the mechanism(s) underlying this phenomenon, Swiss 3T3 cells were cultured in zinc-deficient (D, 0.5 microM zinc), zinc-supplemented (S, 50 microM zinc), or control (C, 4 microM zinc) media. After 24 h of culture, cells in the D group were characterized by a 50% decrease in intracellular zinc and a 35% increase in intracellular iron relative to cells in the S and C groups. The increase in cellular iron was associated with increased transferrin receptor 1 protein and mRNA levels and increased ferritin light chain expression. The divalent metal transporter 1(+)iron-responsive element isoform mRNA was decreased during zinc deficiency-induced iron accumulation. Examination of zinc-deficient cells revealed increased binding of iron regulatory protein 2 (IRP2) and decreased binding of IRP1 to a consensus iron-responsive element. The increased IRP2-binding activity in zinc-deficient cells coincided with an increased level of IRP2 protein. The accumulation of IRP2 protein was independent of zinc deficiency-induced intracellular nitric oxide production but was attenuated by the addition of the antioxidant N-acetylcysteine or ascorbate to the D medium. These data support the concept that zinc deficiency can result in alterations in iron transporter, storage, and regulatory proteins, which facilitate iron accumulation.

  7. Arabidopsis Intracellular NHX-Type Sodium-Proton Antiporters are Required for Seed Storage Protein Processing.

    Science.gov (United States)

    Ashnest, Joanne R; Huynh, Dung L; Dragwidge, Jonathan M; Ford, Brett A; Gendall, Anthony R

    2015-11-01

    The Arabidopsis intracellular sodium-proton exchanger (NHX) proteins AtNHX5 and AtNHX6 have a well-documented role in plant development, and have been used to improve salt tolerance in a variety of species. Despite evidence that intracellular NHX proteins are important in vacuolar trafficking, the mechanism of this role is poorly understood. Here we show that NHX5 and NHX6 are necessary for processing of the predominant seed storage proteins, and also influence the processing and activity of a vacuolar processing enzyme. Furthermore, we show by yeast two-hybrid and bimolecular fluorescence complementation (BiFC) technology that the C-terminal tail of NHX6 interacts with a component of Retromer, another component of the cell sorting machinery, and that this tail is critical for NHX6 activity. These findings demonstrate that NHX5 and NHX6 are important in processing and activity of vacuolar cargo, and suggest a mechanism by which NHX intracellular (IC)-II antiporters may be involved in subcellular trafficking. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  8. Chlamydia trachomatis Is Responsible for Lipid Vacuolation in the Amniotic Epithelium of Fetal Gastroschisis.

    Science.gov (United States)

    Feldkamp, Marcia L; Ward, Diane M; Pysher, Theodore J; Chambers, Christina T

    2017-07-17

    Vacuolated amniotic epithelium with lipid droplets in gastroschisis placentas is an unusual finding. Mass spectrometry of lipid droplets identified triglycerides, ester-linked to an unusual pattern of fatty acids. We hypothesize that these findings result from a Chlamydia trachomatis infection during the periconceptional period. The rising incidence of chlamydia infections has paralleled the increasing prevalence of gastroschisis among women less than 25 years of age. Histologically, young women are at greatest risk for a chlamydia infection due to their immature columnar epithelium, the preferential site for attachment of Chlamydia trachomatis infectious particle (elementary body). Chlamydia trachomatis survive in an inclusion, relying on its host to acquire essential nutrients, amino acids, and nucleotides for survival and replication. If essential nutrients are not available, the bacteria cannot replicate and may be trafficked to the lysosome for degradation or remain quiescent, within the inclusion, subverting innate immunologic clearance. Chlamydiae synthesize several lipids (phosphatidylethanolamine, phosphatidylserine, and phosphoatidylglycerol); however, their lipid content reveal eukaryotic lipids (sphingomyelin, cholesterol, phosphatidylcholine, and phosphatidylinositol), evidence that chlamydiae "hijack" host lipids for expansion and replication. The abnormal amniotic epithelial findings are supported by experimental evidence of the trafficking of host lipids into the chlamydiae inclusion. If not lethal, what harm will elementary bodies inflict to the developing embryo? Do these women have a greater pro-inflammatory response to an environmental exposure, whether cigarette smoking, change in partner, or a pathogen? Testing the hypothesis that Chlamydia trachomatis is responsible for amniotic epithelium vacuoles will be a critical first step. Birth Defects Research 109:1003-1010, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  9. Physicochemical and functional properties, microstructure, and storage stability of whey protein/polyvinylpyrrolidone based glue sticks

    Directory of Open Access Journals (Sweden)

    Guorong Wang

    2012-11-01

    Full Text Available A glue stick is comprised of solidified adhesive mounted in a lipstick-like push-up tube. Whey is a byproduct of cheese making. Direct disposal of whey can cause environmental pollution. The objective of this study was to use whey protein isolate (WPI as a natural polymer along with polyvinylpyrrolidone (PVP to develop safe glue sticks. Pre-dissolved WPI solution, PVP, sucrose, 1,2-propanediol (PG, sodium stearate, defoamer, and preservative were mixed and dissolved in water at 90 oC and then molded in push-up tubes. Chemical composition, functional properties (bonding strength, glue setting time, gel hardness, extension/retraction, and spreading properties, microstructure, and storage stability of the prototypes were evaluated in comparison with a commercial control. Results showed that all WPI/PVP prototypes had desirable bonding strength and exhibited faster setting than PVP prototypes and control. WPI could reduce gel hardness and form less compact and rougher structures than that of PVP, but there was no difference in bonding strength. PVP and sucrose could increase the hygroscopicity of glue sticks, thus increasing storage stability. Finally, the optimized prototype GS3 (major components: WPI 8.0%, PVP 12.0%, 1,2-propanediol 10.0%, sucrose 10.0%, and stearic sodium 7.0% had a comparable functionality to the commercial control. Results indicated that whey protein could be used as an adhesive polymer for glue stick formulations, which could be used to bond fiber or cellulose derived substrates such as paper.

  10. Antioxidant activities of dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber.

    Science.gov (United States)

    Hou, W C; Lee, M H; Chen, H J; Liang, W L; Han, C H; Liu, Y W; Lin, Y H

    2001-10-01

    Dioscorin, the storage protein of yam (Dioscorea batatas Decne) tuber (which is different from dioscorine found in tubers of Dioscorea hirsuta), was purified to homogeneity after DE-52 ion exchange column according to the methods of Hou et al. (J. Agric. Food Chem. 1999, 47, 2168-2172). A single band of 32 kDa dioscorin was obtained on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel with 2-mercaptoethanol treatment. This purified dioscorin was shown by spectrophotometric method to have scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical in a pH-dependent manner. There is a positive correlation between scavenging effects against DPPH (8-46%) and amounts of 32 kDa dioscorin (5.97-47.80 nmol) added in Tris-HCl buffer (pH 7.9), which are comparable to those of glutathione at the same concentrations. Using electron paramagnetic resonance (EPR) spectrometry for DPPH radical detection, it was found that the intensities of the EPR signal were decreased by 28.6 and 57 nmol of 32 kDa dioscorin in Tris-HCl buffer (pH 7.9) more than in distilled water compared to controls. EPR spectrometry was also used for hydroxyl radical detection. It was found that 32 kDa dioscorin could capture hydroxyl radical, and the intensities of the EPR signal were significantly decreased dose-dependently by 1.79-14.32 nmol of 32 kDa dioscorin (r = 0.975) compared to the control. It is suggested that 32 kDa dioscorin, the storage protein of yam tuber, may play a role as antioxidant in tubers and may be beneficial for health when people take it as a food additive or consume yam tubers.

  11. A novel mutation in PNPLA2 causes neutral lipid storage disease with myopathy and triglyceride deposit cardiomyovasculopathy: a case report and literature review.

    Science.gov (United States)

    Kaneko, Kimihiko; Kuroda, Hiroshi; Izumi, Rumiko; Tateyama, Maki; Kato, Masaaki; Sugimura, Koichiro; Sakata, Yasuhiko; Ikeda, Yoshihiko; Hirano, Ken-Ichi; Aoki, Masashi

    2014-07-01

    Mutations in PNPLA2 cause neutral lipid storage disease with myopathy (NLSDM) or triglyceride deposit cardiomyovasculopathy (TGCV). We report a 59-year-old patient with NLSDM/TGCV presenting marked asymmetric skeletal myopathy and cardiomyovasculopathy. Skeletal muscle and endomyocardial biopsies showed cytoplasmic vacuoles containing neutral lipid. Gene analysis revealed a novel homozygous mutation (c.576delC) in PNPLA2. We reviewed 37 genetically-proven NLSDM/TGCV cases; median age was 30 years; distribution of myopathy was proximal (69%) and distal predominant (16%); asymmetric myopathy (right>left) was reported in 41% of the patients. Frequently-affected muscles were posterior compartment of leg (75%), shoulder girdle to upper arm (50%), and paraspinal (33%). Skeletal muscle biopsies showed lipid accumulation in 100% and rimmed vacuoles in 22%. Frequent comorbidities were cardiomyopathy (44%), hyperlipidemia (23%), diabetes mellitus (24%), and pancreatitis (14%). PNPLA2 mutations concentrated in Exon 4-7 without apparent genotype-phenotype correlations. To know the characteristic features is essential for the early diagnosis of NLSDM/TGCV. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. Influence of cooking methods and storage time on lipid and protein oxidation and heterocyclic aromatic amines production in bacon.

    Science.gov (United States)

    Soladoye, O P; Shand, P; Dugan, M E R; Gariépy, C; Aalhus, J L; Estévez, M; Juárez, M

    2017-09-01

    This study aimed to examine the influence of cooking methods and pre-determined refrigerated storage days on the production of lipid oxidation (TBARS), protein oxidation (PROTOX) and heterocyclic aromatic amines (HAA) in bacon. Forty-four pork bellies selected from pigs varying in breed, sex and diets to introduce variability in composition were processed as bacon. Sliced-bacon was stored at 4°C either for 2 or 28days and these storage groups were cooked either with microwave or frying pan. Microwave led to significantly higher PROTOX (P0.05) by the cooking methods and storage times. Similarly, the fatty acid composition of pork belly did not significantly influence the production of HAA, TBARS and PROTOX produced in bacon during cooking. Overall, microwave cooking had lesser impact on the production of carcinogenic compounds in bacon with only minor impact on sensory attributes. Copyright © 2017. Published by Elsevier Ltd.

  13. Oxidative deterioration of pork during superchilling storage.

    Science.gov (United States)

    Pomponio, Luigi; Ruiz-Carrascal, Jorge

    2017-12-01

    In superchilling (SC), meat is kept at temperatures around 1 °C below its initial freezing point, leading to a significant increase in shelf life. This study aimed to address the oxidative changes taking place in pork loins during prolonged storage at SC temperature. Loins were stored either at chilling (CH) conditions (2-4 °C) for 4 weeks or at SC temperature (around -1 °C) for 12 weeks. Storage at SC temperature diminished the rate of lipid and protein oxidation and discoloration in pork loins, so that final levels of most oxidation products and instrumental color values after 12 weeks of SC storage were similar to those after 4 weeks at CH conditions. However, hexanal content peaked by the end of SC storage, pointing to a potential accumulation of compounds from lipid oxidation during SC storage. SC storage of pork slows down the rate of lipid and protein oxidation. However, accumulation of volatile compounds from lipid oxidation could be a limiting factor for shelf life. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  14. Expression of Hsp27 and Hsp70 and vacuolization in the pituitary glands in cases of fatal hypothermia.

    Science.gov (United States)

    Doberentz, Elke; Markwerth, Philipp; Wagner, Rebecca; Madea, Burkhard

    2017-09-01

    Hypothermia causes systemic cellular stress. The pituitary gland is an endocrine gland and plays an important role in thermoregulation. When the core body temperature drops, the pituitary gland is activated by stimulation of hypothalamic hormones. In this study, we investigated morphological alterations of the pituitary gland in cases of fatal hypothermia. Several morphological alterations of the anterior lobe of the pituitary gland, such as hemorrhage, vacuolization, and hyperemia, have been previously described in fatal hypothermia. However, the diagnostic value of these findings is controversial. We compared 11 cases of fatal hypothermia with 10 cases lacking antemortem hypothermic influences. In the presence of thermal cellular stress, the expression of heat shock proteins increases to protect cellular structures. Therefore, we immunohistochemically analyzed Hsp27 and Hsp70. Hsp27 expression was detected in 27.3% of the cases of fatal hypothermia and in 10.0% of the control cases, whereas Hsp70 expression was not detected in any case. Additionally, Sudan staining was performed to quantify fatty degeneration. A positive reaction was found in 45.5% of the study group and in 10.0% of the control group. This indicates that fatty degeneration might be a valuable marker when other macroscopic signs of hypothermia are absent.

  15. Acetonitrile as a buffer additive for free zone capillary electrophoresis separation and characterization of maize (Zeamays L. ) and sorghum (Sorghum bicolor L. Moench) storage proteins.

    Science.gov (United States)

    Bean, S R; Lookhart, G L; Bietz, J A

    2000-02-01

    An improved method for separating and characterizing maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) storage proteins by free zone capillary electrophoresis (FZCE) was developed. Previous electrophoretic methods for analyzing these proteins required high concentrations of urea to maintain protein solubility during separation. To overcome disadvantages of urea, we developed a FZCE method that mimicked reversed-phase high-performance liquid chromatography (RP-HPLC) in that it used high levels of acetonitrile (ACN) at low pH. The optimized FZCE buffer system consisted of 80 mM phosphate-glycine buffer, nominal pH 2.5, containing 60% ACN and a cellulose derivative to dynamically coat capillary walls. Resolution was similar to or higher than that previously achieved by FZCE buffers utilizing 8 M urea as a buffer additive. ACN concentrations of at least 50% were necessary to achieve acceptable separations; this ACN concentration is approximately that necessary to extract these storage proteins. ACN was equally effective as traditional ethanol solvents and 8 M urea for solubilizing maize and sorghum proteins. The ACN-based FZCE buffer system gave high repeatability (buffers. This FZCE method may be applicable for the analysis of other hydrophobic proteins without the use of urea.

  16. Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

    International Nuclear Information System (INIS)

    Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

    2010-01-01

    Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C 60 OH x ), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

  17. Physico-chemical characterization of grain dust in storage air of Bangalore.

    Science.gov (United States)

    Mukherjee, A K; Nag, D P; Kakde, Y; Babu, K R; Prdkash, M N; Rao, S R

    1998-06-01

    An Anderson personal cascade impactor was used to study the particle mass size distribution in the storage air of two major grain storage centers in Bangalore. Dust levels in storage air as well as the personal exposures of workers were determined along with a detailed study on the particle size distribution. Protein and carbohydrate content of the dust were also determined respectively in the phosphate buffer saline (PBS) and water extracts by using the standard analytical techniques. Personal exposures in both of the grain storage centers have been found to be much above the limit prescribed by ACGIH (1995-96). But the results of particle size analysis showed a higher particle mass distribution in the non-respirable size range. The mass median diameters (MMD) of the storage air particulate of both the centers were found to be beyond the respirable range. Presence of protein and carbohydrate in the storage air dust is indicative of the existence of glyco-proteins, mostly of membrane origin.

  18. Lysosomal lipid storage diseases.

    Science.gov (United States)

    Schulze, Heike; Sandhoff, Konrad

    2011-06-01

    Lysosomal lipid storage diseases, or lipidoses, are inherited metabolic disorders in which typically lipids accumulate in cells and tissues. Complex lipids, such as glycosphingolipids, are constitutively degraded within the endolysosomal system by soluble hydrolytic enzymes with the help of lipid binding proteins in a sequential manner. Because of a functionally impaired hydrolase or auxiliary protein, their lipid substrates cannot be degraded, accumulate in the lysosome, and slowly spread to other intracellular membranes. In Niemann-Pick type C disease, cholesterol transport is impaired and unesterified cholesterol accumulates in the late endosome. In most lysosomal lipid storage diseases, the accumulation of one or few lipids leads to the coprecipitation of other hydrophobic substances in the endolysosomal system, such as lipids and proteins, causing a "traffic jam." This can impair lysosomal function, such as delivery of nutrients through the endolysosomal system, leading to a state of cellular starvation. Therapeutic approaches are currently restricted to mild forms of diseases with significant residual catabolic activities and without brain involvement.

  19. Performance of starter in yogurt supplemented with soy protein isolate and biotransformation of isoflavones during storage period.

    Science.gov (United States)

    Pham, T Thuy; Shah, Nagendra P

    2009-01-01

    In this study, soy protein isolate (SPI) (4%, v/w) was supplemented to the yogurt mix to increase the amount of biologically active isoflavone in yogurt (SY). The control yogurt was without any SPI supplementation (USY). The supplementation significantly (P yogurt starter including Lactobacillus delbrueckii ssp. bulgaricus ATCC 11842 (Lb 11842) and Streptococcus thermophilus ST 1342 (ST 1342) during the fermentation process by 4.7%. The starter produced more acetic acid and less lactic acid in SY than that in USY and altered the ratio of lactic and acetic acid during the entire storage period. The viability of both Lb 11842 and ST 1342 in SY was significantly (P < 0.05) lower than that in USY from 14 d of the storage period, however, their concentration still remained high (8.11 to 8.84 log CFU/g). The starter transformed 72.8% of total inactive isoflavone glycosides (IG) to active isoflavone aglycones (IA), increasing the IA content from 1.35 to 15.01 mg/100 g sample. During the storage period, IA concentration slowly rose from 15.02 to 15.51 mg/100 g sample.

  20. Subcellular localization of acyl-CoA binding protein in Aspergillus oryzae is regulated by autophagy machinery.

    Science.gov (United States)

    Kawaguchi, Kouhei; Kikuma, Takashi; Higuchi, Yujiro; Takegawa, Kaoru; Kitamoto, Katsuhiko

    2016-11-04

    In eukaryotic cells, acyl-CoA binding protein (ACBP) is important for cellular activities, such as in lipid metabolism. In the industrially important fungus Aspergillus oryzae, the ACBP, known as AoACBP, has been biochemically characterized, but its physiological function is not known. In the present study, although we could not find any phenotype of AoACBP disruptants in the normal growth conditions, we examined the subcellular localization of AoACBP to understand its physiological function. Using an enhanced green fluorescent protein (EGFP)-tagged AoACBP construct we showed that AoACBP localized to punctate structures in the cytoplasm, some of which moved inside the cells in a microtubule-dependent manner. Further microscopic analyses showed that AoACBP-EGFP co-localized with the autophagy marker protein AoAtg8 tagged with red fluorescent protein (mDsRed). Expression of AoACBP-EGFP in disruptants of autophagy-related genes revealed aggregation of AoACBP-EGFP fluorescence in the cytoplasm of Aoatg1, Aoatg4 and Aoatg8 disruptant cells. However, in cells harboring disruption of Aoatg15, which encodes a lipase for autophagic body, puncta of AoACBP-EGFP fluorescence accumulated in vacuoles, indicating that AoACBP is transported to vacuoles via the autophagy machinery. Collectively, these results suggest the existence of a regulatory mechanism between AoACBP localization and autophagy. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. A mouse model for fucosidosis recapitulates storage pathology and neurological features of the milder form of the human disease

    Directory of Open Access Journals (Sweden)

    Heike Wolf

    2016-09-01

    Full Text Available Fucosidosis is a rare lysosomal storage disorder caused by the inherited deficiency of the lysosomal hydrolase α-L-fucosidase, which leads to an impaired degradation of fucosylated glycoconjugates. Here, we report the generation of a fucosidosis mouse model, in which the gene for lysosomal α-L-fucosidase (Fuca1 was disrupted by gene targeting. Homozygous knockout mice completely lack α-L-fucosidase activity in all tested organs leading to highly elevated amounts of the core-fucosylated glycoasparagine Fuc(α1,6-GlcNAc(β1-N-Asn and, to a lesser extent, other fucosylated glycoasparagines, which all were also partially excreted in urine. Lysosomal storage pathology was observed in many visceral organs, such as in the liver, kidney, spleen and bladder, as well as in the central nervous system (CNS. On the cellular level, storage was characterized by membrane-limited cytoplasmic vacuoles primarily containing water-soluble storage material. In the CNS, cellular alterations included enlargement of the lysosomal compartment in various cell types, accumulation of secondary storage material and neuroinflammation, as well as a progressive loss of Purkinje cells combined with astrogliosis leading to psychomotor and memory deficits. Our results demonstrate that this new fucosidosis mouse model resembles the human disease and thus will help to unravel underlying pathological processes. Moreover, this model could be utilized to establish diagnostic and therapeutic strategies for fucosidosis.

  2. Storage beyond Three Hours at Ambient Temperature Alters the ...

    African Journals Online (AJOL)

    The effect of storage on stability of human breast milk was investigated in 30 lactating mothers. Samples stored for 3, 6 and 24 hours at ambient temperature of 302K (29°) were analysed for protein, lactose, pH, and microbial content. There were significant (p < 0.01) decreases in protein, lactose and pH upon storage for 6 ...

  3. Effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage

    Science.gov (United States)

    The objective of this study was to evaluate effects of heat stress and probiotic supplementation on protein functionality and oxidative stability of ground chicken leg meat during display storage. Two hundred and forty 1-day-old male chicks (5 bird per pen) were randomly subjected to four treatments...

  4. Acyl-CoA binding proteins; structural and functional conservation over 2000 MYA

    DEFF Research Database (Denmark)

    Faergeman, Nils J; Wadum, Majken; Feddersen, Søren

    2007-01-01

    -CoA binding protein, ACBP, has been proposed to play a pivotal role in the intracellular trafficking and utilization of long-chain fatty acyl-CoA esters. Depletion of acyl-CoA binding protein in yeast results in aberrant organelle morphology incl. fragmented vacuoles, multi-layered plasma membranes...... and accumulation of vesicles of variable sizes. In contrast to synthesis and turn-over of glycerolipids, the levels of very-long-chain fatty acids, long-chain bases and ceramide are severely affected by Acb1p depletion, suggesting that Acb1p, rather than playing a general role, serves specific roles in cellular...

  5. Lysosomal exocytosis and lipid storage disorders.

    Science.gov (United States)

    Samie, Mohammad Ali; Xu, Haoxing

    2014-06-01

    Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  6. Lysosomal exocytosis and lipid storage disorders

    Science.gov (United States)

    Samie, Mohammad Ali; Xu, Haoxing

    2014-01-01

    Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. PMID:24668941

  7. REDOX POTENTIAL AND DYNAMICS OF PROTEIN AND FAT DESTRUCTION DURING STORAGE OF CANNED MEAT IN PIECES

    Directory of Open Access Journals (Sweden)

    V. B. Krylova

    2016-01-01

    Full Text Available The studies on the dynamics of the redox potential of systems and its relationship with the processes of protein and fat destruction in canned foods during their storage are fragmented and not systemized, which highlight their topicality. The aim of the research was to obtain the experimental data on the Eh values and physico-chemical indicators of canned food quality during storage in order to establish their possible correlation. It was shown that the dynamics of Eh, the content of free amino acids and fatty acid fractions in the canned products from beef and pork was different during storage. For example, a decrease in the Eh value and free amino acid content in the canned products from beef had a smooth character, while in the canned products from pork several periods were observed, which differed in the character of the change in the quality indicators.A linear character of the changes in the proportion of fatty acid fractions during storage of the canned products from beef and pork was noticed. With that, both canned food items had an increase in the saturated fatty acid content at the concomitant decrease in the sum of mono- and polyunsaturated fatty acids. The value of an increase in the proportion of saturated fatty acids associated with the process of reduction of mono- and polyunsaturated fatty acids did not depend on the kind of meat in the canned foods and was on average 6%. A decrease in the proportion of mono- and polyunsaturated fatty acids in the canned products from pork was about 4 times more intensive compared to the canned products from beef.

  8. Amino acid transport across the tonoplast of vacuoles isolated from barley mesophyll protoplasts: Uptake of alanine, leucine, and glutamine

    International Nuclear Information System (INIS)

    Dietz, K.J.; Jaeger, R.; Kaiser, G.; Martinoia, E.

    1990-01-01

    Mesophyll protoplasts from leaves of well-fertilized barley (Hordeum vulgare L.) plants contained amino acids at concentrations as high as 120 millimoles per liter. With the exception of glutamic acid, which is predominantly localized in the cytoplasm, a major part of all other amino acids was contained inside the large central vacuole. Alanine, leucine, and glutamine are the dominant vacuolar amino acids in barley. Their transport into isolated vacuoles was studied using 14 C-labeled amino acids. Uptake was slow in the absence of ATP. A three- to sixfold stimulation of uptake was observed after addition of ATP or adenylyl imidodiphosphate an ATP analogue not being hydrolyzed by ATPases. Other nucleotides were ineffective in increasing the rate of uptake. ATP-Stimulated amino acid transport was not dependent on the transtonoplast pH or membrane potential. p-Chloromercuriphenylsulfonic acid and n-ethyl maleimide increased transport independently of ATP. Neutral amino acids such as valine or leucine effectively decreased the rate of alanine transport. Glutamine and glycine were less effective or not effective as competitive inhibitors of alanine transport. The results indicate the existence of a uniport translocator specific for neutral or basic amino acids that is under control of metabolic effectors

  9. CHARACTERIZATION OF SEED STORAGE PROTEINS IN SOME IRANIAN DATE PALM CULTIVARS USING SDS-PAGE

    Directory of Open Access Journals (Sweden)

    Sayed Mohammad Reza Khoshroo

    2013-08-01

    Full Text Available The date palm (Phoenix dactylifera L. is most adapted tree to grow in desert areas. It has always been looked on as a key source of stability, survival and evolution of the oasis agro-system since it constitutes the basic features of the ecological pyramid in desert regions. Determining genetic variability and cultivars identification in date palm are two major important factors in breeding programs, characterization of germplasm, and conservation purposes. The genetic variation of seed proteins was assayed by SDS-PAGE for 9 cultivars in Shahdad region in Iran. A total of 16 alternative protein bands with different mobility rates were identified within a molecular weight range of 11 KDa to 350 KDa. Then, electrophorogram for each cultivar was scored, and Jaccard‘s Similarity Index was calculated. Relying on UPGMA and NJ methods, genetic diversity of cultivars was evaluated by constructing the dendrogram for protein bands. Moreover, genetic distance was calculated for all of the cultivars.  It is concluded that seed storage protein profiles could be useful markers in genetic diversity studies and classification of cultivars. The cultivars from Shahdad were well separated from each other. This might have been done due to their unique genetic build-up. The cluster analysis displayed five major classes. In order to precise this assumption, data were computed to perform a PCA. Cluster analysis and PCA demonstrated their validity in establishing genetic diversity. When PCA was studied, the previously described results about Jaccard Similarity Coefficient dendrogram were also visualized.

  10. Effect of harvest period on the quality of storage roots and protein content of the leaves in five cassava cultivars (Manihot esculenta, Crantz

    Directory of Open Access Journals (Sweden)

    Sagrilo Edvaldo

    2003-01-01

    Full Text Available The effect of harvest period on the quality of storage roots and leaves of cassava cultivars was determined in an experiment carried out in a randomized complete block design with four replications in a split plot scheme, with five cultivars in the plots and ten harvest times in the subplots. The IAC 13 cultivar had the highest rate of dry matter accumulation in the storage roots and the Mico cultivar the lowest. The period of least dry matter content in the storage roots occurred later for the Fécula Branca, Mico and IAC 14 cultivars, and the minimum starch content in the storage roots occurred later for the Fécula Branca and Mico cultivars. In general, the IAC 13, IAC 14 and Fécula Branca cultivars had higher dry matter content in the storage roots, while higher starch content in the dry and fresh matter were obtained in the Fécula Branca cultivar. The crude protein content in the leaves decreased as the plant aged.

  11. Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183

    International Nuclear Information System (INIS)

    Larkins, Brian A.

    2003-01-01

    -kD proteins bind and assemble alpha-zeins in protein bodies. Additional evidence supporting this hypothesis was obtained by showing that the starchy endosperm mutant, Mucuronate, appears to result from a defective 16-kD gamma-zein protein. By deletion mutagenesis, we identified domains within an alpha-zein that cause it to interact with other zein proteins, particularly gamma-zeins. This allowed us to develop a minimal alpha-zein gene construct that can be used as a vector to target heterologous proteins, such as green fluorescent protein, into protein bodies. We characterized the nature of storage proteins synthesized in the endosperm using a genomics analysis of endosperm ESTs. This study identified several new storage proteins and demonstrated the existence of novel protein storage vacuoles. We used mRNA transcript profiling of eight different starchy endosperm (opaque) mutants (o1, o2, o5, o9, o11, Mucronate, Defective endosperm B30, and floury2) to identify patterns of gene expression that are consistently altered in all of them, or that are unique to each one of them. These mutants fall into two subgroups: one systematically manifests an ''unfolded protein'' response (fl2, Mc, DeB30) and the other (o1, o2, o5, o9, o11) does not. Genes encoding cytoskeletal proteins are generally up-regulated in all the mutants, and this may be associated with higher lysine contents in several of them

  12. Effects of Tannic Acid on Lipid and Protein Oxidation, Color, and Volatiles of Raw and Cooked Chicken Breast Meat during Storage

    Directory of Open Access Journals (Sweden)

    Marwan Al-Hijazeen

    2016-06-01

    Full Text Available The objective of this study was to determine the effect of tannic acid (TA on the oxidative stability and the quality characteristics of ground chicken breast meat. Five treatments including (1 control (none added, (2 2.5 ppm TA, (3 5 ppm TA, (4 10 ppm TA, and (5 5 ppm butylated hydroxyanisole (BHA were added to boneless, skinless ground chicken breast meat, and used for both raw and cooked meat studies. For the raw meat study, the ground chicken breast meat was packaged in oxygen-permeable bags and stored at 4 °C for 7 days. For the cooked study, raw ground meat samples were vacuum-packaged in oxygen-impermeable vacuum bags, cooked in-bag to the internal temperature of 75 °C, re-packaged in oxygen-permeable bags, and then stored. Both raw and cooked meats were analyzed for lipid and protein oxidation, color, and volatiles (cooked meat only at 0, 3, and 7 days of storage. Raw meats with 10 ppm of TA added had significantly (p ≤ 0.05 lower lipid and protein oxidation than other treatments during storage. In addition, TA at 10 ppm level maintained the highest color a*- and L*-values during storage. Cooked chicken breast meat with 5 and 10 ppm TA added produced significantly (p ≤ 0.05 lower amounts of off-odor volatiles than other treatments. Among the volatile compounds, the amount of hexanal increased rapidly during storage for cooked meat. However, meats with 5 and 10 ppm TA added showed the lowest amount of hexanal and other aldehydes related to lipid oxidation, indicating a strong antioxidant effect of TA in cooked chicken breast meat. Furthermore, the differences in aldehydes among the treatments were bigger in cooked than in raw meat, indicating that the antioxidant effect of TA in cooked meat was greater than that in raw meat. Therefore, TA at >5 ppm can be used as a good natural preservative in cooked chicken meat to maintain its quality during storage.

  13. Sensory properties and storage stability of salad cream from ...

    African Journals Online (AJOL)

    Sensory properties and storage stability of salad cream prepared from cassava starch and soy protein concentrates was studied. Cassava starch and soy protein concentrate blends were prepared with 30% soy protein concentrate and incorporated into 70% cassava starch. The cassava-soy protein concentrate blend was ...

  14. Effect of storage temperature on survival and recovery of thermal and extrusion injured Escherichia coli populations in whey protein concentrate and corn meal

    Science.gov (United States)

    In a previous study, we reported viability loss of Escherichia coli populations in corn (CP) and whey protein products (WPP) extruded at different temperatures. However, information on the effect of storage temperatures on injured bacterial populations was not addressed. The objective of this study ...

  15. Influence of prolonged storage process, pasteurization, and heat treatment on biologically-active human milk proteins.

    Science.gov (United States)

    Chang, Jih-Chin; Chen, Chao-Huei; Fang, Li-Jung; Tsai, Chi-Ren; Chang, Yu-Chuan; Wang, Teh-Ming

    2013-12-01

    The bioactive proteins in human milk may be influenced by prolonged storage process, pasteurization, and heat treatment. This study was conducted to evaluate the effects of these procedures. Three forms of human milk - freshly expressed, frozen at -20°C for a prolonged duration, and pasteurized milk - were collected from 14 healthy lactating mothers and a milk bank. The concentrations of major bioactive proteins (secretory immunoglobulin A, lactoferrin, lysozyme, and leptin) were quantified using enzyme-linked immunosorbent assay kits. Changes in these proteins by heat treatment at 40°C or 60°C for 30 minutes were further evaluated. The mean concentrations of lactoferrin and secretory immunoglobulin A were significantly reduced by 66% and 25.9%, respectively, in pasteurized milk compared with those in freshly-expressed milk. Heat treatment at 40°C or 60°C did not cause significant changes in lactoferrin and secretory immunoglobulin A, but there was an apparent increase in lysozyme (p = 0.016). There were no significant differences in leptin level among these three forms of milk prior to (p = 0.153) or after heat treatment (p = 0.053). Various freezing/heating/pasteurization processes applied to human milk prior to delivery to neonates could affect the concentration of immunomodulatory proteins, especially lactoferrin, secretory immunoglobulin A, and lysozyme. Leptin was unaffected by the various handling processes tested. Fresh milk was found to be the best food for neonates. Further studies are warranted to evaluate the functional activity of these proteins and their effects on infants' immunological status. Copyright © 2013. Published by Elsevier B.V.

