WorldWideScience

Sample records for microbial protein formation

  1. Microbial processes in banded iron formation deposition

    DEFF Research Database (Denmark)

    Posth, Nicole; Konhauser, Kurt; Kappler, Andreas

    2013-01-01

    , remains unresolved. Evidence of an anoxic Earth with only localized oxic areas until the Great Oxidation Event ca 2·45 to 2·32 Ga makes the investigation of O2-independent mechanisms for banded iron formation deposition relevant. Recent studies have explored the long-standing proposition that Archean......Banded iron formations have been studied for decades, particularly regarding their potential as archives of the Precambrian environment. In spite of this effort, the mechanism of their deposition and, specifically, the role that microbes played in the precipitation of banded iron formation minerals...... banded iron formations may have been formed, and diagenetically modified, by anaerobic microbial metabolisms. These efforts encompass a wide array of approaches including isotope, ecophysiological and phylogeny studies, molecular and mineral marker analysis, and sedimentological reconstructions. Herein...

  2. Fermentable sugars and microbial inhibitors formation from two ...

    African Journals Online (AJOL)

    ... under low severity factor and its enzymatic degradability was investigated in this ... The highest glucan conversion and recovery at the optimum conditions were ... reduce microbial inhibitors formation and excessive biomass processing cost.

  3. Spatial & Temporal Geophysical Monitoring of Microbial Growth and Biofilm Formation

    Science.gov (United States)

    Previous studies have examined the effect of biogenic gases and biomineralization on the acoustic properties of porous media. In this study, we investigated the spatiotemporal effect of microbial growth and biofilm formation on compressional waves and complex conductivity in sand...

  4. Selenite reduction by anaerobic microbial aggregates: Microbial community structure, and proteins associated to the produced selenium spheres.

    Directory of Open Access Journals (Sweden)

    Graciela eGonzalez-Gil

    2016-04-01

    Full Text Available Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0, insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20 % and Pseudomonadaceae (c.a.10 % were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (~200 m of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

  5. Selenite Reduction by Anaerobic Microbial Aggregates: Microbial Community Structure, and Proteins Associated to the Produced Selenium Spheres

    KAUST Repository

    Gonzalez-Gil, Graciela

    2016-04-26

    Certain types of anaerobic granular sludge, which consists of microbial aggregates, can reduce selenium oxyanions. To envisage strategies for removing those oxyanions from wastewater and recovering the produced elemental selenium (Se0), insights into the microbial community structure and synthesis of Se0 within these microbial aggregates are required. High-throughput sequencing showed that Veillonellaceae (c.a. 20%) and Pseudomonadaceae (c.a.10%) were the most abundant microbial phylotypes in selenite reducing microbial aggregates. The majority of the Pseudomonadaceae sequences were affiliated to the genus Pseudomonas. A distinct outer layer (∼200 μm) of selenium deposits indicated that bioreduction occurred in the outer zone of the microbial aggregates. In that outer layer, SEM analysis showed abundant intracellular and extracellular Se0 (nano)spheres, with some cells having high numbers of intracellular Se0 spheres. Electron tomography showed that microbial cells can harbor a single large intracellular sphere that stretches the cell body. The Se0 spheres produced by the microorganisms were capped with organic material. X-ray photoelectron spectroscopy (XPS) analysis of extracted Se0 spheres, combined with a mathematical approach to analyzing XPS spectra from biological origin, indicated that proteins and lipids were components of the capping material associated to the Se0 spheres. The most abundant proteins associated to the spheres were identified by proteomic analysis. Most of the proteins or peptide sequences capping the Se0 spheres were identified as periplasmic outer membrane porins and as the cytoplasmic elongation factor Tu protein, suggesting an intracellular formation of the Se0 spheres. In view of these and previous findings, a schematic model for the synthesis of Se0 spheres by the microorganisms inhabiting the granular sludge is proposed.

  6. Coexistence facilitates interspecific biofilm formation in complex microbial communities

    DEFF Research Database (Denmark)

    Madsen, Jonas Stenløkke; Røder, Henriette Lyng; Russel, Jakob

    2016-01-01

    Social interactions in which bacteria respond to one another by modifying their phenotype are central determinants of microbial communities. It is known that interspecific interactions influence the biofilm phenotype of bacteria; a phenotype that is central to the fitness of bacteria. However......, the underlying role of fundamental ecological factors, specifically coexistence and phylogenetic history, in biofilm formation remains unclear. This study examines how social interactions affect biofilm formation in multi-species co-cultures from five diverse environments. We found prevalence of increased...

  7. Investigating Microbial Biofilm Formations on Crustal Rock Substrates

    Science.gov (United States)

    Weiser, M.; D'Angelo, T.; Carr, S. A.; Orcutt, B.

    2017-12-01

    Ocean crust hosts microbial life that, in some cases, alter the component rocks as a means of obtaining energy. Variations in crust lithology, included trace metal and mineral content, as well as the chemistry of the fluids circulating through them, provide substrates for some microbes to metabolize, leading to formation of biofilm community structures. Microbes have different parameters for the situations in which they will form biofilms, but they must have some source of energy in excess at the site of biofilm formation for them to become stationary and form the carbohydrate-rich structures connecting the cells to one another and the substrate. Generally, the requirements for microbes to form biofilms on crustal minerals are unclear. We designed two experiments to test (1) mineral preference and biofilm formation rates by natural seawater microbial communities, and (2) biofilm development as a function of phosphate availability for an organism isolated from subseafloor ocean crust. In Experiment 1, we observed that phyric basalt groundmass is preferentially colonized over aphyric basalt or metal sulfides in a shallow water and oxic seawater environment. In experiment 2, tests of the anaerobic heterotroph Thalassospira bacteria isolated from oceanic crustal fluids showed that they preferentially form biofilms, lose motility, and increase exponentially in number over time in higher-PO4 treatments (50 micromolar), including with phosphate-doped basalts, than in treatments with low phosphate concentrations (0.5 micromolar) often found in crustal fluids. These observations suggest phosphate as a main driver of biofilm formation in subsurface crust. Overall, these data suggest that the drivers of microbial biofilm formation on crustal substrates are selective to the substrate conditions, which has important implications for estimating the global biomass of life harbored in oceanic crust.

  8. Microbial communities in bentonite formations and their interactions with uranium

    International Nuclear Information System (INIS)

    López-Fernández, Margarita; Fernández-Sanfrancisco, Omar; Moreno-García, Alberto; Martín-Sánchez, Inés; Sánchez-Castro, Iván; Merroun, Mohamed Larbi

    2014-01-01

    Highlights: • Microbial diversity of Spanish bentonites was studied. • High number of aerobe and facultative anaerobe microbes were isolated from bentonites. • Natural bentonite microbes are able to tolerate high U concentrations. • U is immobilized by the cells of the strain Rhodotorula mucilaginosa BII-R8 as U(VI) phosphates. - Abstract: A reliable performance assessment of deep geological disposal of nuclear waste depends on better knowledge of radionuclide interactions with natural microbes of geological formations (granitic rock, clay, salts) used to host these disposal systems. In Spain, clay deposits from Cabo de Gata region, Almeria, are investigated for this purpose. The present work characterizes the culture-dependent microbial diversity of two bentonite samples (BI and BII) recovered from Spanish clay deposits. The evaluation of aerobe and facultative anaerobe microbial populations shows the presence of a high number of cultivable bacteria (e.g. Stenotrophomonas, Micrococcus, Arthrobacter, Kocuria, Sphingomonas, Bacillus, Pseudomonas, etc.) affiliated to three phyla Proteobacteria, Actinobacteria, and Firmicutes. In addition, a pigmented yeast strain BII-R8 related to Rhodotorula mucilaginosa was also recovered from these formations. The minimal inhibitory concentrations of uranium for the growth of these natural isolates were found to range from 4 to 10.0 mM. For instance, strain R. mucilaginosa BII-R8 was shown to tolerate up to 8 mM of U. Flow cytometry studies indicated that the high U tolerance of this yeast isolate is a biologically mediated process. Microscopically dense intracellular and cell wall-bound precipitates were observed by Scanning Transmission Electron Microscopy-High-Angle Annular Dark-Field (STEM-HAADF). Energy Dispersive X-ray (EDX) element-distribution maps showed the presence of U and P within these accumulates, indicating the ability of cells to precipitate U as U(VI) phosphate minerals. Fundamental understanding of the

  9. Microbial communities in bentonite formations and their interactions with uranium

    Energy Technology Data Exchange (ETDEWEB)

    López-Fernández, Margarita; Fernández-Sanfrancisco, Omar; Moreno-García, Alberto; Martín-Sánchez, Inés; Sánchez-Castro, Iván; Merroun, Mohamed Larbi, E-mail: merroun@ugr.es

    2014-10-15

    Highlights: • Microbial diversity of Spanish bentonites was studied. • High number of aerobe and facultative anaerobe microbes were isolated from bentonites. • Natural bentonite microbes are able to tolerate high U concentrations. • U is immobilized by the cells of the strain Rhodotorula mucilaginosa BII-R8 as U(VI) phosphates. - Abstract: A reliable performance assessment of deep geological disposal of nuclear waste depends on better knowledge of radionuclide interactions with natural microbes of geological formations (granitic rock, clay, salts) used to host these disposal systems. In Spain, clay deposits from Cabo de Gata region, Almeria, are investigated for this purpose. The present work characterizes the culture-dependent microbial diversity of two bentonite samples (BI and BII) recovered from Spanish clay deposits. The evaluation of aerobe and facultative anaerobe microbial populations shows the presence of a high number of cultivable bacteria (e.g. Stenotrophomonas, Micrococcus, Arthrobacter, Kocuria, Sphingomonas, Bacillus, Pseudomonas, etc.) affiliated to three phyla Proteobacteria, Actinobacteria, and Firmicutes. In addition, a pigmented yeast strain BII-R8 related to Rhodotorula mucilaginosa was also recovered from these formations. The minimal inhibitory concentrations of uranium for the growth of these natural isolates were found to range from 4 to 10.0 mM. For instance, strain R. mucilaginosa BII-R8 was shown to tolerate up to 8 mM of U. Flow cytometry studies indicated that the high U tolerance of this yeast isolate is a biologically mediated process. Microscopically dense intracellular and cell wall-bound precipitates were observed by Scanning Transmission Electron Microscopy-High-Angle Annular Dark-Field (STEM-HAADF). Energy Dispersive X-ray (EDX) element-distribution maps showed the presence of U and P within these accumulates, indicating the ability of cells to precipitate U as U(VI) phosphate minerals. Fundamental understanding of the

  10. Manufacturing of recombinant therapeutic proteins in microbial systems.

    Science.gov (United States)

    Graumann, Klaus; Premstaller, Andreas

    2006-02-01

    Recombinant therapeutic proteins have gained enormous importance for clinical applications. The first recombinant products have been produced in E. coli more than 20 years ago. Although with the advent of antibody-based therapeutics mammalian expression systems have experienced a major boost, microbial expression systems continue to be widely used in industry. Their intrinsic advantages, such as rapid growth, high yields and ease of manipulation, make them the premier choice for expression of non-glycosylated peptides and proteins. Innovative product classes such as antibody fragments or alternative binding molecules will further expand the use of microbial systems. Even more, novel, engineered production hosts and integrated technology platforms hold enormous potential for future applications. This review summarizes current applications and trends for development, production and analytical characterization of recombinant therapeutic proteins in microbial systems.

  11. Acoustic and Electrical Property Changes Due to Microbial Growth and Biofilm Formation in Porous Media

    Science.gov (United States)

    A laboratory study was conducted to investigate the effect of microbial growth and biofilm formation on compressional waves, and complex conductivity during stimulated microbial growth. Over the 29 day duration of the experiment, compressional wave amplitudes and arrival times f...

  12. COMPOSITION AND METHOD FOR CONTROLLING MICROBIAL ADHESION AND BIOFILM FORMATION OF SURFACES

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention describes how coating of surfaces with an extract, particularly a fish extract, can significantly reduce microbial adhesion, attachment, colonization and biofilm formation on surfaces. Such reduction of microbial adherence, attachment and colonization will be applicable...

  13. [Formation of microbial populations on the surface of protective coatings].

    Science.gov (United States)

    Kopteva, Zh P; Zanina, V V; Piliashenko-Novokhatnyĭ, A I; Kopteva, A E; Kozlova, I A

    2001-01-01

    Formation of microbial cenosis on the surface of polyethylene-, polyurethane- and oil-bitumen-based protective coatings was studied in dynamics during 1, 3, 7, 14 and 21 days. It has been shown that the biofilm was formed on the protective materials during 14 days and consisted of ammonifying, denitrifying, hydrocarbon-oxidizing and sulphate-reducing bacteria referred to Pseudomonas, Arthrobacter, Bacillus and Kesulfovibrio genera. The bacteria which form the biofilm on coatings possess high denitrifying and sulphate-reducing activities. Corrosion inhibitors-biocydes, introduced in composition of oil-bitumen coatings suppressed growth and metabolic activity of corrosion-active bacteria.

  14. Spatial & Temporal Geophysical Monitoring of Microbial Growth and Biofilm Formation

    Science.gov (United States)

    Davis, C. A.; Pyrak-Nolte, L. J.; Atekwana, E. A.; Werkema, D. D.; Haugen, M. E.

    2009-12-01

    Previous studies have examined the effect of biogenic gases and biomineralization on the acoustic properties of porous media. In this study, we investigated the spatiotemporal effect of microbial growth and biofilm formation on compressional waves and complex conductivity in sand columns. A control column (non-biostimulated) and a biostimulated column were studied in a 2D acoustic scanning apparatus, and a second set of columns were constructed with Ag-AgCl electrodes for complex conductivity measurements. At the completion of the 29-day experiment, compressional wave amplitudes and arrival times for the control column were observed to be relatively uniform over the scanned 2D region. However, the biostimulated sample exhibited a high degree of spatial variability within the column for both the amplitude and arrival times. Furthermore, portions of the sample exhibited increased attenuation (~ 80%) concurrent with an increase in the arrival times, while other portions exhibited decreased attenuation (~ 45%) and decreased arrival time. The acoustic amplitude and arrival times changed significantly in the biostimulated column between Days 5 and 7 of the experiment and are consistent with a peak in the imaginary conductivity (σ”) values. The σ” response corresponds to different stages of biofilm development. That is, we interpret the peak σ” with the maximum biofilm thickness and decreasing σ” due to cell death or detachment. Environmental scanning electron microscope (ESEM) imaging confirmed microbial cell attachment to sand surfaces in the biostimulated columns, showed apparent differences in the morphology of attached biomass between regions of increased and decreased attenuation, and indicated no mineral precipitation or biomineralization. The heterogeneity in the elastic properties arises from the differences in the morphology and structure of attached biofilms. These results suggest that combining acoustic imaging and complex conductivity techniques

  15. Chimeric microbial rhodopsins for optical activation of Gs-proteins

    Science.gov (United States)

    Yoshida, Kazuho; Yamashita, Takahiro; Sasaki, Kengo; Inoue, Keiichi; Shichida, Yoshinori; Kandori, Hideki

    2017-01-01

    We previously showed that the chimeric proteins of microbial rhodopsins, such as light-driven proton pump bacteriorhodopsin (BR) and Gloeobacter rhodopsin (GR) that contain cytoplasmic loops of bovine rhodopsin, are able to activate Gt protein upon light absorption. These facts suggest similar protein structural changes in both the light-driven proton pump and animal rhodopsin. Here we report two trials to engineer chimeric rhodopsins, one for the inserted loop, and another for the microbial rhodopsin template. For the former, we successfully activated Gs protein by light through the incorporation of the cytoplasmic loop of β2-adrenergic receptor (β2AR). For the latter, we did not observe any G-protein activation for the light-driven sodium pump from Indibacter alkaliphilus (IndiR2) or a light-driven chloride pump halorhodopsin from Natronomonas pharaonis (NpHR), whereas the light-driven proton pump GR showed light-dependent G-protein activation. This fact suggests that a helix opening motion is common to G protein coupled receptor (GPCR) and GR, but not to IndiR2 and NpHR. Light-induced difference FTIR spectroscopy revealed similar structural changes between WT and the third loop chimera for each light-driven pump. A helical structural perturbation, which was largest for GR, was further enhanced in the chimera. We conclude that similar structural dynamics that occur on the cytoplasmic side of GPCR are needed to design chimeric microbial rhodopsins. PMID:29362703

  16. Fibril formation from pea protein and subsequent gel formation.

    Science.gov (United States)

    Munialo, Claire Darizu; Martin, Anneke H; van der Linden, Erik; de Jongh, Harmen H J

    2014-03-19

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20 h at pH 2.0. Following heating of pea proteins, it was observed that all of the proteins were hydrolyzed into peptides and that 50% of these peptides were assembled into fibrils. Changes on a structural level in pea proteins were studied using circular dichroism, transmission electron microscopy, and particle size analysis. During the fibril assembly process, an increase in aggregate size was observed, which coincided with an increase in thioflavin T binding, indicating the presence of β-sheet aggregates. Fibrils made using pea proteins were more branched and curly. Gel formation of preformed fibrils was induced by slow acidification from pH 7.0 to a final pH of around pH 5.0. The ability of pea protein-based fibrillar gels to fracture during an amplitude sweep was comparable to those of soy protein and whey protein-based fibrillar gels, although gels prepared from fibrils made using pea protein and soy protein were weaker than those of whey protein. The findings show that fibrils can be prepared from pea protein, which can be incorporated into protein-based fibrillar gels.