  16. Autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

    Science.gov (United States)

    Xie, Xiaolei; Le, Li; Fan, Yanxin; Lv, Lin; Zhang, Junjie

    2012-07-01

    Mitoribosome in mammalian cells is responsible for synthesis of 13 mtDNA-encoded proteins, which are integral parts of four mitochondrial respiratory chain complexes (I, III, IV and V). ERAL1 is a nuclear-encoded GTPase important for the formation of the 28S small mitoribosomal subunit. Here, we demonstrate that knockdown of ERAL1 by RNA interference inhibits mitochondrial protein synthesis and promotes reactive oxygen species (ROS) generation, leading to autophagic vacuolization in HeLa cells. Cells that lack ERAL1 expression showed a significant conversion of LC3-I to LC3-II and an enhanced accumulation of autophagic vacuoles carrying the LC3 marker, all of which were blocked by the autophagy inhibitor 3-MA as well as by the ROS scavenger NAC. Inhibition of mitochondrial protein synthesis either by ERAL1 siRNA or chloramphenicol (CAP), a specific inhibitor of mitoribosomes, induced autophagy in HTC-116 TP53 (+/+) cells, but not in HTC-116 TP53 (-/-) cells, indicating that tumor protein 53 (TP53) is essential for the autophagy induction. The ROS elevation resulting from mitochondrial protein synthesis inhibition induced TP53 expression at transcriptional levels by enhancing TP53 promoter activity, and increased TP53 protein stability by suppressing TP53 ubiquitination through MAPK14/p38 MAPK-mediated TP53 phosphorylation. Upregulation of TP53 and its downstream target gene DRAM1, but not CDKN1A/p21, was required for the autophagy induction in ERAL1 siRNA or CAP-treated cells. Altogether, these data indicate that autophagy is induced through the ROS-TP53-DRAM1 pathway in response to mitochondrial protein synthesis inhibition.

  17. Effects of Starter Culture and Storage Temperature on Functional, Microbial and Sensory Characteristics of Kefir during Storage

    Directory of Open Access Journals (Sweden)

    Tayyebeh Sarlak

    2017-03-01

    Full Text Available The aim of this study was to investigate the effects of starters and storage temperature (4 ºC, 25 ºC on microbiological and physicochemical properties, volatile compounds and sensory evaluation of kefir. Kefirs produced by KFA and Chr. Hansen starters were stored at 4 ºC and 25 ºC for 40 days. pH and acidity at 4 ºC did not change (p ≥ 0.05, while at 25 ºC pH and total solid decreased as well as acidity. Concentrations of acetaldehyde and ethanol increased (p < 0.05. No significant differences (p ≥ 0.05 were observed in protein and non-protein-nitrogen for both samples. Sensory evaluation revealed that storage did not affect texture and color of samples at 4 ºC. Kefir produced by Chr. Hansen starter stored at 4 ºC had the highest acceptability until 40-day storage and was preferred by the panelists.

  18. Self-assembly of caseinomacropeptide as a potential key mechanism in the formation of visible storage induced aggregates in acidic whey protein isolate dispersions

    DEFF Research Database (Denmark)

    Villumsen, Nanna Stengaard; Jensen, Hanne Bak; Thu Le, Thao Thi

    2015-01-01

    Visible aggregates formed during storage in acidic whey protein isolate (WPI) dispersions represent a challenge to the beverage industry. Batch-to-batch variations are observed that prevents consistent quality and shelf-life prediction. Heat-treatment of WPI dispersions at 120°C for 20s instead...

  19. Studies on RBC lipid and protein phosphorylation during blood bank storage

    International Nuclear Information System (INIS)

    Dumaswala, U.J.; Bryan, D.J.; Greenwalt, T.J.

    1986-01-01

    Recent evidence has suggested that phosphoinositides play a significant role in maintaining membrane structure and function. Their importance during blood bank storage is not understood. They have performed preliminary studies of the phosphoinositide synthetic pathway enzymes of RBC during blood bank storage. At 0 and 35 days of storage leaky ghosts were prepared and incubated with [γ- 32 P]ATP for 5 minutes at 30 C. One aliquot was subjected to acidified solvent extraction and thin layer chromatography. The labeled phosphoinositide -4,5 biphosphate (PIP 2 ), phosphoinositide-4 phosphate (PIP) and phosphatidic acid (PA) spots were scraped and counted by liquid scintillation spectrometry. Another aliquot was used for SDS-PAGE and the radioactivity associated with the β-spectrin was measured. These experiments suggest a decrease in RBC phosphoinositol and PIP-Kinases and β-spectrin kinase activities during blood bank storage. Further studies are being done to evaluate significance of these observations

  20. Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 B139

    Energy Technology Data Exchange (ETDEWEB)

    Brian A. Larkins

    2003-03-21

    -kD proteins bind and assemble alpha-zeins in protein bodies. Additional evidence supporting this hypothesis was obtained by showing that the starchy endosperm mutant, Mucuronate, appears to result from a defective 16-kD gamma-zein protein. By deletion mutagenesis, we identified domains within an alpha-zein that cause it to interact with other zein proteins, particularly gamma-zeins. This allowed us to develop a minimal alpha-zein gene construct that can be used as a vector to target heterologous proteins, such as green fluorescent protein, into protein bodies. We characterized the nature of storage proteins synthesized in the endosperm using a genomics analysis of endosperm ESTs. This study identified several new storage proteins and demonstrated the existence of novel protein storage vacuoles. We used mRNA transcript profiling of eight different starchy endosperm (opaque) mutants (o1, o2, o5, o9, o11, Mucronate, Defective endosperm B30, and floury2) to identify patterns of gene expression that are consistently altered in all of them, or that are unique to each one of them. These mutants fall into two subgroups: one systematically manifests an ''unfolded protein'' response (fl2, Mc, DeB30) and the other (o1, o2, o5, o9, o11) does not. Genes encoding cytoskeletal proteins are generally up-regulated in all the mutants, and this may be associated with higher lysine contents in several of them.

  1. Immunological demonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri).

    Science.gov (United States)

    Georgopoulou, U; Sire, M F; Vernier, J M

    1986-01-01

    An immunofluorescence technique using antibodies against the Fc and Fab fragments of human IgG (IgGH) was used to study the absorption of proteins by the intestinal epithelial cells of rainbow trout after oral or anal administration. Cellular absorption of a high molecular weight protein, hepatitis-B surface antigen (HBsAg), was also studied by using two monoclonal antibodies, one specific for the confirmation of the antigen (implying disulfide bridges), and the other that reacts with the constituent polypeptides. Both absorbed IgGH and HBsAg were seen to be segregated in the apical vacuolar system, a characteristic feature of intestinal epithelial cells. The same antibodies were used with an everted sac technique in conjunction with immunofluorescence, to show the intravacuolar degradation of IgGH and HBsAg following absorption. By using an antibody against cathepsin D, it was possible to demonstrate, by immunofluorescence, the localization of this enzyme in the same vacuolar system. After coupling the antibody to peroxidase or to the protein A/colloidalgold complex, the ultrastructural antigenic sites of cathepsin D could be seen to be localized in the interior of the vacuoles. The vacuolar localization of a cathepsin B activity was determined by incubating sections of intestinal mucosa, or isolated epithelial cells, with a specific synthetic substrate (Z-Ala-Arg-Arg-methoxynaphthylamide). The supranuclear hyaloplasmic vacuoles of intestinal epithelial cells may be considered to be phagolysosomes that assure the degradation of absorbed proteins. This function may be of fundamental importance in the in the nutritional processes of this species.

  2. Wild blueberry polyphenol-protein food ingredients produced by three drying methods: Comparative physico-chemical properties, phytochemical content, and stability during storage.

    Science.gov (United States)

    Correia, Roberta; Grace, Mary H; Esposito, Debora; Lila, Mary Ann

    2017-11-15

    Particulate colloidal aggregate food ingredients were prepared by complexing wheat flour, chickpea flour, coconut flour and soy protein isolate with aqueous wild blueberry pomace extracts, then spray drying, freeze drying, or vacuum oven drying to prepare dry, flour-like matrices. Physico-chemical attributes, phytochemical content and stability during storage were compared. Eighteen anthocyanins peaks were identified for samples. Spray dried matrices produced with soy protein isolate had the highest concentration of polyphenols (156.2mg GAE/g) and anthocyanins (13.4mg/g) and the most potent DPPH scavenging activity (714.1μmolesTE/g). Spray dried blueberry polyphenols complexed with protein were protected from degradation during 16weeks at 4°C and 20°C. Soy protein isolate more efficiently captured and stabilized wild blueberry pomace phytochemicals than other protein sources. Overall, spray drying the blueberry extracts complexed with protein proved to be an environment-friendly strategy to produce stable functional ingredients with multiple applications for the food industry. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Identification of Proteins Involved in Salinity Tolerance in Salicornia bigelovii

    KAUST Repository

    Salazar Moya, Octavio Ruben

    2017-11-01

    With a global growing demand in food production, agricultural output must increase accordingly. An increased use of saline soils and brackish water would contribute to the required increase in world food production. Abiotic stresses, such as salinity and drought, are also major limiters of crop growth globally - most crops are relatively salt sensitive and are significantly affected when exposed to salt in the range of 50 to 200 mM NaCl. Genomic resources from plants that naturally thrive in highly saline environments have the potential to be valuable in the generation of salt tolerant crops; however, these resources have been largely unexplored. Salicornia bigelovii is a plant native to Mexico and the United States that grows in salt marshes and coastal regions. It can thrive in environments with salt concentrations higher than seawater. In contrast to most crops, S. bigelovii is able to accumulate very high concentrations (in the order of 1.5 M) of Na+ and Cl- in its photosynthetically active succulent shoots. Part of this tolerance is likely to include the storage of Na+ in the vacuoles of the shoots, making S. bigelovii a good model for understanding mechanisms of Na+ compartmentalization in the vacuoles and a good resource for gene discovery. In this research project, phenotypic, genomic, transcriptomic, and proteomic approaches have been used for the identification of candidate genes involved in salinity tolerance in S. bigelovii. The genomes and transcriptomes of three Salicornia species have been sequenced. This information has been used to support the characterization of the salt-induced transcriptome of S. bigelovii shoots and the salt-induced proteome of various organellar membrane enriched fractions from S. bigelovii shoots, which led to the creation of organellar membrane proteomes. Yeast spot assays at different salt concentrations revealed several proteins increasing or decreasing yeast salt tolerance. This work aims to create the basis for

  4. A study of biodiversity using DSS method and seed storage protein comparison of populations in two species of Achillea L. in the west of Iran

    Directory of Open Access Journals (Sweden)

    Hajar Salehi

    2013-11-01

    Full Text Available Intarspecific and interspecific variations are the main reserves of biodiversity and both are important sources of speciation. On this basis, identifing and recognizing the intra and interspecific variations is important in order to recognition of biodiversity. This research was done to study biodiversity and electrophoresis comparison of seed storage proteins in the populations of the two species of the genus Achillea in Hamadan and Kurdistan provinces using of the method of determination of special station (DSS. For this purpose, 12 and 9 special stations were selected for the species A. tenuifolia and A. biebresteinii using the data published in the related flora. Seed storage proteins were extracted and then studied using electrophoresis techniques (SDS-PAGE. In survey of all special stations, 120 plant species were distinguished as associated species. The results of the floristic data for the both species determined six distinctive groups that indicated the existence of intraspecific diversity in this species. The result of analysis of ecological data and seed storage proteins for the two species was in accordance with the floristic data and showed six distinctive groups. The existence of the bands of no. 4, 5, 8, 12 and 13 in the special stations of A. tenuifolia and the bands of 14, 15 and 16 in the special stations of A. biebresteinii o separated the populations of the species in two quite different and distinctive groups.

  5. Sequence Classification: 889075 [

    Lifescience Database Archive (English)

    Full Text Available localizes to vacuolar membranes and punctate structures attached to the vacuole; Atg22p || http://www.ncbi.nlm.nih.gov/protein/6319811 ... ...he breakdown of autophagic vesicles in the vacuole during autophagy, putative integral membrane protein that

  6. The sugarcane defense protein SUGARWIN2 causes cell death in Colletotrichum falcatum but not in non-pathogenic fungi.

    Directory of Open Access Journals (Sweden)

    Flávia P Franco

    Full Text Available Plants respond to pathogens and insect attacks by inducing and accumulating a large set of defense-related proteins. Two homologues of a barley wound-inducible protein (BARWIN have been characterized in sugarcane, SUGARWIN1 and SUGARWIN2 (sugarcane wound-inducible proteins. Induction of SUGARWINs occurs in response to Diatraea saccharalis damage but not to pathogen infection. In addition, the protein itself does not show any effect on insect development; instead, it has antimicrobial activities toward Fusarium verticillioides, an opportunistic fungus that usually occurs after D. saccharalis borer attacks on sugarcane. In this study, we sought to evaluate the specificity of SUGARWIN2 to better understand its mechanism of action against phytopathogens and the associations between fungi and insects that affect plants. We used Colletotrichum falcatum, a fungus that causes red rot disease in sugarcane fields infested by D. saccharalis, and Ceratocystis paradoxa, which causes pineapple disease in sugarcane. We also tested whether SUGARWIN2 is able to cause cell death in Aspergillus nidulans, a fungus that does not infect sugarcane, and in the model yeast Saccharomyces cerevisiae, which is used for bioethanol production. Recombinant SUGARWIN2 altered C. falcatum morphology by increasing vacuolization, points of fractures and a leak of intracellular material, leading to germling apoptosis. In C. paradoxa, SUGARWIN2 showed increased vacuolization in hyphae but did not kill the fungi. Neither the non-pathogenic fungus A. nidulans nor the yeast S. cerevisiae was affected by recombinant SUGARWIN2, suggesting that the protein is specific to sugarcane opportunistic fungal pathogens.

  7. Proteomics of apheresis platelet supernatants during routine storage: Gender-related differences.

    Science.gov (United States)

    Dzieciatkowska, Monika; D'Alessandro, Angelo; Burke, Timothy A; Kelher, Marguerite R; Moore, Ernest E; Banerjee, Anirban; Silliman, Christopher C; West, Bernadette F; Hansen, Kirk C

    2015-01-01

    Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender. The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet

  8. Psychromonas boydii sp. nov., a gas-vacuolate, psychrophilic bacterium isolated from an Arctic sea-ice core.

    Science.gov (United States)

    Auman, Ann J; Breezee, Jennifer L; Gosink, John J; Schumann, Peter; Barnes, Carmen R; Kämpfer, Peter; Staley, James T

    2010-01-01

    A gas-vacuolate bacterium, strain 174(T), was isolated from a sea-ice core collected from Point Barrow, Alaska, USA. Comparative analysis of 16S rRNA gene sequences showed that this bacterium was most closely related to Psychromonas ingrahamii 37(T), with a similarity of >99 %. However, strain 174(T) could be clearly distinguished from closely related species by DNA-DNA hybridization; relatedness values determined by two different methods between strain 174(T) and P. ingrahamii 37(T) were 58.4 and 55.7 % and those between strain 174(T) and Psychromonas antarctica DSM 10704(T) were 46.1 and 33.1 %, which are well below the 70 % level used to define a distinct species. Phenotypic analysis, including cell size (strain 174(T) is the largest member of the genus Psychromonas, with rod-shaped cells, 8-18 microm long), further differentiated strain 174(T) from other members of the genus Psychromonas. Strain 174(T) could be distinguished from its closest relative, P. ingrahamii, by its utilization of D-mannose and D-xylose as sole carbon sources, its ability to ferment myo-inositol and its inability to use fumarate and glycerol as sole carbon sources. In addition, strain 174(T) contained gas vacuoles of two distinct morphologies and grew at temperatures ranging from below 0 to 10 degrees C and its optimal NaCl concentration for growth was 3.5 %. The DNA G+C content was 40 mol%. Whole-cell fatty acid analysis showed that 16 : 1omega7c and 16 : 0 comprised 44.9 and 26.4 % of the total fatty acid content, respectively. The name Psychromonas boydii sp. nov. is proposed for this novel species, with strain 174(T) (=DSM 17665(T) =CCM 7498(T)) as the type strain.

  9. Sterylglucoside catabolism in Cryptococcus neoformans with endoglycoceramidase-related protein 2 (EGCrP2), the first steryl-β-glucosidase identified in fungi.

    Science.gov (United States)

    Watanabe, Takashi; Ito, Tomoharu; Goda, Hatsumi M; Ishibashi, Yohei; Miyamoto, Tomofumi; Ikeda, Kazutaka; Taguchi, Ryo; Okino, Nozomu; Ito, Makoto

    2015-01-09

    Cryptococcosis is an infectious disease caused by pathogenic fungi, such as Cryptococcus neoformans and Cryptococcus gattii. The ceramide structure (methyl-d18:2/h18:0) of C. neoformans glucosylceramide (GlcCer) is characteristic and strongly related to its pathogenicity. We recently identified endoglycoceramidase-related protein 1 (EGCrP1) as a glucocerebrosidase in C. neoformans and showed that it was involved in the quality control of GlcCer by eliminating immature GlcCer during the synthesis of GlcCer (Ishibashi, Y., Ikeda, K., Sakaguchi, K., Okino, N., Taguchi, R., and Ito, M. (2012) Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1). J. Biol. Chem. 287, 368-381). We herein identified and characterized EGCrP2, a homologue of EGCrP1, as the enzyme responsible for sterylglucoside catabolism in C. neoformans. In contrast to EGCrP1, which is specific to GlcCer, EGCrP2 hydrolyzed various β-glucosides, including GlcCer, cholesteryl-β-glucoside, ergosteryl-β-glucoside, sitosteryl-β-glucoside, and para-nitrophenyl-β-glucoside, but not α-glucosides or β-galactosides, under acidic conditions. Disruption of the EGCrP2 gene (egcrp2) resulted in the accumulation of a glycolipid, the structure of which was determined following purification to ergosteryl-3β-glucoside, a major sterylglucoside in fungi, by mass spectrometric and two-dimensional nuclear magnetic resonance analyses. This glycolipid accumulated in vacuoles and EGCrP2 was detected in vacuole-enriched fraction. These results indicated that EGCrP2 was involved in the catabolism of ergosteryl-β-glucoside in the vacuoles of C. neoformans. Distinct growth arrest, a dysfunction in cell budding, and an abnormal vacuole morphology were detected in the egcrp2-disrupted mutants, suggesting that EGCrP2 may be a promising target for anti-cryptococcal drugs. EGCrP2, classified into glycohydrolase family 5, is the first steryl

  10. Maillard-reaction-induced modification and aggregation of proteins and hardening of texture in protein bar model systems.

    Science.gov (United States)

    Zhou, Peng; Guo, Mufan; Liu, Dasong; Liu, Xiaoming; Labuza, Teodore P

    2013-03-01

    The hardening of high-protein bars causes problems in their acceptability to consumers. The objective of this study was to determine the progress of the Maillard reaction in model systems of high-protein nutritional bars containing reducing sugars, and to illustrate the influences of the Maillard reaction on the modification and aggregation of proteins and the hardening of bar matrices during storage. The progress of the Maillard reaction, glycation, and aggregation of proteins, and textural changes in bar matrices were investigated during storage at 25, 35, and 45 °C. The initial development of the Maillard reaction caused little changes in hardness; however, further storage resulted in dramatic modification of protein with formation of high-molecular-weight polymers, resulting in the hardening in texture. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula minimized the changes in texture. The hardening of high-protein bars causes problems in their acceptability to consumers. Maillard reaction is one of the mechanisms contributing to the hardening of bar matrix, particularly for the late stage of storage. The replacement of reducing sugars with nonreducing ingredients such as sugar alcohols in the formula will minimize the changes in texture. © 2013 Institute of Food Technologists®

  11. The oviducal protein, heat-shock 70-kDa protein 8, improves the long-term survival of ram spermatozoa during storage at 17°C in a commercial extender.

    Science.gov (United States)

    Lloyd, R E; Fazeli, A; Watson, P F; Holt, W V

    2012-01-01

    Poor fertility rates are often observed when fresh ram semen stored in conventional extenders is used for cervical artificial insemination (AI). Heat-shock 70-kDa protein 8 (HSPA8), found within the oviduct, prolongs boar, ram and bull sperm survival at body temperatures in vitro. Here, we aimed to determine whether supplementing extenders (INRA-96 and RSD-1) with HSPA8 (4 µg mL⁻¹) would improve their performance in maintaining freshly collected ram sperm viability and sperm nuclear DNA integrity during storage over 48 h at 17°C. Sperm function was assessed at 1, 6, 24 and 48h and this experiment was repeated using 25 × 10⁶ and 800 × 10⁶ spermatozoa mL⁻¹. INRA96 supplemented with HSPA8 maintained sperm viability significantly better than INRA96 alone at both sperm concentrations. However, sperm nuclear DNA fragmentation (DF) increased significantly during storage using the higher sperm concentration, irrespective of the extender and the protein treatment used. Increasing levels of sperm nuclear DF over time could explain why poor fertility rates are often observed following cervical AI using stored ram semen. However, further research is required to ascertain whether supplementing the commercially available INRA96 extender with HSPA8 will improve fertility rates following cervical AI using stored ram semen.

  12. Reticulophagy and Ribophagy: Regulated Degradation of Protein Production Factories

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    Eduardo Cebollero

    2012-01-01

    Full Text Available During autophagy, cytosol, protein aggregates, and organelles are sequestered into double-membrane vesicles called autophagosomes and delivered to the lysosome/vacuole for breakdown and recycling of their basic components. In all eukaryotes this pathway is important for adaptation to stress conditions such as nutrient deprivation, as well as to regulate intracellular homeostasis by adjusting organelle number and clearing damaged structures. For a long time, starvation-induced autophagy has been viewed as a nonselective transport pathway; however, recent studies have revealed that autophagy is able to selectively engulf specific structures, ranging from proteins to entire organelles. In this paper, we discuss recent findings on the mechanisms and physiological implications of two selective types of autophagy: ribophagy, the specific degradation of ribosomes, and reticulophagy, the selective elimination of portions of the ER.

  13. Protein aggregation in aqueous casein solution. Effect of irradiation, dose level, concentration, storage and additives (carbohydrate and lipid)

    Energy Technology Data Exchange (ETDEWEB)

    Yousri, R M

    1980-06-01

    From the vast amount of research efforts dealing with various aspects of radiation effects on foods and food components, it is apparent up to now that much remains to be studied in depth, much may have to be added or corrected about radiation-induced physico-chemical changes in foods. A great many reactions that take place when foodstuffs are subjected to ionizing radiation are still not fully understood. The better understanding of some of the radiation-induced changes in pure proteins as such or in mixture with other food constituents could yield much data which could be meaningfully extrapolated to intact foods and consequently could help to improve the assessment of the wholesomeness of irradiated foods. It was the purpose of our investigations to elucidate some of the changes in the chemical structure of a pure protein (casein), irradiated as such or with added carbohydrate and/or lipid. The effect of subsequent storage of the irradiated solutions has been also examined. The formation of protein aggregates was studied by gel filtration technique. The application of thin-layer gel filtration, its speed and adaptability to very small samples facilitated the measurements of the extent of aggregation which occurred in protein molecules after irradiation.

  14. Serotonin storage pools in basophil leukemia and mast cells: characterization of two types of serotonin binding protein and radioautographic analysis of the intracellular distribution of [3H]serotonin

    International Nuclear Information System (INIS)

    Tamir, H.; Theoharides, T.C.; Gershon, M.D.; Askenase, P.W.

    1982-01-01

    The binding of serotonin to protein(s) derived from rat basophil leukemia (RBL) cells and mast cells was studied. Two types of serotonin binding protein in RBL cells was found. These proteins differed from one another in molecular weight and eluted in separate peaks from sephadex G-200 columns. Peak I protein (KD = 1.9 x 10 -6 M) was a glycoprotein that bound to concanavalin A (Con A); Peak II protein (KD 1 = 4.5 x 10 - 8 M; KD 2 = 3.9 x 10 -6 M) did not bind to Con A. Moreover, binding of [ 3 H]serotonin to protein of Peak I was sensitive to inhibition by reserpine, while binding of [ 3 H]serotonin to protein of Peak II resisted inhibition by that drug. Other differences between the two types of binding protein were found, the most significant of which was the far more vigorous conditions of homogenization required to extract Peak I than Peak II protein. Electron microscope radioautographic analysis of the intracellular distribution of [ 3 H] serotonin taken up in vitro by RBL cells or in vivo by murine mast cells indicated that essentially all of the labeled amine was located in cytoplasmic granules.No evidence for a pool in the cytosol was found and all granules were capable of becoming labeled. The presence of two types of intracellular serotonin binding proteins in these cells may indicate that there are two intracellular storage compartments for the amine. Both may be intragranular, but Peak I protein may be associated with the granular membrane while Peak II protein may be more free within the granular core. Different storage proteins may help to explain the differential release of amines from mast cell granules

  15. Expression and Clinical Significance of the Autophagy Proteins BECLIN 1 and LC3 in Ovarian Cancer

    Directory of Open Access Journals (Sweden)

    Guido Valente

    2014-01-01

    Full Text Available Autophagy is dysregulated in cancer and might be involved in ovarian carcinogenesis. BECLIN-1, a protein that interacts with either BCL-2 or PI3k class III, plays a critical role in the regulation of both autophagy and cell death. Induction of autophagy is associated with the presence of vacuoles characteristically labelled with the protein LC3. We have studied the biological and clinical significance of BECLIN 1 and LC3 in ovary tumours of different histological types. The positive expression of BECLIN 1 was well correlated with the presence of LC3-positive autophagic vacuoles and was inversely correlated with the expression of BCL-2. The latter inhibits the autophagy function of BECLIN 1. We found that type I tumours, which are less aggressive than type II, were more frequently expressing high level of BECLIN 1. Of note, tumours of histologic grade III expressed low level of BECLIN 1. Consistently, high level of expression of BECLIN 1 and LC3 in tumours is well correlated with the overall survival of the patients. The present data are compatible with the hypotheses that a low level of autophagy favours cancer progression and that ovary cancer with upregulated autophagy has a less aggressive behaviour and is more responsive to chemotherapy.

  16. Quality of black beans as a function of long-term storage and moldy development: Chemical and functional properties of flour and isolated protein.

    Science.gov (United States)

    Ferreira, Cristiano Dietrich; Ziegler, Valmor; Lindemann, Igor da Silva; Hoffmann, Jessica Fernanda; Vanier, Nathan Levien; Oliveira, Maurício de

    2018-04-25

    The aim of this study was to evaluate the effects of moisture content and storage temperature on the percentage of moldy and fermented beans, mycotoxins levels, phenolic acids content, pasting properties of whole flour, as well as functional and thermal properties of protein isolates from black beans stored for 12 months. Beans stored under 14%/32 °C exhibited 16% of fermented grains, while at 17%/25 °C (42.3%) and 17%/32 °C (93.5%) of moldy plus fermented grains, named drastic conditions (DC). Mycotoxins were not present in grains from all storage conditions. Reduction of gallic, caffeic, and p-hydroxybenzoic acid contents, and increase of sinapic acid were observed in DC. Reduction of peak, final, and setback viscosities of bean flours in DC indicate the application in refrigerated and frozen products. The increase in foaming and reduction in foam degradation of the proteins highlights their use in beverages where the foam is an important factor. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel

    2013-05-13

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  18. Molecular cloning and characterization of Polygalacturonase-Inhibiting Protein and Cinnamoyl-Coa Reductase genes and their association with fruit storage conditions in blueberry (Vaccinium corymbosum)

    KAUST Repository

    Khraiwesh, Basel; Harb, Jamil; Qudeimat, Enas

    2013-01-01

    Blueberry is a widely grown and easily perishable fruit crop. An efficient post-harvest handling is critical, and for that purpose gene technology methods have been part of ongoing programmes to improve crops with high food values such as blueberry. Here we report the isolation, cloning, characterization and differential expression levels of two cDNAs encoding Polygalacturonase-Inhibitor Protein (PGIP) and Cinnamoyl-Coa Reductase (CCR) from blueberry fruits in relation to various storage conditions. The open reading frame of PGIP and CCR encodes a polypeptide of 329 and 347 amino acids, respectively. To assess changes in the expression of blueberry PGIP and CCR after harvest, a storage trial was initiated. The northern blots hybridization showed a clear differential expression level of PGIP and CCR between freshly harvested and stored fruits as well as between fruits stored under various storage conditions. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the quality over the storage period at the molecular level.

  19. Increasing Sucrose Uptake Capacity of Wheat Grains Stimulates Storage Protein Synthesis1[W

    Science.gov (United States)

    Weichert, Nicola; Saalbach, Isolde; Weichert, Heiko; Kohl, Stefan; Erban, Alexander; Kopka, Joachim; Hause, Bettina; Varshney, Alok; Sreenivasulu, Nese; Strickert, Marc; Kumlehn, Jochen; Weschke, Winfriede; Weber, Hans

    2010-01-01

    Increasing grain sink strength by improving assimilate uptake capacity could be a promising approach toward getting higher yield. The barley (Hordeum vulgare) sucrose transporter HvSUT1 (SUT) was expressed under control of the endosperm-specific Hordein B1 promoter (HO). Compared with the wild type, transgenic HOSUT grains take up more sucrose (Suc) in vitro, showing that the transgene is functional. Grain Suc levels are not altered, indicating that Suc fluxes are influenced rather than steady-state levels. HOSUT grains have increased percentages of total nitrogen and prolamins, which is reflected in increased levels of phenylalanine, tyrosine, tryptophan, isoleucine, and leucine at late grain development. Transcript profiling indicates specific stimulation of prolamin gene expression at the onset of storage phase. Changes in gene expression and metabolite levels related to carbon metabolism and amino acid biosynthesis suggest deregulated carbon-nitrogen balance, which together indicate carbon sufficiency and relative depletion of nitrogen. Genes, deregulated together with prolamin genes, might represent candidates, which respond positively to assimilate supply and are related to sugar-starch metabolism, cytokinin and brassinosteroid functions, cell proliferation, and sugar/abscisic acid signaling. Genes showing inverse expression patterns represent potential negative regulators. It is concluded that HvSUT1 overexpression increases grain protein content but also deregulates the metabolic status of wheat (Triticum aestivum) grains, accompanied by up-regulated gene expression of positive and negative regulators related to sugar signaling and assimilate supply. In HOSUT grains, alternating stimulation of positive and negative regulators causes oscillatory patterns of gene expression and highlights the capacity and great flexibility to adjust wheat grain storage metabolism in response to metabolic alterations. PMID:20018590

  20. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    International Nuclear Information System (INIS)

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-01-01

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  1. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Amr [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619 (Egypt); Hutchens, Heather M. [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Howard Berg, R. [Integrated Microscopy Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132 (United States); Sue Loesch-Fries, L., E-mail: loeschfr@purdue.edu [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States)

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  2. Red blood cell aging markers during storage in citrate-phosphate-dextrose-saline-adenine-glucose-mannitol.

    Science.gov (United States)

    Antonelou, Marianna H; Kriebardis, Anastasios G; Stamoulis, Konstantinos E; Economou-Petersen, Effrosini; Margaritis, Lukas H; Papassideri, Issidora S

    2010-02-01

    It has been suggested that red blood cell (RBC) senescence is accelerated under blood bank conditions, although neither protein profile of RBC aging nor the impact of additive solutions on it have been studied in detail. RBCs and vesicles derived from RBCs in both citrate-phosphate-dextrose (CPD)-saline-adenine-glucose-mannitol (SAGM) and citrate-phosphate-dextrose-adenine (CPDA) were evaluated for the expression of cell senescence markers (vesiculation, protein aggregation, degradation, activation, oxidation, and topology) through immunoblotting technique and immunofluorescence or immunoelectron microscopy study. A group of cellular stress proteins exhibited storage time- and storage medium-related changes in their membrane association and exocytosis. The extent, the rate, and the expression of protein oxidation, Fas oligomerization, caspase activation, and protein modifications in Band 3, hemoglobin, and immunoglobulin G were less conspicuous and/or exhibited significant time retardation under storage in CPD-SAGM, compared to the CPDA storage. There was evidence for the localization of activated caspases near to the membrane of both cells and vesicles. We provide circumstantial evidence for a lower protein oxidative damage in CPD-SAGM-stored RBCs compared to the CPDA-stored cells. The different expression patterns of the senescence markers in the RBCs seem to be accordingly related to the oxidative stress management of the cells. We suggest that the storage of RBCs in CPD-SAGM might be more alike the in vivo RBC aging process, compared to storage in CPDA, since it is characterized by a slower stimulation of the recognition signaling pathways that are already known to trigger the erythrophagocytosis of senescent RBCs.

  3. A storage-protein marker associated with the suppressor of Pm8 for powdery mildew resistance in wheat.

    Science.gov (United States)

    Ren, S X; McIntosh, R A; Sharp, P J; The, T T

    1996-11-01

    A suppressor of resistance to powdery mildew conferred by Pm8 showed complete association with the presence of a storage-protein marker resolved by electrophoresis on SDS-PAGE gels. This marker was identified as the product of the gliadin allele Gli-A1a. The mildewresponse phenotypes of wheats possessing the 1BL.1RS translocation were completely predictable from electrophoretograms. The suppressor, designated SuPm8, was located on chromosome 1AS. It was specific in its suppression of Pm8, and did not affect the rye-derived resistance phenotypes of wheat lines with Pm17, also located in 1RS, or of lines with Pm7.