  17. Digestion and microbial protein synthesis in sheep as affected by ...

    African Journals Online (AJOL)

    Useni , Alain

    enzyme (EFE) on the in vitro gas production (GP) and ANKOM digestion systems on the mixture of milled ... determine the EFE effect on the DM, CP and NDF digestion of a mixture of lucerne hay and wheat straw .... and the microbial protein synthesis (MPS) measured as purine derivates (RNA equivalent in µg/DM g) on.

  18. Microbial degradation of dissolved proteins in seawater

    International Nuclear Information System (INIS)

    Hollibaugh, J.T.; Azam, F.

    1983-01-01

    An experimental protocol using radiolabeled proteins was developed to investigate the rates and mechanisms whereby dissolved proteins are degraded in natural marine plankton communities. The results of field observations and laboratory experiments indicate that proteins are degraded by a particle-bound, thermolabile system, presumably bacteria-associated enzymes, with an apparent half-saturation constant of ca. 25 μg bovine serum albumin (BSA) per liter. Gel permeation chromatography indicated that peptides of chain length intermediate between BSA and the final products of degradation (MW<700) do not accumulate in the medium. Competition experiments indicate that the system is relatively nonspecific. Turnover rates for the protein pool in samples collected in the Southern California Bight were of the same order of magnitude as the turnover rate of the L-leucine pool and were correlated with primary productivity, chlorophyll a concentrations, bacterial abundance and biomass, and L-leucine turnover rate. These data suggest that amino acids derived from proteins are utilized preferentially and do not completely mix with the amino acids in the bulk phase

  19. Truly Absorbed Microbial Protein Synthesis, Rumen Bypass Protein, Endogenous Protein, and Total Metabolizable Protein from Starchy and Protein-Rich Raw Materials

    NARCIS (Netherlands)

    Parand, Ehsan; Vakili, Alireza; Mesgaran, Mohsen Danesh; Duinkerken, Van Gert; Yu, Peiqiang

    2015-01-01

    This study was carried out to measure truly absorbed microbial protein synthesis, rumen bypass protein, and endogenous protein loss, as well as total metabolizable protein, from starchy and protein-rich raw feed materials with model comparisons. Predictions by the DVE2010 system as a more

  20. Interaction between Protein, Phytate, and Microbial Phytase. In Vitro Studies

    NARCIS (Netherlands)

    Kies, A.K.; Jonge, de L.H.; Kemme, P.A.; Jongbloed, A.W.

    2006-01-01

    The interaction between protein and phytate was investigated in vitro using proteins extracted from five common feedstuffs and from casein. The appearance of naturally present soluble protein-phytate complexes in the feedstuffs, the formation of complexes at different pHs, and the degradation of

  1. PATtyFams: Protein families for the microbial genomes in the PATRIC database

    Directory of Open Access Journals (Sweden)

    James J Davis

    2016-02-01

    Full Text Available The ability to build accurate protein families is a fundamental operation in bioinformatics that influences comparative analyses, genome annotation and metabolic modeling. For several years we have been maintaining protein families for all microbial genomes in the PATRIC database (Pathosystems Resource Integration Center, patricbrc.org in order to drive many of the comparative analysis tools that are available through the PATRIC website. However, due to the burgeoning number of genomes, traditional approaches for generating protein families are becoming prohibitive. In this report, we describe a new approach for generating protein families, which we call PATtyFams. This method uses the k-mer-based function assignments available through RAST (Rapid Annotation using Subsystem Technology to rapidly guide family formation, and then differentiates the function-based groups into families using a Markov Cluster algorithm (MCL. This new approach for generating protein families is rapid, scalable and has properties that are consistent with alignment-based methods.

  2. Pharmaceutical protein production by yeast: towards production of human blood proteins by microbial fermentation

    DEFF Research Database (Denmark)

    Martinez Ruiz, José Luis; Liu, Lifang; Petranovic, Dina

    2012-01-01

    Since the approval of recombinant insulin from Escherichia coli for its clinical use in the early 1980s, the amount of recombinant pharmaceutical proteins obtained by microbial fermentations has significantly increased. The recent advances in genomics together with high throughput analysis...... of recombinant therapeutics using yeast Saccharomyces cerevisiae as a model platform, and discusses the future potential of this platform for production of blood proteins and substitutes....

  3. Ammonia treatment of wheat straw. 2. Efficiency of microbial protein synthesis, rumen microbial protein pool size and turnover, and small intestinal protein digestion in sheep.

    NARCIS (Netherlands)

    Oosting, S.J.; Viets, T.C.; Lammers-Wienhoven, S.C.W.; Bruchem, van J.

    1993-01-01

    Ammonia-treated wheat straw (AWS) was compared with untreated wheat straw (UWS) and untreated wheat straw supplemented with urea (SWS) in an experiment with 6 wether sheep. Microbial protein synthesis increased after ammonia treatment due to the higher intake of rumen degradable organic matter (OM).

  4. Formation and Stability of Microbially Derived Soil Organic Matter

    Science.gov (United States)

    Waldrop, M. P.; Creamer, C.; Foster, A. L.; Lawrence, C. R.; Mcfarland, J. W.; Schulz, M. S.

    2017-12-01

    Soil carbon is vital to soil health, food security, and climate change mitigation, but the underlying mechanisms controlling the stabilization and destabilization of soil carbon are still poorly understood. There has been a conceptual paradigm shift in how soil organic matter is formed which now emphasizes the importance of microbial activity to build stable (i.e. long-lived) and mineral-associated soil organic matter. In this conceptual model, the consumption of plant carbon by microorganisms, followed by subsequent turnover of microbial bodies closely associated with mineral particles, produces a layering of amino acid and lipid residues on the surfaces of soil minerals that remains protected from destabilization by mineral-association and aggregation processes. We tested this new model by examining how isotopically labeled plant and microbial C differ in their fundamental stabilization and destabilization processes on soil minerals through a soil profile. We used a combination of laboratory and field-based approaches to bridge multiple spatial scales, and used soil depth as well as synthetic minerals to create gradients of soil mineralogy. We used Raman microscopy as a tool to probe organic matter association with mineral surfaces, as it allows for the simultaneous quantification and identification of living microbes, carbon, minerals, and isotopes through time. As expected, we found that the type of minerals present had a strong influence on the amount of C retained, but the stabilization of new C critically depends on growth, death, and turnover of microbial cells. Additionally, the destabilization of microbial residue C on mineral surfaces was little affected by flushes of DOC relative to wet-dry cycles alone. We believe this new insight into microbial mechanisms of C stabilization in soils will eventually lead to new avenues for measuring and modeling SOM dynamics in soils, and aid in the management of soil C to mediate global challenges.

  5. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

    2006-01-01

    Microbial biofilm formation often causes problems in medical and industrial settings, and knowledge about the factors that are involved in biofilm development and dispersion is useful for creating strategies to control the processes. In this report, we present evidence that proteins with GGDEF...... and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli Yhj......H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

  6. Microbial biofilm formation and its consequences for the CELSS program

    Science.gov (United States)

    Mitchell, R.

    1994-01-01

    A major goal of the Controlled Ecology Life Support System (CELSS) program is to provide reliable and efficient life support systems for long-duration space flights. A principal focus of the program is on the growth of higher plants in growth chambers. These crops should be grown without the risk of damage from microbial contamination. While it is unlikely that plant pathogens will pose a risk, there are serious hazards associated with microorganisms carried in the nutrient delivery systems and in the atmosphere of the growth chamber. Our experience in surface microbiology showed that colonization of surfaces with microorganisms is extremely rapid even when the inoculum is small. After initial colonization extensive biofilms accumulate on moist surfaces. These microbial films metabolize actively and slough off continuously to the air and water. During plant growth in the CELSS program, microbial biofilms have the potential to foul sensors and to plug nutrient delivery systems. In addition both metabolic products of microbial growth and degradation products of materials being considered for use as nutrient reservoirs and for delivery are likely sources of chemicals known to adversly affect plant growth.

  7. Microbial Formation of Ethane in Anoxic Estuarine Sediments

    OpenAIRE

    Oremland, Ronald S.

    1981-01-01

    Estuarine sediment slurries produced methane and traces of ethane when incubated under hydrogen. Formation of methane occurred over a broad temperature range with an optimum above 65°C. Ethane formation had a temperature optimum at 40°C. Formation of these two gases was inhibited by air, autoclaving, incubation at 4 and 80°C, and by the methanogenic inhibitor, 2-bromoethanesulfonic acid. Ethane production was stimulated by addition of ethylthioethanesulfonic acid, and production from ethylthi...

  8. A fermented meat model system for studies of microbial aroma formation

    DEFF Research Database (Denmark)

    Tjener, Karsten; Stahnke, Louise Heller; Andersen, L.

    2003-01-01

    A fermented meat model system was developed, by which microbial formation of volatiles could be examined The model was evaluated against dry, fermented sausages with respect to microbial growth, pH and volatile profiles. Fast and slowly acidified sausages and models were produced using the starte......H, microbial growth and volatile profiles was similar to sausage production. Based on these findings, the model system was considered valid for studies of aroma formation of meat cultures for fermented sausage.......A fermented meat model system was developed, by which microbial formation of volatiles could be examined The model was evaluated against dry, fermented sausages with respect to microbial growth, pH and volatile profiles. Fast and slowly acidified sausages and models were produced using the starter...... cultures Pediococcus pentosaceus and Staphylococcus xylosus. Volatiles were collected and analysed by dynamic headspace sampling and GC MS. The analysis was primarily focused on volatiles arising from amino acid degradation and a total of 24 compounds, of which 19 were quantified, were used...

  9. Formation of palladium(0) nanoparticles at microbial surfaces

    DEFF Research Database (Denmark)

    Bunge, Michael; Søbjerg, Lina S; Rotaru, Amelia-Elena

    2010-01-01

    ) nanoparticles were still deposited on autoclaved cells of C. necator that had no hydrogenase activity, suggesting a hydrogenase-independent formation mechanism. The catalytic properties of Pd(0) and bioPd(0) were determined by the amount of hydrogen released in a reaction with hypophosphite. Generally, bioPd(0...... potential. Hitherto, bacteria with the property of dissimilatory metal reduction have been in focus, although the biochemical reactions linking enzymatic Pd(II) reduction and Pd(0) deposition have not yet been identified. In this study we investigated Pd(II) reduction with formate as the electron donor......) demonstrated a lower level of activity than the Pd(0) control, possibly due to the inaccessibility of the Pd(0) fraction embedded in the cell envelope. Our results demonstrate the suitability of bacterial cells for the recovery of Pd(0), and formation and immobilization of Pd(0) nanoparticles inside the cell...

  10. A fermented meat model system for studies of microbial aroma formation

    DEFF Research Database (Denmark)

    Tjener, Karsten; Stahnke, Louise Heller; Andersen, L.

    2003-01-01

    A fermented meat model system was developed, by which microbial formation of volatiles could be examined The model was evaluated against dry, fermented sausages with respect to microbial growth, pH and volatile profiles. Fast and slowly acidified sausages and models were produced using the starter......H, microbial growth and volatile profiles was similar to sausage production. Based on these findings, the model system was considered valid for studies of aroma formation of meat cultures for fermented sausage....... for multivariate data analysis. Growth of lactic acid bacteria was comparable for model and sausages, whereas survival of S. xylosus was better in the model. Multivariate analysis of volatiles showed that differences between fast and slowly acidified samples were identical for model and sausage. For both sausage...

  11. High-throughput metagenomic technologies for complex microbial community analysis: open and closed formats.

    Science.gov (United States)

    Zhou, Jizhong; He, Zhili; Yang, Yunfeng; Deng, Ye; Tringe, Susannah G; Alvarez-Cohen, Lisa

    2015-01-27

    Understanding the structure, functions, activities and dynamics of microbial communities in natural environments is one of the grand challenges of 21st century science. To address this challenge, over the past decade, numerous technologies have been developed for interrogating microbial communities, of which some are amenable to exploratory work (e.g., high-throughput sequencing and phenotypic screening) and others depend on reference genes or genomes (e.g., phylogenetic and functional gene arrays). Here, we provide a critical review and synthesis of the most commonly applied "open-format" and "closed-format" detection technologies. We discuss their characteristics, advantages, and disadvantages within the context of environmental applications and focus on analysis of complex microbial systems, such as those in soils, in which diversity is high and reference genomes are few. In addition, we discuss crucial issues and considerations associated with applying complementary high-throughput molecular technologies to address important ecological questions. Copyright © 2015 Zhou et al.

  12. Fibril Formation from Pea Protein and Sesequent Gel Formation

    NARCIS (Netherlands)

    Munialo, C.D.; Martin, A.H.; Linden, van der E.; Jongh, de H.H.J.

    2014-01-01

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20

  13. Fibril Formation from Pea Protein and Subsequent Gel Formation

    NARCIS (Netherlands)

    Munialo, XC.D.; Martin, A.H.; Linden, E. van der; Jongh, H.H.J de

    2014-01-01

    The objective of this study was to characterize fibrillar aggregates made using pea proteins, to assemble formed fibrils into protein-based gels, and to study the rheological behavior of these gels. Micrometer-long fibrillar aggregates were observed after pea protein solutions had been heated for 20

  14. Determination of microbial protein in perennial ryegrass silage

    NARCIS (Netherlands)

    Driehuis, F.; Wikselaar, van P.G.