  4. Lack of Population Structure in Coriander Populations Based on SDS (Seed Storage Protein Page Analysis

    Directory of Open Access Journals (Sweden)

    Gülsüm Yaldiz

    2016-08-01

    Full Text Available Genetic variation is prerequisite for plant breeding. Nothing information existed in the literature for available diversity of Coriander accession in Turkey. Plant breeding activities are negligible in Turkey. So in order to start effective plant breeding program in Turkey, information on the available genetic diversity is viable. Therefore we planned to study the genetic variation and population structure of 29 Coriander accessions by seed storage protein (SDS. SDS analysis elaborated the lack of population structure and genetic bottleneck in the Coriander accessions in Turkey. Based on the results of this study, it was clear that sampling strategy was not appropriate and plant introduction should be made from different sources and diverse genotypes should be used as parents to initialize the effective Turkish Coriander breeding program.

  5. Activation of Ran GTPase by a Legionella effector promotes microtubule polymerization, pathogen vacuole motility and infection.

    Directory of Open Access Journals (Sweden)

    Eva Rothmeier

    2013-09-01

    Full Text Available The causative agent of Legionnaires' disease, Legionella pneumophila, uses the Icm/Dot type IV secretion system (T4SS to form in phagocytes a distinct "Legionella-containing vacuole" (LCV, which intercepts endosomal and secretory vesicle trafficking. Proteomics revealed the presence of the small GTPase Ran and its effector RanBP1 on purified LCVs. Here we validate that Ran and RanBP1 localize to LCVs and promote intracellular growth of L. pneumophila. Moreover, the L. pneumophila protein LegG1, which contains putative RCC1 Ran guanine nucleotide exchange factor (GEF domains, accumulates on LCVs in an Icm/Dot-dependent manner. L. pneumophila wild-type bacteria, but not strains lacking LegG1 or a functional Icm/Dot T4SS, activate Ran on LCVs, while purified LegG1 produces active Ran(GTP in cell lysates. L. pneumophila lacking legG1 is compromised for intracellular growth in macrophages and amoebae, yet is as cytotoxic as the wild-type strain. A downstream effect of LegG1 is to stabilize microtubules, as revealed by conventional and stimulated emission depletion (STED fluorescence microscopy, subcellular fractionation and Western blot, or by microbial microinjection through the T3SS of a Yersinia strain lacking endogenous effectors. Real-time fluorescence imaging indicates that LCVs harboring wild-type L. pneumophila rapidly move along microtubules, while LCVs harboring ΔlegG1 mutant bacteria are stalled. Together, our results demonstrate that Ran activation and RanBP1 promote LCV formation, and the Icm/Dot substrate LegG1 functions as a bacterial Ran activator, which localizes to LCVs and promotes microtubule stabilization, LCV motility as well as intracellular replication of L. pneumophila.

  6. Rab32 is important for autophagy and lipid storage in Drosophila.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.

  7. Effect of high carbon dioxide storage and gamma irradiation on membrane deterioration in cauliflower florets

    International Nuclear Information System (INIS)

    Voisine, R.; Hombourger, C.; Willemot, C.; Castaigne, F.; Makhlouf, J.

    1993-01-01

    Controlled atmospheres and gamma irradiation are technologies which extend storage-life of fruits and vegetables. Separate and combined effects of high CO 2 storage and gamma irradiation on cell membranes from cauliflower florets (Brassica oleracea L., Botrytis group) were investigated. Storage of the florets for 8 days at 13°C, either under 15% carbon dioxide or in air after irradiation at 2 kGy, accelerated the deterioration of microsomal membranes during storage. Both treatments caused an early loss in lipid phosphate. Irradiation enhanced the free fatty acid content of the membranes during storage and caused an extensive protein loss. When irradiation and high CO 2 storage were combined, electrolyte leakage significantly increased while protein loss was considerably reduced. The results indicate that high CO 2 and irradiation accelerate membrane degradation through different mechanisms. The combined effects of the treatments were not additive, but membrane yield was apparently reduced. CO 2 protected the membranes from protein loss induced by irradiation. The apparent increase in electrolyte leakage after irradiation may be caused by the release of ions following cell wall deterioration

  8. Vacuolar processing enzyme plays an essential role in the crystalline structure of glutelin in rice seed.

    Science.gov (United States)

    Kumamaru, Toshihiro; Uemura, Yuji; Inoue, Yoshimi; Takemoto, Yoko; Siddiqui, Sadar Uddin; Ogawa, Masahiro; Hara-Nishimura, Ikuko; Satoh, Hikaru

    2010-01-01

    To identify the function of genes that regulate the processing of proglutelin, we performed an analysis of glup3 mutants, which accumulates excess amounts of proglutelin and lack the vacuolar processing enzyme (VPE). VPE activity in developing seeds from glup3 lines was reduced remarkably compared with the wild type. DNA sequencing of the VPE gene in glup3 mutants revealed either amino acid substitutions or the appearance of a stop codon within the coding region. Microscopic observations showed that alpha-globulin and proglutelin were distributed homogeneously within glup3 protein storage vacuoles (PSVs), and that glup3 PSVs lacked the crystalline lattice structure typical of wild-type PSVs. This suggests that the processing of proglutelin by VPE in rice is essential for proper PSV structure and compartmentalization of storage proteins. Growth retardation in glup3 seedlings was also observed, indicating that the processing of proglutelin influences early seedling development. These findings indicate that storage of glutelin in its mature form as a crystalline structure in PSVs is required for the rapid use of glutelin as a source of amino acids during early seedling development. In conclusion, VPE plays an important role in the formation of protein crystalline structures in PSVs.

  9. Iron storage proteins are essential for the survival and pathogenesis of Mycobacterium tuberculosis in THP-1 macrophages and the guinea pig model of infection.

    Science.gov (United States)

    Reddy, P Vineel; Puri, Rupangi Verma; Khera, Aparna; Tyagi, Anil K

    2012-02-01

    Iron is one of the crucial elements required for the growth of Mycobacterium tuberculosis. However, excess free iron becomes toxic for the cells because it catalyzes the production of reactive oxygen radicals, leading to oxidative damage. Hence, it is essential for the pathogen to have the ability to store intracellular iron in an iron-rich environment and utilize it under iron depletion. M. tuberculosis has two iron storage proteins, namely BfrA (Rv1876; a bacterioferritin) and BfrB (Rv3841; a ferritin-like protein). However, the demonstration of biological significance requires the disruption of relevant genes and the evaluation of the resulting mutant for its ability to survive in the host and cause disease. In this study, we have disrupted bfrA and bfrB of M. tuberculosis and demonstrated that these genes are crucial for the storage and supply of iron for the growth of bacteria and to withstand oxidative stress in vitro. In addition, the bfrA bfrB double mutant (H37Rv ΔbfrA ΔbfrB) exhibited a marked reduction in its ability to survive inside human macrophages. Guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited a marked diminution in the dissemination of the bacilli to spleen compared to that of the parental strain. Moreover, guinea pigs infected with H37Rv ΔbfrA ΔbfrB exhibited significantly reduced pathological damage in spleen and lungs compared to that of animals infected with the parental strain. Our study clearly demonstrates the importance of these iron storage proteins in the survival and pathogenesis of M. tuberculosis in the host and establishes them as attractive targets for the development of new inhibitors against mycobacterial infections.

  10. Subversion of the Endocytic and Secretory Pathways by Bacterial Effector Proteins

    Directory of Open Access Journals (Sweden)

    Mary M. Weber

    2018-01-01

    Full Text Available Intracellular bacteria have developed numerous strategies to hijack host vesicular trafficking pathways to form their unique replicative niches. To promote intracellular replication, the bacteria must interact with host organelles and modulate host signaling pathways to acquire nutrients and membrane for the growing parasitophorous vacuole all while suppressing activation of the immune response. To facilitate host cell subversion, bacterial pathogens use specialized secretion systems to deliver bacterial virulence factors, termed effectors, into the host cell that mimic, agonize, and/or antagonize the function of host proteins. In this review we will discuss how bacterial effector proteins from Coxiella burnetii, Brucella abortus, Salmonella enterica serovar Typhimurium, Legionella pneumophila, Chlamydia trachomatis, and Orientia tsutsugamushi manipulate the endocytic and secretory pathways. Understanding how bacterial effector proteins manipulate host processes not only gives us keen insight into bacterial pathogenesis, but also enhances our understanding of how eukaryotic membrane trafficking is regulated.

  11. A signaling pathway contributing to platelet storage lesion development: targeting PI3-kinase–dependent Rap1 activation slows storage-induced platelet deterioration

    Science.gov (United States)

    Schubert, Peter; Thon, Jonathan N.; Walsh, Geraldine M.; Chen, Cindy H.I.; Moore, Edwin D.; Devine, Dana V.; Kast, Juergen

    2015-01-01

    BACKGROUND The term platelet storage lesion (PSL) describes the structural and biochemical changes in platelets (PLTs) during storage. These are typified by alterations of morphologic features and PLT metabolism leading to reduced functionality and hence reduced viability for transfusion. While the manifestations of the storage lesion are well characterized, the biochemical pathways involved in the initiation of this process are unknown. STUDY DESIGN AND METHODS A complementary proteomic approach has recently been applied to analyze changes in the PLT proteome during storage. By employing stringent proteomic criteria, 12 proteins were identified as significantly and consistently changing in relative concentration over a 7-day storage period. Microscopy, Western blot analysis, flow cytometry, and PLT functionality analyses were used to unravel the involvement of a subset of these 12 proteins, which are connected through integrin signaling in one potential signaling pathway underlying storage lesion development. RESULTS Microscopic analysis revealed changes in localization of glycoprotein IIIa, Rap1, and talin during storage. Rap1 activation was observed to correlate with expression of the PLT activation marker CD62P. PLTs incubated for 7 days with the PI3-kinase inhibitor LY294002 showed diminished Rap1 activation as well as a moderate reduction in integrin αIIbβ3 activation and release of α-granules. Furthermore, this inhibitor seemed to improve PLT integrity and quality during storage as several in vitro probes showed a deceleration of PLT activation. CONCLUSION These results provide the first evidence for a signaling pathway mediating PSL in which PI3-kinase–dependent Rap1 activation leads to integrin αIIbβ3 activation and PLT degranulation. PMID:19497060

  12. Experimental and computational analysis of a large protein network that controls fat storage reveals the design principles of a signaling network.

    Science.gov (United States)

    Al-Anzi, Bader; Arpp, Patrick; Gerges, Sherif; Ormerod, Christopher; Olsman, Noah; Zinn, Kai

    2015-05-01

    An approach combining genetic, proteomic, computational, and physiological analysis was used to define a protein network that regulates fat storage in budding yeast (Saccharomyces cerevisiae). A computational analysis of this network shows that it is not scale-free, and is best approximated by the Watts-Strogatz model, which generates "small-world" networks with high clustering and short path lengths. The network is also modular, containing energy level sensing proteins that connect to four output processes: autophagy, fatty acid synthesis, mRNA processing, and MAP kinase signaling. The importance of each protein to network function is dependent on its Katz centrality score, which is related both to the protein's position within a module and to the module's relationship to the network as a whole. The network is also divisible into subnetworks that span modular boundaries and regulate different aspects of fat metabolism. We used a combination of genetics and pharmacology to simultaneously block output from multiple network nodes. The phenotypic results of this blockage define patterns of communication among distant network nodes, and these patterns are consistent with the Watts-Strogatz model.

  13. Oxidative alterations during human platelet storage

    OpenAIRE

    Göker, Bahar; Özsavcı, Derya; Şener, Azize; Aksoy, Halil; Bağışgil, Vedat; Yanıkkaya Demirel, Gülderen; Uras, Fikriye

    2014-01-01

    SUMMARY: During storage of platelet obtained by apheresis several changes occur. The aimof this study was to investigate the effect of storage on activation, apoptosis, protein pattern,lipid peroxidation, and the levels of nitric oxide (NO) and glutathione (GSH) of platelets. In thisstudy, platelets obtained from healty donors (n=7) by apheresis were kept in an agitator fornine days at 20-24°C. The samples were taken on the 1st, 3 rd, 5 th and 9 th days and plateletswere precipitated. Platele...

  14. Genetic diversity in radish germplasm for morphological traits and seed storage proteins

    International Nuclear Information System (INIS)

    Jatoi, S.A.; Siddiqui, S.U.; Masood, M.S.; Javaid, A.; Iqbal, M.; Sayal, O.U.

    2011-01-01

    Genetic variation of forty-nine local and exotic radish genotypes including two checks was studied for morphological traits and seed storage protein electrophoresis using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) markers. A high variation in germplasm for root shape, root length, root colour (internal and external), flesh texture and root type was observed. Among these genotypes, the genetic variation was apparent for most of the characters like plant biomass, root weight, leaf length, root length and root diameter that indicated the potential for crop improvement in these traits through simple selection. Exotic germplasm exhibited higher variation for plant biomass, root weight and root length which could be utilized through breeding programme. Cluster analysis on the basis of genetic diversity for seven quantitative traits resulted into four clusters. No clustering was found on the basis of origin. Low level of variance was observed for SDS-PAGE electrophoresis that suggested acquisition of more germplasm. On the basis of high yield and crispy root texture some genotypes (10076, 10362, 10429, 10658, 10662 and 10667) were identified for further testing under wide range of agro-ecological conditions. (author)

  15. Knowns and unknowns of plasma membrane protein degradation in plants.

    Science.gov (United States)

    Liu, Chuanliang; Shen, Wenjin; Yang, Chao; Zeng, Lizhang; Gao, Caiji

    2018-07-01

    Plasma membrane (PM) not only creates a physical barrier to enclose the intracellular compartments but also mediates the direct communication between plants and the ever-changing environment. A tight control of PM protein homeostasis by selective degradation is thus crucial for proper plant development and plant-environment interactions. Accumulated evidences have shown that a number of plant PM proteins undergo clathrin-dependent or membrane microdomain-associated endocytic routes to vacuole for degradation in a cargo-ubiquitination dependent or independent manner. Besides, several trans-acting determinants involved in the regulation of endocytosis, recycling and multivesicular body-mediated vacuolar sorting have been identified in plants. More interestingly, recent findings have uncovered the participation of selective autophagy in PM protein turnover in plants. Although great progresses have been made to identify the PM proteins that undergo dynamic changes in subcellular localizations and to explore the factors that control the membrane protein trafficking, several questions remain to be answered regarding the molecular mechanisms of PM protein degradation in plants. In this short review article, we briefly summarize recent progress in our understanding of the internalization, sorting and degradation of plant PM proteins. More specifically, we focus on discussing the elusive aspects underlying the pathways of PM protein degradation in plants. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Macroautophagy-generated increase of lysosomal amyloid β-protein mediates oxidant-induced apoptosis of cultured neuroblastoma cells

    DEFF Research Database (Denmark)

    Zheng, Lin; Terman, Alexei; Hallbeck, Martin

    2011-01-01

    and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production...... and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration....

  17. Property of lysosomal storage disease associated with midbrain pathology in the central nervous system of Lamp-2-deficient mice.

    Science.gov (United States)

    Furuta, Akiko; Kikuchi, Hisae; Fujita, Hiromi; Yamada, Daisuke; Fujiwara, Yuuki; Kabuta, Tomohiro; Nishino, Ichizo; Wada, Keiji; Uchiyama, Yasuo

    2015-06-01

    Lysosome-associated membrane protein-2 (LAMP-2) is the gene responsible for Danon disease, which is characterized by cardiomyopathy, autophagic vacuolar myopathy, and variable mental retardation. To elucidate the function of LAMP-2 in the central nervous system, we investigated the neuropathological changes in Lamp-2-deficient mice. Immunohistochemical observations revealed that Lamp-1 and cathepsin D-positive lysosomal structures increased in the large neurons of the mouse brain. Ubiquitin-immunoreactive aggregates and concanavalin A-positive materials were detected in these neurons. By means of ultrastructural studies, we found various-shaped accumulations, including lipofuscin, glycolipid-like materials, and membranous structures, in the neurons and glial cells of Lamp-2-deficient brains. In deficient mice, glycogen granules accumulated in hepatocyte lysosomes but were not observed in neurons. These pathological features indicate lysosomal storage disease; however, the findings are unlikely a consequence of deficiency of a single lysosomal enzyme. Although previous study results have shown a large amount of autophagic vacuoles in parenchymal cells of the visceral organs, these findings were rarely detected in the brain tissue except for some axons in the substantia nigra, in which abundant activated microglial cells with increased lipid peroxidation were observed. Thus, LAMP-2 in the central nervous system has a possible role in the degradation of the various macromolecules in lysosomes and an additional function concerning protection from oxidative stress, especially in the substantia nigra. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  18. Improved biochemical preservation of heart slices during cold storage.

    Science.gov (United States)

    Bull, D A; Reid, B B; Connors, R C; Albanil, A; Stringham, J C; Karwande, S V

    2000-01-01

    Development of myocardial preservation solutions requires the use of whole organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that myocardial slices could be used to assess preservation of biochemical function during cold storage. Whole rat hearts were precision cut into slices with a thickness of 200 microm and preserved at 4 degrees C in one of the following solutions: Columbia University (CU), University of Wisconsin (UW), D5 0.2% normal saline with 20 meq/l KCL (QNS), normal saline (NS), or a novel cardiac preservation solution (NPS) developed using this model. Myocardial biochemical function was assessed by ATP content (etamoles ATP/mg wet weight) and capacity for protein synthesis (counts per minute (cpm)/mg protein) immediately following slicing (0 hours), and at 6, 12, 18, and 24 hours of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as the mean +/- standard deviation. ATP content was higher in the heart slices stored in the NPS compared to all other solutions at 6, 12, 18 and 24 hours of cold storage (p cold storage (p cold storage.

  19. Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia, Capparis, Drypetes.

    Science.gov (United States)

    Jørgensen, L B; Behnke, H D; Mabry, T J

    1977-01-01

    Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.

  20. Proteolysis in salmon ( Salmo salar ) during cold storage : Effects of storage time and smoking process

    DEFF Research Database (Denmark)

    Lund, K.E.; Nielsen, Henrik Hauch

    2001-01-01

    Changes in free amino acids (FAAs), small peptides and myofibrillar proteins were investigated in salmon (Salmo salar) muscle stored at OC for up to 23 days and after the stored salmon was smoked. Storage time and smoking process did not increase the formation of FAAs and small peptides indicating...

  1. Effect of temperature and time of pasteurization on the milk quality during storage

    Directory of Open Access Journals (Sweden)

    Abubakar

    2001-03-01

    Full Text Available A study on the effect of temperature and time of pasteurization on the milk quality during storage was carried out using fresh milk. The aim of the experiment was to asses the storage time of pasteurized milk for consumption without nutrient losses. A completely randomized factorial design, 2 x 8 was used, with pasteurization temperature (T, consisted of 2 levels, the low temperature long time (LTLT, i.e. fresh milk was warmed at 65oC for 30 minutes (T1 and the high temperature short time (HTST, i.e. fresh milk was warmed at 71oC for 15 seconds (T2; and storage time (S, consisted of 8 levels, i.e. 0, 3, 6, 9, 12, 15, 18, and 21 hours respectively, as the factors, with 3 replicates. Parameters measured were alcohol test, water, fat, and protein concentrations, and microbial population of pasteurized milk during storage. Data were analyzed using analysis of variance and simple linear regression. The result showed that water and fat concentrations and microbial population was not significantly different (P>0.05 in pasteurization temperature treatment, but was significantly different (P<0.05 due to storage time treatment. Meanwhile, the protein concentration was significantly different (P<0.05 either in pasteurization temperature or storage time. It was concluded that pasteurized milk was still suitable for consumption at 15-21 hours storage, while protein concentration tended to be better when was pasteurized at 65oC.

  2. Effect of ice storage on muscle protein properties and qualities of emulsion fish sausage from bigeye snapper (Priacanthus tayenus and lizardfish (Saurida undosquamis

    Directory of Open Access Journals (Sweden)

    Vittayanont, M.

    2005-01-01

    Full Text Available The chemical changes in fish muscle and natural actomyosin (NAM from bigeye snapper (Priacanthus tayenus and lizardfish (Saurida undosquamis muscle during 0, 2, 4, 6, 8, 10, 12 and 14 days of iced storage were studied. Myosin heavy chain (MHC of NAM extracted from two fish species was degraded throughout iced storage. However, no changes in actin were observed. The total volatile base (TVB. trimethylamine (TMA and surface hydrophobicity increased, while the total sulfhydryl content and emulsion capacity of NAM from both fish species decreased significantly as the storage time increased (p<0.05. A Texture ProfileAnalysis (TPA and shear force of emulsion fish sausages prepared from two fish species kept in ice for 0, 4, 8 and 12 days were investigated. The results showed that hardness, cohesiveness, gumminess, chewiness and shear force of sausage prepared from fish kept in ice were lower than those produced from fresh fish. However, no significant differences in adhesiveness were observed. Cooking loss of emulsion fish sausage from two fish species increased throughout storage time (p<0.05. The texture of bigeye snapper sausage was better than that of lizardfish sausages. Scanning electron microscopy (SEM micrographs of emulsion fish sausage from two fish species revealed bigger voids, thicker strands and less continuity of protein strands with increasing storage time. More microstructural changes were observed in sausages from lizardfish, compared to those in sausages from bigeye snapper.

  3. Single Cell Synchrotron FT-IR Microspectroscopy Reveals a Link between Neutral Lipid and Storage Carbohydrate Fluxes in S. cerevisiae

    Science.gov (United States)

    Jamme, Frédéric; Vindigni, Jean-David; Méchin, Valérie; Cherifi, Tamazight; Chardot, Thierry; Froissard, Marine

    2013-01-01

    In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins). We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated. PMID:24040242

  4. Single cell synchrotron FT-IR microspectroscopy reveals a link between neutral lipid and storage carbohydrate fluxes in S. cerevisiae.

    Directory of Open Access Journals (Sweden)

    Frédéric Jamme

    Full Text Available In most organisms, storage lipids are packaged into specialized structures called lipid droplets. These contain a core of neutral lipids surrounded by a monolayer of phospholipids, and various proteins which vary depending on the species. Hydrophobic structural proteins stabilize the interface between the lipid core and aqueous cellular environment (perilipin family of proteins, apolipoproteins, oleosins. We developed a genetic approach using heterologous expression in Saccharomyces cerevisiae of the Arabidopsis thaliana lipid droplet oleosin and caleosin proteins AtOle1 and AtClo1. These transformed yeasts overaccumulate lipid droplets, leading to a specific increase in storage lipids. The phenotype of these cells was explored using synchrotron FT-IR microspectroscopy to investigate the dynamics of lipid storage and cellular carbon fluxes reflected as changes in spectral fingerprints. Multivariate statistical analysis of the data showed a clear effect on storage carbohydrates and more specifically, a decrease in glycogen in our modified strains. These observations were confirmed by biochemical quantification of the storage carbohydrates glycogen and trehalose. Our results demonstrate that neutral lipid and storage carbohydrate fluxes are tightly connected and co-regulated.

  5. Ultrastructural and biochemical investigations of protein mobilization of Mucuna pruriens (L.) DC. cotyledons and embryo axis.

    Science.gov (United States)

    Muccifora, Simonetta; Guerranti, Roberto; Muzzi, Chiara; Hope-Onyekwere, Nnadozies S; Pagani, Roberto; Leoncini, Roberto; Bellani, Lorenza M

    2010-03-01

    The mobilization of storage reserves, with particular emphasis on storage proteins of Mucuna pruriens (L.) DC., cotyledons, and embryo was investigated from the ultrastructural and biochemical points of view. Proteins and starch were the two main storage substances in cotyledons, and proteins and lipids were the main ones in the embryo. Embryo protein bodies were smaller and fewer in number than those of cotyledons. Structural and ultrastructural data determined between 24 and 48 h after imbibition and between 48 and 72 h after imbibition, the end of significant embryo and cotyledon protein mobilization, respectively, indicating more precocious storage protein mobilization in the axis than cotyledons. Moreover, storage protein mobilization in embryo and cotyledons occurred before the end of germination. Water soluble proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, producing 29 bands with molecular weights from 14 to 90 KDa. Embryo extract contained more proteins than cotyledon extract, contained seven characteristic bands, and showed a higher variability of the optical density trend than cotyledon.

  6. ORFer--retrieval of protein sequences and open reading frames from GenBank and storage into relational databases or text files.

    Science.gov (United States)

    Büssow, Konrad; Hoffmann, Steve; Sievert, Volker

    2002-12-19

    Functional genomics involves the parallel experimentation with large sets of proteins. This requires management of large sets of open reading frames as a prerequisite of the cloning and recombinant expression of these proteins. A Java program was developed for retrieval of protein and nucleic acid sequences and annotations from NCBI GenBank, using the XML sequence format. Annotations retrieved by ORFer include sequence name, organism and also the completeness of the sequence. The program has a graphical user interface, although it can be used in a non-interactive mode. For protein sequences, the program also extracts the open reading frame sequence, if available, and checks its correct translation. ORFer accepts user input in the form of single or lists of GenBank GI identifiers or accession numbers. It can be used to extract complete sets of open reading frames and protein sequences from any kind of GenBank sequence entry, including complete genomes or chromosomes. Sequences are either stored with their features in a relational database or can be exported as text files in Fasta or tabulator delimited format. The ORFer program is freely available at http://www.proteinstrukturfabrik.de/orfer. The ORFer program allows for fast retrieval of DNA sequences, protein sequences and their open reading frames and sequence annotations from GenBank. Furthermore, storage of sequences and features in a relational database is supported. Such a database can supplement a laboratory information system (LIMS) with appropriate sequence information.

  7. Analysis of iron storage proteins in chicken liver and spleen tissues in comparison with human liver ferritin by Moessbauer spectroscopy

    International Nuclear Information System (INIS)

    Oshtrakh, M.I.; Milder, O.B.; Semionkin, V.A.; Malakheeva, L.I.; Prokopenko, P.G.

    2006-01-01

    Characterization of iron storage proteins in liver and spleen from normal chicken and chicken with lymphoid leukemia in comparison with human liver ferritin were considered by Moessbauer spectroscopy (preliminary results). Small differences in Moessbauer hyperfine parameters for both normal and lymphoid leukemia chicken liver and spleen were observed. The value of quadrupole splitting for human liver ferritin was higher than those for chicken tissues. A decrease of iron content in lymphoid leukemia chicken tissues was also found, however, the reason of this fact (pathology or feeding) was not clear yet. (author)

  8. RHEOLOGY OF CHICKPEA PROTEIN CONCENTRATE DISPERSIONS

    Directory of Open Access Journals (Sweden)

    Aurelia Ionescu

    2011-12-01

    Full Text Available Chickpea proteins are used as ingredients in comminuted sausage products and many oriental textured foods. Rheological behaviour of chickpea protein concentrate was studied using a controlled stress rheometer. The protein dispersion prepared with phosphate buffer at pH 7.0 presented non-Newtonian shear thinning behaviour and rheological data well fitted to the Sisko, Carreau and Cross models. The viscoelastic properties of the chickpea protein suspensions were estimated by measuring the storage and loss moduli in oscillatory frequency conditions (0.1-10 Hz at 20°C. Moreover, thermally induced gelation of the chickpea proteins (16, 24 and 36% was studied at pH 7.0 and 4.5 in the temperature range 50 to 100oC and salt concentration ranging from 0 to 1 M. Gelling behaviour was quantified by means of dynamic rheological measurements. Gels formation was preceded by the decrease of storage modulus and loss moduli, coupled with the increase of the phase angle (delta. The beginning of thermal gelation was influenced by protein concentration, pH and salt level. In all studied cases, storage modulus increased rapidly in the temperature range 70-90°C. All rheological parameters measured at 90°C were significantly higher at pH 4.5 compared to pH 7.0.

  9. Determination of soluble protein contents from RVNRL

    International Nuclear Information System (INIS)

    Wan Manshol Wan Zin; Nurulhuda Othman

    1996-01-01

    This project was carried out to determine the soluble protein contents on RVNRL film vulcanisates, with respect to the RVNRL storage time, gamma irradiation dose absorbed by the latex and the effect of different leaching time and leaching conditions. These three factors are important in the hope to determine the best possible mean of minimizing the soluble protein contents in products made from RVNRL. Within the nine months storage period employed in the study, the results show that, the longer the storage period the less the soluble protein extracted from the film samples. Gamma irradiation dose absorbed by the samples, between 5.3 kGy to 25.2 kGy seems to influence the soluble protein contents of the RVNRL films vulcanisates. The higher the dose the more was the soluble protein extracted from the film samples. At an absorbed dose of 5.3 kGy and 25.2 kGy, the soluble contents were 0. 198 mg/ml and 0.247 mg/ml respectively. At a fixed leaching temperature, the soluble proteins increases with leaching time and at a fixed leaching time, the soluble proteins increases with leaching temperature. ne highest extractable protein contents was determined at a leaching time of 10 minutes and leaching temperature of 90'C The protein analysis were done by using Modified Lowry Method

  10. Corn Storage Protein - A Molecular Genetic Model

    Energy Technology Data Exchange (ETDEWEB)

    Messing, Joachim [Rutgers Univ., Piscataway, NJ (United States)

    2013-05-31

    Corn is the highest yielding crop on earth and probably the most valuable agricultural product of the United States. Because it converts sun energy through photosynthesis into starch and proteins, we addressed energy savings by focusing on protein quality. People and animals require essential amino acids derived from the digestion of proteins. If proteins are relatively low in certain essential amino acids, the crop becomes nutritionally defective and has to be supplemented. Such deficiency affects meat and fish production and countries where corn is a staple. Because corn seed proteins have relatively low levels of lysine and methionine, a diet has to be supplemented with soybeans for the missing lysine and with chemically synthesized methionine. We therefore have studied genes expressed during maize seed development and their chromosomal organization. A critical technical requirement for the understanding of the molecular structure of genes and their positional information was DNA sequencing. Because of the length of sequences, DNA sequencing methods themselves were insufficient for this type of analysis. We therefore developed the so-called “DNA shotgun sequencing” strategy, where overlapping DNA fragments were sequenced in parallel and used to reconstruct large DNA molecules via overlaps. Our publications became the most frequently cited ones during the decade of 1981-1990 and former Associate Director of Science for the Office of Basic Energy Sciences Patricia M. Dehmer presented our work as one of the great successes of this program. A major component of the sequencing strategy was the development of bacterial strains and vectors, which were also used to develop the first biotechnology crops. These crops possessed new traits thanks to the expression of foreign genes in plants. To enable such expression, chimeric genes had to be constructed using our materials and methods by the industry. Because we made our materials and methods freely available to

  11. Function and regulation of plant major intrinsic proteins

    DEFF Research Database (Denmark)

    Popovic, Milan

    ;1 in Arabidopsis. That led to the discovery that tip4;1 is gametophytic lethal- gene essential for normal seed set. ICP-MS analyses of the elemental composition of tip4;1 heterozygous T-DNA insert mutant plants and 35S::TIP4;1 over-expression plants indicate that AtTIP4;1 has a role in arsenic distribution...... inorganic forms of arsenic in the environment, can be taken up by plants and thus enter the food chain. Once inside the root cells, As(V) is reduced to As(III) which is then extruded to the soil solution or bound to phytochelatins (PCs) and transported to the vacuole in an effort to accomplish...... detoxification. Plant Noduline 26-like Intrinsic Proteins (NIPs) can channel As(III) and consequently influence the detoxification process. The role of the Tonoplast Intrinsic Proteins (TIPs) in As(III) detoxification remains to be clarified, yet TIPs could have an impact on As(III) accumulation in plant cell...

  12. Experimental and computational analysis of a large protein network that controls fat storage reveals the design principles of a signaling network.

    Directory of Open Access Journals (Sweden)

    Bader Al-Anzi

    2015-05-01

    Full Text Available An approach combining genetic, proteomic, computational, and physiological analysis was used to define a protein network that regulates fat storage in budding yeast (Saccharomyces cerevisiae. A computational analysis of this network shows that it is not scale-free, and is best approximated by the Watts-Strogatz model, which generates "small-world" networks with high clustering and short path lengths. The network is also modular, containing energy level sensing proteins that connect to four output processes: autophagy, fatty acid synthesis, mRNA processing, and MAP kinase signaling. The importance of each protein to network function is dependent on its Katz centrality score, which is related both to the protein's position within a module and to the module's relationship to the network as a whole. The network is also divisible into subnetworks that span modular boundaries and regulate different aspects of fat metabolism. We used a combination of genetics and pharmacology to simultaneously block output from multiple network nodes. The phenotypic results of this blockage define patterns of communication among distant network nodes, and these patterns are consistent with the Watts-Strogatz model.

  13. Prion protein immunocytochemistry helps to establish the true incidence of prion diseases.

    Science.gov (United States)

    Lantos, P L; McGill, I S; Janota, I; Doey, L J; Collinge, J; Bruce, M T; Whatley, S A; Anderton, B H; Clinton, J; Roberts, G W

    1992-11-23

    Creutzfeldt-Jakob disease (CJD) and Gerstmann-Strüssler-Scheinker disease (GSSD) are transmissible spongiform encephalopathies or prion diseases affecting man. It has been reported that prion diseases may occur without the histological hallmarks of spongiform encephalopathies: vacuolation of the cerebral grey matter, neuronal loss and astrocytosis. These cases without characteristic neuropathology may go undiagnosed and consequently the true incidence of transmissible dementias is likely to have been under-estimated. Immunocytochemistry using antibodies to prion protein gives positive staining of these cases, albeit the pattern of immunostaining differs from that seen in typical forms. Accumulation of prion protein is a molecular hallmark of prion diseases, and thus a reproducible, speedy and cost-efficient immunocytochemical screening of unusual dementias may help to establish the true incidence of prion diseases.

  14. Polymorphism in the Plasmodium falciparum chloroquine-resistance transporter protein links verapamil enhancement of chloroquine sensitivity with the clinical efficacy of amodiaquine

    Directory of Open Access Journals (Sweden)

    Warhurst David C

    2003-09-01

    Full Text Available Abstract Background Chloroquine accumulates in the acidic digestive vacuole of the intraerythrocytic malaria parasite, and prevents the detoxication of haematin released during haemoglobin digestion. Changes in protein PfCRT in the digestive vacuole membrane of growing intra-erythrocytic stages of Plasmodium falciparum are crucial for resistance. Expressed in yeast, PfCRT resembles an anion channel. Depressed anion channel function could increase intralysosomal pH to reduce entry of basic drug, or enhanced function could reduce drug interaction with target haematin. The most important resistance-associated change is from positively-charged lysine-76 to neutral threonine which could facilitate drug efflux through a putative channel. It has been proposed that the resistance-reversing effect of verapamil is due to hydrophobic binding to the mutated PfCRT protein, and replacement of the lost positive charge, which repels the access of 4-aminoquinoline cations, thus partially restoring sensitivity. Desethylamodiaquine, the active metabolite of amodiaquine, which has significant activity in chloroquine-resistance, may also act similarly on its own. Methods Changes in physicochemical parameters in different CQ-resistant PfCRT sequences are analysed, and correlations with drug activity on lines transfected with different alleles of the pfcrt gene are examined. Results and conclusions The results support the idea that PfCRT is a channel which, in resistant parasites, can allow efflux of chloroquine from the digestive vacuole. Activity of the chloroquine/verapamil combination and of desethylamodiaquine both correlate with the mean hydrophobicity of PfCRT residues 72-76. This may partly explain clinical-resistance to amodiaquine found in the first chloroquine-resistant malaria cases from South America and enables tentative prediction of amodiaquine's clinical activity against novel haplotypes of PfCRT.

  15. Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

    Directory of Open Access Journals (Sweden)

    Jorge E Vidal

    2009-02-01

    Full Text Available OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+ and a non-toxigenic Mexican strain (CM 91-3, ctxAB-. Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+ y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-. El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un

  16. Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

    Science.gov (United States)

    Yoo, Byung-Chun; Lee, Jung-Youn; Lucas, William J

    2002-05-03

    In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

  17. Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

    Science.gov (United States)

    Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

    2014-11-24

    Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

  18. Effect of gamma irradiation and cold storage at 4±1 on components of buffalo meat

    International Nuclear Information System (INIS)

    MAHMOUD, A. A.; SHALABY, M. T.; DOMA, M. B.; HUSSEIN, M. A.; EMAM, O.

    1989-01-01

    The effect of gamma irradiation with 2.5, 5.0 and 7.5 KGy and cold storage at 4± 1 on some components of buffalo meat was studied. The results showed that no significant differences effect on the chemical composition of buffalo meat such as moisture content, crude protein, crude fat and ash content. While cold storage caused a slight decrease in the above mentioned parameters. irradiation treatment did not affect significantly on the total nitrogen and non-protein nitrogen. Gradual increase in the solubility of protein and slight decrease in the total nitrogen was observed during cold storage. Total volatile nitrogen and free amino nitrogen increased during cold storage. Total free amino acids increased gradually with different rates depending on the applied irradiation dose. Directly after irradiation isoleucine, phenylalanine, tryptophan and arginine exhibit a proportional relationship with the dose level used with the buffalo meat tissues.4 tab

  19. The use of recombinant nAG protein In spinal cord crush injury in a rat model

    International Nuclear Information System (INIS)

    Al-Qattan, M.M.; Al-Motairi, M.; Ah-Habib, A.

    2017-01-01

    Objective: To evaluate the therapeutic properties of nAG protein during the recovery following acute spinal cord injuries in the rat. Study Design: An experimental study. Place and Duration of Study: King Saud University, Riyadh, Saudi Arabia, from September 2014 to September 2015. Methodology: Eight rats were studied (4 control rats and 4 experimental rats; and hence 50% were controls and 50% were experimental). All rats were subjected to an acute spinal cord injury using the aneurysmal clip injury model. Immediately after the injury, a single intra-dural injection of either normal saline (in the control group) or the nAG protein (in the experimental group) was done. Assessment of both groups was done over a 6-week period with regard to weight maintenance, motor recovery scores, MRI and histopathology of the injury site. Results: Weight maintenance was seen in the experimental and not in the control rats. Starting at 3 weeks after injury, the motor recovery was significantly (p<0.05) better in the experimental group. MRI assessment at 6 weeks showed better maintenance of cord continuity and less fluid accumulation at the injury site in the nAG-treated group. Just proximal to the injury site, there was less gliosis in the experimental group compared to the control group. At the crush injury site, there was less tissue architecture distortion, less vacuole formation, and less granulation tissue formation in the experimental group. Conclusion: The local injection nAG protein enhances neuro-restoration, reduces gliosis, and reduces vacuole/ granulation tissue formation following acute spinal cord crush injury in the rat aneurysmal clip animal model. (author)

  20. Irradiation and storage effect on some characteristics of soy seeds

    International Nuclear Information System (INIS)

    Ramirez Ascheri, Diego Palmiro; Devilla, Ivano Alessandro

    2008-01-01

    The irradiation has been applied frequently in seeds conservation to obtain reduction of losses caused by physiologic processes, besides reducing the microbial load. However, the irradiation process for X-rays is not a common practice in seeds; for that, it is necessary to study that process, in order to know irradiation effect on the soy seeds quality. The objective of this work was to verify the irradiation effect of X-rays and the storage period on the water, oil and protein contends of soy seeds (Glycine max L.) variety Emgopa 302. The experiment was represented by the combination of two factors: X-rays dosage with four irradiation levels [0, 50, 65 and 70 kV] and storage period in laboratory atmosphere with five levels (0, 15, 30, 45 and 60 days). The water content, oil and protein in seeds were assayed in the beginning and every 15 days of storage. The results showed the seeds quality stayed unaffected in irradiation function in the beginning of the experience, with alterations after 15 days, was verified a quality decreasing with the increasing of the X-rays dosage. The soy seeds irradiated had reduced quality in elapsing of the storage period. (author)

  1. Lysosome-related organelles as mediators of metal homeostasis.

    Science.gov (United States)

    Blaby-Haas, Crysten E; Merchant, Sabeeha S

    2014-10-10

    Metal ion assimilation is essential for all forms of life. However, organisms must properly control the availability of these nutrients within the cell to avoid inactivating proteins by mismetallation. To safeguard against an imbalance between supply and demand in eukaryotes, intracellular compartments contain metal transporters that load and unload metals. Although the vacuoles of Saccharomyces cerevisiae and Arabidopsis thaliana are well established locales for the storage of copper, zinc, iron, and manganese, related compartments are emerging as important mediators of metal homeostasis. Here we describe these compartments and review their metal transporter complement. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

    Science.gov (United States)

    Zick, Michael; Stroupe, Christopher; Orr, Amy; Douville, Deborah; Wickner, William T

    2014-01-01

    Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.

  3. Erythrocyte endogenous proteinase activity during blood bank storage.

    Science.gov (United States)

    de Angelis, V; de Matteis, M C; Orazi, B M; Santarossa, L; Della Toffola, L; Raineri, A; Vettore, L

    1990-01-01

    We studied proteolytic alterations of membrane proteins in ghosts derived from human red blood cells, preserved up to 35 days in the liquid state either as whole blood or with additive solution. The study was carried out by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis of stromal proteins from erythrocytes, either previously treated with proteinase inhibitors or previously incubated in conditions promoting proteolysis. To differentiate the effect of erythrocyte from granulocyte proteinases, the investigation was also carried out in leukocyte-free red cell preparations. The results show: (1) the effects of endogenous proteinases on membrane proteins derived from red cells stored under blood bank conditions; (2) a decrease of proteolytic effects in ghosts derived from red cells which have been submitted to a longer storage; (3) a relevant influence of the red cell resuspending medium before lysis on the time-dependent onset and exhaustion of proteolysis in ghosts. The presence of increased proteolysis in ghosts could be regarded as a marker of molecular lesions induced in red cells by storage under blood bank conditions.

  4. Sequence Classification: 893876 [

    Lifescience Database Archive (English)

    Full Text Available TMB Non-TMH TMB TMB Non-TMB TMB >gi|6325306|ref|NP_015374.1| Peripheral membrane protein required for delive...ry of aminopeptidase I (Lap4p) to the vacuole in the cytoplasm-to-vacuole targeting

  5. The effects of feed composition on the sensory quality of organic rainbow trout during ice storage

    DEFF Research Database (Denmark)

    Green-Pedersen, Ditte; Hyldig, Grethe; Jacobsen, Charlotte

    fishmeal and a mixture of protein from organic vegetable, while the lipid sources were fish oil and organic oil from linseed, sunflower, rapeseed and grape seed. Sensory analysis was performed after 3, 5, 7 and 14 days of storage in ice. The results showed that both protein and lipid source in the feed can...... after 14 days of storage, indicating that vegetable protein in the feed increases the self-life of organic rainbow trout.......The focus of this work was to study which effects the type of protein and lipid source in the feed for organic Rainbow trout influences had on the sensory quality of final product. Two and four different protein and lipid sources were used in the experiment respectively. The protein sources were...

  6. The diverse and dynamic nature of Leishmania parasitophorous vacuoles studied by multidimensional imaging.

    Directory of Open Access Journals (Sweden)

    Fernando Real

    Full Text Available An important area in the cell biology of intracellular parasitism is the customization of parasitophorous vacuoles (PVs by prokaryotic or eukaryotic intracellular microorganisms. We were curious to compare PV biogenesis in primary mouse bone marrow-derived macrophages exposed to carefully prepared amastigotes of either Leishmania major or L. amazonensis. While tight-fitting PVs are housing one or two L. major amastigotes, giant PVs are housing many L. amazonensis amastigotes. In this study, using multidimensional imaging of live cells, we compare and characterize the PV biogenesis/remodeling of macrophages i hosting amastigotes of either L. major or L. amazonensis and ii loaded with Lysotracker, a lysosomotropic fluorescent probe. Three dynamic features of Leishmania amastigote-hosting PVs are documented: they range from i entry of Lysotracker transients within tight-fitting, fission-prone L. major amastigote-housing PVs; ii the decrease in the number of macrophage acidic vesicles during the L. major PV fission or L. amazonensis PV enlargement; to iii the L. amazonensis PV remodeling after homotypic fusion. The high content information of multidimensional images allowed the updating of our understanding of the Leishmania species-specific differences in PV biogenesis/remodeling and could be useful for the study of other intracellular microorganisms.

  7. Increased serum C-reactive protein level is associated with increased storage lower urinary tract symptoms in men with benign prostatic hyperplasia.

    Directory of Open Access Journals (Sweden)

    Shun-Fa Hung

    Full Text Available OBJECTIVE: Chronic inflammation is considered as one of the contributing mechanisms of lower urinary tract symptoms (LUTS. Serum C-reactive protein (CRP level is the widely used biomarker of inflammatory status. This study investigated the association between serum CRP level in men with benign prostatic hyperplasia (BPH and lower urinary tract symptoms (LUTS before and after medical treatment. METHODS: A total of 853 men with BPH and LUTS were enrolled. All patients completed the International Prostate Symptoms Score (IPSS questionnaire and urological examinations. The parameters of uroflowmetry (maximum flow rate, Qmax; voided volume, VV, post-void residual (PVR, total prostate volume (TPV and transition zone index (TZI, serum prostate specific antigen (PSA, and serum CRP levels were obtained. All patients were treated with alpha-blocker or antimuscarinic agent based on the IPSS voiding to storage subscore ratio (IPSS-V/S. Correlation analyses were performed between serum CRP levels with age, IPSS, TPV, TZI, Qmax, PVR, VV, PSA and between baseline and post treatment. RESULTS: The mean age was 66.9 ± 11.6 years old and the mean serum CRP levels were 0.31 ± 0.43 mg/dL. Univariate analyses revealed serum CRP levels were significantly associated with age (p<0.001, PSA levels (p = 0.005 and VV (p = 0.017, but not significantly associated with TPV (p = 0.854 or PVR (p = 0.068. CRP levels were positively associated with urgency (p<0.001 and nocturia (p<0.001 subscore of IPSS, total IPSS (p = 0.008 and storage IPSS (p<0.001 and negatively associated with IPSS- V/S ratio (p = 0.014. Multivariate analyses revealed that serum CRP levels were significantly associated with age (p = 0.004 and storage IPSS subscore p<0.001. Patients with IPSS-V/S<1 and treated with tolterodine for 3 months had significant decrease of CRP levels after treatment. CONCLUSION: Serum CRP levels are associated with storage LUTS and sensory bladder disorders, suggesting chronic

  8. Low-density Lipoprotein Receptor-related Protein-1 (LRP1) Mediates Autophagy and Apoptosis Caused by Helicobacter pylori VacA*

    OpenAIRE

    Yahiro, Kinnosuke; Satoh, Mamoru; Nakano, Masayuki; Hisatsune, Junzo; Isomoto, Hajime; Sap, Jan; Suzuki, Hidekazu; Nomura, Fumio; Noda, Masatoshi; Moss, Joel; Hirayama, Toshiya

    2012-01-01

    In Helicobacter pylori infection, vacuolating cytotoxin (VacA)-induced mitochondrial damage leading to apoptosis is believed to be a major cause of cell death. It has also been proposed that VacA-induced autophagy serves as a host mechanism to limit toxin-induced cellular damage. Apoptosis and autophagy are two dynamic and opposing processes that must be balanced to regulate cell death and survival. Here we identify the low-density lipoprotein receptor-related protein-1 (LRP1) as the VacA rec...

  9. Seed Storage Proteins as a System for Teaching Protein Identification by Mass Spectrometry in Biochemistry Laboratory

    Science.gov (United States)

    Wilson, Karl A.; Tan-Wilson, Anna

    2013-01-01

    Mass spectrometry (MS) has become an important tool in studying biological systems. One application is the identification of proteins and peptides by the matching of peptide and peptide fragment masses to the sequences of proteins in protein sequence databases. Often prior protein separation of complex protein mixtures by 2D-PAGE is needed,…

  10. Effect of different fining agents and additives in white wine protein stability

    OpenAIRE

    Ribeiro, Tânia Isabel Monteiro; Cosme, Fernanda; Filipe-Ribeiro, L.; Fernandes, Conceição; Mendes-Faia, Arlete

    2012-01-01

    Proteins in white wine could become insoluble and precipitate causing the appearance of a haze in bottled wine. Protein instability may be due to intrinsically or extrinsically factor such as protein molecular weight, isoelectric point, ionic strength, alcohol degree and wine pH or storage temperature. These modifications may occur during aging, storage or when diverse wines are blended. The type and concentration of proteins in the wine depends on grape variety, maturation deg...

  11. Characterization of the yam tuber storage proteins from Dioscorea batatas exhibiting unique lectin activities.

    Science.gov (United States)

    Gaidamashvili, Mariam; Ohizumi, Yuki; Iijima, Shinichiro; Takayama, Tomo; Ogawa, Tomohisa; Muramoto, Koji

    2004-06-18

    Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic

  12. Identification of the chemical forms of selenium in soy protein

    International Nuclear Information System (INIS)

    Rodibaugh, R.

    1989-01-01

    Soybeans (Glycine max. L. Merr., Century) were grown hydroponically and intrinsically radiolabeled with 75 Se, an isotope of selenium (Se). The isotope was provided as 75 Se-Na 2 SeO 3 during the reproductive stage of growth until onset of senescence. Harvested seeds were processed into defatted soy meal. Soluble proteins were extracted in 20mM Tris-HCl buffer and fractionated into 11S, 7S, and 2S protein fractions by isoelectric precipitation. The 11S and 7S globulins, containing the glycinin and conglycinin storage proteins respectively, constitute the majority of extractable soy proteins. These storage proteins are the predominant proteins in soy protein isolate frequently used in food for human consumption. Approximately 24% of the defatted meal was soluble protein and accounted for 65% of the radioactivity associated with the soybean meal. The 11S fraction contained approximately 31% of the extracted protein and 27% of the extracted radioactivity. The 7S fraction contained approximately 32% and 35% of the extractable protein and radioactivity, respectively. The 2S fraction, containing the sulfur (S)-rich trypsin inhibitors, accounted for 17% of the protein and 27% of the radioactivity extracted from the defatted soy meal. Purification of the storage proteins by gel filtration and affinity chromatography showed higher levels of radioactivity associated with glycinin than conglycinin. Purified 11S proteins contained 1.09 ng Se per mg protein while 7S proteins contained 0.36 ng Se per mg protein

  13. Improved protein extraction and protein identification from archival formalin-fixed paraffin-embedded human aortas.

    Science.gov (United States)

    Fu, Zongming; Yan, Kun; Rosenberg, Avraham; Jin, Zhicheng; Crain, Barbara; Athas, Grace; Heide, Richard S Vander; Howard, Timothy; Everett, Allen D; Herrington, David; Van Eyk, Jennifer E

    2013-04-01

    Evaluate combination of heat and elevated pressure to enhance protein extraction and quality of formalin-fixed (FF), and FF paraffin-embedded (FFPE) aorta for proteomics. Proteins were extracted from fresh frozen aorta at room temperature (RT). FF and FFPE aortas (3 months and 15 years) were extracted at RT, heat alone, or a combination of heat and high pressure. Protein yields were compared, and digested peptides from the extracts were analyzed with MS. Combined heat and elevated pressure increased protein yield from human FF or FFPE aorta compared to matched tissues with heat alone (1.5-fold) or at RT (8.3-fold), resulting in more proteins identified and with more sequence coverage. The length of storage did adversely affect the quality of proteins from FF tissue. For long-term storage, aorta was preserved better with FFPE than FF alone. Periostin and MGF-E8 were demonstrated suitable for MRM assays from FFPE aorta. Combination of heat and high pressure is an effective method to extract proteins from FFPE aorta for downstream proteomics. This method opens the possibility for use of archival and often rare FFPE aortas and possibly other tissues available to proteomics for biomarker discovery and quantification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Drying and storage effects on poly(ethylene glycol) hydrogel mechanical properties and bioactivity.

    Science.gov (United States)

    Luong, P T; Browning, M B; Bixler, R S; Cosgriff-Hernandez, E

    2014-09-01

    Hydrogels based on poly(ethylene glycol) (PEG) are increasingly used in biomedical applications because of their ability to control cell-material interactions by tuning hydrogel physical and biological properties. Evaluation of stability after drying and storage are critical in creating an off-the-shelf biomaterial that functions in vivo according to original specifications. However, there has not been a study that systematically investigates the effects of different drying conditions on hydrogel compositional variables. In the first part of this study, PEG-diacrylate hydrogels underwent common processing procedures (vacuum-drying, lyophilizing, hydrating then vacuum-drying), and the effect of this processing on the mechanical properties and swelling ratios was measured. Significant changes in compressive modulus, tensile modulus, and swelling ratio only occurred for select processed hydrogels. No consistent trends were observed after processing for any of the formulations tested. The effect of storage conditions on cell adhesion and spreading on collagen- and streptococcal collagen-like protein (Scl2-2)-PEG-diacrylamide hydrogels was then evaluated to characterize bioactivity retention after storage. Dry storage conditions preserved bioactivity after 6 weeks of storage; whereas, storage in PBS significantly reduced bioactivity. This loss of bioactivity was attributed to ester hydrolysis of the protein linker, acrylate-PEG-N-hydroxysuccinimide. These studies demonstrate that these processing methods and dry storage conditions may be used to prepare bioactive PEG hydrogel scaffolds with recoverable functionality after storage. © 2013 Wiley Periodicals, Inc.

  15. Protein and solute distribution in drug substance containers during frozen storage and post-thawing: a tool to understand and define freezing-thawing parameters in biotechnology process development.

    Science.gov (United States)

    Kolhe, Parag; Badkar, Advait

    2011-01-01

    Active pharmaceutical ingredient for biotechnology-based drugs, commonly known as drug substance (DS), is often stored frozen for longer shelf-life. Freezing DS enhances stability by slowing down reaction rates that lead to protein instability, minimizes the risk of microbial growth, and eliminates the risk of transport-related stress. High density polyethylene bottles are commonly used for storing monoclonal antibody DS due to good mechanical stress/strain resistant properties even at low temperatures. Despite the aforementioned advantages for frozen storage of DS, this is not devoid of risks. Proteins are known to undergo ice-water surface denaturation, cryoconcentration, and cold denaturation during freezing. A systematic investigation was performed to better understand the protein and solute distribution along with potential of aggregate formation during freeze and thaw process. A significant solute and protein concentration gradient was observed for both frozen and thawed DS bottles. In case of thawed DS, cryoconcentration was localized in the bottom layer and a linear increase in concentration as a function of liquid depth was observed. On the other hand, for frozen DS, a "bell shaped" cryoconcentration distribution was observed between the bottom layers and centre position. A cryoconcentration of almost three-fold was observed for frozen DS in the most concentrated part when freezing was conducted at -20 and -40 °C and 2.5-fold cryoconcentration was observed in the thawed DS before mixing. The information obtained in this study is critical to design freeze thaw experiments, storage condition determination, and process improvement in manufacturing environment. Copyright © 2011 American Institute of Chemical Engineers (AIChE).

  16. characterisation of common bean genotypes based on storage

    African Journals Online (AJOL)

    ACSS

    of pliers and then ground to fine powder with a ... segregation of genotypes were Rm 23.75, 32.50,. 33.75, 22.50 ... Figure 1. Positions of Phaseolus vulgarisL. genotypes on the first and second correspondence scores based on storage protein.

  17. Subcellular localization and vacuolar targeting of sorbitol dehydrogenase in apple seed.

    Science.gov (United States)

    Wang, Xiu-Ling; Hu, Zi-Ying; You, Chun-Xiang; Kong, Xiu-Zhen; Shi, Xiao-Pu

    2013-09-01

    Sorbitol is the primary photosynthate and translocated carbohydrate in fruit trees of the Rosaceae family. NAD(+)-dependent sorbitol dehydrogenase (NAD-SDH, EC 1.1.1.14), which mainly catalyzes the oxidation of sorbitol to fructose, plays a key role in regulating sink strength in apple. In this study, we found that apple NAD-SDH was ubiquitously distributed in epidermis, parenchyma, and vascular bundle in developing cotyledon. NAD-SDH was localized in the cytosol, the membranes of endoplasmic reticulum and vesicles, and the vacuolar lumen in the cotyledon at the middle stage of seed development. In contrast, NAD-SDH was mainly distributed in the protein storage vacuoles in cotyledon at the late stage of seed development. Sequence analysis revealed there is a putative signal peptide (SP), also being predicated to be a transmembrane domain, in the middle of proteins of apple NAD-SDH isoforms. To investigate whether the putative internal SP functions in the vacuolar targeting of NAD-SDH, we analyzed the localization of the SP-deletion mutants of MdSDH5 and MdSDH6 (two NAD-SDH isoforms in apple) by the transient expression system in Arabidopsis protoplasts. MdSDH5 and MdSDH6 were not localized in the vacuoles after their SPs were deleted, suggesting the internal SP functions in the vacuolar targeting of apple NAD-SDH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  18. Energy storage

    International Nuclear Information System (INIS)

    Anon.

    1992-01-01

    This chapter discusses the role that energy storage may have on the energy future of the US. The topics discussed in the chapter include historical aspects of energy storage, thermal energy storage including sensible heat storage, latent heat storage, thermochemical heat storage, and seasonal heat storage, electricity storage including batteries, pumped hydroelectric storage, compressed air energy storage, and superconducting magnetic energy storage, and production and combustion of hydrogen as an energy storage option

  19. Effects of long-term storage on the quality of soybean, Glycine max ...

    African Journals Online (AJOL)

    Soybean, Glycine max (L.) Merrill, is one of the five most important legumes in the tropics and provides the protein eaten by most people in the region. One of the major constraints to soybean production is that the seed quality deteriorates rapidly during storage. This study was undertaken to assess the effect of some storage ...

  20. Gold nanoparticles administration induces disarray of heart muscle, hemorrhagic, chronic inflammatory cells infiltrated by small lymphocytes, cytoplasmic vacuolization and congested and dilated blood vessels

    Directory of Open Access Journals (Sweden)

    Abdelhalim Mohamed Anwar K

    2011-12-01

    GNPs. These alterations may indicate scattered cytoplasmic vacuolization, which may induce the toxicity effect through an inability to deal with the accumulated residues resulting from metabolic and structural disturbances caused by these NPs. These histological alterations were more prominent with 10 nm size particles than with the larger ones. The interaction of GNPs with proteins and various cell types should be considered as part of the toxicological evaluation. Additional experiments related to plasma, tissues cytokine, antioxidant defense mechanism, lipid peroxidation, histomorphologcal and ultrastructure will be performed to identify and understand the toxicity and the potential use of GNPs as therapeutic and diagnostic tools.

  1. Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

    Science.gov (United States)

    Werner, Antonia; Herzog, Britta; Frey, Stefan; Pöggeler, Stefanie

    2016-01-01

    In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL) conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Sm)atg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.

  2. Autophagy-Associated Protein SmATG12 Is Required for Fruiting-Body Formation in the Filamentous Ascomycete Sordaria macrospora.

    Directory of Open Access Journals (Sweden)

    Antonia Werner

    Full Text Available In filamentous fungi, autophagy functions as a catabolic mechanism to overcome starvation and to control diverse developmental processes under normal nutritional conditions. Autophagy involves the formation of double-membrane vesicles, termed autophagosomes that engulf cellular components and bring about their degradation via fusion with vacuoles. Two ubiquitin-like (UBL conjugation systems are essential for the expansion of the autophagosomal membrane: the UBL protein ATG8 is conjugated to the lipid phosphatidylethanolamine and the UBL protein ATG12 is coupled to ATG5. We recently showed that in the homothallic ascomycete Sordaria macrospora autophagy-related genes encoding components of the conjugation systems are required for fruiting-body development and/or are essential for viability. In the present work, we cloned and characterized the S. macrospora (Smatg12 gene. Two-hybrid analysis revealed that SmATG12 can interact with SmATG7 and SmATG3. To examine its role in S. macrospora, we replaced the open reading frame of Smatg12 with a hygromycin resistance cassette and generated a homokaryotic ΔSmatg12 knockout strain, which displayed slower vegetative growth under nutrient starvation conditions and was unable to form fruiting bodies. In the hyphae of S. macrospora EGFP-labeled SmATG12 was detected in the cytoplasm and as punctate structures presumed to be phagophores or phagophore assembly sites. Delivery of EGFP-labelled SmATG8 to the vacuole was entirely dependent on SmATG12.

  3. Fat storage-inducing transmembrane (FIT or FITM proteins are related to lipid phosphatase/phosphotransferase enzymes

    Directory of Open Access Journals (Sweden)

    Matthew J Hayes

    2017-12-01

    Full Text Available Fat storage-inducing transmembrane (FIT or FITM proteins have been implicated in the partitioning of triacylglycerol to lipid droplets and the budding of lipid droplets from the ER. At the molecular level, the sole relevant interaction is that FITMs directly bind to triacyglycerol and diacylglycerol, but how they function at the molecular level is not known. Saccharomyces cerevisiae has two FITM homologues: Scs3p and Yft2p. Scs3p was initially identified because deletion leads to inositol auxotrophy, with an unusual sensitivity to addition of choline. This strongly suggests a role for Scs3p in phospholipid biosynthesis. Looking at the FITM family as widely as possible, we found that FITMs are widespread throughout eukaryotes, indicating presence in the last eukaryotic common ancestor. Protein alignments also showed that FITM sequences contain the active site of lipid phosphatase/phosphotransferase (LPT enzymes. This large family transfers phosphate-containing headgroups either between lipids or in exchange for water. We confirmed the prediction that FITMs are related to LPTs by showing that single amino-acid substitutions in the presumptive catalytic site prevented their ability to rescue growth of the mutants on low inositol/high choline media when over-expressed. The substitutions also prevented rescue of other phenotypes associated with loss of FITM in yeast, including mistargeting of Opi1p, defective ER morphology, and aberrant lipid droplet budding. These results suggest that Scs3p, Yft2p and FITMs in general are LPT enzymes involved in an as yet unknown critical step in phospholipid metabolism.

  4. Inhibition of primary human T cell proliferation by Helicobacter pylori vacuolating toxin (VacA) is independent of VacA effects on IL-2 secretion

    OpenAIRE

    Sundrud, Mark S.; Torres, Victor J.; Unutmaz, Derya; Cover, Timothy L.

    2004-01-01

    Recent evidence indicates that the secreted Helicobacter pylori vacuolating toxin (VacA) inhibits the activation of T cells. VacA blocks IL-2 secretion in transformed T cell lines by suppressing the activation of nuclear factor of activated T cells (NFAT). In this study, we investigated the effects of VacA on primary human CD4+ T cells. VacA inhibited the proliferation of primary human T cells activated through the T cell receptor (TCR) and CD28. VacA-treated Jurkat T cells secreted markedly ...

  5. Improved biochemical preservation of lung slices during cold storage.

    Science.gov (United States)

    Bull, D A; Connors, R C; Reid, B B; Albanil, A; Stringham, J C; Karwande, S V

    2000-05-15

    Development of lung preservation solutions typically requires whole-organ models which are animal and labor intensive. These models rely on physiologic rather than biochemical endpoints, making accurate comparison of the relative efficacy of individual solution components difficult. We hypothesized that lung slices could be used to assess preservation of biochemical function during cold storage. Whole rat lungs were precision cut into slices with a thickness of 500 microm and preserved at 4 degrees C in the following solutions: University of Wisconsin (UW), Euro-Collins (EC), low-potassium-dextran (LPD), Kyoto (K), normal saline (NS), or a novel lung preservation solution (NPS) developed using this model. Lung biochemical function was assessed by ATP content (etamol ATP/mg wet wt) and capacity for protein synthesis (cpm/mg protein) immediately following slicing (0 h) and at 6, 12, 18, and 24 h of cold storage. Six slices were assayed at each time point for each solution. The data were analyzed using analysis of variance and are presented as means +/- SD. ATP content was significantly higher in the lung slices stored in NPS compared with all other solutions at each time point (P cold storage. Copyright 2000 Academic Press.

  6. Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

    Science.gov (United States)

    Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

    2014-09-01

    Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

  7. Lysosomal storage diseases: current diagnostic and therapeutic options

    International Nuclear Information System (INIS)

    Malinova, V.; Honzik, T.

    2013-01-01

    Lysosomal storage diseases are rare genetic diseases caused by insufficient activity of some of the lysosomal enzymes and/or transport proteins. Initial symptoms may appear any time from the neonatal period to late adulthood; early forms tend to have a severe course with rapid progression and unfavorable prognosis. There is multisystem involvement with continuous progression of symptoms and involvement of metabolically active organs or tissues – the bone marrow, liver, bones, skeletal muscles, myocardium, or CNS. The diagnosis is definitively confirmed by demonstration of reduced activity of the particular enzyme and by mutation analysis. Some of the storage diseases can be effectively treated by intravenous administration of recombinant enzymes or by limiting the amount of the substrate stored. In a small number of lysosomal storage diseases, bone marrow transplantation is successful. Multidisciplinary collaboration, including genetic counselling and prenatal diagnosis in patient families, is required. The first part of the paper deals with general characteristics of lysosomal storage diseases and the most common diseases that are currently treatable in the Czech Republic (Gaucher’s disease, Pompe disease, Fabry disease, Niemann–Pick disease, cholesterol ester storage disease). The second part of the paper deals with mucopolysaccharidase, another group of rare lysosomal storage diseases. (author)

  8. The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

    Directory of Open Access Journals (Sweden)

    Kris E Spaeth

    2009-09-01

    Full Text Available In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone. Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion". The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

  9. Proteomic Analysis of Lettuce Seed Germination and Thermoinhibition by Sampling of Individual Seeds at Germination and Removal of Storage Proteins by Polyethylene Glycol Fractionation1

    Science.gov (United States)

    Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-01-01

    Germination and thermoinhibition in lettuce (Lactuca sativa ‘Jianyexianfeng No. 1’) seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (P lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. PMID:25736209

  10. Yeast Interacting Proteins Database: YPL151C, YOR036W [Yeast Interacting Proteins Database

    Lifescience Database Archive (English)

    Full Text Available t-SNARE) for vesicular intermediates traveling between the Golgi apparatus and the vacuole; controls entry o...e PEP12 Prey description Target membrane receptor (t-SNARE) for vesicular intermediates traveling between th

  11. Viability and Biochemical Content Changes in Seed Storage of Jabon Putih (Anthocephalus Cadamba (Roxb Miq.

    Directory of Open Access Journals (Sweden)

    Naning Yuniarti

    2015-08-01

    Full Text Available Seed deterioration is the process of deteriorated seed in view of viability that changed the physiological and chemical content. The aim of this research is to find out the effect of moisture content and storage on viability and biochemical content of jabon putih seed. Completely Randomized Design was used for decreasing moisture content based on seed drying time for 0, 24, 48, 72, 96, 120 hours, and Completely Randomized Factorial Design was used for the combination of moisture content (drying time for 0, 24, 48, 72, 96, 120 hours and room storage (ambient room, air-conditioned room, refrigerator. The results of this research are: (1 seed drying time and seed storage affected the change of seed viability and biochemical content, (2 seed drying time and seed storage influence significantly the value of moisture content, germination percentage, and biochemical content (lipid, carbohydrate, protein, (3 The longer time of seed drying and seed storage has decreased the seed moisture content, germination percentage, and the carbohydrate content, but it has increased the content of lipid and protein, and (4 the seeds were stored in refrigerator has better viability compared to dry cold storage and ambient room.