    2001-01-01

    The microbial matter fraction was determined in perennial ryegrass silages of different dry-matter (DM) contents, ensiled with or without Lactobacillus plantarum. 15N-Leucine and the bacterial cell wall constituent diaminopimelic acid (DAPA) were used as markers for microbial-N. Perennial ryegrass

  15. Syringe test screening of microbial gas production activity: Cases denitrification and biogas formation.

    Science.gov (United States)

    Østgaard, Kjetill; Kowarz, Viktoria; Shuai, Wang; Henry, Ingrid A; Sposob, Michal; Haugen, Hildegunn Hegna; Bakke, Rune

    2017-01-01

    Mass produced plastic syringes may be applied as vessels for cheap, simple and large scale batch culture testing. As illustrated for the cases of denitrification and of biogas formation, metabolic activity was monitored by direct reading of the piston movement due to the gas volume formed. Pressure buildup due to friction was shown to be moderate. A piston pull and slide back routine can be applied before recording gas volume to minimize experimental errors due to friction. Inoculum handling and activity may be conveniently standardized as illustrated by applying biofilm carriers. A robust set of positive as well as negative controls ("blanks") should be included to ensure quality of the actual testing. The denitrification test showed saturation response at increasing amounts of inoculum in the form of adapted moving bed biofilm reactor (MBBR) carriers, with well correlated nitrate consumption vs. gas volume formed. As shown, the denitrification test efficiently screened different inocula at standardized substrates. Also, different substrates were successfully screened and compared at standardized inocula. The biogas potential test showed efficient screening of different substrates with effects of relative amounts of carbohydrate, protein, fat. A second case with CO 2 capture reclaimer waste as substrate demonstrated successful use of co-feeding to support waste treatment and how temperature effects on kinetics and stoichiometry can be observed. In total, syringe test screening of microbial gas production seems highly efficient at a low cost when properly applied. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Examination of protein degradation in continuous flow, microbial electrolysis cells treating fermentation wastewater

    KAUST Repository

    Nam, Joo-Youn; Yates, Matthew D.; Zaybak, Zehra; Logan, Bruce E.

    2014-01-01

    © 2014 Elsevier Ltd. Cellulose fermentation wastewaters (FWWs) contain short chain volatile fatty acids and alcohols, but they also have high concentrations of proteins. Hydrogen gas production from FWW was examined using continuous flow microbial

  17. Selection of methanogenic microbial by gamma irradiation on improvement of unaerobic digestion efficiency on biogas formation

    International Nuclear Information System (INIS)

    M Yazid; Aris Bastianudin

    2011-01-01

    Selection of methanogenic microbial by gamma irradiation as an effort on improvement of efficiency process on biogas formation has been done. The objectives of this research is to obtain the methanogenic microbial isolate with high specific growth constant (μ), there for will be applicable for increasing the efficiency of biogas formation process. The microbial content sludge sample was taken from the digester tank conventional biogas installation located in Marangan village, Bokoharjo, Prambanan, Sleman and the sludge was irradiated using Co-60 gamma irradiator with varied dosage dose of 0-25 KGy. Microbial culture formation is conducted in growing media with 30% liquid rumen content in un-aerobe condition by addition of 80% H2 and 20% CO_2 gas mixture. Analysis of colony growth was performed by observation using long-wave ultraviolet rays (UV rays), while the microbial growth was by spectro-photometric analysis. Determination of gas methane product was done using gas chromatographic method. The result shown that 4 isolated methanogenic microbial (RB10, RB15, RB20 and RB25) that grown on 10-25 kGy gamma irradiation. The identification result shows that isolate RB10 and RB25 are belong to Methanobacterium genus, while isolate RB15 and RB20 are belong to Methanosarcina and Methanospirillum genus respectively. The specific growth constant (μ) values of the 4 bacterial isolates are in the range between 0.022 - 0.031. On the other hand, the efficiency of methane gas production for each isolates is in the range of 53.4%. - 67.6%. It can be concluded that isolate RB25 was the isolate with the highest specific growth constant (μ) value 0.031 and its efficiency of methane gas production was 67.6%. (author)

  18. Effects of hydraulic frac fluids and formation waters on groundwater microbial communities

    Science.gov (United States)

    Krueger, Martin; Jimenez, Nuria

    2017-04-01

    Shale gas is being considered as a complementary energy resource to other fossil fuels. Its exploitation requires using advanced drilling techniques and hydraulic stimulation (fracking). During fracking operations, large amounts of fluids (fresh water, proppants and chemicals) are injected at high pressures into the formations, to create fractures and fissures, and thus to release gas from the source rock into the wellbore. The injected fluid partly remains in the formation, while up to 40% flows back to the surface, together with reservoir waters, sometimes containing dissolved hydrocarbons, high salt concentrations, etc. The aim of our study was to investigate the potential impacts of frac or geogenic chemicals, frac fluid, formation water or flowback on groudnwater microbial communities. Laboratory experiments under in situ conditions (i.e. at in situ temperature, high pressure) were conducted using groundwater samples from three different locations. Series of microcosms containing R2 broth medium or groundwater spiked with either single frac chemicals (including biocides), frac fluids, artificial reservoir water, NaCl, or different mixtures of reservoir water and frac fluid (to simulate flowback) were incubated in the dark. Controls included non-amended and non-inoculated microcosms. Classical microbiological methods and molecular analyses were used to assess changes in the microbial abundance, community structure and function in response to the different treatments. Microbial communities were quite halotolerant and their growth benefited from low concentrations of reservoir waters or salt, but they were negatively affected by higher concentrations of formation waters, salt, biocides or frac fluids. Changes on the microbial community structure could be detected by T-RFLP. Single frac components like guar gum or choline chloride were used as substrates, while others like triethanolamine or light oil distillate hydrogenated prevented microbial growth in

  19. Soil Microbes and soil microbial proteins: interactions with clay minerals

    International Nuclear Information System (INIS)

    Spence, A.; Kelleher, B. P.

    2009-01-01

    Bacterial enumeration in soil environments estimates that the population may reach approximately 10 1 0 g - 1 of soil and comprise up to 90% of the total soil microbial biomass. Bacteria are present in soils as single cells or multicell colonies and often strongly adsorb onto mineral surfaces such as sand and clay. The interactions of microbes and microbial biomolecules with these minerals have profound impacts on the physical, chemical and biological properties of soils. (Author)

  20. Production of microbial biomass protein by sequential culture fermentation of Arachniotus sp., and Candida utilis

    International Nuclear Information System (INIS)

    Ahmed, S.; Ahmad, F.; Hashmi, A.S.

    2010-01-01

    Sequential culture fermentation by Arachniotus sp. at 35 deg. C for 72 h and followed by Candida utilis fermentation at 35 deg. C for 72 h more resulted in higher production of microbial biomass protein. 6% (w/v) corn stover, 0.0075% CaCl/sub 2/.2H/sub 2/O, 0.005% MgSO/sub 4/.7H/sub 2/O, 0.01% KH/sub 2/PO/sub 4/, C:N ratio of 30:1 and 1% molasses gave higher microbial biomass protein production by the sequential culture fermentation of Arachniotus sp., and C. utilis. The mixed microbial biomass protein produced in the 75-L fermentor contained 16.41%, 23.51%, 10.9%, 12.11% and 0.12% true protein, crude protein, crude fiber, ash and RNA content, respectively. The amino acid profile of final mixed microbial biomass protein showed that it was enriched with essential amino acids. Thus, the potential utilization of corn stover can minimize the cost for growth of these microorganisms and enhance microbial biomass protein production by sequential culture fermentation. (author)

  1. Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

    Science.gov (United States)

    von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

    2013-10-01

    The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

  2. The formation and deformation of protein structures with viscoelastic properties

    NARCIS (Netherlands)

    Riemsdijk, van L.E.

    2011-01-01

    This study describes the formation of a gluten substitute.

    Chapter 1 describes the properties that are necessary to obtain a gluten substitute.

    Chapter 2 describes the formation and properties of protein particle suspensions. Two proteins with different

  3. Modeling the formation of soluble microbial products (SMP in drinking water biofiltration

    Directory of Open Access Journals (Sweden)

    Yu Xin

    2008-09-01

    Full Text Available Both a theoretical and an empirical model were developed for predicting the formation of soluble microbial products (SMP during drinking water biofiltration. Four pilot-scale biofilters with ceramsite as the medium were fed with different acetate loadings for the determination of SMP formation. Using numerically simulated and measured parameters, the theoretical model was developed according to the substrate and biomass balance. The results of this model matched the measured data better for higher SMP formation but did not fit well when SMP formation was lower. In order to better simulate the reality and overcome the difficulties of measuring the kinetic parameters, a simpler empirical model was also developed. In this model, SMP formation was expressed as a function of fed organic loadings and the depth of the medium, and a much better fit was obtained.

  4. Formation of industrial mixed culture biofilm in chlorophenol cultivated medium of microbial fuel cell

    Science.gov (United States)

    Hassan, Huzairy; Jin, Bo; Dai, Sheng; Ngau, Cornelius

    2016-11-01

    The formation of microbial biofilm while maintaining the electricity output is a challenging topic in microbial fuel cell (MFC) studies. This MFC critical factor becomes more significant when handling with industrial wastewater which normally contains refractory and toxic compounds. This study explores the formation of industrial mixed culture biofilm in chlorophenol cultivated medium through observing and characterizing microscopically its establishment on MFC anode surface. The mixed culture was found to develop its biofilm on the anode surface in the chlorophenol environment and established its maturity and dispersal stages with concurrent electricity generation and phenolic degradation. The mixed culture biofilm engaged the electron transfer roles in MFC by generating current density of 1.4 mA/m2 and removing 53 % of 2,4-dichlorophenol. The results support further research especially on hazardous wastewater treatment using a benign and sustainable method.

  5. In vitro estimation of rumen microbial protein synthesis of water buffaloes using 30S as tracer

    International Nuclear Information System (INIS)

    Hendratno, C.; Abidin, Z.; Bahaudin, R.; Sastrapradja, D.

    1977-01-01

    An experiment to study the effect of diet and individual differences of animals on the in vitro estimation of rumen microbial protein synthesis in young female water buffaloes using the technique of inorganic 35 S incorporation, is described. The dietary treatments were four combinations of roughage supplemented with cassava meal. From the value of rate constant for dilution of radioactivity in the sulphide pool and percentage of inorganic 35 S incorporated into microbial protein, it can be concluded that individual differences of animals have no influence on the efficiency of microbial protein synthesis. Feed composition, on the other hand, tends to have some influence on the efficiency of protein synthesis(P3O.15). (author)

  6. Purine derivative excretion and microbial protein synthesis in sheep ...

    African Journals Online (AJOL)

    In a 3 x 3 Latin square design experiment, urinary excretions of purine derivatives (allantoin N, Uric acid N, Xanthine + Hypoxanthine N) were measured and used to estimate microbial N yield in 9 sheep fed roughage- based diet supplemented with 0, 150 and 300g DM grass silage respectively. Daily urinary excretions of ...

  7. Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis.

    Directory of Open Access Journals (Sweden)

    Devendra H Dusane

    Full Text Available BACKGROUND: Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. METHODOLOGY/PRINCIPAL FINDINGS: B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275 derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. CONCLUSION/SIGNIFICANCE: We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

  8. Disruption of Microbial Biofilms by an Extracellular Protein Isolated from Epibiotic Tropical Marine Strain of Bacillus licheniformis

    Science.gov (United States)

    Dusane, Devendra H.; Damare, Samir R.; Nancharaiah, Yarlagadda V.; Ramaiah, N.; Venugopalan, Vayalam P.; Kumar, Ameeta Ravi; Zinjarde, Smita S.

    2013-01-01

    Background Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. Methodology/Principal Findings B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. Conclusion/Significance We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent. PMID:23691235

  9. Protein modification by acrolein: Formation and stability of cysteine adducts

    OpenAIRE

    Cai, Jian; Bhatnagar, Aruni; Pierce, William M.

    2009-01-01

    The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine-acrolein adducts are difficult to iden...

  10. Chemotactic preferences govern competition and pattern formation in simulated two-strain microbial communities.

    Science.gov (United States)

    Centler, Florian; Thullner, Martin

    2015-01-01

    Substrate competition is a common mode of microbial interaction in natural environments. While growth properties play an important and well-studied role in competition, we here focus on the influence of motility. In a simulated two-strain community populating a homogeneous two-dimensional environment, strains competed for a common substrate and only differed in their chemotactic preference, either responding more sensitively to a chemoattractant excreted by themselves or responding more sensitively to substrate. Starting from homogeneous distributions, three possible behaviors were observed depending on the competitors' chemotactic preferences: (i) distributions remained homogeneous, (ii) patterns formed but dissolved at a later time point, resulting in a shifted community composition, and (iii) patterns emerged and led to the extinction of one strain. When patterns formed, the more aggregating strain populated the core of microbial aggregates where starving conditions prevailed, while the less aggregating strain populated the more productive zones at the fringe or outside aggregates, leading to a competitive advantage of the less aggregating strain. The presence of a competitor was found to modulate a strain's behavior, either suppressing or promoting aggregate formation. This observation provides a potential mechanism by which an aggregated lifestyle might evolve even if it is initially disadvantageous. Adverse effects can be avoided as a competitor hinders aggregate formation by a strain which has just acquired this ability. The presented results highlight both, the importance of microbial motility for competition and pattern formation, and the importance of the temporal evolution, or history, of microbial communities when trying to explain an observed distribution.

  11. Effects of Synchronicity of Carbohydrate and Protein Degradation on Rumen Fermentation Characteristics and Microbial Protein Synthesis

    Directory of Open Access Journals (Sweden)

    J. K. Seo

    2013-03-01

    Full Text Available A series of in vitro studies were carried out to determine i the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS in in vitro experiments. Untreated corn (C and enzyme-treated corn (EC were combined with soy bean meal with (ES and without (S enzyme treatment or formaldehyde treatment (FS. Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS. The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity.

  12. Discriminating Microbial Species Using Protein Sequence Properties and Machine Learning

    NARCIS (Netherlands)

    Shahib, Ali Al-; Gilbert, David; Breitling, Rainer

    2007-01-01

    Much work has been done to identify species-specific proteins in sequenced genomes and hence to determine their function. We assumed that such proteins have specific physico-chemical properties that will discriminate them from proteins in other species. In this paper, we examine the validity of this

  13. [Formation of microbial biofilms in causative agents of acute and chronic pyelonephritis].

    Science.gov (United States)

    Lagun, L V; Atanasova, Iu V; Tapal'skiĭ, D V

    2013-01-01

    Study the intensity of formation of microbial biofilms by Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus strains isolated during various forms of pyelonephritis. 150 clinical isolates of microorganisms isolated from urine ofpatientswith acute and chronic pyelonephritiswere included into the study. Determination of intensity of film-formation was carried out by staining of the formed biofilms by crystal violet with consequent extraction of the dye and measurement of its concentration in washout solution. Among causative agents ofpyelonephritis P. aeruginosa isolates had the maximum film-forming ability. The intensity of biofilm formation of these isolates was 2-3 time higher than staphylococcus and enterobacteria strains. Strains isolated from patients with chronic pyelonephritis by ability to form biofilms significantly surpassed strains isolated from acute pyelonephritis patients. A higher ability to form microbial biofilms for microorganisms--causative agents of pyelonephritis progressing against the background ofurolithiasis was noted. The ability to form biofilms is determined by both causative agent species and character of the infectious process in which this microorganism participates. Intensive formation of biofilms by E. coli, P. aeruginosa, K. pneumoniae, S. aureus clinical isolates may be an important factor of chronization of urinary tract infections.

  14. Effects of grain source, grain processing, and protein degradability on rumen kinetics and microbial protein synthesis in Boer kids.

    Science.gov (United States)

    Brassard, M-E; Chouinard, P Y; Berthiaume, R; Tremblay, G F; Gervais, R; Martineau, R; Cinq-Mars, D

    2015-11-01

    Microbial protein synthesis in the rumen would be optimized when dietary carbohydrates and proteins have synchronized rates and extent of degradation. The aim of this study was to evaluate the effect of varying ruminal degradation rate of energy and nitrogen sources on intake, nitrogen balance, microbial protein yield, and kinetics of nutrients in the rumen of growing kids. Eight Boer goats (38.2 ± 3.0 kg) were used. The treatments were arranged in a split-plot Latin square design with grain sources (barley or corn) forming the main plots (squares). Grain processing methods and levels of protein degradability formed the subplots in a 2 × 2 factorial arrangement for a total of 8 dietary treatments. The grain processing method was rolling for barley and cracking for corn. Levels of protein degradability were obtained by feeding untreated soybean meal (SBM) or heat-treated soybean meal (HSBM). Each experimental period lasted 21 d, consisting of a 10-d adaptation period, a 7-d digestibility determination period, and a 4-d rumen evacuation and sampling period. Kids fed with corn had higher purine derivatives (PD) excretion when coupled with SBM compared with HSBM and the opposite occurred with barley-fed kids ( ≤ 0.01). Unprocessed grain offered with SBM led to higher PD excretion than with HSBM whereas protein degradability had no effect when processed grain was fed ( ≤ 0.03). Results of the current experiment with high-concentrate diets showed that microbial N synthesis could be maximized in goat kids by combining slowly fermented grains (corn or unprocessed grains) with a highly degradable protein supplement (SBM). With barley, a more rapidly fermented grain, a greater microbial N synthesis was observed when supplementing a low-degradable protein (HSBM).