  12. Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

    Science.gov (United States)

    Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

    2012-09-01

    Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Clustering of Helicobacter pylori VacA in lipid rafts, mediated by its receptor, receptor-like protein tyrosine phosphatase beta, is required for intoxication in AZ-521 Cells

    DEFF Research Database (Denmark)

    Nakayama, Masaaki; Hisatsune, Jyunzo; Yamasaki, Eiki

    2006-01-01

    Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation of proinflamm......Helicobacter pylori vacuolating cytotoxin, VacA, induces multiple effects on epithelial cells through different cellular events: one involves pore formation, leading to vacuolation, mitochondrial damage, and apoptosis, and the second involves cell signaling, resulting in stimulation...

  14. Antioxidant activity and protein-polyphenol interactions in a pomegranate (Punica granatum L.) yogurt.

    Science.gov (United States)

    Trigueros, Lorena; Wojdyło, Aneta; Sendra, Esther

    2014-07-09

    Pomegranate juice (PGJ) is rich in phenolics which are potent antioxidants but also prone to interact with proteins. A yogurt rich in PGJ (40%) made from arils was elaborated (PGY) to determine the antioxidant activity and to estimate the phenolics-proteins interaction during 28 days of cold storage. Juice, yogurts, and protein-free permeates were analyzed for phenolic composition. Yogurt fermentation modified the anthocyanin profile of the initial PGJ, especially the content in cyanidin-3-O-glucoside. During storage, individual anthocyanin content in PGY decreased but it did not modify yogurt color. The analysis of permeates revealed that the degree of phenol-protein interaction depends on the type of phenolic, ellagic acid and dephinidin-3,5-O-diglucoside being the least bound phenolic compounds. The presence of PGJ in yogurt enhanced radical scavenging performance, whereas all the observed ferric reducing power ability of PGY was strictly due to the PGJ present. The 84.73% of total anthocyanins remained bound to proteins at the first day of storage and 90.06% after 28 days of cold storage, revealing the high affinity of anthocyanins for milk proteins.

  15. The macronutrients in human milk change after storage in various containers.

    Science.gov (United States)

    Chang, Yu-Chuan; Chen, Chao-Huei; Lin, Ming-Chih

    2012-06-01

    The concentrations of macronutrients in human milk can be influenced by various processes, such as storage, freezing, and thawing, that are performed by lactating working mothers and breast milk banks. We evaluated the impact of various containers on the nutrient concentrations in human milk. A total of 42 breast milk samples from 18 healthy lactating mothers were collected. A baseline macronutrient concentration was determined for each sample. Then, the breast milk samples were divided and stored in nine different commercial milk containers. After freezing at -20°C for 2 days, the milk samples were thawed and analyzed again. A midinfrared human milk analyzer (HMA) was used to measure the protein, fat, and carbohydrate contents. There was a significant decrease in the fat content following the storage, freezing, and thawing processes, ranging from 0.27-0.30 g/dL (p=0.02), but no significant decrease in energy content (p=0.069) was noted in the nine different containers. There were statistically significant increases in protein and carbohydrate concentrations in all containers (p=0.021 and 0.001, respectively), however there were no significant differences between the containers in terms of fat, protein, carbohydrate, or energy contents. Human milk, when subjected to storage, freezing, and thawing processes, demonstrated a significant decrease in fat content (up to 9% reduction) in various containers. It is better for infants to receive milk directly from the mother via breastfeeding. More studies are warranted to evaluate the effects of milk storage on infant growth and development. Copyright © 2012. Published by Elsevier B.V.

  16. Towards muscle-specific meat color stability of Chinese Luxi yellow cattle: A proteomic insight into post-mortem storage.

    Science.gov (United States)

    Wu, Wei; Yu, Qian-Qian; Fu, Yu; Tian, Xiao-Jing; Jia, Fei; Li, Xing-Min; Dai, Rui-Tong

    2016-09-16

    Searching for potential predictors of meat color is a challenging task for the meat industry. In this study, the relationship between meat color parameters and the sarcoplasmic proteome of M. longissimuss lumborum (LL) and M. psoas major (PM) from Chinese Luxi yellow cattle during post-mortem storage (0, 5, 10 and 15days) were explored with the aid of the integrated proteomics and bioinformatics approaches. Meat color attributes revealed that LL displayed better color stability than PM during storage. Furthermore, sarcoplasmic proteins of these two muscles were compared between days 5, 10, 15 and day 0. Several proteins were closely correlated with meat color attributes and they were muscle-specific and responsible for the meat color stability at different storage periods. Glycerol-3-phosphate dehydrogenase, fructose-bisphosphate aldolase A isoform, glycogen phosphorylase, peroxiredoxin-2, phosphoglucomutase-1, superoxide dismutase [Cu-Zn], heat shock cognate protein (71kDa) might serve as the candidate predictors of meat color stability during post-mortem storage. In addition, bioinformatics analyses indicated that more proteins were involved in glycolytic metabolism of LL, which contributed to better meat color stability of LL than PM. The present results could provide a proteomic insight into muscle-specific meat color stability of Chinese Luxi yellow cattle during post-mortem storage. Copyright © 2015 Elsevier B.V. All rights reserved.

  17. A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

    Science.gov (United States)

    DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

    2014-07-15

    Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. © 2014 DeMille et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  18. Effect of storage temperature on survival and recovery of thermal and extrusion injured Escherichia coli K-12 in whey protein concentrate and corn meal.

    Science.gov (United States)

    Ukuku, Dike O; Mukhopadhyay, Sudarsan; Onwulata, Charles

    2013-01-01

    Previously, we reported inactivation of Escherichia coli populations in corn product (CP) and whey protein product (WPP) extruded at different temperatures. However, information on the effect of storage temperatures on injured bacterial populations was not addressed. In this study, the effect of storage temperatures on the survival and recovery of thermal death time (TDT) disks and extrusion injured E. coli populations in CP and WPP was investigated. CP and WPP inoculated with E. coli bacteria at 7.8 log(10) CFU/g were conveyed separately into the extruder with a series 6300 digital type T-35 twin screw volumetric feeder set at a speed of 600 rpm and extruded at 35°C, 55°C, 75°C, and 95°C, or thermally treated with TDT disks submerged into water bath set at 35°C, 55°C, 75°C, and 95°C for 120 s. Populations of surviving bacteria including injured cells in all treated samples were determined immediately and every day for 5 days, and up to 10 days for untreated samples during storage at 5°C, 10°C, and 23°C. TDT disks treatment at 35°C and 55°C did not cause significant changes in the population of the surviving bacteria including injured populations. Extrusion treatment at 35°C and 55°C led to significant (pagar plates. The results of this study showed that further inactivation of the injured populations occurred during storage at 5°C for 5 days suggesting the need for immediate storage of 75°C extruded CP and WPP at 5°C for at least 24 h to enhance their microbial safety.

  19. Proteomic analysis of lettuce seed germination and thermoinhibition by sampling of individual seeds at germination and removal of storage proteins by polyethylene glycol fractionation.

    Science.gov (United States)

    Wang, Wei-Qing; Song, Bin-Yan; Deng, Zhi-Jun; Wang, Yue; Liu, Shu-Jun; Møller, Ian Max; Song, Song-Quan

    2015-04-01

    Germination and thermoinhibition in lettuce (Lactuca sativa 'Jianyexianfeng No. 1') seeds were investigated by a proteomic comparison among dry seeds, germinated seeds at 15°C, at 15°C after imbibition at 25°C for 48 h, or at 25°C in KNO3 (all sampled individually at germination), and ungerminated seeds at 25°C, a thermoinhibitory temperature. Before two-dimensional gel electrophoresis analysis, storage proteins (greater than 50% of total extractable protein) were removed by polyethylene glycol precipitation, which significantly improved the detection of less abundant proteins on two-dimensional gels. A total of 108 protein spots were identified to change more than 2-fold (Pseeds than in ungerminated 25°C seeds. Gene expression of 12 of those proteins correlated well with the protein accumulation. Methionine metabolism, ethylene production, lipid mobilization, cell elongation, and detoxification of aldehydes were revealed to be potentially related to lettuce seed germination and thermoinhibition. Accumulation of three proteins and expression of five genes participating in the mevalonate (MVA) pathway of isoprenoid biosynthesis correlated positively with seed germinability. Inhibition of this pathway by lovastatin delayed seed germination and increased the sensitivity of germination to abscisic acid. MVA pathway-derived products, cytokinins, partially reversed the lovastatin inhibition of germination and released seed thermoinhibition at 25°C. We conclude that the MVA pathway for isoprenoid biosynthesis is involved in lettuce seed germination and thermoinhibition. © 2015 American Society of Plant Biologists. All Rights Reserved.

  20. Neuronal vacuolation of the trigeminal nuclei in goats caused by ingestion of Prosopis juliflora pods (mesquite beans).

    Science.gov (United States)

    Tabosa, I M; Souza, J C; Graça, D L; Barbosa-Filho, J M; Almeida, R N; Riet-Correa, F

    2000-06-01

    Three groups of 6 goats each were fed a ration containing 30, 60, or 90%, on a dry matter base, of Prosopis juliflora pods. A control group of 4 goats ingested only the basic ration. Two hundred and ten days after the start of the experiment 3 goats that ingested 60% pods in and 4 that ingested 90% had mandibular tremors, mainly during chewing. All animals were killed after 270 d of ingestion. No gross lesions were observed. Histologic lesions were characterized by fine vacuolation of the pericaryon of neurons from the trigeminal nuclei. Occasionally neurons of the oculomotor nuclei were also affected. Wallerian degeneration was occasionally observed in the mandibular and trigeminal nerves. Denervation atrophy of the masseter, temporal, hypoglossus, genioglossus, styloglossus, medial pterygoid and lateral pterygoid muscles was seen. The clinical signs from feeding the P juliflora pods were caused by a selective toxicity to neurons of some cranial nerve nuclei.

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

    Science.gov (United States)

    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.

  2. Vitellogenin RNAi halts ovarian growth and diverts reproductive proteins and lipids in young grasshoppers.

    Science.gov (United States)

    Tokar, Derek R; Veleta, Katherine A; Canzano, Joseph; Hahn, Daniel A; Hatle, John D

    2014-11-01

    Reduced reproduction extends lifespan of females in many animals. To test the effects of reproduction on storage of macronutrients, we block reproductive output in the lubber grasshopper by injecting RNAi against the precursor to egg-yolk protein, vitellogenin, in early adulthood. Controls were injected with either buffer or RNAi against the major storage protein in the hemolymph, hexamerin-90. Vitellogenin RNAi greatly reduced both levels of mRNA for vitellogenin and ovarian growth, in comparison to both controls. Fat body mass was increased upon vitellogenin RNAi, but concentrations of the three hexameric storage proteins from the hemolymph were not. Surprisingly, hemolymph vitellogenin levels were increased upon vitellogenin RNAi. Total reproductive protein (hemolymph vitellogenin plus ovarian vitellin) was unchanged by vitellogenin RNAi, as reproductive protein was diverted to the hemolymph. Similarly, the increased lipid storage upon vitellogenin RNAi was largely attributable to the reduction in lipid in the ovary, due to decreased ovarian growth. A BLAST search revealed that the 515 bp sequence of vitellogenin used for RNAi had three 11 bp regions identical to the vitellogenin receptor of the cockroach Leucophaea maderae. This suggests that our treatment, in addition to reducing levels of vitellogenin transcript, may have also blocked transport of vitellogenin from the hemolymph to the ovary. This would be consistent with halted ovarian growth simultaneous with high levels of vitellogenin in the hemolymph. Nonetheless, the accumulation of vitellogenin, instead of hexameric storage proteins, is inconsistent with a simple model of the trade-off between reproduction and storage. This was observed in young females; future studies will address whether investment of proteins may shift to the soma as individuals age. Overall, our results suggest that blockage of reproduction in young grasshoppers redirects lipids to storage and reproductive proteins to the hemolymph

  3. Energy Storage.

    Science.gov (United States)

    Eaton, William W.

    Described are technological considerations affecting storage of energy, particularly electrical energy. The background and present status of energy storage by batteries, water storage, compressed air storage, flywheels, magnetic storage, hydrogen storage, and thermal storage are discussed followed by a review of development trends. Included are…

  4. Helicobacter pylori VacA, acting through receptor protein tyrosine phosphatase ?, is crucial for CagA phosphorylation in human duodenum carcinoma cell line AZ-521

    OpenAIRE

    Nakano, Masayuki; Yahiro, Kinnosuke; Yamasaki, Eiki; Kurazono, Hisao; Akada, Junko; Yamaoka, Yoshio; Niidome, Takuro; Hatakeyama, Masanori; Suzuki, Hidekazu; Yamamoto, Taro; Moss, Joel; Isomoto, Hajime; Hirayama, Toshiya

    2016-01-01

    ABSTRACT Helicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)?, a VacA receptor, reduced VacA-induced Src ph...

  5. ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation

    CSIR Research Space (South Africa)

    Mtwisha, L

    2007-02-01

    Full Text Available ) and the Typha pollen D7 protein was found to stabilise sugar glasses in an in vitro system (Wolkers et al. 2001). The cupin family of proteins comprises a wide variety of proteins from both prokaryotes and eukaryotes and includes the seed storage proteins...–268. Garay-Arroyo A, Colmenero-Flores JM, Garciarrubio A, Covarrubias AA (2000) Highly hydrophilic proteins in prokaryotes and eukaryotes are common during conditions of water deficit. Journal of Biological Chemistry 275, 5668–5674. doi: 10.1074/jbc.275...

  6. Underground Storage Tanks - Storage Tank Locations

    Data.gov (United States)

    NSGIC Education | GIS Inventory — A Storage Tank Location is a DEP primary facility type, and its sole sub-facility is the storage tank itself. Storage tanks are aboveground or underground, and are...

  7. Serratia marcescens Is Able to Survive and Proliferate in Autophagic-Like Vacuoles inside Non-Phagocytic Cells

    Science.gov (United States)

    Colombo, María Isabel; García Véscovi, Eleonora

    2011-01-01

    Serratia marcescens is an opportunistic human pathogen that represents a growing problem for public health, particularly in hospitalized or immunocompromised patients. However, little is known about factors and mechanisms that contribute to S. marcescens pathogenesis within its host. In this work, we explore the invasion process of this opportunistic pathogen to epithelial cells. We demonstrate that once internalized, Serratia is able not only to persist but also to multiply inside a large membrane-bound compartment. This structure displays autophagic-like features, acquiring LC3 and Rab7, markers described to be recruited throughout the progression of antibacterial autophagy. The majority of the autophagic-like vacuoles in which Serratia resides and proliferates are non-acidic and have no degradative properties, indicating that the bacteria are capable to either delay or prevent fusion with lysosomal compartments, altering the expected progression of autophagosome maturation. In addition, our results demonstrate that Serratia triggers a non-canonical autophagic process before internalization. These findings reveal that S. marcescens is able to manipulate the autophagic traffic, generating a suitable niche for survival and proliferation inside the host cell. PMID:21901159

  8. Photoinhibition influences protein utilisation during seed ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... Seed storage proteins are mobilised during germination, especially at ... of this study was to examine changes in protein expression during ... MATERIALS AND METHODS ... System (Pharmacia) were 500V for 4.5 h in Phase I and II and 2000 .... characterisation of photoinhibition and recovery during cold.

  9. Protein mobilities and P-selectin storage in Weibel–Palade bodies

    OpenAIRE

    Kiskin, Nikolai I.; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J.; Carter, Tom

    2010-01-01

    Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P...

  10. Yeast Ivy1p Is a Putative I-BAR-domain Protein with pH-sensitive Filament Forming Ability in vitro.

    Science.gov (United States)

    Itoh, Yuzuru; Kida, Kazuki; Hanawa-Suetsugu, Kyoko; Suetsugu, Shiro

    2016-01-01

    Bin-Amphiphysin-Rvs161/167 (BAR) domains mold lipid bilayer membranes into tubules, by forming a spiral polymer on the membrane. Most BAR domains are thought to be involved in forming membrane invaginations through their concave membrane binding surfaces, whereas some members have convex membrane binding surfaces, and thereby mold membranes into protrusions. The BAR domains with a convex surface form a subtype called the inverse BAR (I-BAR) domain or IRSp53-MIM-homology domain (IMD). Although the mammalian I-BAR domains have been studied, those from other organisms remain elusive. Here, we found putative I-BAR domains in Fungi and animal-like unicellular organisms. The fungal protein containing the putative I-BAR-domain is known as Ivy1p in yeast, and is reportedly localized in the vacuole. The phylogenetic analysis of the I-BAR domains revealed that the fungal I-BAR-domain containing proteins comprise a distinct group from those containing IRSp53 or MIM. Importantly, Ivy1p formed a polymer with a diameter of approximately 20 nm in vitro, without a lipid membrane. The filaments were formed at neutral pH, but disassembled when pH was reverted to basic. Moreover, Ivy1p and the I-BAR domain expressed in mammalian HeLa cells was localized at a vacuole-like structure as filaments as revealed by super-resolved microscopy. These data indicate the pH-sensitive polymer forming ability and the functional conservation of Ivy1p in eukaryotic cells.

  11. Light assisted drying (LAD) for protein stabilization: optical characterization of samples

    Science.gov (United States)

    Young, Madison A.; McKinnon, Madison E.; Elliott, Gloria D.; Trammell, Susan R.

    2018-02-01

    Light-Assisted Drying (LAD) is a novel biopreservation technique which allows proteins to be immobilized in a dry, amorphous solid at room temperature. Indicator proteins are used in a variety of diagnostic assays ranging from highthroughput 96-well plates to new microfluidic devices. A challenge in the development of protein-based assays is preserving the structure of the protein during production and storage of the assay, as the structure of the protein is responsible for its functional activity. Freeze-drying or freezing are currently the standard for the preservation of proteins, but these methods are expensive and can be challenging in some environments due to a lack of available infrastructure. An inexpensive, simple processing method that enables supra-zero temperature storage of proteins used in assays is needed. Light-assisted drying offers a relatively inexpensive method for drying samples. Proteins suspended in a trehalose solution are dehydrated using near-infrared laser light. The laser radiation speeds drying and as water is removed the sugar forms a protective matrix. The goal of this study is optically characterize samples processed with LAD. We use polarized light imaging (PLI) to look at crystallization kinetics of samples and determine optimal humidity. PLI shows a 62.5% chance of crystallization during LAD processing and negligible crystallization during low RH storage.

  12. Soybean protein-based microparticles for oral delivery of probiotics with improved stability during storage and gut resistance.

    Science.gov (United States)

    González-Ferrero, C; Irache, J M; González-Navarro, C J

    2018-01-15

    The present work describes the encapsulation of probiotics using a by-product as wall material and a process feasible to be scaled-up: coacervation of soybean protein concentrate (SPC) by using calcium salts and spray-drying. SPC was extracted from soybean flour, produced during the processing of soybean milk, by alkaline extraction following isoelectric precipitation. Two probiotic strains were selected for encapsulation (Lactobacillus plantarum CECT 220 and Lactobacillus casei CECT 475) in order to evaluate the ability of SPC to encapsulate and protect bacteria from stress conditions. The viability of these encapsulated strains under in vitro gastrointestinal conditions and shelf-life during storage were compared with the most common forms commercialized nowadays. Results show that SPC is a feasible material for the development of probiotic microparticles with adequate physicochemical properties and enhanced significantly both probiotic viability and tolerance against simulated gastrointestinal fluids when compared to current available commercial forms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Immunohistochemistry of LAMP-2 and adipophilin for phospholipidosis in liver and kidney in ketoconazole-treated mice.

    Science.gov (United States)

    Asaoka, Yoshiji; Togashi, Yuko; Imura, Naoko; Sai, Takafumi; Miyoshi, Tomoya; Miyamoto, Yohei

    2013-09-01

    Drug-induced phospholipidosis is an abnormal accumulation of phospholipids in the lysosomes following repeated administration of cationic amphiphilic drugs. Phospholipidosis is detected histopathologically as cytoplasmic vacuolation; however, it is difficult to distinguish from lipid accumulation since their morphological features are similar. In this study, we investigated the usefulness of immunohistochemistry for lysosome-associated membrane protein-2 (LAMP-2) and adipophilin, a membrane protein of cytosolic non-lysosomal lipid droplets, in the liver and kidneys of mice orally administered ketoconazole, an inducer of hepatic phospholipidosis. In 7-week-old mice administered ketoconazole (300 mg/kg/day) for 7 days, cytoplasmic vacuolation was histopathologically observed in centrilobular hepatocytes and proximal tubular epithelial cells under the fasted condition. The cytoplasmic vacuolation consisted of foamy vacuoles, which were revealed to be phospholipidosis-characteristic lamellar bodies by electron microscopy. Furthermore, lipid-like vacuoles were observed in the perilobular hepatocytes, and revealed to be lipid droplets by electron microscopy. In immunohistochemistry, the foamy vacuoles and lipid-like vacuoles were positive for LAMP-2 and adipophilin, respectively. These results indicate that immunohistochemistry for LAMP-2 and adipophilin could distinguish between phospholipidosis and lipid accumulation. Additionally, it could detect ketoconazole-induced phospholipidosis in the glycogen-rich livers of non-fasted mice. In conclusion, ketoconazole induced phospholipidosis in not only the liver but also the kidneys, and immunohistochemistry for LAMP-2 and adipophilin could be useful for the pathological evaluation of drug-induced phospholipidosis in mice. Crown Copyright © 2012. Published by Elsevier GmbH. All rights reserved.

  14. Cell biological characterization of the malaria vaccine candidate trophozoite exported protein 1.

    Directory of Open Access Journals (Sweden)

    Caroline Kulangara

    Full Text Available In a genome-wide screen for alpha-helical coiled coil motifs aiming at structurally defined vaccine candidates we identified PFF0165c. This protein is exported in the trophozoite stage and was named accordingly Trophozoite exported protein 1 (Tex1. In an extensive preclinical evaluation of its coiled coil peptides Tex1 was identified as promising novel malaria vaccine candidate providing the rational for a comprehensive cell biological characterization of Tex1. Antibodies generated against an intrinsically unstructured N-terminal region of Tex1 and against a coiled coil domain were used to investigate cytological localization, solubility and expression profile. Co-localization experiments revealed that Tex1 is exported across the parasitophorous vacuole membrane and located to Maurer's clefts. Change in location is accompanied by a change in solubility: from a soluble state within the parasite to a membrane-associated state after export to Maurer's clefts. No classical export motifs such as PEXEL, signal sequence/anchor or transmembrane domain was identified for Tex1.

  15. Pb-induced cellular defense system in the root meristematic cells of Allium sativum L.

    Science.gov (United States)

    Jiang, Wusheng; Liu, Donghua

    2010-03-02

    Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10(-5) to 10(-3) M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10(-3) to 10(-4) M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum. After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells. Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are

  16. Pb-induced cellular defense system in the root meristematic cells of Allium sativum L

    Directory of Open Access Journals (Sweden)

    Liu Donghua

    2010-03-01

    Full Text Available Abstract Background Electron microscopy (EM techniques enable identification of the main accumulations of lead (Pb in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10-5 to 10-3 M of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10-3 to 10-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum. Results After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells. Conclusions Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification

  17. Protein stability: a crystallographer’s perspective

    Science.gov (United States)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed. PMID:26841758

  18. Effects of gamma irradiation on microbial safety and quality of stir fry chicken dices with hot chili during storage

    International Nuclear Information System (INIS)

    Chen, Qian; Cao, Mei; Chen, Hao; Gao, Peng; Fu, Yi; Liu, Mianxue; Wang, Yan; Huang, Min

    2016-01-01

    The purpose of this study was to investigate effects of irradiation with different doses on microbial safety, sensory quality and protein content of ready-to-eat stir fry chicken dices with hot chili (FCC) during one year storage. Fresh chicken meat was cut into small dices and fried at approximately 180 °C for 10 min for preparation of FCC samples. The samples were vacuum-packaged and gamma irradiated at 10, 20, 30 and 40 kGy. The results suggest that irradiation with the doses of 10 and 20 kGy could ensure microbiological safety of the samples without deterioration of sensory quality. Microbial counts, sensory qualities and protein contents of the samples were investigated during one year storage. No viable cells were observed and the samples were completely sterilized. Sensory qualities showed no significant difference after irradiated at the doses of 10 and 20 kGy during the storage period. Protein contents were also not affected by irradiation at the same doses. Our results indicate that gamma irradiation of 10 and 20 kGy are effective to maintain shelf stability of ready-to-eat FCC products with microbial safety, sensory quality and nutritional value. - Highlights: • Microbial safety and sensory quality of the FCC are ensured by gamma irradiation. • No viable cells in the irradiated FCC are detected during one year storage. • Sensory quality of the irradiated FCC is not changed during one year storage. • Protein content of the FCC is not affected by irradiation during one year storage.

  19. Neutrino Signals in Electron-Capture Storage-Ring Experiments

    Directory of Open Access Journals (Sweden)

    Avraham Gal

    2016-06-01

    Full Text Available Neutrino signals in electron-capture decays of hydrogen-like parent ions P in storage-ring experiments at GSI are reconsidered, with special emphasis placed on the storage-ring quasi-circular motion of the daughter ions D in two-body decays P → D + ν e . It is argued that, to the extent that daughter ions are detected, these detection rates might exhibit modulations with periods of order seconds, similar to those reported in the GSI storage-ring experiments for two-body decay rates. New dedicated experiments in storage rings, or using traps, could explore these modulations.

  20. Gas storage materials, including hydrogen storage materials

    Science.gov (United States)

    Mohtadi, Rana F; Wicks, George G; Heung, Leung K; Nakamura, Kenji

    2013-02-19

    A material for the storage and release of gases comprises a plurality of hollow elements, each hollow element comprising a porous wall enclosing an interior cavity, the interior cavity including structures of a solid-state storage material. In particular examples, the storage material is a hydrogen storage material such as a solid state hydride. An improved method for forming such materials includes the solution diffusion of a storage material solution through a porous wall of a hollow element into an interior cavity.

  1. 19q13.12 microdeletion syndrome fibroblasts display abnormal storage of cholesterol and sphingolipids in the endo-lysosomal system.

    Science.gov (United States)

    Zhao, Kexin; van der Spoel, Aarnoud; Castiglioni, Claudia; Gale, Sarah; Fujiwara, Hideji; Ory, Daniel S; Ridgway, Neale D

    2018-06-01

    Microdeletions in 19q12q13.12 cause a rare and complex haploinsufficiency syndrome characterized by intellectual deficiency, developmental delays, and neurological movement disorders. Variability in the size and interval of the deletions makes it difficult to attribute the complex clinical phenotype of this syndrome to an underlying gene(s). As an alternate approach, we examined the biochemical and metabolic features of fibroblasts from an affected individual to derive clues as to the molecular basis for the syndrome. Immunofluorescence and electron microscopy of affected fibroblasts revealed an abnormal endo-lysosomal compartment that was characterized by rapid accumulation of lysosomotropic dyes, elevated LAMP1 and LAMP2 expression and vacuoles containing membrane whorls, common features of lysosomal lipid storage disorders. The late endosomes-lysosomes (LE/LY) of affected fibroblasts accumulated low-density lipoprotein cholesterol, and displayed reduced cholesterol esterification and increased de novo cholesterol synthesis, indicative of defective cholesterol transport to the endoplasmic reticulum. Affected fibroblasts also had increased ceramide and sphingolipid mass, altered glycosphingolipid species and accumulation of a fluorescent lactosylceramide probe in LE/LY. Autophagosomes also accumulated in affected fibroblasts because of decreased fusion with autolysosomes, a defect associated with other lysosomal storage diseases. Attempts to correct the cholesterol/sphingolipid storage defect in fibroblasts with cyclodextrin, sphingolipid synthesis inhibitors or by altering ion transport were unsuccessful. Our data show that 19q13.12 deletion fibroblasts have abnormal accumulation of cholesterol and sphingolipids in the endo-lysosomal system that compromises organelle function and could be an underlying cause of the clinical features of the syndrome. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Variation of nephrotoxicity biomarkers by urinary storage condition in rats.

    Science.gov (United States)

    Le, Jung-Min; Han, Young-Hwan; Choi, Su-Jeong; Park, Ju-Seong; Jang, Jeong-Jun; Bae, Re-Ji-Na; Lee, Mi Ju; Kim, Myoung Jun; Lee, Yong-Hoon; Kim, Duyeol; Lee, Hye-Young; Park, Sun-Hee; Park, Cheol-Beom; Kang, Jin Seok; Kang, Jong-Koo

    2014-12-01

    Recently, there has been an increase in the use of several nephrotoxicity biomarkers in preclinical experiments. In addition, it has been indicated that the result may have been influenced by secondary factors, such as sample storage condition or storage period. In this study, we have assessed the variation in urinary nephrotoxicity biomarkers as a result of urine storage conditions and storage period of the urine. Urine was sampled from specific pathogen-free Sprague-Dawley rats (19 weeks old), which were housed individually in hanged stainless steel wire mesh cages. Urine was stored at 20℃, at 4℃, or at -70℃ after sampling. The levels of the biomarkers such as beta-2 microglobulin (B2M), cystatin-C (Cys-C), N-acetyl-β- D-glucosaminidase (NAG), micro albumin (MA), micro protein (MP) were measured at 6, 24, 48 and 144 hr after sampling. The B2M level was significantly decreased at 6, 24, 48, and 144 hr compared to 0 hr at -70℃ (p storage conditions. Taken together, B2M and Cys-C levels were modulated by storage temperature and period. For the enhancement of test accuracy, it is suggested that strict protocols be established for samples to minimize the effects of the storage conditions on the detected levels of biomarkers.

  3. Fat-specific protein 27 regulates storage of triacylglycerol

    DEFF Research Database (Denmark)

    Keller, P.; Petrie, J.T.; Rose, P. De

    2008-01-01

    FSP27 (fat-specific protein 27) is a member of the cell death-inducing DNA fragmentation factor-alpha-like effector (CIDE) family. Although Cidea and Cideb were initially characterized as activators of apoptosis, recent studies have demonstrated important metabolic roles for these proteins...... in several cell types without induction of adipocyte genes. Increased triacylglycerol is likely due to decreased beta-oxidation of nonesterified fatty acids. Altered flux of fatty acids into triacylglycerol may be a direct effect of FSP27 function, which is localized to lipid droplets in 293T cells and 3T3-L...... decreases with total fat mass but is not associated with measures of insulin resistance (e.g. homeostasis model assessment). Together, these data indicate that FSP27 binds to lipid droplets and regulates their enlargement Udgivelsesdato: 2008/5/23...

  4. Storage changes in the quality of sound and sprouted flour.

    Science.gov (United States)

    Sur, R; Nagi, H P; Sharma, S; Sekhon, K S

    1993-07-01

    Sound and sprouted flours (24 and 48 hr) from bread wheat (WL-1562), durum wheat (PBW-34) and triticale (TL-1210) were stored at room temperature (34.8 degrees C) and relative humidity (66.7%) for 0, 45, 90 and 135 days to assess the changes in physico-chemical and baking properties. Protein, gluten, sedimentation value, starch and crude fat decreased during storage in all the samples; however, the decrease was more in sprouted flours. Free amino acids, proteolytic activity, diastatic activity and damaged starch decreased with increase in storage period. Total sugars and free fatty acids increased more rapidly in the flours of sprouted wheats during 135 days of storage. Loaf volume of breads decreased during storage in both sound and sprouted flour but the mean percent decrease in loaf volume was more in stored sound flours. Aging of sprouted flour for 45 days improved the cookie and cake making properties but further storage was of no value for these baked products. Chapati making properties of stored sound and sprouted flour were inferior to that of fresh counterparts.

  5. Assessment of genetic diversity among some accessions of sage (Salvia officinalis L. using electrophoresis of seed storage proteins

    Directory of Open Access Journals (Sweden)

    Seyed Abbas Mirjalili

    2016-06-01

    Full Text Available The genus Salvia (Lamiaceae comprises over 900 species in the world, with a relatively wide dispersion in the Iran’s flora. Until now, about 58 species of the genus have been reported and identified in Iran, in which 17 of them were endemic. In order to study, investigate and evaluate the intraspecific diversity, similarity and dissimilarity among Iranian Salvia officinalis accessions, an experiment was carried out using SDS-PAGE technique. In this study, the seeds from five accessions were collected from gene bank and were evaluated. The seed storage proteins were extracted by buffers and were measured. Phylogenetic relationships were analyzed according to presence and absence of bands on the gel. A dendrogram was prepared using calculation of the accession’s similarity index. Mean comparison were done by Tukey’s test. The seed protein contents showed significant differences (p≤0.01 among accessions. A total of 39 bands were indicated on the gel. The maximum diversity was detected in the accession No. 2 while, the lowest band’s number were recorded with the accessions No. 1 and 5. Based on dendrogram, the accessions were divided into two groups; one includes accession No. 1 and 4 other accessions were located in the second group further classified into two subgroup including accessions No. 2 and 3 in one clade and accessions No. 4 and 5 in the other ones.