  15. Recombinant human bone morphogenetic protein induces bone formation

    International Nuclear Information System (INIS)

    Wang, E.A.; Rosen, V.; D'Alessandro, J.S.; Bauduy, M.; Cordes, P.; Harada, T.; Israel, D.I.; Hewick, R.M.; Kerns, K.M.; LaPan, P.; Luxenberg, D.P.; McQuaid, D.; Moutsatsos, I.K.; Nove, J.; Wozney, J.M.

    1990-01-01

    The authors have purified and characterized active recombinant human bone morphogenetic protein (BMP) 2A. Implantation of the recombinant protein in rats showed that a single BMP can induce bone formation in vivo. A dose-response and time-course study using the rat ectopic bone formation assay revealed that implantation of 0.5-115 μg of partially purified recombinant human BMP-2A resulted in cartilage by day 7 and bone formation by day 14. The time at which bone formation occurred was dependent on the amount of BMP-2A implanted; at high doses bone formation could be observed at 5 days. The cartilage- and bone-inductive activity of the recombinant BMP-2A is histologically indistinguishable from that of bone extracts. Thus, recombinant BMP-2A has therapeutic potential to promote de novo bone formation in humans

  16. MULTIFUNCTIONAL ADHESIN PROTEINS AND THEIR DISPLAY IN MICROBIAL CELLS

    DEFF Research Database (Denmark)

    1999-01-01

    Recombinant cells expressing a multifunctional adhesin protein derived from a naturally occurring adhesin, containing a binding domain that is capable of binding to an organic receptor and a binding domain that is capable of binding to a compound to which the naturally occurring adhesin protein...... substantially does not bind. The cells or modified adhesin proteins, optionally in immobilized form, are useful for separating organic and inorganic compounds including toxic or precious metals from an environment....

  17. Non-protein nitrogen utilization and microbial synthesis in the rumen

    International Nuclear Information System (INIS)

    Abou Akkada, A.R.; El-Shazly, K.

    1976-01-01

    The distinction between bacterial and protozoal proteins in the rumen has been a difficult task. The use of diaminopimelic acid (DAP) as a marker for bacterial protein and 2-aminoethanephosphonic acid (AEP) for protozoal protein has been examined in the present studies in sheep fed on a semi-purified diet. The microbial protein predicted from DAP and AEP determination was similar to that obtained by the precipitation of proteins. From rates of VFA determination by the in vitro zero-time rate technique, and from measurements of rumen volume using Cr-EDTA colour determination at 550 nm, the total VFA production in 24 h could be calculated, and the ATP generated (mol/day) was calculated using Baldwin and co-workers' transformation value of 2.44. The yield of microbial cells (g/mol ATP) was found to be 28.15, similar to that suggested by Hobson and Summers and Gunzalus and Schuster. Using a sheep double-fistulated in the duodenum proximal to the pyloric sphincter, and blocking the path of the abomasal fluid by inserting an inflated baloon in the second fistula, the abomasal fluid could be collected for 12 h. The total microbial-N as predicted from DAP and AEP was approximately similar to the total non-ammonia-N in the abomasal digesta. It could be concluded that DAP and AEP determination could be used within reasonable limits to differentiate between bacterial and protozoal proteins. A good agreement was found between rates of outflow of the rumen digesta and microbial growth (percentage hourly) as measured by different techniques. The agreement between values of microbial growth rates and rates of outflow of digesta from the rumen with the differences between production and absorption of VFA is an indication of the validity of the techniques employed in the present studies. (author)

  18. RNA Recognition and Stress Granule Formation by TIA Proteins

    Science.gov (United States)

    Waris, Saboora; Wilce, Matthew Charles James; Wilce, Jacqueline Anne

    2014-01-01

    Stress granule (SG) formation is a primary mechanism through which gene expression is rapidly modulated when the eukaryotic cell undergoes cellular stresses (including heat, oxidative, viral infection, starvation). In particular, the sequestration of specifically targeted translationally stalled mRNAs into SGs limits the expression of a subset of genes, but allows the expression of heatshock proteins that have a protective effect in the cell. The importance of SGs is seen in several disease states in which SG function is disrupted. Fundamental to SG formation are the T cell restricted intracellular antigen (TIA) proteins (TIA-1 and TIA-1 related protein (TIAR)), that both directly bind to target RNA and self-associate to seed the formation of SGs. Here a summary is provided of the current understanding of the way in which TIA proteins target specific mRNA, and how TIA self-association is triggered under conditions of cellular stress. PMID:25522169

  19. Chlorination or monochloramination: Balancing the regulated trihalomethane formation and microbial inactivation in marine aquaculture waters

    KAUST Repository

    Sanawar, Huma

    2017-08-15

    Disinfection methods like chlorination are increasingly used to sanitize the water, equipment, tools and surfaces in aquaculture facilities. This is to improve water quality, and to maintain a hygienic environment for the well-being of aquatic organisms. However, chlorination can result in formation of regulated disinfection byproducts (DBPs) that can be carcinogenic and toxic. This study aims to evaluate if an optimal balance can be achieved between minimal regulated DBP formation and effective microbial inactivation with either chlorination or monochloramination for application in the Red Sea aquaculture waters. Upon chlorination, the concentration of total trihalomethanes (THMs), primarily bromoform, exceeded the regulatory limit of 80μg/L even at the lowest tested concentration of chlorine (1mg/L) and contact time (1h). Comparatively, regulated THMs concentration was only detectable at 30μg/L level in one of the three sets of monochloraminated marine aquaculture waters. The average log reduction of antibiotic-resistant bacteria (ARB) by chlorine ranged from 2.3-log to 3.2-log with different contact time. The average log reduction of ARB by monochloramine was comparatively lower at 1.9 to 2.9-log. Although viable Staphylococcus aureus was recovered from monochloraminated samples as opposed to chlorinated samples, the abundance of S. aureus was not high enough to result in any significant microbial risks. Both chlorination and monochloramination did not provide any significant improvement in the reduction of antibiotic resistance genes (ARGs). This study demonstrates that a systematic evaluation is needed to determine the optimal disinfectant required to balance both microbial and chemical risks. Compared to chlorine, monochloramine may be a more appropriate disinfection strategy for the treatment of aquaculture effluents prior to discharge or for recirculatory use in the aquaculture facility.

  20. Alignment of microbial fitness with engineered product formation: obligatory coupling between acetate production and photoautotrophic growth.

    Science.gov (United States)

    Du, Wei; Jongbloets, Joeri A; van Boxtel, Coco; Pineda Hernández, Hugo; Lips, David; Oliver, Brett G; Hellingwerf, Klaas J; Branco Dos Santos, Filipe

    2018-01-01

    Microbial bioengineering has the potential to become a key contributor to the future development of human society by providing sustainable, novel, and cost-effective production pipelines. However, the sustained productivity of genetically engineered strains is often a challenge, as spontaneous non-producing mutants tend to grow faster and take over the population. Novel strategies to prevent this issue of strain instability are urgently needed. In this study, we propose a novel strategy applicable to all microbial production systems for which a genome-scale metabolic model is available that aligns the production of native metabolites to the formation of biomass. Based on well-established constraint-based analysis techniques such as OptKnock and FVA, we developed an in silico pipeline-FRUITS-that specifically 'Finds Reactions Usable in Tapping Side-products'. It analyses a metabolic network to identify compounds produced in anabolism that are suitable to be coupled to growth by deletion of their re-utilization pathway(s), and computes their respective biomass and product formation rates. When applied to Synechocystis sp. PCC6803, a model cyanobacterium explored for sustainable bioproduction, a total of nine target metabolites were identified. We tested our approach for one of these compounds, acetate, which is used in a wide range of industrial applications. The model-guided engineered strain shows an obligatory coupling between acetate production and photoautotrophic growth as predicted. Furthermore, the stability of acetate productivity in this strain was confirmed by performing prolonged turbidostat cultivations. This work demonstrates a novel approach to stabilize the production of target compounds in cyanobacteria that culminated in the first report of a photoautotrophic growth-coupled cell factory. The method developed is generic and can easily be extended to any other modeled microbial production system.

  1. Geochemical patterns and microbial contribution to iron plaque formation in the rice plant rhizosphere

    Science.gov (United States)

    Maisch, Markus; Murata, Chihiro; Unger, Julia; Kappler, Andreas; Schmidt, Caroline

    2015-04-01

    Rice is the major food source for more than half of the world population and 80 percent of the worldwide rice cultivation is performed on water logged paddy soils. The establishment of reducing conditions in the soil and across the soil-water interface not only stimulates the microbial production and release of the greenhouse gas methane. These settings also create optimal conditions for microbial iron(III) reduction and therefore saturate the system with reduced ferrous iron. Through the reduction and dissolution of ferric minerals that are characterized by their high surface activity, sorbed nutrients and contaminants (e.g. arsenic) will be mobilized and are thus available for uptake by plants. Rice plants have evolved a strategy to release oxygen from their roots in order to prevent iron toxification in highly ferrous environments. The release of oxygen to the reduced paddy soil causes ferric iron plaque formation on the rice roots and finally increases the sorption capacity for toxic metals. To this date the geochemical and microbiological processes that control the formation of iron plaque are not deciphered. It has been hypothesized that iron(II)-oxidizing bacteria play a potential role in the iron(III) mineral formation along the roots. However, not much is known about the actual processes, mineral products, and geochemical gradients that establish within the rhizosphere. In the present study we have developed a growth set-up that allows the co-cultivation of rice plants and iron(II)-oxidizing bacteria, as well as the visual observation and in situ measurement of geochemical parameters. Oxygen and dissolved iron(II) gradients have been measured using microelectrodes and show geochemical hot spots that offer optimal growth conditions for microaerophilic iron(II) oxidizers. First mineral identification attempts of iron plaque have been performed using Mössbauer spectroscopy and microscopy. The obtained results on mineraology and crystallinity have been

  2. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  3. In silico Characterization of Plant and Microbial Antifreeze Proteins

    Directory of Open Access Journals (Sweden)

    Abdul Mohin Sajib

    2012-12-01

    Full Text Available Antifreeze proteins (AFPs are class of proteins that protect organisms from the damage caused by freezing through their ability to inhibit ice growth and effectively lower the temperature at which water freezes. In this study, a total of 25 antifreeze proteins were selected from four different sources (plant, bacteria and fungus where they represent distinct physicochemical and structural features. Several Physico-chemical properties such as grand average hydropathy (GRAVY, aliphatic index (AI, extinction coefficient (EC, isolelectric point (pI, and instability index (II were computed. S-S bridges and secondary structures were analyzed using CYS_REC and SOPMA programs respectively. The three dimensional structure of Antifreeze proteins is predicted by using three homology modelling server Geno3D, Swiss-model and CPHmodels. These models were evaluated with PROCHECK, What If, and ProSA programs. Model visualization and analysis was done with Pymol. These structures will provide a good foundation for functional analysis of experimentally derived crystal structures.

  4. Circulating microbial products and acute phase proteins as markers of pathogenesis in lymphatic filarial disease.

    Directory of Open Access Journals (Sweden)

    R Anuradha

    Full Text Available Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+ or without (CP Ag- active infection; with clinically asymptomatic infections (INF; and in those without infection (endemic normal [EN]. Comparisons between the two actively infected groups (CP Ag+ compared to INF and those without active infection (CP Ag- compared to EN were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein, acute phase proteins (haptoglobin and serum amyloid protein-A, and inflammatory cytokines (IL-1β, IL-12, and TNF-α are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.

  5. DELLA proteins regulate arbuscule formation in arbuscular mycorrhizal symbiosis.

    Science.gov (United States)

    Floss, Daniela S; Levy, Julien G; Lévesque-Tremblay, Véronique; Pumplin, Nathan; Harrison, Maria J

    2013-12-17

    Most flowering plants are able to form endosymbioses with arbuscular mycorrhizal fungi. In this mutualistic association, the fungus colonizes the root cortex and establishes elaborately branched hyphae, called arbuscules, within the cortical cells. Arbuscule development requires the cellular reorganization of both symbionts, and the resulting symbiotic interface functions in nutrient exchange. A plant symbiosis signaling pathway controls the development of the symbiosis. Several components of the pathway have been identified, but transcriptional regulators that control downstream pathways for arbuscule formation are still unknown. Here we show that DELLA proteins, which are repressors of gibberellic acid (GA) signaling and function at the nexus of several signaling pathways, are required for arbuscule formation. Arbuscule formation is severely impaired in a Medicago truncatula Mtdella1/Mtdella2 double mutant; GA treatment of wild-type roots phenocopies the della double mutant, and a dominant DELLA protein (della1-Δ18) enables arbuscule formation in the presence of GA. Ectopic expression of della1-Δ18 suggests that DELLA activity in the vascular tissue and endodermis is sufficient to enable arbuscule formation in the inner cortical cells. In addition, expression of della1-Δ18 restores arbuscule formation in the symbiosis signaling pathway mutant cyclops/ipd3, indicating an intersection between DELLA and symbiosis signaling for arbuscule formation. GA signaling also influences arbuscule formation in monocots, and a Green Revolution wheat variety carrying dominant DELLA alleles shows enhanced colonization but a limited growth response to arbuscular mycorrhizal symbiosis.

  6. AFM characterization of protein net formation on a fibrous medium

    Directory of Open Access Journals (Sweden)

    Assis O.B.G.

    2000-01-01

    Full Text Available Lysozyme protein net is set on a glass fiber support using the self-assembly technique. Enzymatic film formation is followed by surface imaging via atomic force microscopy (AFM. Change in roughness as a function of deposition time is used as an indirect indicator of film formation. The objective was to form a protein film that would have no effect on the permeability of the medium, aiming at its application as a bioactive membrane or reactor suitable for bacteria and chemical interactions in aqueous media.

  7. 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.

  8. Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency.

    Science.gov (United States)

    Pendini, Nicole R; Bailey, Lisa M; Booker, Grant W; Wilce, Matthew C; Wallace, John C; Polyak, Steven W

    2008-01-01

    The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.

  9. Effect of inclusion of different levels of silage on rumen microbial population and microbial protein synthesis in dairy steers fed on rice straw

    Directory of Open Access Journals (Sweden)

    Thien Truong Giang Nguyen

    2017-02-01

    Full Text Available Objective Leucaena leucocephala (Leucaena is a perennial tropical legume that can be directly grazed or harvested and offered to ruminants as hay, silage, or fresh. However, Leucaena contain phenolic compounds, which are considered anti-nutritional factors as these may reduce intake, digestibility and thus animal performance. Therefore, the objective of this experiment was to determine effects of Leucaena silage (LS feeding levels on rumen microbial populations, N-balance and microbial protein synthesis in dairy steers. Methods Four, rumen fistulated dairy steers with initial weight of 167±12 kg were randomly assigned to receive dietary treatments according to a 4×4 Latin square design. Treatments were as followings: T1 = untreated rice straw (RS; Control, T2 = 70% RS+30% LS, T3 = 40% RS+60% LS, and T4 = 100% LS. Dairy steers were fed rice straw and LS ad libitum and supplemented with concentrate at 0.2% of body weight/d. Results Results revealed that the rumen microbial population, especially cellulolytic, proteolytic bacteria and fungal zoospores were enhanced in steers that received 60% of LS (p0.05. Protozoal population was linearly decreased with increasing level of LS (p<0.05. Moreover, N-balance and microbial protein synthesis were enhanced by LS feeding (p<0.05 and were the highest in 60% LS group. Conclusion Based on this study, it could be concluded that replacement of RS with 60% LS significantly improved microbial population and microbial protein synthesis in diary steers.

  10. Formation of Giant Protein Vesicles by a Lipid Cosolvent Method

    DEFF Research Database (Denmark)

    Hansen, Jesper S.; Vararattanavech, Ardcharaporn; Vissing, Thomas

    2011-01-01

    This paper describes a method to create giant protein vesicles (GPVs) of ≥10 μm by solvent‐driven fusion of large vesicles (0.1–0.2 μm) with reconstituted membrane proteins. We found that formation of GPVs proceeded from rotational mixing of protein‐reconstituted large unilamellar vesicles (LUVs)...... of spinach SoPIP2;1 and E. coli AqpZ aquaporins. Our findings show that hydrophobic interactions within the bilayer of formed GPVs are influenced not only by the solvent partitioning propensity, but also by lipid composition and membrane protein isoform....

  11. Biofilm formation and microbial community analysis of the simulated river bioreactor for contaminated source water remediation.

    Science.gov (United States)

    Xu, Xiang-Yang; Feng, Li-Juan; Zhu, Liang; Xu, Jing; Ding, Wei; Qi, Han-Ying

    2012-06-01

    The start-up pattern of biofilm remediation system affects the biofilm characteristics and operating performances. The objective of this study was to evaluate the performances of the contaminated source water remediation systems with different start-up patterns in view of the pollutants removal performances and microbial community succession. The operating performances of four lab-scale simulated river biofilm reactors were examined which employed different start-up methods (natural enrichment and artificial enhancement via discharging sediment with influent velocity gradient increase) and different bio-fillers (Elastic filler and AquaMats® ecobase). At the same time, the microbial communities of the bioreactors in different phases were analyzed by polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. The pollutants removal performances became stable in the four reactors after 2 months' operation, with ammonia nitrogen and permanganate index (COD(Mn)) removal efficiencies of 84.41-94.21% and 69.66-76.60%, respectively. The biomass of mature biofilm was higher in the bioreactors by artificial enhancement than that by natural enrichment. Microbial community analysis indicated that elastic filler could enrich mature biofilm faster than AquaMats®. The heterotrophic bacteria diversity of biofilm decreased by artificial enhancement, which favored the ammonia-oxidizing bacteria (AOB) developing on the bio-fillers. Furthermore, Nitrosomonas- and Nitrosospira-like AOB coexisted in the biofilm, and Pseudomonas sp., Sphaerotilus sp., Janthinobacterium sp., Corynebacterium aurimucosum were dominant in the oligotrophic niche. Artificial enhancement via the combination of sediment discharging and influent velocity gradient increasing could enhance the biofilm formation and autotrophic AOB enrichment in oligotrophic niche.

  12. Effect of Stirring and Seeding on Whey Protein Fibril Formation

    NARCIS (Netherlands)

    Bolder, S.G.; Sagis, L.M.C.; Venema, P.; Linden, van der E.

    2007-01-01

    The effect of stirring and seeding on the formation of fibrils in whey protein isolate (WPI) solutions was studied. More fibrils of a similar length are formed when WPI is stirred during heating at pH 2 and 80 C compared to samples that were heated at rest. Addition of seeds did not show an

  13. Disulphide bond formation in food protein aggregation and gelation

    NARCIS (Netherlands)

    Visschers, R.W.; Jongh, de H.H.J.

    2005-01-01

    In this short review we discuss the role of cysteine residues and cystine bridges for the functional aggregation of food proteins. We evaluate how formation and cleavage of disulphide bonds proceeds at a molecular level, and how inter- and intramolecular disulfide bonds can be detected and modified.

  14. In situ formation of graphene layers on graphite surfaces for efficient anodes of microbial fuel cells.

    Science.gov (United States)

    Tang, Jiahuan; Chen, Shanshan; Yuan, Yong; Cai, Xixi; Zhou, Shungui

    2015-09-15

    Graphene can be used to improve the performance of the anode in a microbial fuel cell (MFC) due to its good biocompatibility, high electrical conductivity and large surface area. However, the chemical production and modification of the graphene on the anode are environmentally hazardous because of the use of various harmful chemicals. This study reports a novel method based on the electrochemical exfoliation of a graphite plate (GP) for the in situ formation of graphene layers on the surface of a graphite electrode. When the resultant graphene-layer-based graphite plate electrode (GL/GP) was used as an anode in an MFC, a maximum power density of 0.67 ± 0.034 W/m(2) was achieved. This value corresponds to 1.72-, 1.56- and 1.26-times the maximum power densities of the original GP, exfoliated-graphene-modified GP (EG/GP) and chemically-reduced-graphene-modified GP (rGO/GP) anodes, respectively. Electrochemical measurements revealed that the high performance of the GL/GP anode was attributable to its macroporous structure, improved electron transfer and high electrochemical capacitance. The results demonstrated that the proposed method is a facile and environmentally friendly synthesis technique for the fabrication of high-performance graphene-based electrodes for use in microbial energy harvesting. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Stochastic calculus of protein filament formation under spatial confinement

    Science.gov (United States)

    Michaels, Thomas C. T.; Dear, Alexander J.; Knowles, Tuomas P. J.

    2018-05-01

    The growth of filamentous aggregates from precursor proteins is a process of central importance to both normal and aberrant biology, for instance as the driver of devastating human disorders such as Alzheimer's and Parkinson's diseases. The conventional theoretical framework for describing this class of phenomena in bulk is based upon the mean-field limit of the law of mass action, which implicitly assumes deterministic dynamics. However, protein filament formation processes under spatial confinement, such as in microdroplets or in the cellular environment, show intrinsic variability due to the molecular noise associated with small-volume effects. To account for this effect, in this paper we introduce a stochastic differential equation approach for investigating protein filament formation processes under spatial confinement. Using this framework, we study the statistical properties of stochastic aggregation curves, as well as the distribution of reaction lag-times. Moreover, we establish the gradual breakdown of the correlation between lag-time and normalized growth rate under spatial confinement. Our results establish the key role of spatial confinement in determining the onset of stochasticity in protein filament formation and offer a formalism for studying protein aggregation kinetics in small volumes in terms of the kinetic parameters describing the aggregation dynamics in bulk.

  16. Methane production from formate, acetate and H2/CO2; focusing on kinetics and microbial characterization

    DEFF Research Database (Denmark)

    Pan, Xiaofang; Angelidaki, Irini; Alvarado-Morales, Merlin

    2016-01-01

    For evaluating the methanogenesis from typical methanogenic precursors (formate, acetate and H-2/CO2), CH4 production kinetics were investigated at 37 +/- 1 degrees C in batch anaerobic digestion tests and stimulated by modified Gompertz model. The results showed that maximum methanation rate from...... formate, acetate and H-2/CO2 were 19.58 +/- 0.49, 42.65 +/- 1.17 and 314.64 +/- 3.58 N mL/gVS/d in digested manure system and 6.53 +/- 0.31, 132.04 +/- 3.96 and 640.16 +/- 19.92 N mL/gVS/d in sewage sludge system during second generation incubation. Meanwhile the model could not fit well in granular...... sludge system, while the rate of formate methanation was faster than from H-2/CO2 and acetate. Considering both the kinetic results and microbial assay we could conclude that H-2/CO2 methanation was the fastest methanogenic step in digested manure and sewage sludge system with Methanomicrobiales...

  17. Estimation of microbial protein supply in ruminants using urinary purine derivatives

    International Nuclear Information System (INIS)

    Makkar, H.P.S.; Chen, X.B.

    2004-01-01

    This publication presents various models, describing the quantitative excretion of purine derivatives in urine, developed for various breeds of cattle and for sheep, goat, camel and buffalo and their use for estimation of microbial protein supply in ruminant livestock. It also describes progress made over the last decade in analytical methods for determining purine derivatives, and a unique approach for estimating microbial protein supply using spot urine samples developed under the FAO/IAEA CRP. This approach of using spot urine samples dispenses with quantitative recovery of urine, enabling its use by field and extension workers for evaluation of the nutritional status of farm animals. Future areas of research are also highlighted in the book. This book is a good source of reference for research workers, students and extension workers alike

  18. Crosslinking of milk proteins by microbial transglutaminase: Utilization in functional yogurt products

    DEFF Research Database (Denmark)

    Gharibzahedi, Seyed Mohammad Taghi; Chronakis, Ioannis S.

    2018-01-01

    Key modifying roles of microbial transglutaminase (MTGase) in the development of innovative probiotic and non-probiotic yogurts with improved functional and quality characteristics have been comprehensively reviewed. MTGase crosslinking reactions with milk proteins stabilize the three-dimensional......Key modifying roles of microbial transglutaminase (MTGase) in the development of innovative probiotic and non-probiotic yogurts with improved functional and quality characteristics have been comprehensively reviewed. MTGase crosslinking reactions with milk proteins stabilize the three......-dimensional structure of yogurt. Yogurts treated with MTGase showed decreased syneresis, increased water-holding capacity and viscosity, homogeneous structure, desired texture, and physicochemical high stability during storage time. The utilization of MTGase does not affect negatively the sensory attributes of yogurt...

  19. Biomarker Analysis of Samples Visually Identified as Microbial in the Eocene Green River Formation: An Analogue for Mars.

    Science.gov (United States)

    Olcott Marshall, Alison; Cestari, Nicholas A

    2015-09-01

    One of the major exploration targets for current and future Mars missions are lithofacies suggestive of biotic activity. Although such lithofacies are not confirmation of biotic activity, they provide a way to identify samples for further analyses. To test the efficacy of this approach, we identified carbonate samples from the Eocene Green River Formation as "microbial" or "non-microbial" based on the macroscale morphology of their laminations. These samples were then crushed and analyzed by gas chromatography/mass spectroscopy (GC/MS) to determine their lipid biomarker composition. GC/MS analysis revealed that carbonates visually identified as "microbial" contained a higher concentration of more diverse biomarkers than those identified as "non-microbial," suggesting that this could be a viable detection strategy for selecting samples for further analysis or caching on Mars.

  20. {alpha}-Man monolayer formation via Si-C bond formation and protein recognition

    Energy Technology Data Exchange (ETDEWEB)

    Funato, Koji [School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan); Shirahata, Naoto [National Institute for Materials Science (NIMS), 1-2-1 Sengen, Tsukuba, Ibaraki 305-0047 (Japan); Miura, Yoshiko, E-mail: miuray@jaist.ac.j [School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292 (Japan)

    2009-11-30

    An acetylenyl-terminated saccharide was synthesized and the thin layer formation on the hydrogen-terminated silicon was investigated. The acetylenyl-terminated saccharide was synthesized by the condensation reaction of hexynoic acid and p-aminophenyl saccharide. This was reacted with hydrogen-terminated silicon (Si-H) by a photochemical reaction. The resulting saccharide modified substrate was analyzed by ellipsometry and X-ray photoelectron spectroscopy, which showed the formation of a uniform monolayer. The surface's ability to recognize proteins was analyzed by fluorescent microscopy, and showed specific interactions with sugar recognition proteins.

  1. The Growth Rate and Efficiency of Rumen Microbial Protein Digestion of Red Clover Silage (Trifolium pratense cv. Sabatron)

    International Nuclear Information System (INIS)

    Asih Kurniawati

    2004-01-01

    (Trifolium pratense cv. Sabatron). Red clover silage supplemented with different level of carbohydrates has been examined using the in-vitro gas production technique. Cumulative gas production, hydro.gen sulfite production, and ammonia was followed and used as indicators of microbial growth rate and extent of protein degradation. Microbial nitrogen production, VFA, and efficiency microbial production was used as indicator of nitrogen use efficiency. 15 N was used as a microbial marker to estimate the amount of nitrogen incorporation into microbial protein. Supplementation of Red clover with increasing 5 levels; 0 g; 0.625 g; 0.15 g; 0.225 g and 0.3 g of maize starch led to graded increase in microbial growth and protein degradation. This was reflected in the increasing gas production and the accumulation of hydrogen sulfite. Diurnal change in ammonia production reflected the microbial utilization of ammonia for protein synthesis. Protein microbe (P<0.001) as VFA (P<0.001) increased due to carbohydrate addition as well as utilization of nitrogen (P<0.001). There was also the efficiency of nitrogen utilization which increased significantly. This result suggested that energy supply can increased efficiency of nitrogen use in the rumen and may reduce nitrogen losses into the environment. (author)

  2. Microbial Adhesion and Biofilm Formation on Microfiltration Membranes: A Detailed Characterization Using Model Organisms with Increasing Complexity

    Directory of Open Access Journals (Sweden)

    L. Vanysacker

    2013-01-01

    Full Text Available Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development.

  3. Microbial Adhesion and Biofilm Formation on Microfiltration Membranes: A Detailed Characterization Using Model Organisms with Increasing Complexity

    Science.gov (United States)

    Vanysacker, L.; Denis, C.; Declerck, P.; Piasecka, A.; Vankelecom, I. F. J.

    2013-01-01

    Since many years, membrane biofouling has been described as the Achilles heel of membrane fouling. In the present study, an ecological assay was performed using model systems with increasing complexity: a monospecies assay using Pseudomonas aeruginosa or Escherichia coli separately, a duospecies assay using both microorganisms, and a multispecies assay using activated sludge with or without spiked P. aeruginosa. The microbial adhesion and biofilm formation were evaluated in terms of bacterial cell densities, species richness, and bacterial community composition on polyvinyldifluoride, polyethylene, and polysulfone membranes. The data show that biofouling formation was strongly influenced by the kind of microorganism, the interactions between the organisms, and the changes in environmental conditions whereas the membrane effect was less important. The findings obtained in this study suggest that more knowledge in species composition and microbial interactions is needed in order to understand the complex biofouling process. This is the first report describing the microbial interactions with a membrane during the biofouling development. PMID:23986906

  4. Direct evidence for microbial-derived soil organic matter formation and its ecophysiological controls

    Science.gov (United States)

    Kallenbach, Cynthia M.; Frey, Serita D.; Grandy, A. Stuart

    2016-11-01

    Soil organic matter (SOM) and the carbon and nutrients therein drive fundamental submicron- to global-scale biogeochemical processes and influence carbon-climate feedbacks. Consensus is emerging that microbial materials are an important constituent of stable SOM, and new conceptual and quantitative SOM models are rapidly incorporating this view. However, direct evidence demonstrating that microbial residues account for the chemistry, stability and abundance of SOM is still lacking. Further, emerging models emphasize the stabilization of microbial-derived SOM by abiotic mechanisms, while the effects of microbial physiology on microbial residue production remain unclear. Here we provide the first direct evidence that soil microbes produce chemically diverse, stable SOM. We show that SOM accumulation is driven by distinct microbial communities more so than clay mineralogy, where microbial-derived SOM accumulation is greatest in soils with higher fungal abundances and more efficient microbial biomass production.

  5. Use of Modern Chemical Protein Synthesis and Advanced Fluorescent Assay Techniques to Experimentally Validate the Functional Annotation of Microbial Genomes

    Energy Technology Data Exchange (ETDEWEB)

    Kent, Stephen [University of Chicago

    2012-07-20

    The objective of this research program was to prototype methods for the chemical synthesis of predicted protein molecules in annotated microbial genomes. High throughput chemical methods were to be used to make large numbers of predicted proteins and protein domains, based on microbial genome sequences. Microscale chemical synthesis methods for the parallel preparation of peptide-thioester building blocks were developed; these peptide segments are used for the parallel chemical synthesis of proteins and protein domains. Ultimately, it is envisaged that these synthetic molecules would be ‘printed’ in spatially addressable arrays. The unique ability of total synthesis to precision label protein molecules with dyes and with chemical or biochemical ‘tags’ can be used to facilitate novel assay technologies adapted from state-of-the art single molecule fluorescence detection techniques. In the future, in conjunction with modern laboratory automation this integrated set of techniques will enable high throughput experimental validation of the functional annotation of microbial genomes.