  6. PC-Cluster based Storage System Architecture for Cloud Storage

    OpenAIRE

    Yee, Tin Tin; Naing, Thinn Thu

    2011-01-01

    Design and architecture of cloud storage system plays a vital role in cloud computing infrastructure in order to improve the storage capacity as well as cost effectiveness. Usually cloud storage system provides users to efficient storage space with elasticity feature. One of the challenges of cloud storage system is difficult to balance the providing huge elastic capacity of storage and investment of expensive cost for it. In order to solve this issue in the cloud storage infrastructure, low ...

  7. Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

    Science.gov (United States)

    Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

    2005-01-01

    The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

  8. Structural markers of the evolution of whey protein isolate powder during aging and effects on foaming properties.

    Science.gov (United States)

    Norwood, E-A; Le Floch-Fouéré, C; Briard-Bion, V; Schuck, P; Croguennec, T; Jeantet, R

    2016-07-01

    The market for dairy powders, including high added-value products (e.g., infant formulas, protein isolates) has increased continuously over the past decade. However, the processing and storage of whey protein isolate (WPI) powders can result in changes in their structural and functional properties. It is therefore of great importance to understand the mechanisms and to identify the structural markers involved in the aging of WPI powders to control their end use properties. This study was performed to determine the effects of different storage conditions on protein lactosylations, protein denaturation in WPI, and in parallel on their foaming and interfacial properties. Six storage conditions involving different temperatures (θ) and water activities (aw) were studied for periods of up to 12mo. The results showed that for θ≤20°C, foaming properties of powders did not significantly differ from nonaged whey protein isolates (reference), regardless of the aw. On the other hand, powders presented significant levels of denaturation/aggregation and protein modification involving first protein lactosylation and then degradation of Maillard reaction products, resulting in a higher browning index compared with the reference, starting from the early stage of storage at 60°C. These changes resulted in a higher foam density and a slightly better foam stability (whisking) at 6mo. At 40°C, powders showed transitional evolution. The findings of this study will make it possible to define maximum storage durations and to recommend optimal storage conditions in accordance with WPI powder end-use properties. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  9. Regulation of transport processes across the tonoplast

    Science.gov (United States)

    Neuhaus, H. Ekkehard; Trentmann, Oliver

    2014-01-01

    In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g., due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation. PMID:25309559

  10. Regulation of transport processes across the tonoplast membrane

    Directory of Open Access Journals (Sweden)

    Oliver eTrentmann

    2014-09-01

    Full Text Available In plants, the vacuole builds up the cellular turgor and represents an important component in cellular responses to diverse stress stimuli. Rapid volume changes of cells, particularly of motor cells, like guard cells, are caused by variation of osmolytes and consequently of the water contents in the vacuole. Moreover, directed solute uptake into or release out of the large central vacuole allows adaptation of cytosolic metabolite levels according to the current physiological requirements and specific cellular demands. Therefore, solute passage across the vacuolar membrane, the tonoplast, has to be tightly regulated. Important principles in vacuolar transport regulation are changes of tonoplast transport protein abundances by differential expression of genes or changes of their activities, e.g. due to post-translational modification or by interacting proteins. Because vacuolar transport is in most cases driven by an electro-chemical gradient altered activities of tonoplast proton pumps significantly influence vacuolar transport capacities. Intense studies on individual tonoplast proteins but also unbiased system biological approaches have provided important insights into the regulation of vacuolar transport. This short review refers to selected examples of tonoplast proteins and their regulation, with special focus on protein phosphorylation.

  11. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma

    Directory of Open Access Journals (Sweden)

    Lourdes Gómez-Gómez

    2017-01-01

    Full Text Available Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined “crocinoplast” were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-d-xylulose 5-phosphate synthase (DXS, 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, carotenoid isomerase (CRTISO, Crocetin glucosyltransferase 2 (UGT2, etc. and crocin (e.g., ζ-carotene desaturase (ZDS and plastid-lipid-associated proteins (PLAP2 accumulation; in addition, candidate aldehyde dehydrogenase (ADH genes were highlighted.

  12. Unraveling Massive Crocins Transport and Accumulation through Proteome and Microscopy Tools during the Development of Saffron Stigma

    Science.gov (United States)

    Gómez-Gómez, Lourdes; Parra-Vega, Verónica; Rivas-Sendra, Alba; Seguí-Simarro, Jose M.; Molina, Rosa Victoria; Pallotti, Claudia; Rubio-Moraga, Ángela; Diretto, Gianfranco; Prieto, Alicia; Ahrazem, Oussama

    2017-01-01

    Crocins, the glucosides of crocetin, are present at high concentrations in saffron stigmas and accumulate in the vacuole. However, the biogenesis of the saffron chromoplast, the changes during the development of the stigma and the transport of crocins to the vacuole, are processes that remain poorly understood. We studied the process of chromoplast differentiation in saffron throughout stigma development by means of transmission electron microscopy. Our results provided an overview of a massive transport of crocins to the vacuole in the later developmental stages, when electron dense drops of a much greater size than plastoglobules (here defined “crocinoplast”) were observed in the chromoplast, connected to the vacuole with a subsequent transfer of these large globules inside the vacuole. A proteome analysis of chromoplasts from saffron stigma allowed the identification of several well-known plastid proteins and new candidates involved in crocetin metabolism. Furthermore, expressions throughout five developmental stages of candidate genes responsible for carotenoid and apocarotenoid biogenesis, crocins transport to the vacuole and starch metabolism were analyzed. Correlation matrices and networks were exploited to identify a series of transcripts highly associated to crocetin (such as 1-Deoxy-d-xylulose 5-phosphate synthase (DXS), 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), carotenoid isomerase (CRTISO), Crocetin glucosyltransferase 2 (UGT2), etc.) and crocin (e.g., ζ-carotene desaturase (ZDS) and plastid-lipid-associated proteins (PLAP2)) accumulation; in addition, candidate aldehyde dehydrogenase (ADH) genes were highlighted. PMID:28045431

  13. SOLID-STATE STORAGE DEVICE WITH PROGRAMMABLE PHYSICAL STORAGE ACCESS

    DEFF Research Database (Denmark)

    2017-01-01

    a storage device action request, and the storage device evaluating a first rule of the one or more rules by determining if the received request fulfills request conditions comprised in the first rule, and in the affirmative the storage device performing request actions comprised in the first rule......Embodiments of the present invention includes a method of operating a solid-state storage device, comprising a storage device controller in the storage device receiving a set of one or more rules, each rule comprising (i) one or more request conditions to be evaluated for a storage device action...... request received from a host computer, and (ii) one or more request actions to be performed on a physical address space of a non-volatile storage unit in the solid-state storage device in case the one or more request conditions are fulfilled; the method further comprises: the storage device receiving...

  14. Annual cycle of sperm storage in spermathecae of the red-spotted newt, Notophthalmus viridescens (Amphibia: Salamandridae).

    Science.gov (United States)

    Sever, David M; Rania, Lisa C; Krenz, John D

    1996-02-01

    Female sperm storage was studied in a population of Notophthalmus viridescens from South Carolina. Spermathecae initiate production of a glycoprotein secretory product in October. At this time ovarian follicles are immature (0.5-0.9 mm dia), and mating does not occur despite spermiation in males. Six of the 10 females collected in December had sperm in their spermathecae, indicating onset of mating. Unmated females collected in October and sacrificed in February and March possessed mature ovarian follicles (1.3-1.4 mm dia), and the spermathecae contained large secretory vacuoles 2-3 μm dia. Release of secretory product is concomitant with the appearance of sperm in the spermathecae. Thus mated females lack secretory vacuoles in the spermathecal epithelium, and additional synthesis of secretory product does not occur. All females collected in February and March have mated. Sperm are embedded in the spermathecal epithelium and are undergoing degradation in February. Degradation of sperm in the lumen and epithelium is evident in specimens examined from May and June. Atresia of ovarian follicles begins in April in captive specimens, and specimens captured from the bay in May are spent. A general postbreeding emigration from the pond occurs in summer. Fourteen females collected 7 March were injected with human chorionic gonadotropin (hCG) on 9 March and laid fertile eggs 10-18 March. Two of these females were sacrificed each month from April-September; all retained some sperm in their spermathecae, but further oviposition did not occur. Four females were kept 1 year after oviposition of fertile eggs, and oviposition again was induced with hCG; these eggs were infertile, and spermathecae lacked sperm. Spermathecae are inactive from June-September in captive and wild-caught specimens. Sperm may be stored effectively up to 6 months (December-May), and no evidence was found for retention of viable sperm from one breeding season to the next. © 1996 Wiley-Liss, Inc

  15. A sporozoite asparagine-rich protein controls initiation of Plasmodium liver stage development.

    Directory of Open Access Journals (Sweden)

    Olivier Silvie

    2008-06-01

    Full Text Available Plasmodium sporozoites invade host hepatocytes and develop as liver stages (LS before the onset of erythrocytic infection and malaria symptoms. LS are clinically silent, and constitute ideal targets for causal prophylactic drugs and vaccines. The molecular and cellular mechanisms underlying LS development remain poorly characterized. Here we describe a conserved Plasmodium asparagine-rich protein that is specifically expressed in sporozoites and liver stages. Gene disruption in Plasmodium berghei results in complete loss of sporozoite infectivity to rodents, due to early developmental arrest after invasion of hepatocytes. Mutant sporozoites productively invade host cells by forming a parasitophorous vacuole (PV, but subsequent remodelling of the membrane of the PV (PVM is impaired as a consequence of dramatic down-regulation of genes encoding PVM-resident proteins. These early arrested mutants confer only limited protective immunity in immunized animals. Our results demonstrate the role of an asparagine-rich protein as a key regulator of Plasmodium sporozoite gene expression and LS development, and suggest a requirement of partial LS maturation to induce optimal protective immune responses against malaria pre-erythrocytic stages. These findings have important implications for the development of genetically attenuated parasites as a vaccine approach.

  16. CRP-Mediated Carbon Catabolite Regulation of Yersinia pestis Biofilm Formation Is Enhanced by the Carbon Storage Regulator Protein, CsrA.

    Directory of Open Access Journals (Sweden)

    Stephan P Willias

    Full Text Available The natural transmission of Yersinia pestis is reliant upon biofilm blockage of the flea vector. However, the environmentally-responsive adaptive regulators which facilitate Y. pestis biofilm production in accordance with the flea midgut milieu are not well understood. We seek to establish the impact of available carbon source metabolism and storage upon Y. pestis biofilm production. Our findings demonstrate that Y. pestis biofilm production is subject to carbon catabolite regulation in which the presence of glucose impairs biofilm production; whereas, the sole metabolism of alternate carbon sources promotes robust biofilm formation. This observation is facilitated by the cAMP receptor protein, CRP. In accordance with a stark growth defect, deletion of crp in both CO92 and KIM6+ Y. pestis strains significantly impaired biofilm production when solely utilizing alternate carbon sources. Media supplementation with cAMP, a small-molecule activator of CRP, did not significantly alter Y. pestis biofilm production. Furthermore, CRP did not alter mRNA abundance of previously-characterized hms biofilm synthesis and regulation factors. Therefore, our findings indicate CRP does not confer a direct stimulatory effect, but may indirectly promote Y. pestis biofilm production by facilitating the alternate carbon source expression profile. Additionally, we assessed the impact of the carbon storage regulator protein, CsrA, upon Y. pestis biofilm production. Contrary to what has been described for E. coli, Y. pestis biofilm formation was found to be enhanced by CsrA. Regardless of media composition and available carbon source, deletion of csrA significantly impaired Y. pestis biofilm production. CsrA was found to promote Y. pestis biofilm production independent of glycogen regulation. Loss of csrA did not significantly alter relative hmsH, hmsP, or hmsT mRNA abundance. However, deletion of hmsP in the csrA-deficient mutant enabled excessive biofilm production

  17. Protein landmarks for diversity assessment in wheat genotypes ...

    African Journals Online (AJOL)

    Grain proteins from 20 Indian wheat genotypes were evaluated for diversity assessment based seed storage protein profiling on sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE). Genetic diversity was evaluated using Nei's index, Shannon index and Unweighted pair group method with arithmetic ...

  18. Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

    Science.gov (United States)

    Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

    2011-02-01

    Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

  19. The balance of protein expression and degradation: an ESCRTs point of view.

    Science.gov (United States)

    Babst, Markus; Odorizzi, Greg

    2013-08-01

    Endosomal sorting complexes required for transport (ESCRTs) execute the biogenesis of late endosomal multivesicular bodies (MVBs). The ESCRT pathway has traditionally been viewed as a means by which transmembrane proteins are degraded in vacuoles/lysosomes. More recent studies aimed at understanding the broader functions of ESCRTs have uncovered unexpected links with pathways that control cellular metabolism. Central to this communication is TORC1, the kinase complex that controls many of the catabolic and anabolic systems. The connection between TORC1 activity and ESCRTs allows cells to quickly adapt to the stress of nutrient limitations until the longer-term autophagic pathway is activated. Increasing evidence also points to ESCRTs regulating RNA interference (RNAi) pathways that control translation. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Developing new transportable storage casks for interim dry storage

    International Nuclear Information System (INIS)

    Hayashi, K.; Iwasa, K.; Araki, K.; Asano, R.

    2004-01-01

    Transportable storage metal casks are to be consistently used during transport and storage for AFR interim dry storage facilities planning in Japan. The casks are required to comply with the technical standards of regulations for both transport (hereinafter called ''transport regulation'') and storage (hereafter called ''storage regulation'') to maintain safety functions (heat transfer, containment, shielding and sub-critical control). In addition to these requirements, it is not planned in normal state to change the seal materials during storage at the storage facility, therefore it is requested to use same seal materials when the casks are transported after storage period. The dry transportable storage metal casks that satisfy the requirements have been developed to meet the needs of the dry storage facilities. The basic policy of this development is to utilize proven technology achieved from our design and fabrication experience, to carry out necessary verification for new designs and to realize a safe and rational design with higher capacity and efficient fabrication

  1. A missense change in the ATG4D gene links aberrant autophagy to a neurodegenerative vacuolar storage disease.

    Directory of Open Access Journals (Sweden)

    Kaisa Kyöstilä

    2015-04-01

    Full Text Available Inherited neurodegenerative disorders are debilitating diseases that occur across different species. We have performed clinical, pathological and genetic studies to characterize a novel canine neurodegenerative disease present in the Lagotto Romagnolo dog breed. Affected dogs suffer from progressive cerebellar ataxia, sometimes accompanied by episodic nystagmus and behavioral changes. Histological examination revealed unique pathological changes, including profound neuronal cytoplasmic vacuolization in the nervous system, as well as spheroid formation and cytoplasmic aggregation of vacuoles in secretory epithelial tissues and mesenchymal cells. Genetic analyses uncovered a missense change, c.1288G>A; p.A430T, in the autophagy-related ATG4D gene on canine chromosome 20 with a highly significant disease association (p = 3.8 x 10-136 in a cohort of more than 2300 Lagotto Romagnolo dogs. ATG4D encodes a poorly characterized cysteine protease belonging to the macroautophagy pathway. Accordingly, our histological analyses indicated altered autophagic flux in affected tissues. The knockdown of the zebrafish homologue atg4da resulted in a widespread developmental disturbance and neurodegeneration in the central nervous system. Our study describes a previously unknown canine neurological disease with particular pathological features and implicates the ATG4D protein as an important autophagy mediator in neuronal homeostasis. The canine phenotype serves as a model to delineate the disease-causing pathological mechanism(s and ATG4D function, and can also be used to explore treatment options. Furthermore, our results reveal a novel candidate gene for human neurodegeneration and enable the development of a genetic test for veterinary diagnostic and breeding purposes.

  2. Constitutive protein secretion from the exocrine pancreas of fetal rats

    International Nuclear Information System (INIS)

    Arvan, P.; Chang, A.

    1987-01-01

    Two general kinds of exocytotic secretion of proteins are known: that which is stimulated by secretagogues; and constitutive exocytosis, which is unable to be stimulated. The exocrine pancreas has often been cited as a model system for the first kind of secretion. However, the release of digestive enzymes from the exocrine pancreas of 1-day prenatal rats cannot be stimulated by secretagogues; therefore, its secretion is constitutive. To gain insight into the intracellular pathways which mediate secretion in the fetal gland, we examined the kinetics of release of newly synthesized proteins. We find that fetal pancreas in a steady state of secretion releases pulse-labeled secretory proteins in two kinetically distinct phases. The first phase occurring during 0-6.5 h of chase comprises approximately 12% of total incorporated radioactivity, the second phase beginning at greater than 7 h of chase comprises the remainder. Based on analysis by electron microscope autoradiography, radiolabel is localized during the first phase of secretion in immature granules/condensing vacuoles, Golgi compartments, and few mature granules. The second phase of secretion occurs when radiolabel is predominantly in mature granules. We propose that secretion occurs via (at least) 2 exocytotic routes, both of which are constitutive in fetal pancreatic tissue

  3. Seed protein improvement in cereals and grain legumes. Proceedings series

    Energy Technology Data Exchange (ETDEWEB)

    1979-01-01

    The following topics were discussed in the five sections of the conference: the world protein and nutritional situation; the need for and use of variability in protein characteristics; genetics, biochemistry, and physiology of seed storage proteins; analytical and nutritional techniques; and coordinated research programs. (HLW)

  4. Effect of storage duration on cytokine stability in human serum and plasma.

    Science.gov (United States)

    Vincent, Fabien B; Nim, Hieu T; Lee, Jacinta P W; Morand, Eric F; Harris, James

    2018-06-14

    Quantification of analytes such as cytokines in serum samples is intrinsic to translational research in immune diseases. Optimising pre-analytical conditions is critical for ensuring study quality, including evaluation of cytokine stability. We aimed to evaluate the effect on cytokine stability of storage duration prior to freezing of serum, and compare to plasma samples obtained from patients with systemic lupus erythematosus (SLE). Protein stability was analysed by simultaneously quantifying 18 analytes using a custom multi-analyte profile in SLE patient serum and plasma samples that had been prospectively stored at 4 °C for pre-determined periods between 0 and 30 days, prior to freezing. Six analytes were excluded from analysis, because most tested samples were above or below the limit of detection. Amongst the 12 analysed proteins, 11 did not show significant signal degradation. Significant signal degradation was observed from the fourth day of storage for a single analyte, CCL19. Proteins levels were more stable in unseparated serum compared to plasma for most analytes, with the exception of IL-37 which appears slightly more stable in plasma. Based on this, a maximum 3 days of storage at 4 °C for unseparated serum samples is recommended for biobanked samples intended for cytokine analysis in studies of human immune disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Quality evaluation of polypropylene packaged corn yogurt during storage

    Science.gov (United States)

    Aini, Nur; Prihananto, V.; Sustriawan, B.; Astuti, Y.; Maulina, M. R.

    2018-01-01

    Packaging is an important factor to control the process of quality decrease of any food product, including to determine the shelf life. The objective of this study was to determine changes quality of corn yogurt packaged using polypropylene. The method were using was package yogurt polypropylene, then it was stored in a refrigerator at 5, 10, or 15°C during 21 days. The yogurt was analysed every 7 days over a 21-day period. The results indicate that protein content decreased during storage, while the lactic acid bacteria, total acid, pH, viscosity, and total solids were increased. At the end of storage, the amount of lactic acid bacteria still fulfil the minimum requirements of a probiotic food, with a count of 6.407 log CFU/g. Overal scoring by panelist (scores ranged from 0 to 5) have a 4.78 at the beginning of storage. By the 21st day of storage, yogurt was packaging using transparent polypropylene having a score of 3.85, and that stored in opaque white packaging having a value of 3.95.

  6. The endoplasmic reticulum is a hub to sort proteins toward unconventional traffic pathways and endosymbiotic organelles.

    Science.gov (United States)

    Bellucci, Michele; De Marchis, Francesca; Pompa, Andrea

    2017-12-18

    The discovery that much of the extracellular proteome in eukaryotic cells consists of proteins lacking a signal peptide, which cannot therefore enter the secretory pathway, has led to the identification of alternative protein secretion routes bypassing the Golgi apparatus. However, proteins harboring a signal peptide for translocation into the endoplasmic reticulum can also be transported along these alternative routes, which are still far from being well elucidated in terms of the molecular machineries and subcellular/intermediate compartments involved. In this review, we first try to provide a definition of all the unconventional protein secretion pathways in eukaryotic cells, as those pathways followed by proteins directed to an 'external space' bypassing the Golgi, where 'external space' refers to the extracellular space plus the lumen of the secretory route compartments and the inner space of mitochondria and plastids. Then, we discuss the role of the endoplasmic reticulum in sorting proteins toward unconventional traffic pathways in plants. In this regard, various unconventional pathways exporting proteins from the endoplasmic reticulum to the vacuole, plasma membrane, apoplast, mitochondria, and plastids are described, including the short routes followed by the proteins resident in the endoplasmic reticulum. © The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  7. Mechanism of Stabilization of Labile Compounds by Silk Fibroin Proteins

    Science.gov (United States)

    2017-04-05

    saliva, or urine , and their collection and storage is critical to obtain reliable results. Without proper temperature regulation protein biomarkers in... samples for long-term ambient storage and subsequent on-demand recovery and laboratory analysis. Air dried silks provide a protective barrier that...silk in the stabilization of a range of different analytes, including entrapment, storage and recovery. Here, we successfully used silk fibroin as a

  8. Storage and recycling utilization of leaf-nitrogen of jujube tree

    International Nuclear Information System (INIS)

    Zeng Xiang; Hao Zhongning

    1991-01-01

    16 N-urea was foliarly applied on bearing or young jujube tree in autumn of 1987. The effects of leaf-nitrogen retranslocation in the trees, positions of the N stored, forms of reserved N, and reutilization of storage N in the next year were studied. The results were as follows: 15 N returned and stored in all parts of the tree following foliar application of 15 N-urea. Root could use the nitrogen not only absorbed from soil but also transported from leaves. The above-ground organs and roots of jujube tree played the same important roles on nitrogen storage in winter. The main forms of storage nitrogen were protein-N, which was 2-3 fold more than non-protein-N. The storage nitrogen existed in above-ground parts was used first in early spring, and that returned from leaves last year could be prior used for the developments of leaves, branchlets and infloresences. The relative distribution of nitrogen in floresence was more in bearing tree than in young tree. In the next year, 15 N was redistributed in branchlets, leaves, flowers, young fruits and perennial spurs in autumn. The 15 N transported out of the treated spurs reappeared in next spring and further transported to neighboring spurs. The local storage N was prior used for growth. There was 21.49% of fertilizer-N stored in the young jujube trees when treated foliarly with urea in autumn of 1987. After one years's use, there was still 18.91% of fertilizer-N existed in the trees, which indicated a characteristic of circulatory utilization of nitrogen for a long period and the reutilized nitrogen was mainly from jujube leaves and deciduous branchlets

  9. Developing new transportable storage casks for interim dry storage

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, K.; Iwasa, K.; Araki, K.; Asano, R. [Hitachi Zosen Diesel and Engineering Co., Ltd., Tokyo (Japan)

    2004-07-01

    Transportable storage metal casks are to be consistently used during transport and storage for AFR interim dry storage facilities planning in Japan. The casks are required to comply with the technical standards of regulations for both transport (hereinafter called ''transport regulation'') and storage (hereafter called ''storage regulation'') to maintain safety functions (heat transfer, containment, shielding and sub-critical control). In addition to these requirements, it is not planned in normal state to change the seal materials during storage at the storage facility, therefore it is requested to use same seal materials when the casks are transported after storage period. The dry transportable storage metal casks that satisfy the requirements have been developed to meet the needs of the dry storage facilities. The basic policy of this development is to utilize proven technology achieved from our design and fabrication experience, to carry out necessary verification for new designs and to realize a safe and rational design with higher capacity and efficient fabrication.

  10. Human stefin B role in cell's response to misfolded proteins and autophagy.

    Directory of Open Access Journals (Sweden)

    Mira Polajnar

    Full Text Available Alternative functions, apart from cathepsins inhibition, are being discovered for stefin B. Here, we investigate its role in vesicular trafficking and autophagy. Astrocytes isolated from stefin B knock-out (KO mice exhibited an increased level of protein aggregates scattered throughout the cytoplasm. Addition of stefin B monomers or small oligomers to the cell medium reverted this phenotype, as imaged by confocal microscopy. To monitor the identity of proteins embedded within aggregates in wild type (wt and KO cells, the insoluble cell lysate fractions were isolated and analyzed by mass spectrometry. Chaperones, tubulins, dyneins, and proteosomal components were detected in the insoluble fraction of wt cells but not in KO aggregates. In contrast, the insoluble fraction of KO cells exhibited increased levels of apolipoprotein E, fibronectin, clusterin, major prion protein, and serpins H1 and I2 and some proteins of lysosomal origin, such as cathepsin D and CD63, relative to wt astrocytes. Analysis of autophagy activity demonstrated that this pathway was less functional in KO astrocytes. In addition, synthetic dosage lethality (SDL gene interactions analysis in Saccharomyces cerevisiae expressing human stefin B suggests a role in transport of vesicles and vacuoles These activities would contribute, directly or indirectly to completion of autophagy in wt astrocytes and would account for the accumulation of protein aggregates in KO cells, since autophagy is a key pathway for the clearance of intracellular protein aggregates.

  11. Effect of Gamma Radiation and temperature on storage quality of Pea (Pisum sativum L)

    International Nuclear Information System (INIS)

    Islam, M.S.; Hossain, M. M.; Hossain, M. A.; Alam, M. K.; Sarder, A. H.

    2004-01-01

    Effect of radiation, storage temperature and storage period of pea was investigated. During the entire storage period of 12 months, no major changes occurred in weight loss, insect infestation, moisture content, water activity (a w ) and protein content of the pea stored at room temperature (RT) and at 4 0 C. Reconstitution properties and tenderness after cooking were affected at room temperature storage. Both reconstitution properties and tenderness gradually decreased with the increase of storage period when pea was stored at RT. The initial reconstitution properties (94%) decreased to 77% at the end of 12 months and the initial tenderness (97%) decreased to 13% when pea was stored at room temperature. On the other hand the reconstitution properties and tenderness were found 92% and 83% respectively in pea stored at 4 0 C.(author)

  12. Yam storage protein dioscorins from Dioscorea alata and Dioscorea japonica exhibit distinct immunomodulatory activities in mice.

    Science.gov (United States)

    Lin, Pei-Lan; Lin, Kuo-Wei; Weng, Ching-Feng; Lin, Kuo-Chih

    2009-06-10

    The aim of this study was to elucidate the effect of the major storage protein dioscorin isolated from two different yam species, Tainong No. 1 (TN1-dioscorins) and Japanese yam (Dj-dioscorins), on the immune activities of mice. Dj-dioscorins, like TN1-dioscorins, could induce expression of the pro-inflammatory cytokines and stimulate phagocytosis of RAW 264.7. Intraperitoneal injection of the TN1-dioscorins into mice stimulated phagocytosis of bone marrow, spleen, and thymic cells. In contrast, the T and B cells in bone marrow, spleen, and thymus isolated from mice injected with Dj-dioscorins had higher proliferative responses to mitogens. Furthermore, Dj-dioscorins enhanced proliferation of CD4(+), CD8(+), and Tim3(+) (Th1) cells in spleen and CD19(+) cells in both spleen and thymus. Supplement of Dj-dioscorins in the lymphoid cells isolated from Dj-dioscorins primed mice induced cell proliferation of both spleen and thymic cells. These findings indicated that TN1-dioscorins have a higher ability to stimulate the phagocytic activity of the lymphoid cells than Dj-dioscorins, whereas Dj-dioscorins possess more abilities than TN1-dioscorins to enhance the proliferation of the lymphoid cells.

  13. Energy storage

    Science.gov (United States)

    Kaier, U.

    1981-04-01

    Developments in the area of energy storage are characterized, with respect to theory and laboratory, by an emergence of novel concepts and technologies for storing electric energy and heat. However, there are no new commercial devices on the market. New storage batteries as basis for a wider introduction of electric cars, and latent heat storage devices, as an aid for solar technology applications, with satisfactory performance standards are not yet commercially available. Devices for the intermediate storage of electric energy for solar electric-energy systems, and for satisfying peak-load current demands in the case of public utility companies are considered. In spite of many promising novel developments, there is yet no practical alternative to the lead-acid storage battery. Attention is given to central heat storage for systems transporting heat energy, small-scale heat storage installations, and large-scale technical energy-storage systems.

  14. Structure and Composition of Protein Bodies from Wild-Type and High-Lysine Barley Endosperm

    DEFF Research Database (Denmark)

    Ingversen, J.

    1975-01-01

    Protein bodies were isolated from 13 and 28 day old endosperms of barley mutant 1508 and its wild type, Bomi barley. The fine structure of the isolated protein bodies was determined by electron microscopy, and the proteins present in the preparations characterized by amino-acid analysis and SDS......-polyacrylamidegel electrophoresis. Sections through pellets of isolated protein bodies from both the mutant and the wild type revealed protein body structures corresponding with those observed in sections through the intact starchy endosperms. The majority of the wild-type protein bodies was homogeneous spheres accompanied...... that the wild-type protein bodies contained large amounts of prolamines (the storage protein group which is soluble in 55 % isopropanol) and some glutelins (the storage proteins soluble in dilute alkali), whereas the mutant protein bodies have glutelin as the major component and little prolamines...

  15. Investigations on Mushroom Storage and Quality Parameters

    Directory of Open Access Journals (Sweden)

    Ömür Dündar

    2016-03-01

    Full Text Available In this study, researchers on storage and quality properties of mushrooms cultivated in the world and Turkey have been investigated. Mushrooms contain some important minerals and vitamins such as iron, calcium, phosphorus, potassium, copper and folate, thiamine, riboflavin, niacin, vitamin B, C, D and also they are a good source of carbohydrate and protein. After harvest, to extend the shelf life of mushrooms, some applications such as pre-cooling, storage in appropriate temperature, use of different types of polyethylene packaging, modified atmosphere packaging, nitric oxide and UV light applications were done on mushrooms. The effects of these applications on physical and chemical features such as like weight loss, firmness, cap opening rate, cap diameter, stem diameter, browning, colour, respiration rate, enzymatic reactions, total phenols, total sugars, aminoacid content were investigated.

  16. Toxoplasma gondii-derived synthetic peptides containing B- and T-cell epitopes from GRA2 protein are able to enhance mice survival in a model of experimental toxoplasmosis

    Directory of Open Access Journals (Sweden)

    Luciana Machado Bastos

    2016-06-01

    Full Text Available Toxoplasmosis is a zoonosis distributed all over the world, which the etiologic agent is an intracellular protozoan parasite, Toxoplasma gondii. This disease may cause abortions and severe diseases in many warm-blood hosts, including humans, particularly the immunocompromised patients. The parasite specialized secretory organelles, as micronemes, rhoptries and dense granules, are critical for the successful parasitism. The dense granule protein 2 (GRA2 is a parasite immunogenic protein secreted during infections and previous studies have been shown that this parasite component is crucial for the formation of intravacuolar membranous nanotubular network (MNN, as well as for secretion into the vacuole and spatial organization of the parasites within the vacuole. In the present study, we produced a monoclonal antibody to GRA2 (C3C5 mAb, isotype IgG2b, mapped the immunodominant epitope of the protein by phage display and built GRA2 synthetic epitopes to evaluate their ability to protect mice in a model of experimental infection. Our results showed that synthetic peptides for B- and T-cell epitopes are able to improve survival of immunized animals. In contrast with non-immunized animals, the immunized mice with both B- and T-cell epitopes had a better balance of cytokines and demonstrated higher levels of IL-10, IL-4 and IL-17 production, though similar levels of TNF-alpha and IL-6 were observed. The immunization with both B- and T-cell epitopes resulted in survival rate higher than 85% of the challenged mice. Overall, these results demonstrate that immunization with synthetic epitopes for both B- and T-cells from GRA2 protein can be more effective to protect against infection by T. gondii.

  17. Mitochondrial dysfunction in human skeletal muscle biopsies of lipid storage disorder.

    Science.gov (United States)

    Debashree, Bandopadhyay; Kumar, Manish; Keshava Prasad, Thottethodi Subrahmanya; Natarajan, Archana; Christopher, Rita; Nalini, Atchayaram; Bindu, Parayil Sankaran; Gayathri, Narayanappa; Srinivas Bharath, Muchukunte Mukunda

    2018-02-09

    Mitochondria regulate the balance between lipid metabolism and storage in the skeletal muscle. Altered lipid transport, metabolism and storage influence the bioenergetics, redox status and insulin signalling, contributing to cardiac and neurological diseases. Lipid storage disorders (LSDs) are neurological disorders which entail intramuscular lipid accumulation and impaired mitochondrial bioenergetics in the skeletal muscle causing progressive myopathy with muscle weakness. However, the mitochondrial changes including molecular events associated with impaired lipid storage have not been completely understood in the human skeletal muscle. We carried out morphological and biochemical analysis of mitochondrial function in muscle biopsies of human subjects with LSDs (n = 7), compared to controls (n = 10). Routine histology, enzyme histochemistry and ultrastructural analysis indicated altered muscle cell morphology and mitochondrial structure. Protein profiling of the muscle mitochondria from LSD samples (n = 5) (vs. control, n = 5) by high-throughput mass spectrometric analysis revealed that impaired metabolic processes could contribute to mitochondrial dysfunction and ensuing myopathy in LSDs. We propose that impaired fatty acid and respiratory metabolism along with increased membrane permeability, elevated lipolysis and altered cristae entail mitochondrial dysfunction in LSDs. Some of these mechanisms were unique to LSD apart from others that were common to dystrophic and inflammatory muscle pathologies. Many differentially regulated mitochondrial proteins in LSD are linked with other human diseases, indicating that mitochondrial protection via targeted drugs could be a treatment modality in LSD and related metabolic diseases. © 2018 International Society for Neurochemistry.