  6. PanCoreGen - Profiling, detecting, annotating protein-coding genes in microbial genomes.

    Science.gov (United States)

    Paul, Sandip; Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V; Chattopadhyay, Sujay

    2015-12-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing the pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen - a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for a species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars - Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. PanCoreGen – profiling, detecting, annotating protein-coding genes in microbial genomes

    Science.gov (United States)

    Bhardwaj, Archana; Bag, Sumit K; Sokurenko, Evgeni V.

    2015-01-01

    A large amount of genomic data, especially from multiple isolates of a single species, has opened new vistas for microbial genomics analysis. Analyzing pan-genome (i.e. the sum of genetic repertoire) of microbial species is crucial in understanding the dynamics of molecular evolution, where virulence evolution is of major interest. Here we present PanCoreGen – a standalone application for pan- and core-genomic profiling of microbial protein-coding genes. PanCoreGen overcomes key limitations of the existing pan-genomic analysis tools, and develops an integrated annotation-structure for species-specific pan-genomic profile. It provides important new features for annotating draft genomes/contigs and detecting unidentified genes in annotated genomes. It also generates user-defined group-specific datasets within the pan-genome. Interestingly, analyzing an example-set of Salmonella genomes, we detect potential footprints of adaptive convergence of horizontally transferred genes in two human-restricted pathogenic serovars – Typhi and Paratyphi A. Overall, PanCoreGen represents a state-of-the-art tool for microbial phylogenomics and pathogenomics study. PMID:26456591

  8. Empirical evidence that soil carbon formation from plant inputs is positively related to microbial growth

    Science.gov (United States)

    Mark A. Bradford; Ashley D. Keiser; Christian A. Davies; Calley A. Mersmann; Michael S. Strickland

    2012-01-01

    Plant-carbon inputs to soils in the form of dissolved sugars, organic acids and amino acids fuel much of heterotrophic microbial activity belowground. Initial residence times of these compounds in the soil solution are on the order of hours, with microbial uptake a primary removal mechanism. Through microbial biosynthesis, the dissolved compounds become dominant...

  9. Formation and functions of aerobic microbial granula; Entstehung und Funktionen aerober mikrobieller Granula

    Energy Technology Data Exchange (ETDEWEB)

    Etterer, T.; Wilderer, P.A. [Technische Univ. Muenchen, Garching (Germany). Lehrstuhl und Pruefamt fuer Wasserguete- und Abfallwirtschaft

    1999-07-01

    The present project investigates the phenomenon of the formation of aerobic microbial granula and their properties. To generate granula, sequencing batch reactors fed in batches were used. As shown by microbiological assays, fungi played an above-average role in granula formation and build-up. In first degradation experiments, furthermore, chemical oxygen demand (COD) could be reduced by over 90 %. The determination yielded comparable values to activated sludge, standing on average at 1.044g/ml. (orig.) [German] Im Rahmen des hier vorgestellten Projekts wurde das Phaenomen der Bildung aerober mikrobieller Granula sowie deren Eigenschaften untersucht. Zur Erzeugung von Granula wurden schubweise beschickte Reaktoren, sogenannte Sequencing-Batch-Reaktoren (SBR) verwendet. Wie mikrobiologische Untersuchungen zeigten spielen Pilze bei der Entstehung und beim Aufbau eine ueberdurchschnittliche Rolle. Des weiteren konnte in ersten Abbauversuchen der chemische Sauerstoff-Bedarf (CSB) um ueber 90% gesenkt werden. Die Dichtebestimmung ergab vergleichbare Werte zu Belebtschlamm und zwar im Durchschnitt 1,044 g/ml. (orig.)

  10. Gene identification and protein classification in microbial metagenomic sequence data via incremental clustering

    Directory of Open Access Journals (Sweden)

    Li Weizhong

    2008-04-01

    Full Text Available Abstract Background The identification and study of proteins from metagenomic datasets can shed light on the roles and interactions of the source organisms in their communities. However, metagenomic datasets are characterized by the presence of organisms with varying GC composition, codon usage biases etc., and consequently gene identification is challenging. The vast amount of sequence data also requires faster protein family classification tools. Results We present a computational improvement to a sequence clustering approach that we developed previously to identify and classify protein coding genes in large microbial metagenomic datasets. The clustering approach can be used to identify protein coding genes in prokaryotes, viruses, and intron-less eukaryotes. The computational improvement is based on an incremental clustering method that does not require the expensive all-against-all compute that was required by the original approach, while still preserving the remote homology detection capabilities. We present evaluations of the clustering approach in protein-coding gene identification and classification, and also present the results of updating the protein clusters from our previous work with recent genomic and metagenomic sequences. The clustering results are available via CAMERA, (http://camera.calit2.net. Conclusion The clustering paradigm is shown to be a very useful tool in the analysis of microbial metagenomic data. The incremental clustering method is shown to be much faster than the original approach in identifying genes, grouping sequences into existing protein families, and also identifying novel families that have multiple members in a metagenomic dataset. These clusters provide a basis for further studies of protein families.

  11. Effects of gene-augmentation on the formation, characteristics and microbial community of 2,4-dichlorophenoxyacetic acid degrading aerobic microbial granules

    International Nuclear Information System (INIS)

    Quan, Xiang-chun; Ma, Jing-yun; Xiong, Wei-cong; Yang, Zhi-feng

    2011-01-01

    Highlights: ► The first study to cultivate aerobic granules capable of utilizing 2,4-D as the sole carbon source. ► Granules cultivated through gene-augmentation were first compared systematically with the control on granule formation, degradation kinetics, morphology, and microbial community. ► The first report on the fate of transconjugats in the granules during long term operation after bioaugmentation. ► The first study to isolate in dominant bacteria in 2,4-D degrading microbial granules. - Abstract: Development of 2,4-dichlorophenoxyacetic acid (2,4-D) degrading aerobic granular sludge was conducted in two sequencing batch reactors (SBR) with one bioaugmented with a plasmid pJP4 donor strain Pseudomonas putida SM1443 and the other as a control. Half-matured aerobic granules pre-grown on glucose were used as the starting seeds and a two-stage operation strategy was applied. Granules capable of utilizing 2,4-D (about 500 mg/L) as the sole carbon source was successfully cultivated in both reactors. Gene-augmentation resulted in the enhancement of 2,4-D degradation rates by the percentage of 65–135% for the granules on Day 18, and 6–24% for the granules on Day 105. Transconjugants receiving plasmid pJP4 were established in the granule microbial community after bioaugmentation and persisted till the end of operation. Compared with the control granules, the granules in the bioaugmented reactor demonstrated a better settling ability, larger size, more abundant microbial diversity and stronger tolerance to 2,4-D. The finally obtained granules in the bioaugmented and control reactor had a granule size of around 600 μm and 500 μm, a Shannon–Weaver diversity index (H) of 0.96 and 0.55, respectively. A shift in microbial community was found during the granulation process.

  12. Effect of monensin on in vitro fermentation of silages and microbial protein synthesis.

    Science.gov (United States)

    Wischer, Gerald; Boguhn, Jeannette; Steingaß, Herbert; Schollenberger, Margit; Hartung, Karin; Rodehutscord, Markus

    2013-06-01

    The objective of the study was to investigate the effects of monensin on silage fermentation and microbial net protein synthesis. In Experiment 1, monensin (0.5, 1, 2, 4, 6, or 10 µg) was added to syringes that contained 120 mg of grass silage (GS), grass silage and concentrate (GS + C), or maize silage (MS), resulting in concentrations of 4.2, 8.3, 16.7, 33.3, 50.0 and 83.3 mg monensin/kg feed. Samples were incubated for 24 h to determine the monensin concentration that resulted in the maximum reduction in methane production without effects on the total gas production. In Experiment 2, GS and GS + C were incubated in a rumen simulation technique (Rusitec) to assess the monensin effects (133 and 266 mg/kg feed) on the production of total gas, methane and volatile fatty acids (VFA), degradation of nutrients and microbial net protein synthesis. In Experiment 1, methane production was reduced without significant effects on the total gas production; the reductions were 17% (GS), 10% (GS + C) and 13% (MS) with 16.7 (GS), 50.0 (GS + C) and 33.3 (MS) mg monensin/kg feed. Monensin reduced the total gas and methane production in GS and GS + C in Experiment 2. Propionate production was enhanced by monensin, accompanied by a decrease in acetate production. Along with a reduction in crude protein (CP) degradation, monensin reduced the ammonia nitrogen concentration in the effluent of both treatments. While the protein produced by liquid-associated microbes increased with monensin, protein production by solid-associated microbes was reduced. Total microbial net protein synthesis increased in the presence of monensin. Monensin influenced the production of total gas, methane and VFA from the silages without an effect on the degradation of organic matter (OM). Different microbial fractions were affected differently by monensin supplementation. If monensin is used as a tool to reduce methane emission, the supplementation level must be carefully chosen to avoid negative effects on

  13. Protein kinase C involvement in focal adhesion formation

    DEFF Research Database (Denmark)

    Woods, A; Couchman, J R

    1992-01-01

    Matrix molecules such as fibronectin can promote cell attachment, spreading and focal adhesion formation. Although some interactions of fibronectin with cell surface receptors have now been identified, the consequent activation of intracellular messenger systems by cell/matrix interactions have...... still to be elucidated. We show here that the kinase inhibitors H7 and HA1004 reduce focal adhesion and stress fiber formation in response to fibronectin in a dose-dependent manner, and that activators of protein kinase C can promote their formation under conditions where they do not normally form....... Fibroblasts spread within 1h on substrata composed of fibronectin and formed focal adhesions by 3h, as monitored by interference reflection microscopy (IRM) and by labeling for talin, vinculin and integrin beta 1 subunits. In addition, stress fibers were visible. When cells were allowed to spread for 1h...

  14. Blocking of bacterial biofilm formation by a fish protein coating

    DEFF Research Database (Denmark)

    Vejborg, Rebecca Munk; Klemm, Per

    2008-01-01

    Bacterial biofilm formation on inert surfaces is a significant health and economic problem in a wide range of environmental, industrial, and medical areas. Bacterial adhesion is generally a prerequisite for this colonization process and, thus, represents an attractive target for the development......, this proteinaceous coating is characterized with regards to its biofilm-reducing properties by using a range of urinary tract infectious isolates with various pathogenic and adhesive properties. The antiadhesive coating significantly reduced or delayed biofilm formation by all these isolates under every condition...... examined. The biofilm-reducing activity did, however, vary depending on the substratum physicochemical characteristics and the environmental conditions studied. These data illustrate the importance of protein conditioning layers with respect to bacterial biofilm formation and suggest that antiadhesive...

  15. Microbial growth yield estimates from thermodynamics and its importance for degradation of pesticides and formation of biogenic non-extractable residues

    DEFF Research Database (Denmark)

    Brock, Andreas Libonati; Kästner, M.; Trapp, Stefan

    2017-01-01

    NER. Formation of microbial mass can be estimated from the microbial growth yield, but experimental data is rare. Instead, we suggest using prediction methods for the theoretical yield based on thermodynamics. Recently, we presented the Microbial Turnover to Biomass (MTB) method that needs a minimum...... and using the released CO2 as a measure for microbial activity, we predicted a range for the formation of biogenic NER. For the majority of the pesticides, a considerable fraction of the NER was estimated to be biogenic. This novel approach provides a theoretical foundation applicable to the evaluation...

  16. Microbial Protein Production from Candida tropicalis ATCC13803 in a Submerged Batch Fermentation Process

    Directory of Open Access Journals (Sweden)

    Sahar Golaghaiee

    2017-01-01

    Full Text Available Background and Objective: Microbial protein production can resolve one of the major world challenges, i.e. lack of protein sources. Candida tropicalis growth was investigated to specify a medium to reach the highest cell proliferation and protein production.Material and Methods: Fractional factorial design and the index of signal to noise ratio were applied for optimization of microbial protein production. Optimization process was conducted based on the experimental results of Taguchi approach designs. Fermentationwas performed at 25oC and the agitation speed of 300 rpm for 70 h. Ammonium sulfate, iron sulfate, glycine and glucose concentrations were considered as process variables. Optimization of the culture medium composition was conducted in order to obtain the highest cell biomass concentration and protein content. Experiment design was performed based on the Taguchi approach and L-16 orthogonal arrays using Qualitek-4 software.Results and Conclusion: Maximum biomass of 8.72 log (CFU ml-1 was obtained using the optimized medium with 0.3, 0.15, 2 and 80 g l-1 of ammonium sulfate, iron sulfate, glycine and glucose, respectively. Iron sulfate and ammonium sulfate with 41.76% (w w-1 and 35.27% (w w-1 contributions, respectively, were recognized as the main components for cell growth. Glucose and glycine with 17.12% and 5.86% (w w-1 contributions,respectively, also affected cell production. The highest interaction severity index of +54.16% was observed between glycine and glucose while the least one of +0.43% was recorded for ammonium sulfate and glycine. A deviation of 7% between the highestpredicted cell numbers and the experimented count confirms the suitability of the applied statistical method. High protein content of 52.16% (w w-1 as well as low fat and nucleic acids content suggest that Candida tropicalis is a suitable case for commercial processes.Conflict of interest: The authors declare that there is no conflict of interest.

  17. Role of Streptococcus mutans surface proteins for biofilm formation

    Directory of Open Access Journals (Sweden)

    Michiyo Matsumoto-Nakano

    2018-02-01

    Full Text Available Summary: Streptococcus mutans has been implicated as a primary causative agent of dental caries in humans. An important virulence property of the bacterium is its ability to form biofilm known as dental plaque on tooth surfaces. In addition, this organism also produces glucosyltransferases, multiple glucan-binding proteins, protein antigen c, and collagen-binding protein, surface proteins that coordinate to produce dental plaque, thus inducing dental caries. Bacteria utilize quorum-sensing systems to modulate environmental stress responses. A major mechanism of response to signals is represented by the so called two-component signal transduction system, which enables bacteria to regulate their gene expression and coordinate activities in response to environmental stress. As for S. mutans, a signal peptide-mediated quorum-sensing system encoded by comCDE has been found to be a regulatory system that responds to cell density and certain environmental stresses by excreting a peptide signal molecule termed CSP (competence-stimulating peptide. One of its principal virulence factors is production of bacteriocins (peptide antibiotics referred to as mutacins. Two-component signal transduction systems are commonly utilized by bacteria to regulate bacteriocin gene expression and are also related to biofilm formation by S. mutans. Keywords: Streptococcus mutans, Surface proteins, Biofilm, Signal transduction

  18. Nanocoatings for Chronic Wound Repair—Modulation of Microbial Colonization and Biofilm Formation

    Directory of Open Access Journals (Sweden)

    Mara Mădălina Mihai

    2018-04-01

    Full Text Available Wound healing involves a complex interaction between immunity and other natural host processes, and to succeed it requires a well-defined cascade of events. Chronic wound infections can be mono- or polymicrobial but their major characteristic is their ability to develop a biofilm. A biofilm reduces the effectiveness of treatment and increases resistance. A biofilm is an ecosystem on its own, enabling the bacteria and the host to establish different social interactions, such as competition or cooperation. With an increasing incidence of chronic wounds and, implicitly, of chronic biofilm infections, there is a need for alternative therapeutic agents. Nanotechnology shows promising openings, either by the intrinsic antimicrobial properties of nanoparticles or their function as drug carriers. Nanoparticles and nanostructured coatings can be active at low concentrations toward a large variety of infectious agents; thus, they are unlikely to elicit emergence of resistance. Nanoparticles might contribute to the modulation of microbial colonization and biofilm formation in wounds. This comprehensive review comprises the pathogenesis of chronic wounds, the role of chronic wound colonization and infection in the healing process, the conventional and alternative topical therapeutic approaches designed to combat infection and stimulate healing, as well as revolutionizing therapies such as nanotechnology-based wound healing approaches.

  19. The role of microbial iron reduction in the formation of Proterozoic molar tooth structures

    Science.gov (United States)

    Hodgskiss, Malcolm S. W.; Kunzmann, Marcus; Poirier, André; Halverson, Galen P.

    2018-01-01

    Molar tooth structures are poorly understood early diagenetic, microspar-filled voids in clay-rich carbonate sediments. They are a common structure in sedimentary successions dating from 2600-720 Ma, but do not occur in rocks older or younger, with the exception of two isolated Ediacaran occurrences. Despite being locally volumetrically significant in carbonate rocks of this age, their formation and disappearance in the geological record remain enigmatic. Here we present iron isotope data, supported by carbon and oxygen isotopes, major and minor element concentrations, and total organic carbon and sulphur contents for 87 samples from units in ten different basins spanning ca. 1900-635 Ma. The iron isotope composition of molar tooth structures is almost always lighter (modal depletion of 2‰) than the carbonate or residue components in the host sediment. We interpret the isotopically light iron in molar tooth structures to have been produced by dissimilatory iron reduction utilising Fe-rich smectites and Fe-oxyhydroxides in the upper sediment column. The microbial conversion of smectite to illite results in a volume reduction of clay minerals (∼30%) while simultaneously increasing pore water alkalinity. When coupled with wave loading, this biogeochemical process is a viable mechanism to produce voids and subsequently precipitate carbonate minerals. The disappearance of molar tooth structures in the mid-Neoproterozoic is likely linked to a combination of a decrease in smectite abundance, a decline in the marine DIC reservoir, and an increase in the concentration of O2 in shallow seawater.

  20. Characteristics of microbial community involved in early biofilms formation under the influence of wastewater treatment plant effluent.

    Science.gov (United States)

    Peng, Yuke; Li, Jie; Lu, Junling; Xiao, Lin; Yang, Liuyan

    2018-04-01

    Effluents from wastewater treatment plants (WWTPs) containing microorganisms and residual nutrients can influence the biofilm formation. Although the process and mechanism of bacterial biofilm formation have been well characterized, little is known about the characteristics and interaction of bacteria, archaea and eukaryotes in the early colonization, especially under the influence of WWTP effluent. The aim of this study was to characterize the important bacterial, archaeal and eukaryotic species in the early stage of biofilm formation downstream of the WWTP outlet. Water and biofilm samples were collected 24 and 48hr after the deposition of bio-cords in the stream. Illumina Miseq sequencing of the 16S and 18S rDNA showed that, among the three domains, the bacterial biofilm community had the largest alpha and beta diversity. The early bacterial colonizers appeared to be "biofilm-specific", with only a few dominant operational taxonomic units (OTUs) shared between the biofilm and the ambient water environment. Alpha-proteobacteria and Ciliophora tended to dominate the bacterial and eukaryotic communities, respectively, of the early biofilm already at 24hr, whereas archaea played only a minor role during the early stage of colonization. The network analysis showed that the three domains of microbial community connected highly during the early colonization and it might be a characteristic of the microbial communities in the biofilm formation process where co-occurrence relationships could drive coexistence and diversity maintenance within the microbial communities. Copyright © 2017. Published by Elsevier B.V.

  1. Microbial Physiology of the Conversion of Residual Oil to Methane: A Protein Prospective

    Science.gov (United States)

    Morris, Brandon E. L.; Bastida-Lopez, Felipe; von Bergen, Martin; Richnow, Hans-Hermann; Suflita, Joseph M.

    2010-05-01

    Traditional petroleum recovery techniques are unable to extract the majority of oil in most petroliferous deposits. The recovery of even a fraction of residual hydrocarbon in conventional reserves could represent a substantive energy supply. To this end, the microbial conversion of residual oil to methane has gained increasing relevance in recent years [1,2]. Worldwide demand for methane is expected to increase through 2030 [3], as it is a cleaner-burning alternative to traditional fuels [4]. To investigate the microbial physiology of hydrocarbon-decomposition and ultimate methanogenesis, we initiated a two-pronged approach. First, a model alkane-degrading sulfate-reducing bacterium, Desulfoglaeba alkanexedens, was used to interrogate the predominant metabolic pathway(s) differentially expressed during growth on either n-decane or butyrate. A total of 81 proteins were differentially expressed during bacterial growth on butyrate, while 100 proteins were unique to the alkane-grown condition. Proteins related to alkylsuccinate synthase, or the homologous 1-methyl alkylsuccinate synthase, were identified only in the presence of the hydrocarbon. Secondly, we used a newly developed stable isotope probing technique [5] targeted towards proteins to monitor the flux of carbon through a residual oil-degrading bacterial consortium enriched from a gas-condensate contaminated aquifer [1]. Combined carbon and hydrogen stable isotope fractionation identified acetoclastic methanogenesis as the dominant process in this system. Such findings agree with the previous clone library characterization of the consortium. Furthermore, hydrocarbon activation was determined to be the rate-limiting process during the net conversion of residual oil to methane. References 1. Gieg, L.M., K.E. Duncan, and J.M. Suflita, Bioenegy production via microbial conversion of residual oil to natural gas. Appl Environ Micro, 2008. 74(10): p. 3022-3029. 2. Jones, D.M., et al., Crude-oil biodegradation via

  2. Microbial and Chemical Enhancement of In-Situ Carbon Mineralization in Geological Formation

    Energy Technology Data Exchange (ETDEWEB)

    Matter, J.; Chandran, K.

    2013-05-31

    Predictions of global energy usage suggest a continued increase in carbon emissions and rising concentrations of CO{sub 2} in the atmosphere unless major changes are made to the way energy is produced and used. Various carbon capture and storage (CCS) technologies are currently being developed, but unfortunately little is known regarding the fundamental characteristics of CO{sub 2}-mineral reactions to allow a viable in-situ carbon mineralization that would provide the most permanent and safe storage of geologically-injected CO{sub 2}. The ultimate goal of this research project was to develop a microbial and chemical enhancement scheme for in-situ carbon mineralization in geologic formations in order to achieve long-term stability of injected CO{sub 2}. Thermodynamic and kinetic studies of CO{sub 2}-mineral-brine systems were systematically performed to develop the in-situ mineral carbonation process that utilizes organic acids produced by a microbial reactor. The major participants in the project are three faculty members and their graduate and undergraduate students at the School of Engineering and Applied Science and at the Lamont-Doherty Earth Observatory at Columbia University: Alissa Park in Earth and Environmental Engineering & Chemical Engineering (PI), Juerg Matter in Earth and Environmental Science (Co-PI), and Kartik Chandran in Earth and Environmental Engineering (Co-PI). Two graduate students, Huangjing Zhao and Edris Taher, were trained as a part of this project as well as a number of graduate students and undergraduate students who participated part-time. Edris Taher received his MS degree in 2012 and Huangjing Zhao will defend his PhD on Jan. 15th, 2014. The interdisciplinary training provided by this project was valuable to those students who are entering into the workforce in the United States. Furthermore, the findings from this study were and will be published in referred journals to disseminate the results. The list of the papers is given at

  3. New directions towards structure formation and stability of protein-rich foods from globular proteins

    NARCIS (Netherlands)

    Purwanti, N.; Goot, van der A.J.; Boom, R.M.; Vereijken, J.M.

    2010-01-01

    Concentrated protein-rich foods have strong potential to be developed in terms of health and well-being roles. Unfortunately, limitations in creating products with the rights texture and stability hinder the use of those products by consumers. Main reason is that the formation of micro- and

  4. Protein Topology Determines Cysteine Oxidation Fate: The Case of Sulfenyl Amide Formation among Protein Families

    Science.gov (United States)

    Defelipe, Lucas A.; Lanzarotti, Esteban; Gauto, Diego; Marti, Marcelo A.; Turjanski, Adrián G.

    2015-01-01

    Cysteine residues have a rich chemistry and play a critical role in the catalytic activity of a plethora of enzymes. However, cysteines are susceptible to oxidation by Reactive Oxygen and Nitrogen Species, leading to a loss of their catalytic function. Therefore, cysteine oxidation is emerging as a relevant physiological regulatory mechanism. Formation of a cyclic sulfenyl amide residue at the active site of redox-regulated proteins has been proposed as a protection mechanism against irreversible oxidation as the sulfenyl amide intermediate has been identified in several proteins. However, how and why only some specific cysteine residues in particular proteins react to form this intermediate is still unknown. In the present work using in-silico based tools, we have identified a constrained conformation that accelerates sulfenyl amide formation. By means of combined MD and QM/MM calculation we show that this conformation positions the NH backbone towards the sulfenic acid and promotes the reaction to yield the sulfenyl amide intermediate, in one step with the concomitant release of a water molecule. Moreover, in a large subset of the proteins we found a conserved beta sheet-loop-helix motif, which is present across different protein folds, that is key for sulfenyl amide production as it promotes the previous formation of sulfenic acid. For catalytic activity, in several cases, proteins need the Cysteine to be in the cysteinate form, i.e. a low pKa Cys. We found that the conserved motif stabilizes the cysteinate by hydrogen bonding to several NH backbone moieties. As cysteinate is also more reactive toward ROS we propose that the sheet-loop-helix motif and the constraint conformation have been selected by evolution for proteins that need a reactive Cys protected from irreversible oxidation. Our results also highlight how fold conservation can be correlated to redox chemistry regulation of protein function. PMID:25741692

  5. Expanded microbial genome coverage and improved protein family annotation in the COG database.

    Science.gov (United States)

    Galperin, Michael Y; Makarova, Kira S; Wolf, Yuri I; Koonin, Eugene V

    2015-01-01

    Microbial genome sequencing projects produce numerous sequences of deduced proteins, only a small fraction of which have been or will ever be studied experimentally. This leaves sequence analysis as the only feasible way to annotate these proteins and assign to them tentative functions. The Clusters of Orthologous Groups of proteins (COGs) database (http://www.ncbi.nlm.nih.gov/COG/), first created in 1997, has been a popular tool for functional annotation. Its success was largely based on (i) its reliance on complete microbial genomes, which allowed reliable assignment of orthologs and paralogs for most genes; (ii) orthology-based approach, which used the function(s) of the characterized member(s) of the protein family (COG) to assign function(s) to the entire set of carefully identified orthologs and describe the range of potential functions when there were more than one; and (iii) careful manual curation of the annotation of the COGs, aimed at detailed prediction of the biological function(s) for each COG while avoiding annotation errors and overprediction. Here we present an update of the COGs, the first since 2003, and a comprehensive revision of the COG annotations and expansion of the genome coverage to include representative complete genomes from all bacterial and archaeal lineages down to the genus level. This re-analysis of the COGs shows that the original COG assignments had an error rate below 0.5% and allows an assessment of the progress in functional genomics in the past 12 years. During this time, functions of many previously uncharacterized COGs have been elucidated and tentative functional assignments of many COGs have been validated, either by targeted experiments or through the use of high-throughput methods. A particularly important development is the assignment of functions to several widespread, conserved proteins many of which turned out to participate in translation, in particular rRNA maturation and tRNA modification. The new version of the

  6. The influence of nitrogen supplementation on microbial protein synthesis on water-buffaloes

    International Nuclear Information System (INIS)

    Abidin, Zainal; Hendratno, C.; Suharjono; Rustam, B.

    1982-01-01

    This work was carried out to observe the effects of nitrogen supplementation from urea and soybean meal on microbial protein synthesis, and other parameters of rumen functions of the waterbuffalo. Four rations were given to four water-buffaloes assigned in 4x4 latin square design. Ration A consisted of local grass+0% urea, ration B local grass+0.7% urea, ration C local grass+1.4% urea and ration D local grass+8.5% soybean meal. The result indicated that microbial protein synthesis was significantly affected (P/0.05) by the supplementation of urea, and the utilization of N in ration B was more efficient compared to the other rations. The ammonia concentration in the rumen fluid also increased (P/0.05) as a result of urea supplementation. However, no changes were found in the total volatile fatty acids production and total protozoal counts. An increased (P/0.05) of pH in the rumen fluid was also observed in the rations B and C. (author)

  7. Microbial Protein Production and Nitrogen Balance of Local Steer Fed Ammoniated Rice Straws Added

    Directory of Open Access Journals (Sweden)

    H Hindratiningrum

    2009-05-01

    Full Text Available The objective of the experiment was to investigate the kind of energy source feedstuffs on nutrient balance and microbial protein synthesis in local male beef cattle fed with ammoniated rice straws Twenty steers Peranakan Ongole (PO with average age 1-2 years old were used. They were divided 5 groups based on initial body weight as block. Therefore, Completely Randomised Block Design (CBRD was used for this experiment. Data were analysed by analysis variance and continued honestly significant different (HSD to test the differences between means. The result showed that the range MCP and eficiency MCP were 154,61 g/d until 226,54 g/d and 54,08 gMCP/kg DOMR until 62,64 gMCP/kg DOMR. The range of nitrogen balance were 72,28 gram until 111,67 gram. MCP and efficiency MCP were not affected (P>0,05 by the treatments but balance of nitrogen was affected (P<0,05. Diet containing fresh cassava waste as energy source (R2 was lower (P<0,05 than R1 and R4 while between R1,R3 and R4 was similar. This results indicate that feed source of energy (rice brand, wet cassava waste, dry cassava waste and corn can be used in steers with rice straw ensilage as forage. (Animal Production 11(2: 116-121 (2009 Key Words : Microbial protein production, nitrogen balance, rice straw, ensilage

  8. Modelling of microbial polyhydroxyalkanoate surface binding protein PhaP for rational mutagenesis.

    Science.gov (United States)

    Zhao, Hongyu; Yao, Zhenyu; Chen, Xiangbin; Wang, Xinquan; Chen, Guo-Qiang

    2017-11-01

    Phasins are unusual amphiphilic proteins that bind to microbial polyhydroxyalkanoate (PHA) granules in nature and show great potential for various applications in biotechnology and medicine. Despite their remarkable diversity, only the crystal structure of PhaP A h from Aeromonas hydrophila has been solved to date. Based on the structure of PhaP A h , homology models of PhaP A z from Azotobacter sp. FA-8 and PhaP TD from Halomonas bluephagenesis TD were successfully established, allowing rational mutagenesis to be conducted to enhance the stability and surfactant properties of these proteins. PhaP A z mutants, including PhaP A z Q38L and PhaP A z Q78L, as well as PhaP TD mutants, including PhaP TD Q38M and PhaP TD Q72M, showed better emulsification properties and improved thermostability (6-10°C higher melting temperatures) compared with their wild-type homologues under the same conditions. Importantly, the established PhaP homology-modelling approach, based on the high-resolution structure of PhaP A h , can be generalized to facilitate the study of other PhaP members. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  9. Exploitation of the host cell ubiquitin machinery by microbial effector proteins.

    Science.gov (United States)

    Lin, Yi-Han; Machner, Matthias P

    2017-06-15

    Pathogenic bacteria are in a constant battle for survival with their host. In order to gain a competitive edge, they employ a variety of sophisticated strategies that allow them to modify conserved host cell processes in ways that favor bacterial survival and growth. Ubiquitylation, the covalent attachment of the small modifier ubiquitin to target proteins, is such a pathway. Ubiquitylation profoundly alters the fate of a myriad of cellular proteins by inducing changes in their stability or function, subcellular localization or interaction with other proteins. Given the importance of ubiquitylation in cell development, protein homeostasis and innate immunity, it is not surprising that this post-translational modification is exploited by a variety of effector proteins from microbial pathogens. Here, we highlight recent advances in our understanding of the many ways microbes take advantage of host ubiquitylation, along with some surprising deviations from the canonical theme. The lessons learned from the in-depth analyses of these host-pathogen interactions provide a fresh perspective on an ancient post-translational modification that we thought was well understood.This article is part of a Minifocus on Ubiquitin Regulation and Function. For further reading, please see related articles: 'Mechanisms of regulation and diversification of deubiquitylating enzyme function' by Pawel Leznicki and Yogesh Kulathu ( J. Cell Sci. 130 , 1997-2006). 'Cell scientist to watch - Mads Gyrd-Hansen' ( J. Cell Sci. 130 , 1981-1983). © 2017. Published by The Company of Biologists Ltd.