  18. Microbiological and physicochemical profile of traditional Salsiccia toscana during storage

    Directory of Open Access Journals (Sweden)

    Giovanna Preziuso

    2012-07-01

    Full Text Available The aim of this study was to evaluate the evolution during refrigerated storage of the main microbiological and physicochemical parameters of a traditional Italian fresh sausage, salsiccia toscana. The following parameters were analyzed at Days 0, 6 and 9 of storage: total aerobic mesophilic and psychrotrophic counts, Brochothrix thermosphacta, lactic acid bacteria, Pseudomonadaceae, Enterobacteriaceae, Escherichia coli, Micrococcaceae, yeasts and moulds, Salmonella spp., Listeria monocytogenes; pH, Aw, moisture, protein, total lipids, ash, fatty acid composition, Thiobarbituric Acid Reactive Substances, colour measurement. The results revealed a statistically significant increase in the main microbiological parameters between Days 0 and 9 of refrigerated storage, with total bacterial counts exceeding 107 CFU/g after nine days and Brochothrix thermosphacta representing the main spoilage microorganism. Among physicochemical parameters, discolouration was noted during the time of storage, with a significantly paler colour on the surface of sausages with casings after nine days of storage. Considering that the initial bacterial counts were higher than 105 CFU/g for Brochothrix thermosphacta, Pseudomonadaceae, lactic acid bacteria and Enterobacteriaceae, and that the product is traditionally purchased loose without any packaging, it is fundamental to improve the good manufacturing practices, particularly measures to control processing temperatures.

  19. The use of protein patterns in genetic diversity analysis in some Brassica napus cultivars

    Directory of Open Access Journals (Sweden)

    Roya Razavizadeh

    2013-11-01

    Full Text Available In this study, protein variations of seeds and five-day old cotyledonal leaves of four selected Brassica napus cultivars including Elite, Ocapy, Tasilo and Zarfam were analyzed by SDS-PAGE to identify protein markers. The amount of total soluble protein of seed storage proteins did not show significant differences in all cultivars whereas it was different in cotyledonal leaves. Protein patterns of seeds and cotyledonal leaves showed significant differences using SDS-PAGE and consequence analysis of bands by ImageJ program. Relative expression of six protein bands in seeds and five-day old cotyledonal leaves were significantly different. Three protein markers were identified by protein patterns of seed and cotyledonal leaves. The results of relationship analysis based on presence and absence of the specific protein bands in protein pattern of seed storage proteins showed that Tasilo and Elite cultivars had the highest similarities.

  20. Chemical composition and nutritional value of the freezing consolidated burgers (Kilka–Silver carp during cold storage

    Directory of Open Access Journals (Sweden)

    S Fathi

    2014-05-01

    Full Text Available Consolidated fish burger is a new product which is a combination of common Kilka (Clupeonellacultriventriscaspia and Silver carp (Hypophthalmichthys molitrix minced with flavors, fillers, vegetables and tofu dressing. Consolidated fish burger is produced in order to boost the nutritional value and to reduce the cost of end product. This study aimed to investigate the variations in the composition of consolidated burger during 4 months of storage at -18 °C. For this purpose, 4 types of burgers with a combination of a various percentages of Kilka and Silver carp were produced. The chemical composition by means of total protein, fat, moisture and ash contents were evaluated during preparation (zero phase and 4 months of storage. Results showed that at zero-phase protein% and moisture% in raw Silver carp was higher, whereas fat% and ash% in Kilka was found higher. Protein content in all groups was decreased during 4 months of storage. The decreasing rate was more rapid in control group as well as treatment 3. Fat percentage was dropped during the storage period and the decreasing trend in treatment 2 was found higher. In the case of moisture, the percentage was declined in all groups and in treatment 1, in particular. Considering the results, it was concluded that freezing could significantly decrease the nutritional value of the consolidated Burgers.

  1. Integrated and comparative proteomics of high-oil and high-protein soybean seeds.

    Science.gov (United States)

    Xu, Xiu Ping; Liu, Hui; Tian, Lihong; Dong, Xiang Bai; Shen, Shi Hua; Qu, Le Qing

    2015-04-01

    We analysed the global protein expression in seeds of a high-oil soybean cultivar (Jiyu 73, JY73) by proteomics. More than 700 protein spots were detected and 363 protein spots were successfully identified. Comparison of the protein profile of JY73 with that of a high-protein cultivar (Zhonghuang 13, ZH13) revealed 40 differentially expressed proteins, including oil synthesis, redox/stress, hydrolysis and storage-related proteins. All redox/stress proteins were less or not expressed in JY73, whereas the expression of the major storage proteins, nitrogen and carbon metabolism-related proteins was higher in ZH13. Biochemical analysis of JY73 revealed that it was in a low oxidation state, with a high content of polyunsaturated fatty acids and vitamin E. Vitamin E was more active than antioxidant enzymes and protected the soybean seed in a lower oxidation state. The characteristics of high oil and high protein in soybean, we revealed, might provide a reference for soybean nutrition and soybean breeding. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. Variations in grain lipophilic phytochemicals, proteins and resistance to Fusarium spp. growth during grain storage as affected by biological plant protection with Aureobasidium pullulans (de Bary).

    Science.gov (United States)

    Wachowska, Urszula; Tańska, Małgorzata; Konopka, Iwona

    2016-06-16

    Modern agriculture relies on an integrated approach, where chemical treatment is reduced to a minimum and replaced by biological control that involves the use of active microorganisms. The effect of the antagonistic yeast-like fungus Aureobasidium pullulans on proteins and bioactive compounds (alkylresorcinols, sterols, tocols and carotenoids) in winter wheat grain and on the colonization of wheat kernels by fungal microbiota, mainly Fusarium spp. pathogens, was investigated. Biological treatment contributed to a slight increase contents of tocols, alkylresorcinols and sterols in grain. At the same time, the variation of wheat grain proteins was low and not significant. Application of A. pullulans enhanced the natural yeast colonization after six months of grain storage and inhibited growth of F. culmorum pathogens penetrating wheat kernel. This study demonstrated that an integrated approach of wheat grain protection with the use of the yeast-like fungus A. pullulans reduced kernel colonization by Fusarium spp. pathogens and increased the content of nutritionally beneficial phytochemicals in wheat grain without a loss of gluten proteins responsible for baking value. Copyright © 2016. Published by Elsevier B.V.

  3. Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein.

    Science.gov (United States)

    Gaur, Vikram Singh; Kumar, Lallan; Gupta, Supriya; Jaiswal, J P; Pandey, Dinesh; Kumar, Anil

    2018-03-01

    In this study, we report the isolation and characterization of the mRNA encoding OPAQUE2 (O2) like TF of finger millet (FM) ( Eleusine coracana) ( EcO2 ). Full-length EcO2 mRNA was isolated using conserved primers designed by aligning O2 mRNAs of different cereals followed by 3' and 5' RACE (Rapid Amplification of cDNA Ends). The assembled full-length EcO2 mRNA was found to contain an ORF of 1248-nt coding the 416 amino acids O2 protein. Domain analysis revealed the presence of the BLZ and bZIP-C domains which is a characteristic feature of O2 proteins. Phylogenetic analysis of EcO2 protein with other bZIP proteins identified using finger millet transcriptome data and O2 proteins of other cereals showed that EcO2 shared high sequence similarity with barley BLZ1 protein. Transcripts of EcO2 were detected in root, stem, leaves, and seed development stages. Furthermore, to investigate nitrogen responsiveness and the role of EcO2 in regulating seed storage protein gene expression, the expression profiles of EcO2 along with an α-prolamin gene were studied during the seed development stages of two FM genotypes (GE-3885 and GE-1437) differing in grain protein content (13.8 and 6.2%, respectively) grown under increasing nitrogen inputs. Compared to GE-1437, the EcO2 was relatively highly expressed during the S2 stage of seed development which further increased as nitrogen input was increased. The Ecα - prolamin gene was strongly induced in the high protein genotype (GE-3885) at all nitrogen inputs. These results indicate the presence of nitrogen responsiveness regulatory elements which might play an important role in accumulating protein in FM genotypes through modulating EcO2 expression by sensing plant nitrogen status.

  4. Immunogenicity of biopharmaceuticals and biosimilars in relation to storage, handling and stability

    International Nuclear Information System (INIS)

    Hincal, F.

    2009-01-01

    Therapeutic proteins or biopharmaceuticals provide effective treatment for many diseases and medical conditions, and vaccines, immunoglobulins and monoclonal antibodies are critical biodefense biopharmaceuticals which constitute an indispensable part of biodefense stockpiles. The manufacturing process for biopharmaceuticals and their generic forms which are called biosimilars is far more complex than for low molecular weight drugs and generics. Any minor change made at any stage may have a critical effect on the clinical efficacy and safety. Potential immunogenicity is the key issue for biopharmaceuticals and biosimilars and may have serious clinical consequences ranging from allergy and anaphylaxis, as well as loss of efficacy of the product. Immunogenicity may be influenced by factors related to manufacturing process, formulation, aggregate formation, contaminants and impurities, and also by the factors related to the storage and handling. Stability is particularly important with larger protein molecules, because their in vivo effects often depend on their three-dimensional structure. Proteins usually aggregate from partially unfolded molecules and aggregates can enhance immunogenicity. Although product formulations are developed to maximize and maintain the fraction of the protein molecules present in the native state, significant amounts of aggregates can form, especially over pharmaceutically relevant time scales and under stress conditions. Exposure to air-liquid and solid-liquid interfaces, light, temperature fluctuations or minor impurities can induce aggregation. Such exposure can occur during processing steps, as well as in the final product container during storage, shipment and handling. Biopharmaceuticals are particularly sensitive to temperature changes and/or shaking. Strict storage and handling conditions and timely and effective stability/shelf-life testing are therefore essential for maintaining product integrity and stability, and hence efficacy

  5. A histone-like protein of mycobacteria possesses ferritin superfamily protein-like activity and protects against DNA damage by Fenton reaction.

    Directory of Open Access Journals (Sweden)

    Masaki Takatsuka

    Full Text Available Iron is an essential metal for living organisms but its level must be strictly controlled in cells, because ferrous ion induces toxicity by generating highly active reactive oxygen, hydroxyl radicals, through the Fenton reaction. In addition, ferric ion shows low solubility under physiological conditions. To overcome these obstacles living organisms possess Ferritin superfamily proteins that are distributed in all three domains of life: bacteria, archaea, and eukaryotes. These proteins minimize hydroxyl radical formation by ferroxidase activity that converts Fe(2+ into Fe(3+ and sequesters iron by storing it as a mineral inside a protein cage. In this study, we discovered that mycobacterial DNA-binding protein 1 (MDP1, a histone-like protein, has similar activity to ferritin superfamily proteins. MDP1 prevented the Fenton reaction and protects DNA by the ferroxidase activity. The K(m values of the ferroxidase activity by MDP1 of Mycobacterium bovis bacillus Calmette-Guérin (BCG-3007c, Mycobacterium tuberculosis (Rv2986c, and Mycobacterium leprae (ML1683; ML-LBP were 0.292, 0.252, and 0.129 mM, respectively. Furthermore, one MDP1 molecule directly captured 81.4±19.1 iron atoms, suggesting the role of this protein in iron storage. This study describes for the first time a ferroxidase-iron storage protein outside of the ferritin superfamily proteins and the protective role of this bacterial protein from DNA damage.

  6. A concept of an electricity storage system with 50 MWh storage capacity

    Directory of Open Access Journals (Sweden)

    Józef Paska

    2012-06-01

    Full Text Available Electricity storage devices can be divided into indirect storage technology devices (involving electricity conversion into another form of energy, and direct storage (in an electric or magnetic fi eld. Electricity storage technologies include: pumped-storage power plants, BES Battery Energy Storage, CAES Compressed Air Energy Storage, Supercapacitors, FES Flywheel Energy Storage, SMES Superconducting Magnetic Energy Storage, FC Fuel Cells reverse or operated in systems with electrolysers and hydrogen storage. These technologies have diff erent technical characteristics and economic parameters that determine their usability. This paper presents two concepts of an electricity storage tank with a storage capacity of at least 50 MWh, using the BES battery energy storage and CAES compressed air energy storage technologies.

  7. Leading survey and research report for fiscal 1999. New technology based on functions involved in intracellular protein transport; 1999 nendo saibonai tanpakushitsu yuso kino riyo gijutsu kenkyu hokokusho

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    2000-03-01

    An intercellular transport technology (artificial manipulation of an intracellular protein transport system in eucaryotes) is studied for the accumulation of cytotoxic proteins, whose expression has so far been difficult, and activated proteins, which have avoided decomposition, in appropriate intracellular minute organs. The aim is to construct a system to allow foreign proteins high in productivity and quality to express themselves for production in eucaryotes. Basic surveys were conducted of the intracellular biological functions of single-membrane organelles (endoplasmic reticulum, peroxisome, vacuole/lysosome, and Golgi body), the molecular mechanism of protein transport to each organelle, and protein activation and quality control, and element technologies were extracted. For the development of novel pharmaceuticals making use of the intracellular protein transport technology, an activated protein production system was built and a search was made for transport activity impeding substances. Research tasks relative to the development of the new technologies were isolated, such as the visualization of intercellular transport. A survey was made of the market for pharmaceuticals, cosmetics, enzymes, and visualizing equipment (fluorescence microscope provided with new functions), etc. (NEDO)

  8. SDS-Page Seed Storage Protein Profiles in Chili Peppers (Capsicum L.

    Directory of Open Access Journals (Sweden)

    Owk ANIEL KUMAR

    2010-09-01

    Full Text Available Seed protein banding patterns (SDS-PAGE were studied from eighteen genotypes of chili pepper (Capsicum L. A total of 21 protein polypeptide bands with molecular weight ranging from 18.6 to 72.0 kD were recorded. Among the genotypes CA18, CA21 and CA27 represented maximum number of protein bands (12. Band no. (11 and (5,12 are exclusive to C. annuum L. and C. frutescens L. genotypes respectively. Average percent similarity was highest (100% between CA2 and CA8 genotypes and the UPGMA dendrogram represented low genetic diversity. The study revealed that considerable intra and inter-specific differences were found in the genotypes. The variability of protein profiles in the genotypes suggested that these selected genotypes can be a good source for crop improvement through hybridization programs.

  9. Vhc1, a novel transporter belonging to the family of electroneutral cation–Cl− cotransporters, participates in the regulation of cation content and morphology of Saccharomyces cerevisiae vacuoles

    Czech Academy of Sciences Publication Activity Database

    Petrezsélyová, Silvia; Kinclová-Zimmermannová, Olga; Sychrová, Hana

    2013-01-01

    Roč. 1828, č. 2 (2013), s. 623-631 ISSN 0005-2736 R&D Projects: GA AV ČR(CZ) IAA500110801; GA MŠk(CZ) LC531; GA MŠk(CZ) OC10012 Grant - others:Rada Programu interní podpory projektů mezinárodní spolupráce AV ČR(CZ) M200110901 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : salt tolerance * yeast vacuole * potassium homeostasis * cation- chloride cotransport Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.431, year: 2013

  10. Tritium storage

    International Nuclear Information System (INIS)

    Hircq, B.

    1990-01-01

    This document represents a synthesis relative to tritium storage. After indicating the main storage particularities as regards tritium, storages under gaseous and solid form are after examined before establishing choices as a function of the main criteria. Finally, tritium storage is discussed regarding tritium devices associated to Fusion Reactors and regarding smaller devices [fr

  11. Cuticular proteins from the horseshoe crab, Limulus polyphemus

    DEFF Research Database (Denmark)

    Ditzel, Nicholas; Andersen, Svend Olav; Højrup, Peter

    2003-01-01

    Proteins were purified from the carapace cuticle of a juvenile horseshoe crab, Limulus polyphemus, and several of them were characterized by amino acid sequence determination. The proteins are small (7-16 kDa) and their isoelectric points range from 6.5 to 9.2. They have high contents of tyrosine......, ranging from 13.5 to 35.4%. Some of the proteins show sequence similarity to cuticular proteins from other arthropod groups, with the most pronounced similarity to proteins from the cuticle of the spider Araneus diadematus. Two proteins show sequence similarity to a hexamerin storage protein from Blaberus...

  12. Energy storage

    International Nuclear Information System (INIS)

    2012-01-01

    After having outlined the importance of energy storage in the present context, this document outlines that it is an answer to economic, environmental and technological issues. It proposes a brief overview of the various techniques of energy storage: under the form of chemical energy (hydrocarbons, biomass, hydrogen production), thermal energy (sensitive or latent heat storage), mechanical energy (potential energy by hydraulic or compressed air storage, kinetic energy with flywheels), electrochemical energy (in batteries), electric energy (super-capacitors, superconductor magnetic energy storage). Perspectives are briefly evoked

  13. Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

    Science.gov (United States)

    Kuech, Eva-Maria; Brogden, Graham; Naim, Hassan Y

    2016-11-01

    Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Transgenic biofortification of the starchy staple cassava (Manihot esculenta generates a novel sink for protein.

    Directory of Open Access Journals (Sweden)

    Mohammad Abhary

    Full Text Available Although calorie dense, the starchy, tuberous roots of cassava provide the lowest sources of dietary protein within the major staple food crops (Manihot esculenta Crantz. (Montagnac JA, Davis CR, Tanumihardjo SA. (2009 Compr Rev Food Sci Food Saf 8:181-194. Cassava was genetically modified to express zeolin, a nutritionally balanced storage protein under control of the patatin promoter. Transgenic plants accumulated zeolin within de novo protein bodies localized within the root storage tissues, resulting in total protein levels of 12.5% dry weight within this tissue, a fourfold increase compared to non-transgenic controls. No significant differences were seen for morphological or agronomic characteristics of transgenic and wild type plants in the greenhouse and field trials, but relative to controls, levels of cyanogenic compounds were reduced by up to 55% in both leaf and root tissues of transgenic plants. Data described here represent a proof of concept towards the potential transformation of cassava from a starchy staple, devoid of storage protein, to one capable of supplying inexpensive, plant-based proteins for food, feed and industrial applications.

  15. Fluorescent Proteins for Investigating Biological Events in Acidic Environments

    Directory of Open Access Journals (Sweden)

    Hajime Shinoda

    2018-05-01

    Full Text Available The interior lumen of acidic organelles (e.g., endosomes, secretory granules, lysosomes and plant vacuoles is an important platform for modification, transport and degradation of biomolecules as well as signal transduction, which remains challenging to investigate using conventional fluorescent proteins (FPs. Due to the highly acidic luminal environment (pH ~ 4.5–6.0, most FPs and related sensors are apt to lose their fluorescence. To address the need to image in acidic environments, several research groups have developed acid-tolerant FPs in a wide color range. Furthermore, the engineering of pH insensitive sensors, and their concomitant use with pH sensitive sensors for the purpose of pH-calibration has enabled characterization of the role of luminal ions. In this short review, we summarize the recent development of acid-tolerant FPs and related functional sensors and discuss the future prospects for this field.

  16. The Effect of Herbicides on Hydrogen Peroxide Generation in Isolated Vacuoles of Red Beet Root (Beta vulgaris L.

    Directory of Open Access Journals (Sweden)

    E.V. Pradedova

    2015-12-01

    Full Text Available Influence of herbicides on the hydrogen peroxide generation in vacuolar extracts of red beet root (Beta vulgaris L. was investigated. Belonging to different chemical classes of herbicide compounds have been used. Herbicides differ from each other in the mechanism of effects on plants. Clopyralid (aromatic acid herbicide, derivative of picolinic acid and 2.4-D (phenoxyacetic herbicide, characterized by hormone-like effects, contributed to the formation of H2O2 in vacuolar extracts. Fluorodifen (nitrophenyl ether herbicide and diuron (urea herbicide also have increased contents H2O2. These compounds inhibit the electron transport, photosynthesis, and photorespiration in sensitive plants. Herbicidal effect of glyphosate (organophosphorus herbicide is due to the inhibition of amino acid synthesis in plant cells. Glyphosate did not affect the content of H2O2 in vacuolar extracts. Herbicide dependent H2O2-generation did not occur with oxidoreductase inhibitors, potassium cyanide and sodium azide. The results suggest that the formation of ROS in the vacuoles due to activity of oxidoreductases, which could interact with herbicides.

  17. A PQL (protein quantity loci) analysis of mature pea seed proteins identifies loci determining seed protein composition.

    Science.gov (United States)

    Bourgeois, Michael; Jacquin, Françoise; Cassecuelle, Florence; Savois, Vincent; Belghazi, Maya; Aubert, Grégoire; Quillien, Laurence; Huart, Myriam; Marget, Pascal; Burstin, Judith

    2011-05-01

    Legume seeds are a major source of dietary proteins for humans and animals. Deciphering the genetic control of their accumulation is thus of primary significance towards their improvement. At first, we analysed the genetic variability of the pea seed proteome of three genotypes over 3 years of cultivation. This revealed that seed protein composition variability was under predominant genetic control, with as much as 60% of the spots varying quantitatively among the three genotypes. Then, by combining proteomic and quantitative trait loci (QTL) mapping approaches, we uncovered the genetic architecture of seed proteome variability. Protein quantity loci (PQL) were searched for 525 spots detected on 2-D gels obtained for 157 recombinant inbred lines. Most protein quantity loci mapped in clusters, suggesting that the accumulation of the major storage protein families was under the control of a limited number of loci. While convicilin accumulation was mainly under the control of cis-regulatory regions, vicilins and legumins were controlled by both cis- and trans-regulatory regions. Some loci controlled both seed protein composition and protein content and a locus on LGIIa appears to be a major regulator of protein composition and of protein in vitro digestibility. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Protein Correlation Profiles Identify Lipid Droplet Proteins with High Confidence*

    Science.gov (United States)

    Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora; Wilfling, Florian; Stoehr, Gabriele; Mann, Matthias; Farese, Robert V.; Walther, Tobias C.

    2013-01-01

    Lipid droplets (LDs) are important organelles in energy metabolism and lipid storage. Their cores are composed of neutral lipids that form a hydrophobic phase and are surrounded by a phospholipid monolayer that harbors specific proteins. Most well-established LD proteins perform important functions, particularly in cellular lipid metabolism. Morphological studies show LDs in close proximity to and interacting with membrane-bound cellular organelles, including the endoplasmic reticulum, mitochondria, peroxisomes, and endosomes. Because of these close associations, it is difficult to purify LDs to homogeneity. Consequently, the confident identification of bona fide LD proteins via proteomics has been challenging. Here, we report a methodology for LD protein identification based on mass spectrometry and protein correlation profiles. Using LD purification and quantitative, high-resolution mass spectrometry, we identified LD proteins by correlating their purification profiles to those of known LD proteins. Application of the protein correlation profile strategy to LDs isolated from Drosophila S2 cells led to the identification of 111 LD proteins in a cellular LD fraction in which 1481 proteins were detected. LD localization was confirmed in a subset of identified proteins via microscopy of the expressed proteins, thereby validating the approach. Among the identified LD proteins were both well-characterized LD proteins and proteins not previously known to be localized to LDs. Our method provides a high-confidence LD proteome of Drosophila cells and a novel approach that can be applied to identify LD proteins of other cell types and tissues. PMID:23319140

  19. Protein stability: a crystallographer’s perspective

    International Nuclear Information System (INIS)

    Deller, Marc C.; Kong, Leopold; Rupp, Bernhard

    2016-01-01

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed

  20. Protein stability: a crystallographer’s perspective

    Energy Technology Data Exchange (ETDEWEB)

    Deller, Marc C., E-mail: mdeller@stanford.edu [Stanford University, Shriram Center, 443 Via Ortega, Room 097, MC5082, Stanford, CA 94305-4125 (United States); Kong, Leopold [National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Building 8, Room 1A03, 8 Center Drive, Bethesda, MD 20814 (United States); Rupp, Bernhard [k.-k. Hofkristallamt, 91 Audrey Place, Vista, CA 92084 (United States); Medical University of Innsbruck, Schöpfstrasse 41, A-6020 Innsbruck (Austria)

    2016-01-26

    An understanding of protein stability is essential for optimizing the expression, purification and crystallization of proteins. In this review, discussion will focus on factors affecting protein stability on a somewhat practical level, particularly from the view of a protein crystallographer. Protein stability is a topic of major interest for the biotechnology, pharmaceutical and food industries, in addition to being a daily consideration for academic researchers studying proteins. An understanding of protein stability is essential for optimizing the expression, purification, formulation, storage and structural studies of proteins. In this review, discussion will focus on factors affecting protein stability, on a somewhat practical level, particularly from the view of a protein crystallographer. The differences between protein conformational stability and protein compositional stability will be discussed, along with a brief introduction to key methods useful for analyzing protein stability. Finally, tactics for addressing protein-stability issues during protein expression, purification and crystallization will be discussed.

  1. 'Candidatus Liberibacter asiaticus' Accumulates inside Endoplasmic Reticulum Associated Vacuoles in the Gut Cells of Diaphorina citri.

    Science.gov (United States)

    Ghanim, Murad; Achor, Diann; Ghosh, Saptarshi; Kontsedalov, Svetlana; Lebedev, Galina; Levy, Amit

    2017-12-05

    Citrus greening disease known also as Huanglongbing (HLB) caused by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus' (CLas) has resulted in tremendous losses and the death of millions of trees worldwide. CLas is transmitted by the Asian citrus psyllid Diaphorina citri. The closely-related bacteria 'Candidatus Liberibacter solanacearum' (CLso), associated with vegetative disorders in carrots, is transmitted by the carrot psyllid Bactericera trigonica. A promising approach to prevent the transmission of these pathogens is to interfere with the vector-pathogen interactions, but our understanding of these processes is limited. It was recently reported that CLas induced changes in the nuclear architecture, and activated programmed cell death, in D. citri midgut cells. Here, we used electron and fluorescent microscopy and show that CLas induces the formation of endoplasmic reticulum (ER)-associated bodies. The bacterium recruits those ER structures into Liberibacter containing vacuoles (LCVs), in which bacterial cells seem to propagate. ER- associated LCV formation was unique to CLas, as we could not detect these bodies in B. trigonica infected with CLso. ER recruitment is hypothesized to generate a safe replicative body to escape cellular immune responses in the insect gut. Understanding the molecular interactions that undelay these responses will open new opportunities for controlling CLas.

  2. Ovary histology and quantification of hemolymph proteins of Rhipicephalus (Boophilusmicroplus treated with Melia azedarach

    Directory of Open Access Journals (Sweden)

    Lorena Alessandra Dias de Sousa

    Full Text Available This study aimed to analyze ovary histology and quantify total protein in the hemolymph of Rhipicephalus (Boophilusmicroplus females treated with hexane extracts from green fruits of Melia azedarach. Eight engorged females were immersed in the extract at 0.25% concentration, and eight in water containing 5% acetone (control. The females were dissected 72 hours after treatment, and the ovaries were weighed and subjected to standard histological techniques. The total protein concentration was measured in the hemolymph of 200 females, of which 100 were treated as described above and 100 served as a control. In the treated group, ovary weight reduction and predominance of immature oocytes were observed. In addition, there were decreases in the diameters of the cytoplasm and germ vesicle of the oocytes in the treated group, compared with the controls. The protein concentration in the hemolymph was higher in the treated group than in the controls. The morphological changes observed in the treated ovaries included: presence of vacuolization; alteration of oocyte morphology, which changed from rounded to elongated; deformation of the chorion; and disorganization of the yolk granules. These results demonstrate the action ofM. azedarach fruit extracts on R.(B. microplus oogenesis.

  3. Effects of Extended Freezer Storage on the Integrity of Human Milk.

    Science.gov (United States)

    Ahrabi, Ali Faraghi; Handa, Deepali; Codipilly, Champa N; Shah, Syed; Williams, Janet E; McGuire, Mark A; Potak, Debra; Aharon, Grace Golda; Schanler, Richard J

    2016-10-01

    To examine the integrity (pH, bacterial counts, host defense factors, nutrient contents, and osmolality) of freshly expressed and previously refrigerated human milk subjected to long-term freezer storage. Mothers donated 100 mL of freshly expressed milk. Samples were divided into baseline, storage at -20°C (fresh frozen) for 1, 3, 6, and 9 months, and prior storage at +4°C for 72 hours (refrigerated frozen) before storage at -20°C for 1 to 9 months. Samples were analyzed for pH, total bacterial colony count, gram-positive and gram-negative colony counts, and concentrations of total protein, fat, nonesterified fatty acids, lactoferrin, secretory IgA, and osmolality. Milk pH, total bacterial colony count, and Gram-positive colony counts decreased significantly with freezer storage (P negative colony count decreased significantly over time (P human milk for 9 months at -20°C is associated with decreasing pH and bacterial counts, but preservation of key macronutrients and immunoactive components, with or without prior refrigeration for 72 hours. These data support current guidelines for freezer storage of human milk for up to 9 months for both freshly expressed and refrigerated milk. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. A saposin deficiency model in Drosophila: Lysosomal storage, progressive neurodegeneration and sensory physiological decline.

    Science.gov (United States)

    Hindle, Samantha J; Hebbar, Sarita; Schwudke, Dominik; Elliott, Christopher J H; Sweeney, Sean T

    2017-02-01

    Saposin deficiency is a childhood neurodegenerative lysosomal storage disorder (LSD) that can cause premature death within three months of life. Saposins are activator proteins that promote the function of lysosomal hydrolases that mediate the degradation of sphingolipids. There are four saposin proteins in humans, which are encoded by the prosaposin gene. Mutations causing an absence or impaired function of individual saposins or the whole prosaposin gene lead to distinct LSDs due to the storage of different classes of sphingolipids. The pathological events leading to neuronal dysfunction induced by lysosomal storage of sphingolipids are as yet poorly defined. We have generated and characterised a Drosophila model of saposin deficiency that shows striking similarities to the human diseases. Drosophila saposin-related (dSap-r) mutants show a reduced longevity, progressive neurodegeneration, lysosomal storage, dramatic swelling of neuronal soma, perturbations in sphingolipid catabolism, and sensory physiological deterioration. Our data suggests a genetic interaction with a calcium exchanger (Calx) pointing to a possible calcium homeostasis deficit in dSap-r mutants. Together these findings support the use of dSap-r mutants in advancing our understanding of the cellular pathology implicated in saposin deficiency and related LSDs. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  5. High-level production of human interleukin-10 fusions in tobacco cell suspension cultures

    Science.gov (United States)

    Kaldis, Angelo; Ahmad, Adil; Reid, Alexandra; McGarvey, Brian; Brandle, Jim; Ma, Shengwu; Jevnikar, Anthony; Kohalmi, Susanne E; Menassa, Rima

    2013-01-01

    The production of pharmaceutical proteins in plants has made much progress in recent years with the development of transient expression systems, transplastomic technology and humanizing glycosylation patterns in plants. However, the first therapeutic proteins approved for administration to humans and animals were made in plant cell suspensions for reasons of containment, rapid scale-up and lack of toxic contaminants. In this study, we have investigated the production of human interleukin-10 (IL-10) in tobacco BY-2 cell suspension and evaluated the effect of an elastin-like polypeptide tag (ELP) and a green fluorescent protein (GFP) tag on IL-10 accumulation. We report the highest accumulation levels of hIL-10 obtained with any stable plant expression system using the ELP fusion strategy. Although IL-10-ELP has cytokine activity, its activity is reduced compared to unfused IL-10, likely caused by interference of ELP with folding of IL-10. Green fluorescent protein has no effect on IL-10 accumulation, but examining the trafficking of IL-10-GFP over the cell culture cycle revealed fluorescence in the vacuole during the stationary phase of the culture growth cycle. Analysis of isolated vacuoles indicated that GFP alone is found in vacuoles, while the full-size fusion remains in the whole-cell extract. This indicates that GFP is cleaved off prior to its trafficking to the vacuole. On the other hand, IL-10-GFP-ELP remains mostly in the ER and accumulates to high levels. Protein bodies were observed at the end of the culture cycle and are thought to arise as a consequence of high levels of accumulation in the ER. PMID:23297698

  6. Feasibility of extending storage life of sheep-milk cheese using ionizing radiation

    Energy Technology Data Exchange (ETDEWEB)

    Krcal, Z; Prekoppova, J; Slottova, A [Vyskumny Ustav Mliekarensky, Zilina (Czechoslovakia)

    1978-12-01

    3 types of winter full-fat sheep cheese were /sup 60/Co gamma irradiated with doses of 75, 100, 200, and 500 krads, this 48 hours after production. Within a week-long storage the organoleptic properties of the cheeses were repeatedly evaluated. The irradiation was found to significantly reduce all microorganism groups. Doses above 100 krads were found to be unsuitable; cheese taste deteriorated and was defined as ''scorched, strange, impure''. Although during storage these defects disappear, the individual main components of proteins and lipids decompose due to enzyme activity, which results in a rapid deterioration of the product. The dose of 75 krads did not cause any taste defects and storage life was extended by four days as against the stated guarantee period.

  7. Feasibility of extending storage life of sheep-milk cheese using ionizing radiation

    International Nuclear Information System (INIS)

    Krcal, Z.; Prekoppova, J.; Slottova, A.

    1978-01-01

    3 types of winter full-fat sheep cheese were 60 Co gamma irradiated with doses of 75, 100, 200, and 500 krads, this 48 hours after production. Within a week-long storage the organoleptic properties of the cheeses were repeatedly evaluated. The irradiation was found to significantly reduce all microorganism groups. Doses above 100 krads were found to be unsuitable; cheese taste deteriorated and was defined as ''scorched, strange, impure''. Although during storage these defects disappear, the individual main components of proteins and lipids decompose due to enzyme activity, which results in a rapid deterioration of the product. The dose of 75 krads did not cause any taste defects and storage life was extended by four days as against the stated guarantee period. (B.S.)

  8. Thermochemical Heat Storage: from Reaction Storage Density to System Storage Density

    NARCIS (Netherlands)

    Jong, A.J. de; Vliet, L.D. van; Hoegaerts, C.L.G.; Roelands, C.P.M.; Cuypers, R.

    2016-01-01

    Long-term and compact storage of solar energy is crucial for the eventual transition to a 100% renewable energy economy. For this, thermochemical materials provide a promising solution. The compactness of a long-term storage system is determined by the thermochemical reaction, operating conditions,

  9. Malaria parasite cGMP-dependent protein kinase regulates blood stage merozoite secretory organelle discharge and egress.

    Directory of Open Access Journals (Sweden)

    Christine R Collins

    2013-05-01

    Full Text Available The malaria parasite replicates within an intraerythrocytic parasitophorous vacuole (PV. Eventually, in a tightly regulated process called egress, proteins of the PV and intracellular merozoite surface are modified by an essential parasite serine protease called PfSUB1, whilst the enclosing PV and erythrocyte membranes rupture, releasing merozoites to invade fresh erythrocytes. Inhibition of the Plasmodium falciparum cGMP-dependent protein kinase (PfPKG prevents egress, but the underlying mechanism is unknown. Here we show that PfPKG activity is required for PfSUB1 discharge into the PV, as well as for release of distinct merozoite organelles called micronemes. Stimulation of PfPKG by inhibiting parasite phosphodiesterase activity induces premature PfSUB1 discharge and egress of developmentally immature, non-invasive parasites. Our findings identify the signalling pathway that regulates PfSUB1 function and egress, and raise the possibility of targeting PfPKG or parasite phosphodiesterases in therapeutic approaches to dysregulate critical protease-mediated steps in the parasite life cycle.

  10. Subunit composition of seed storage proteins in high-protein soybean genotypes Composição de subunidades de proteínas de reserva em genótipos de soja com alto teor de proteína

    Directory of Open Access Journals (Sweden)

    Ksenija Taski-Ajdukovic

    2010-07-01

    Full Text Available The objective of this work was to quantify the accumulation of the major seed storage protein subunits, β-conglycinin and glycinin, and how they influence yield and protein and oil contents in high-protein soybean genotypes. The relative accumulation of subunits was calculated by scanning SDS-PAGE gels using densitometry. The protein content of the tested genotypes was higher than control cultivar in the same maturity group. Several genotypes with improved protein content and with unchanged yield or oil content were developed as a result of new breeding initiatives. This research confirmed that high-protein cultivars accumulate higher amounts of glycinin and β-conglycinin. Genotypes KO5427, KO5428, and KO5429, which accumulated lower quantities of all subunits of glycinin and β-conglycinin, were the only exceptions. Attention should be given to genotypes KO5314 and KO5317, which accumulated significantly higher amounts of both subunits of glycinin, and to genotypes KO5425, KO5319, KO539 and KO536, which accumulated significantly higher amounts of β-conglycinin subunits. These findings suggest that some of the tested genotypes could be beneficial in different breeding programs aimed at the production of agronomically viable plants, yielding high-protein seed with specific composition of storage proteins for specific food applications.O objetivo deste trabalho foi quantificar o acúmulo das principais subunidades de proteínas de reserva da soja, β-conglicinina e glicinina, e como elas influenciam a produtividade e os conteúdos de proteína e de óleo em genótipos de soja com alto conteúdo de proteína. A acumulação relativa de subunidades foi calculada por escaneamento em géis SDS-PAGE, com uso de densitometria. O conteúdo de proteínas dos genótipos testados foi maior que o da cultivar controle dentro do mesmo grupo de maturação. Vários genótipos com conteúdo de proteína aumentado, mas com produtividade ou conteúdo de

  11. Structural and enzymatic characterization of a host-specificity determinant from Salmonella

    Energy Technology Data Exchange (ETDEWEB)

    Kohler, Amanda C. [Rockefeller University, New York, NY 10065 (United States); Spanò, Stefania; Galán, Jorge E. [Yale University School of Medicine, New Haven, CT 06536 (United States); Stebbins, C. Erec, E-mail: stebbins@rockefeller.edu [Rockefeller University, New York, NY 10065 (United States)

    2014-02-01

    The Salmonella effector protein GtgE functions as a cysteine protease to cleave a subset of the Rab-family GTPases and to prevent delivery of antimicrobial agents to the Salmonella-containing vacuole. GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65 Å resolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine protease inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.

  12. Ultrasonication, lyophilization, freezing and storage effects on fat loss during mechanical infusion of expressed human milk

    International Nuclear Information System (INIS)

    Dhar, J.; Davidson, A.G.F.; Martinez, F.E.; Barr, S.; Desai, I.D.; Nakai, S.

    1995-01-01

    Ultrasonic homogenization was extended to situations where expressed human milk needs to be stored before being administered. We investigated whether the effect of ultrasonication would persist during storage in the frozen or lyophilized form. Recovery of fat was higher in ultrasonicated and frozen milk (stored for both 1 and 4 mo), than in milk stored following ultrasonication and lyophilization. The low tat recovery from stored lyophilized milk was increased by ultrasonicating the milk after storage and reconstitution (instead of prior to storage). Protein recovery was virtually complete with both methods

  13. Studies on protein biosynthesis during grain development in relation to protein quality using tracers

    International Nuclear Information System (INIS)

    Mehta, S.L.; Lodha, M.L.

    1975-01-01

    The amino acid imbalance of storage proteins in grains is mainly due to the synthesis of nutritionally poor quality proteins during late maturation stage. In maize, zein fraction which is extremely deficient in lysine but rich in leucine accumulates during late maturation. Opqaue-2 mutant of maize is characterised by an increase in lysine and a decrease in leucine in the endospem protein which is a result of suppression of zein synthesis. In the investigation carried out using H 3 -UTP, it was found that the opaque-2 gene exerts a regulatory central on MRNA synthesis required for zein formation at early stages of maturation. (M.G.B.)

  14. Alternatives for water basin spent fuel storage using pin storage

    International Nuclear Information System (INIS)

    Viebrock, J.M.; Carlson, R.W.

    1979-09-01

    The densest tolerable form for storing spent nuclear fuel is storage of only the fuel rods. This eliminates the space between the fuel rods and frees the hardware to be treated as non-fuel waste. The storage density can be as much as 1.07 MTU/ft 2 when racks are used that just satisfy the criticality and thermal limitations. One of the major advantages of pin storage is that it is compatible with existing racks; however, this reduces the storage density to 0.69 MTU/ft 2 . Even this is a substantial increase over the 0.39 MTU/ft 2 that is achievable with current high capacity stainless steel racks which have been selected as the bases for comparison. Disassembly requires extensive operation on the fuel assembly to remove the upper end fitting and to extract the fuel rods from the assembly skeleton. These operations will be performed with the aid of an elevator to raise the assembly where each fuel rod is grappled. Lowering the elevator will free the fuel rod for transfer to the storage canister. A storage savings of $1510 per MTU can be realized if the pin storage concept is incorporated at a new away-from-reactor facility. The storage cost ranges from $3340 to $7820 per MTU of fuel stored with the lower cost applying to storage at an existing away-from-reactor storage facility and the higher cost applying to at-reactor storage

  15. PROTEIN COMPLEX OF WHEAT, BUCKWHEAT AND MAIZE IN RELATION TO CELIAC DISEASE

    Directory of Open Access Journals (Sweden)

    Milan Chňapek

    2014-02-01

    Full Text Available Cereals are the most wide spread and very important plants utilized as a food source for mankind and for animals where they play role in energetical metabolism and proteosynthesis. Cereals contain proteins with unique properties. These properties allow us to produce leavened bread. Technological characteristic of cereal grain is determined by quantity and quality of storage proteins which represent alcohol soluble prolamins and glutenins soluble in acids and basis solutions. Celiac disease is one of the most frequent food intolerance caused by cereal storage proteins. Therapy consists of strict diet without consumptions of cereals or gluten. Pseudocereals are very perspective groups of plants in gluten free diet, due to absence of celiac active proteins, but on the other hand, flour from pseudocereals is not very suitable for baking. There are a lot of analytical methods applicable for detection of celiac active proteins in cereal and pseudocereal grain. Electrophoretical and immunochemical methods are the most utilized. Genotypes of wheat and maize were homogeneous and singlelined in contrast with genotypes of buckwheat. Average content of HMW-GS was highest in genotypes of bread wheat and lowest in buckwheat varieties. A celiac active fraction of storage proteins (LMW-GS and gliadins was detected at the highest content level in wheat genotypes. Genotypes of buckwheat and maize showed similar low content of this protein fraction. Presence of residual albumins and globulins in buckwheat varieties showed the highest value.

  16. Ectopic expression of Arabidopsis ABC transporter MRP7 modifies cadmium root-to-shoot transport and accumulation

    International Nuclear Information System (INIS)

    Wojas, Sylwia; Hennig, Jacek; Plaza, Sonia; Geisler, Markus; Siemianowski, Oskar; Sklodowska, Aleksandra; Ruszczynska, Anna; Bulska, Ewa; Antosiewicz, Danuta M.

    2009-01-01

    Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications. - AtMRP7 expression in tobacco enhances Cd-tolerance and increases Cd storage in vacuoles

  17. Volume 9 No. 2 2009 March 2009 678 EFFECTS OF STORAGE ...

    African Journals Online (AJOL)

    and storage period on the nutritional and other qualities of stored yam tubers were investigated. .... The nutritional parameters evaluated were moisture, ash, crude protein, crude fat, crude fibre, and phosphorus, calcium and carbohydrate contents. Three tubers from each ..... Asia, Africa and Latin America. 1985: 16, 55 – 58.

  18. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    Science.gov (United States)

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

  19. Long-term aversive taste memory requires insular and amygdala protein degradation.

    Science.gov (United States)

    Rodriguez-Ortiz, Carlos J; Balderas, Israela; Saucedo-Alquicira, Fernando; Cruz-Castañeda, Paulina; Bermudez-Rattoni, Federico

    2011-03-01

    Some reports have shown that the ubiquitin-proteasome system (UPS) is necessary to degrade repressor factors to produce new proteins essential to memory consolidation. Furthermore, recent evidence suggests that memory updating also relies on protein degradation through the UPS. To evaluate whether degradation of proteins is part of the cellular events needed for long-term storage of taste aversion, we injected lactacystin--an UPS inhibitor--into the amygdala and/or insular cortex 30 min before the first or second training trials. The results revealed that degradation of proteins in either the amygdala or insular cortex suffices for long-term stabilization of first-time encounter taste aversion. On the other hand, lactacystin applied in the insula, but not in the amygdala, before the second training prevented long-term storage of updated information. Our results support that degradation of proteins by means of the UPS is required every time taste aversion is to be stored in long-term memory. Copyright © 2010 Elsevier Inc. All rights reserved.

  20. Vps1 in the late endosome-to-vacuole traffic

    Indian Academy of Sciences (India)

    2013-01-13

    Jan 13, 2013 ... the plasma membrane lipids and follows the endocytic inter- nalization pathways ... plasm and found also as punctuate structures (figure 1A). Our explanation ..... 1992 Morphological classification of the yeast vacuolar protein.

  1. Retrievable surface storage: interim storage of solidified high-level waste

    International Nuclear Information System (INIS)

    LaRiviere, J.R.; Nelson, D.C.

    1976-01-01

    Studies have been conducted on retrievable-surface-storage concepts for the interim storage of solidified high-level wastes. These studies have been reviewed by the Panel on Engineered Storage, convened by the Committee on Radioactive Waste Management of the National Research Council-National Academy of Sciences. The Panel has concluded that ''retrievable surface storage is an acceptable interim stage in a comprehensive system for managing high-level radioactive wastes.'' The scaled storage cask concept, which was recommended by the Panel on Engineered Storage, consists of placing a canister of waste inside a carbon-steel cask, which in turn is placed inside a thick concrete cylinder. The waste is cooled by natural convection air flow through an annulus between the cask and the inner wall of the concrete cylinder. The complete assembly is placed above ground in an outdoor storage area

  2. Optimal design and application of a compound cold storage system combining seasonal ice storage and chilled water storage

    NARCIS (Netherlands)

    Yan, C.; Shi, W.; Li, X.; Zhao, Y.

    2016-01-01

    Seasonal cold storage using natural cold sources for cooling is a sustainable cooling technique. However, this technique suffers from limitations such as large storage space and poor reliability. Combining seasonal storage with short-term storage might be a promising solution while it is not

  3. Dietary Management of the Ketogenic Glycogen Storage Diseases

    Directory of Open Access Journals (Sweden)

    Kaustuv Bhattacharya MBBS, MRCPCH, FRACP, MD

    2016-08-01

    Full Text Available The glycogen storage diseases (GSDs comprise a group of rare inherited disorders of glycogen metabolism. The hepatic glycogenolytic forms of these disorders are typically associated with hypoglycemia and hepatomegaly. For GSD I, secondary metabolic disturbances include fasting hyperlactatemia, hyperuricemia, and hyperlipidemia. Glycogen storage disease III is caused by reduced activity of the debrancher enzyme, GSD VI by phosphorylase, and GSD IX by phosphorylase kinase. It has often been reported that the non-GSD I group of disorders have a benign course. However, myopathy, cardiomyopathy, and cirrhosis have been reported significant clinical morbidities associated with GSD III and IX in particular. There have been a range of reports indicating high-protein diets, high-fat diets, medium chain triglyceride (MCT, modified Atkins diet, and therapeutic ketones as rescuing severe phenotypes of GSD III in particular. The etiology of these severe phenotypes has not been defined. Cases presented in this report indicate potential harm from excessive simple sugar use in GSD IX C. Review of the literature indicates that most interventions have reduced the glycemic load and provide alternate substrates for energy in rescue situations. Prevention of complications is most likely to occur with a mixed balanced low glycemic index diet potentially with relative increases in protein.

  4. Preparing and evaluating delivery systems for proteins

    DEFF Research Database (Denmark)

    Jorgensen, L; Moeller, E H; van de Weert, M

    2006-01-01

    From a formulation perspective proteins are complex and therefore challenging molecules to develop drug delivery systems for. The success of a formulation depends on the ability of the protein to maintain the native structure and activity during preparation and delivery as well as during shipping...... and long-term storage of the formulation. Therefore, the development and evaluation of successful and promising drug delivery systems is essential. In the present review, some of the particulate drug delivery systems for parenteral delivery of protein are presented and discussed. The challenge...... for incorporation of protein in particulate delivery systems is exemplified by water-in-oil emulsions....

  5. Method of storing the fuel storage pot in a fuel storage tank for away-from-reactor-storage

    International Nuclear Information System (INIS)

    Ishiguro, Jun-ichi.

    1980-01-01

    Purpose: To prevent the contact of sodium in the away-from-reactor-storage fuel storage tank with sodium in a fuel storage pool having radioactivity ana always retain clean state therein. Method: Sodium is filled in a container body of the away-from-reactor-storage fuel storage tank, and a conduit, a cycling pump, and cooling means are disposed to form a sodium coolant cycling loop. The fuel storage pool is so stored in the container body that the heat of the pool is projected from the liquid surface of the sodium in the container. Therefore, the sodium in the container is isolated from the sodium in the pool containing strong radioactivity to prevent contact of the former sodium from the latter sodium. (Sekiya, K.)

  6. Computed tomographical findings of skeletal muscles in rimmed vacuole type distal myopathy

    International Nuclear Information System (INIS)

    Kunimoto, Masanari; Kawai, Mitsuru; Goto, Jun; Nakano, Imaharu

    1987-01-01

    Skeletal muscle CT scans of three patients with biopsy-proven rimmed vacuole type distal myopathy(RVDM) from two unrelated families showed unique involvement pattern of the lower extremities. Parents of the patients in both families are first cousins. T.Y. is a 36-year-old woman who noticed mild difficulty in walking at 34 years of age. Now she shows waddling but still indpendent gait. Manual muscle test (MMT) revealed the following results: fair (3+/5) for hip flexion, 3 for hip extension, 3 for knee flexion, normal (5/5) for knee extension, poor (2/5) for ankle dorsi-flexion, good (4/5) for ankle plantar flexion. T.M., a 34-year-old younger sister of T.Y., started dragging her feet on gait at age 27. She could walk neither on toes nor heels. At present, she can walk only with support. MMT showed the following: 3- and trace (1/5) for hip flexion and extension, 3- and 4 for knee flexion and extension, 1 for ankle plantar- and dorsiflexion. K.W., a 33-year-old woman, began to drag her foot tips in walk and became unable to walk on toes at age 21. The leg weakness progressed into the wheelchair-ridden state at age 30. MMT gave the following results: 1 for hip flexion and extension, 2 and 4 for knee flexion and extension, zero for plantar- and dorsi-flexion. The most impressive CT findings common to these three patients are prominent contrast between the quadriceps muscles and the adductor- and hamstring-group: the former is markedly well preserved even in the most advanced patient (K.W.) while the latter are diffusely and severely affected even in the least affected patient (T.Y.). This finding well coincides with the results of MMT: the knee extensor (quadriceps muscles) fairly well keeps its strength even in the most advanced patient but the knee flexors (adductors and hamstrings) are definitely affected in the early stage of the condition. (J.P.N.)

  7. Computed tomographical findings of skeletal muscles in rimmed vacuole type distal myopathy

    Energy Technology Data Exchange (ETDEWEB)

    Kunimoto, Masanari; Kawai, Mitsuru; Goto, Jun; Nakano, Imaharu

    1987-03-01

    Skeletal muscle CT scans of three patients with biopsy-proven rimmed vacuole type distal myopathy(RVDM) from two unrelated families showed unique involvement pattern of the lower extremities. Parents of the patients in both families are first cousins. T.Y. is a 36-year-old woman who noticed mild difficulty in walking at 34 years of age. Now she shows waddling but still indpendent gait. Manual muscle test (MMT) revealed the following results: fair (3+/5) for hip flexion, 3 for hip extension, 3 for knee flexion, normal (5/5) for knee extension, poor (2/5) for ankle dorsi-flexion, good (4/5) for ankle plantar flexion. T.M., a 34-year-old younger sister of T.Y., started dragging her feet on gait at age 27. She could walk neither on toes nor heels. At present, she can walk only with support. MMT showed the following: 3- and trace (1/5) for hip flexion and extension, 3- and 4 for knee flexion and extension, 1 for ankle plantar- and dorsiflexion. K.W., a 33-year-old woman, began to drag her foot tips in walk and became unable to walk on toes at age 21. The leg weakness progressed into the wheelchair-ridden state at age 30. MMT gave the following results: 1 for hip flexion and extension, 2 and 4 for knee flexion and extension, zero for plantar- and dorsi-flexion. The most impressive CT findings common to these three patients are prominent contrast between the quadriceps muscles and the adductor- and hamstring-group: the former is markedly well preserved even in the most advanced patient (K.W.) while the latter are diffusely and severely affected even in the least affected patient (T.Y.). This finding well coincides with the results of MMT: the knee extensor (quadriceps muscles) fairly well keeps its strength even in the most advanced patient but the knee flexors (adductors and hamstrings) are definitely affected in the early stage of the condition. (J.P.N.).

  8. Spent fuel storage - dry storage options and issues

    International Nuclear Information System (INIS)

    Akins, M.J.

    2007-01-01

    The increase in the number of nuclear energy power generation facilities will require the ability to store the spent nuclear fuel for a long period until the host countries develop reprocessing or disposal options. Plants have storage pools which are closely associated with the operating units. These are excellent for short term storage, but require active maintenance and operations support which are not desirable for the long term. Over the past 25 years, dry storage options have been developed and implemented throughout the world. In recent years, protection against terrorist attack has become an increasing source of design objectives for these facilities, as well as the main nuclear plant. This paper explores the current design options of dry storage cask systems and examines some of the current design issues for above ground , in-ground, or below-ground storage of spent fuel in dry casks. (author)

  9. CLC-Nt1, a putative chloride channel protein of tobacco, co-localizes with mitochondrial membrane markers.

    Science.gov (United States)

    Lurin, C; Güclü, J; Cheniclet, C; Carde, J P; Barbier-Brygoo, H; Maurel, C

    2000-06-01

    The voltage-dependent chloride channel (CLC) family of membrane proteins has cognates in animals, yeast, bacteria and plants, and chloride-channel activity has been assigned to most of the animal homologues. Lack of evidence of CLC functions in plants prompted us to characterize the cellular localization of the tobacco CLC-Nt1 protein. Specific polyclonal antibodies were raised against an N-terminal polypeptide of CLC-Nt1. These antibodies were used to probe membrane proteins prepared by various cell-fractionation methods. These included aqueous two-phase partitioning (for plasma membranes), free-flow electrophoresis (for vacuolar and plasma membranes), intact vacuole isolation, Percoll-gradient centrifugation (for plastids and mitochondria) and stepped, linear, sucrose-density-gradient centrifugation (for mitochondria). Each purified membrane fraction was characterized with specific marker enzyme activities or antibodies. Our studies ruled out the possibility that the major cell localization of CLC-Nt1 was the vacuolar or plasma membranes, the endoplasmic reticulum, the Golgi apparatus or the plastids. In contrast, we showed that the tobacco CLC-Nt1 specifically co-localized with the markers of the mitochondrial inner membrane, cytochrome c oxidase and NAD9 protein. CLC-Nt1 may correspond to the inner membrane anion channel ('IMAC') described previously in animal and plant mitochondria.

  10. Morphology and evolutionary implications of the annual cycle of secretion and sperm storage in spermathecae of the salamander Ambystoma opacum (Amphibia: Ambystomatidae).

    Science.gov (United States)

    Sever, David M; Krenz, John D; Johnson, Kristin M; Rania, Lisa C

    1995-01-01

    Females of the marbled salamander, Ambystoma opacum, store sperm in exocrine glands called spermathecae in the roof of the cloaca. Eggs are fertilized by sperm released from the spermathecae during oviposition. Some sperm remain in the spermathecae following oviposition, but these sperm degenerate within a month and none persists more than 6 mo after oviposition. Thus, sperm storage between successive breeding seasons does not occur. Apical secretory vaculoes are abundant during the fall mating season and contain a substance that is alcian blue+ at pH 2.5. Production of secretory vacuoles decreases markedly after oviposition, and the glands are inactive by the summer months. Ambystoma opacum is a terrestrial breeder, and some mating occurs prior to arrival at pond basins where oviposition occurs. Mating prior to arrival at the ovipository site may prolong the breeding season, leading to fitness implications for both males and females. Females have opportunities for more matings, and the possibilities for sperm competition in the spermathecae are enhanced. © 1995 Wiley-Liss, Inc. Copyright © 1995 Wiley-Liss, Inc.

  11. Effect of UHT processing and storage conditions on physico-chemical characteristics of buffalo skim milk

    International Nuclear Information System (INIS)

    Hussain, I.

    2011-01-01

    The obtained results indicated that physico-chemical and nutritional changes in UHT processed buffalo skimmed milk were more pronounced at 45 deg. C than 25 deg. C and 10 deg. C. Duration of storage adversely affected the chemical and nutritional quality of processed milk. A slight decrease in pH, total ash and lactose contents, was observed, whereas acidity was increased on the mentioned storage conditions. Total nitrogen and casein nitrogen contents gradually decreased during storage, whereas non-casein nitrogen (NCN) and non-protein nitrogen (NPN) increased to a great extent in samples stored at higher temperatures. A significant increase in hydroxyl methyl furfural (HMF) values occurred in UHT processed buffalo skim milk at 25 deg. C and 45 deg. C after of 90 days storage. Storage at high temperature (45 deg. C) caused undesirable effects on sensory properties, general quality characteristics and acceptability of UHT buffalo skimmed milk. (author)

  12. The effect of gamma irradiation on the chemical composition and digestible crude protein of poultry excreta

    International Nuclear Information System (INIS)

    Al-Masri, M.R.

    1994-06-01

    The changes in the chemical composition, digestible crude protein and cell-wall constituents for two types of excreta of laying hens were studied. In type I, excreta were dried at 170-180 C for 10 minutes whereas in type II dried at 55-60 C for several days. Each type was divided into two parts, the first stored for 3 months with the control. The second part was irradiated by gamma irradiation at 100 KGy and stored for 3 months with the control. The results indicated that here was significant decrease in the crude fibre (CF), NDF and ADF between the samples and the control, for the types I and II where CF, NDF and ADF values, before and after storage, decreased by 12%, 5% and 6%, respectively, ADL values decreased by 8% (I) and 3%(II). Hemicellulose and soluble carbohydrate (NFE) values increased by 5% and 7% for types I and II respectively as a result of irradiation in comparison to the control before and after storage. Gamma irradiation had no effect on crude protein, crude fat, crude ash and digested crude protein for types I and II before and after storage. Drying type I at high temperature in comparison to type II, before and after storage, decreased the crude protein values by 16%. Digested crude protein and CF, decreased 12% and NDF by 7%. Storage of excreta after drying had no effect on the chemical changes due to irradiation for types I and II. (author). 23 refs., 6 figs., 7 tabs

  13. Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

    Science.gov (United States)

    Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

    2013-01-01

    The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

  14. Chemical Properties and Characteristics of Cow Milk Yogurt with Different Additional Fruit and Storage Time

    Directory of Open Access Journals (Sweden)

    Putri Dian Wulansari

    2016-05-01

    Full Text Available This research was aimed to evaluate the composition (total solids, water content, fat and protein, qualitative properties (color, aroma, and texture and quantitative properties (free fatty acid and lactic acid of cow milk yogurt with different fruits addition and storage time. Experimental method applied Completely Randomized Design with five treatments namely control, dragon fruit, mango, apple and banana (20% v/v, each with 5 replicates. Qualitative characteristic assessment was conducted on 0, 5, 10 and 15 days of storage. Result showed that fruit addition significantly affected the composition and characteristics, while storage time significantly affected quantitative characteristics of yogurt. Apple and banana increased 13% total solids of plain yogurt, while the highest fat content (4,516% was observed in control yogurt which had the lowest protein content (2,564. The highest free fatty acid  was in control yogurt ripen for 15 days (22,885% while the lowest free fatty acid was in mango yogurt ripen for 10 days (13,915%. Fruit addition in yogurt ripen for 15 days at 5C resulted in a safe consumed product.

  15. New challenges for the design of high value plant products: stabilization of anthocyanins in plant vacuoles

    Directory of Open Access Journals (Sweden)

    Valentina ePasseri

    2016-02-01

    Full Text Available In the last decade plant biotechnologists and breeders have made several attempt to improve the antioxidant content of plant-derived food. Most efforts concentrated on increasing the synthesis of antioxidants, in particular anthocyanins, by inducing the transcription of genes encoding the synthesizing enzymes. We present here an overview of economically interesting plant species, both food crops and ornamentals, in which anthocyanin content was improved by traditional breeding or transgenesis. Old genetic studies in petunia and more recent biochemical work in brunfelsia, have shown that after synthesis and compartmentalization in the vacuole, anthocyanins need to be stabilized to preserve the color of the plant tissue over time. The final yield of antioxidant molecules is the result of the balance between synthesis and degradation. Therefore the understanding of the mechanism that determine molecule stabilization in the vacuolar lumen is the next step that needs to be taken to further improve the anthocyanin content in food.In several species a phenomenon known as fading is responsible for the disappearance of pigmentation which in some case can be nearly complete. We discuss the present knowledge about the genetic and biochemical factors involved in pigment preservation/destabilization in plant cells.The improvement of our understanding of the fading process will supply new tools for both biotechnological approaches and marker-assisted breeding.

  16. Identification of an exported heat shock protein 70 in Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Grover Manish

    2013-01-01

    Full Text Available Host cell remodelling is a hallmark of malaria pathogenesis. It involves protein folding, unfolding and trafficking events and thus participation of chaperones such as Hsp70s and Hsp40s is well speculated. Until recently, only Hsp40s were thought to be the sole representative of the parasite chaperones in the exportome. However, based on the re-annotated Plasmodium falciparum genome sequence, a putative candidate for exported Hsp70 has been reported, which otherwise was known to be a pseudogene. We raised a specific antiserum against a C-terminal peptide uniquely present in PfHsp70-x. Immunoblotting and immunofluorescence-based approaches in combination with sub-cellular fractionation by saponin and streptolysin-O have been taken to determine the expression and localization of PfHsp70-x in infected erythrocyte. The re-annotated sequence of PfHsp70-x reveals it to be a functional protein with an endoplasmic reticulum signal peptide. It gets maximally expressed at the schizont stage of intra-erythrocytic life cycle. Majority of the protein localizes to the parasitophorous vacuole and some of it gets exported to the erythrocyte compartment where it associates with Maurer’s clefts. The identification of an exported parasite Hsp70 chaperone presents us with the fact that the parasite has evolved customized chaperones which might be playing crucial roles in aspects of trafficking and host cell remodelling.

  17. Biochemical Aspects of a Serine Protease from Caesalpinia echinata Lam. (Brazilwood Seeds: A Potential Tool to Access the Mobilization of Seed Storage Proteins

    Directory of Open Access Journals (Sweden)

    Priscila Praxedes-Garcia

    2012-01-01

    Full Text Available Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (Km 55.7 μM in an optimum pH of 7.1, and this activity is effectively retained until 50∘C. CeSP remained stable in the presence of kosmotropic anions (PO4 3−, SO4 2−, and CH3COO− or chaotropic cations (K+ and Na+. It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

  18. Application of gamma irradiation for storage potato

    International Nuclear Information System (INIS)

    Rezaee, M.; Almasi, M.

    2009-01-01

    Since deficiency of controlled store in Iran and environmental problems of chemical material the use of gamma irradiation to control sprouting and increase the length of storage time of potatoes has been proposed as an alternative to cold storage or the use of chemical sprout suppressants. In this study potatoes of Agria Variety were irradiated at a dose of 0.10 KGY and stored along with the unirradiated controls at 12±3°C for a period of more than 6 month from October to April .After 4 month of storage the sprouting ranged from 5 to 12% in irradiated potatoes and 45 to 74% in unirradiated samples and after 6 month the unirradiated potatoes were discarded because of heavy sprouting and rotting. The rot attack was approximately double in unirradiated samples. It was found that losses through dehydration were 10.3 to 15.1 % in the irradiated potatoes. Also a comparative study of reducing and non-reducing sugars, vitamin-C content, total sugar, starch and protein was carried out between unirradiated and irradiated samples. The results suggested the efficacy of Gamma irradiation for ensuring availability of the storing quality of potato during lean periods from October to April. (author)

  19. Symplastic Transport of Carboxyfluorescein in Staminal Hairs of Setcreasea purpurea Is Diffusive and Includes Loss to the Vacuole.

    Science.gov (United States)

    Tucker, J E; Mauzerall, D; Tucker, E B

    1989-07-01

    The kinetics of symplastic transport in staminal hairs of Setcreasea purpurea was studied. The tip cell of a staminal hair was microinjected with carboxyfluorescein (CF) and the symplastic transport of this CF was videotaped and the digital data analyzed to produce kinetic curves. Using a finite difference equation for diffusion between cells and for loss of dye into the vacuole, kinetic curves were calculated and fitted to the observed data. These curves were matched with data from actual microinjection experiments by adjusting K (the coefficient of intercellular junction diffusion) and L (the coefficient of intracellular loss) until a minimum in the least squares difference between the curves was obtained. (a) Symplastic transport of CF was governed by diffusion through intercellular pores (plasmodesmata) and intracellular loss. Diffusion within the cell cytoplasm was never limiting. (b) Each cell and its plasmodesmata must be considered as its own diffusion system. Therefore, a diffusion coefficient cannot be calculated for an entire chain of cells. (c) The movement through plasmodesmata in either direction was the same since the data are fit by a diffusion equation. (d) Diffusion through the intercellular pores was estimated to be slower than diffusion through similar pores filled with water.

  20. Approaches to improving the nutritional quality of barley seed proteins

    International Nuclear Information System (INIS)

    Shewry, P.R.; Bright, S.W.J.; Burgess, S.R.; Miflin, B.J.

    1984-01-01

    The poor nutritional quality of barley grain is determined by the low level of lysine in the prolamin storage proteins (hordein). These account for between 35 to 50% of the total grain nitrogen, depending on the nutritional status of the plant. There is a reduced proportion of hordein in mutant high lysine lines but these also have reduced synthesis of storage carbohydrates and hence low yields. Three strategies for improvement are discussed. Increases in the lysine content of hordein may be difficult to achieve because of the presence of complex families of structural genes. It would also be necessary to insert a large number of additional lysine residues. Two more promising approaches are to increase the level of expression of genes coding for lysine-rich globulin storage proteins and to increase the pool of free lysine by selecting mutant lines with relaxed feedback regulation of lysine synthesis. (author)