  10. Quantitative physiology of Penicillium cyclopium grown on whey for production of microbial protein

    Energy Technology Data Exchange (ETDEWEB)

    Kim, J H; Libuchi, S; Lebeault, J M

    1981-01-01

    A filamentous fungus, Penicillium cyclopium, capable of growing on deproteinized whey was isolated and characterized for the purpose of production of microbial protein. This organism has a maximum specific growth rate of 0.2/hour at pH 3.0 to 4.5 and 28 degrees C in a medium containing only ammonium nitrogen and deproteinized whey. The yield coefficients are 0.68 g biomass/g lactose, 12.0 g biomass/g nitrogen, and 2.10 g biomass/g oxygen respectively. Crude protein and total nucleic acid contents of this organism are 47.5% and 7.4% (dry cell weight basis), respectively. The profile of essential amino acids show that it could be a good source of animal feed or food protein. However there are several advantages in using fungal cells (Spicer 1971); their amino acid profile is better, the recovery of biomass from the culture broth is much easier, their filamentous structure facilitates production of texturized foodstuffs without extraction and spinning, and they are already accepted as foods in many parts of the world. The authors have selected a filamentous fungus, Penicillium cyclopium which grows fast on deproteinized whey and has a high protein content. This paper describes the quantitative physiology of this organism and the amino acid profile of its protein. (Refs. 19).

  11. Multi-protein delivery by nanodiamonds promotes bone formation.

    Science.gov (United States)

    Moore, L; Gatica, M; Kim, H; Osawa, E; Ho, D

    2013-11-01

    Bone morphogenetic proteins (BMPs) are well-studied regulators of cartilage and bone development that have been Food and Drug Administration (FDA)-approved for the promotion of bone formation in certain procedures. BMPs are seeing more use in oral and maxillofacial surgeries because of recent FDA approval of InFUSE(®) for sinus augmentation and localized alveolar ridge augmentation. However, the utility of BMPs in medical and dental applications is limited by the delivery method. Currently, BMPs are delivered to the surgical site by the implantation of bulky collagen sponges. Here we evaluate the potential of detonation nanodiamonds (NDs) as a delivery vehicle for BMP-2 and basic fibroblast growth factor (bFGF). Nanodiamonds are biocompatible, 4- to 5-nm carbon nanoparticles that have previously been used to deliver a wide variety of molecules, including proteins and peptides. We find that both BMP-2 and bFGF are readily loaded onto NDs by physisorption, forming a stable colloidal solution, and are triggered to release in slightly acidic conditions. Simultaneous delivery of BMP-2 and bFGF by ND induces differentiation and proliferation in osteoblast progenitor cells. Overall, we find that NDs provide an effective injectable alternative for the delivery of BMP-2 and bFGF to promote bone formation.

  12. Surface proteins and the formation of biofilms by Staphylococcus aureus.

    Science.gov (United States)

    Kim, Sung Joon; Chang, James; Rimal, Binayak; Yang, Hao; Schaefer, Jacob

    2018-03-01

    Staphylococcus aureus biofilms pose a serious clinical threat as reservoirs for persistent infections. Despite this clinical significance, the composition and mechanism of formation of S. aureus biofilms are unknown. To address these problems, we used solid-state NMR to examine S. aureus (SA113), a strong biofilm-forming strain. We labeled whole cells and cell walls of planktonic cells, young biofilms formed for 12-24h after stationary phase, and more mature biofilms formed for up to 60h after stationary phase. All samples were labeled either by (i) [ 15 N]glycine and l-[1- 13 C]threonine, or in separate experiments, by (ii) l-[2- 13 C, 15 N]leucine. We then measured 13 C- 15 N direct bonds by C{N} rotational-echo double resonance (REDOR). The increase in peptidoglycan stems that have bridges connected to a surface protein was determined directly by a cell-wall double difference (biofilm REDOR difference minus planktonic REDOR difference). This procedure eliminates errors arising from differences in 15 N isotopic enrichments and from the routing of 13 C label from threonine degradation to glycine. For both planktonic cells and the mature biofilm, 20% of pentaglycyl bridges are not cross-linked and are potential surface-protein attachment sites. None of these sites has a surface protein attached in the planktonic cells, but one-fourth have a surface protein attached in the mature biofilm. Moreover, the leucine-label shows that the concentration of β-strands in leucine-rich regions doubles in the mature biofilm. Thus, a primary event in establishing a S. aureus biofilm is extensive decoration of the cell surface with surface proteins that are linked covalently to the cell wall and promote cell-cell adhesion. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. MannDB – A microbial database of automated protein sequence analyses and evidence integration for protein characterization

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    Kuczmarski Thomas A

    2006-10-01

    Full Text Available Abstract Background MannDB was created to meet a need for rapid, comprehensive automated protein sequence analyses to support selection of proteins suitable as targets for driving the development of reagents for pathogen or protein toxin detection. Because a large number of open-source tools were needed, it was necessary to produce a software system to scale the computations for whole-proteome analysis. Thus, we built a fully automated system for executing software tools and for storage, integration, and display of automated protein sequence analysis and annotation data. Description MannDB is a relational database that organizes data resulting from fully automated, high-throughput protein-sequence analyses using open-source tools. Types of analyses provided include predictions of cleavage, chemical properties, classification, features, functional assignment, post-translational modifications, motifs, antigenicity, and secondary structure. Proteomes (lists of hypothetical and known proteins are downloaded and parsed from Genbank and then inserted into MannDB, and annotations from SwissProt are downloaded when identifiers are found in the Genbank entry or when identical sequences are identified. Currently 36 open-source tools are run against MannDB protein sequences either on local systems or by means of batch submission to external servers. In addition, BLAST against protein entries in MvirDB, our database of microbial virulence factors, is performed. A web client browser enables viewing of computational results and downloaded annotations, and a query tool enables structured and free-text search capabilities. When available, links to external databases, including MvirDB, are provided. MannDB contains whole-proteome analyses for at least one representative organism from each category of biological threat organism listed by APHIS, CDC, HHS, NIAID, USDA, USFDA, and WHO. Conclusion MannDB comprises a large number of genomes and comprehensive protein

  14. Engineered, highly reactive substrates of microbial transglutaminase enable protein labeling within various secondary structure elements.

    Science.gov (United States)

    Rachel, Natalie M; Quaglia, Daniela; Lévesque, Éric; Charette, André B; Pelletier, Joelle N

    2017-11-01

    Microbial transglutaminase (MTG) is a practical tool to enzymatically form isopeptide bonds between peptide or protein substrates. This natural approach to crosslinking the side-chains of reactive glutamine and lysine residues is solidly rooted in food and textile processing. More recently, MTG's tolerance for various primary amines in lieu of lysine have revealed its potential for site-specific protein labeling with aminated compounds, including fluorophores. Importantly, MTG can label glutamines at accessible positions in the body of a target protein, setting it apart from most labeling enzymes that react exclusively at protein termini. To expand its applicability as a labeling tool, we engineered the B1 domain of Protein G (GB1) to probe the selectivity and enhance the reactivity of MTG toward its glutamine substrate. We built a GB1 library where each variant contained a single glutamine at positions covering all secondary structure elements. The most reactive and selective variants displayed a >100-fold increase in incorporation of a recently developed aminated benzo[a]imidazo[2,1,5-cd]indolizine-type fluorophore, relative to native GB1. None of the variants were destabilized. Our results demonstrate that MTG can react readily with glutamines in α-helical, β-sheet, and unstructured loop elements and does not favor one type of secondary structure. Introducing point mutations within MTG's active site further increased reactivity toward the most reactive substrate variant, I6Q-GB1, enhancing MTG's capacity to fluorescently label an engineered, highly reactive glutamine substrate. This work demonstrates that MTG-reactive glutamines can be readily introduced into a protein domain for fluorescent labeling. © 2017 The Protein Society.

  15. Effect of Plants Containing Secondary Compounds with Palm Oil on Feed Intake, Digestibility, Microbial Protein Synthesis and Microbial Population in Dairy Cows

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    N. Anantasook

    2013-06-01

    Full Text Available The objective of this study was to determine the effect of rain tree pod meal with palm oil supplementation on feed intake, digestibility, microbial protein synthesis and microbial populations in dairy cows. Four, multiparous early-lactation Holstein-Friesian crossbred (75% lactating dairy cows with an initial body weight (BW of 405±40 kg and 36±8 DIM were randomly assigned to receive dietary treatments according to a 4×4 Latin square design. The four dietary treatments were un-supplementation (control, supplementation with rain tree pod meal (RPM at 60 g/kg, supplementation with palm oil (PO at 20 g/kg, and supplementation with RPM at 60 g/kg and PO at 20 g/kg (RPO, of total dry matter intake. The cows were offered concentrates, at a ratio of concentrate to milk production of 1:2, and chopped 30 g/kg of urea treated rice straw was fed ad libitum. The RPM contained condensed tannins and crude saponins at 88 and 141 g/kg of DM, respectively. It was found that supplementation with RPM and/or PO to dairy cows diets did not show negative effects on feed intake and ruminal pH and BUN at any times of sampling (p>0.05. However, RPM supplementation resulted in lower crude protein digestibility, NH3-N concentration and number of proteolytic bacteria. It resulted in greater allantoin absorption and microbial crude protein (p<0.05. In addition, dairy cows showed a higher efficiency of microbial N supply (EMNS in both RPM and RPO treatments. Moreover, NDF digestibility and cellulolytic bacteria numbers were highest in RPO supplementation (p<0.05 while, supplementation with RPM and/or PO decreased the protozoa population in dairy cows. Based on this study, supplementation with RPM and/or PO in diets could improve fiber digestibility, microbial protein synthesis in terms of quantity and efficiency and microbial populations in dairy cows.

  16. Examination of protein degradation in continuous flow, microbial electrolysis cells treating fermentation wastewater

    KAUST Repository

    Nam, Joo-Youn

    2014-11-01

    © 2014 Elsevier Ltd. Cellulose fermentation wastewaters (FWWs) contain short chain volatile fatty acids and alcohols, but they also have high concentrations of proteins. Hydrogen gas production from FWW was examined using continuous flow microbial electrolysis cells (MECs), with a focus on fate of the protein. H2 production rates were 0.49±0.05m3/m3-d for the FWW, compared to 0.63±0.02m3/m3-d using a synthetic wastewater containing only acetate (applied potential of 0.9V). Total organic matter removal was 76±6% for the FWW, compared to 87±5% for acetate. The MEC effluent became relatively enriched in protein (69%) compared to that in the original FWW (19%). Protein was completely removed using higher applied voltages (1.0 or 1.2V), but current generation was erratic due to more positive anode potentials (-113±38mV, Eap=1.2V; -338±38mV, 1.0V; -0.426±4mV, 0.9V). Bacteria on the anodes with FWW were primarily Deltaproteobacteria, while Archaea were predominantly Methanobacterium.

  17. Protein 3-nitrotyrosine formation during Trypanosoma cruzi infection in mice

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    M. Naviliat

    2005-12-01

    Full Text Available Nitric oxide (·NO is a diffusible messenger implicated in Trypanosoma cruzi resistance. Excess production of ·NO and oxidants leads to the generation of nitrogen dioxide (·NO2, a strong nitrating agent. Tyrosine nitration is a post-translational modification resulting from the addition of a nitro (-NO2 group to the ortho-position of tyrosine residues. Detection of protein 3-nitrotyrosine is regarded as a marker of nitro-oxidative stress and is observed in inflammatory processes. The formation and role of nitrating species in the control and myocardiopathy of T. cruzi infection remain to be studied. We investigated the levels of ·NO and protein 3-nitrotyrosine in the plasma of C3H and BALB/c mice and pharmacologically modulated their production during the acute phase of T. cruzi infection. We also looked for protein 3-nitrotyrosine in the hearts of infected animals. Our results demonstrated that C3H animals produced higher amounts of ·NO than BALB/c mice, but their generation of peroxynitrite was not proportionally enhanced and they had higher parasitemias. While N G-nitro-arginine methyl ester treatment abolished ·NO production and drastically augmented the parasitism, mercaptoethylguanidine and guanido-ethyl disulfide, at doses that moderately reduced the ·NO and 3-nitrotyrosine levels, paradoxically diminished the parasitemia in both strains. Nitrated proteins were also demonstrated in myocardial cells of infected mice. These data suggest that the control of T. cruzi infection depends not only on the capacity to produce ·NO, but also on its metabolic fate, including the generation of nitrating species that may constitute an important element in parasite resistance and collateral myocardial damage.

  18. Role of EPS, Dispersant and Nutrients on the Microbial Response and MOS Formation in the Subarctic Northeast Atlantic

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    Tony Gutierrez

    2017-04-01

    Full Text Available In this study we report the formation of marine oil snow (MOS, its associated microbial community, the factors influencing its formation, and the microbial response to crude oil in surface waters of the Faroe-Shetland Channel (FSC. The FSC is a subarctic region that is hydrodynamically complex located in the northeast Atlantic where oil extraction is currently occurring and where exploration is likely to expand into its deeper waters (>500 m. A major oil spill in this region may mirror the aftermath that ensued following the Deepwater Horizon (DWH blowout in the Gulf of Mexico, where the massive influx of Macondo crude oil triggered the formation of copious quantities of rapidly sinking MOS and successional blooms of opportunistic oil-degrading bacteria. In laboratory experiments, we simulated environmental conditions in sea surface waters of the FSC using water collected from this site during the winter of 2015. We demonstrated that the presence of dispersant triggers the formation of MOS, and that nutrient amendments magnify this. Illumina MiSeq sequencing revealed the enrichment on MOS of associated oil-degrading (Cycloclasticus, Thalassolituus, Marinobacter and EPS-producing (Halomonas, Pseudoalteromonas, Alteromonas bacteria, and included major representation by Psychrobacter and Cobetia with putative oil-degrading/EPS-producing qualities. The formation of marine snow, in the absence of crude oil and dispersant, in seawater amended with nutrients alone indicated that the de novo synthesis of bacterial EPS is a key factor in MOS formation, and the glycoprotein composition of the MOS aggregates confirmed that its amorphous biopolymeric matrix was of microbial (likely bacterial origin. The presence of dispersants and crude oil with/without nutrients resulted in distinct microbial responses marked by intermittent, and in some cases short-lived, blooms of opportunistic heterotrophs, principally obligate hydrocarbonoclastic (Alcanivorax

  19. Whey protein isolate edible films with essential oils incorporated to improve the microbial quality of poultry.

    Science.gov (United States)

    Fernández-Pan, Idoya; Mendoza, Mauricio; Maté, Juan I

    2013-09-01

    Whey protein isolate edible films with oregano or clove essential oils (EOs) incorporated as natural antimicrobials have been developed, with the aim of enhancing the microbial quality of poultry. The effectiveness of the films was determined against both the whole and selected microbiota developed during different periods of cold storage on the surface of skinless chicken breast. Tests were conducted by using both turbidimetric and agar disc diffusion methods. The antimicrobial edible films developed showed high effectiveness against the main spoilers developed on the surface of skinless chicken breasts cold-stored for 8 days. The films based on oregano EO showed greater effectiveness than those based on clove EO. Still, clove EO could be part of an effective antimicrobial edible film. Enterobacteriaceae was the most susceptible to the effect of the films when lower concentrations of EO were incorporated. The largest inhibition surfaces obtained were provoked by films with the highest concentration of oregano EO incorporated against lactic acid bacteria. The antimicrobial edible films developed in this study inhibited the growth of the microbial populations that developed through storage of the chicken breast and caused its spoilage. The results of this research have direct application in the food industry to enhance the control of the development of spoilers such as Pseudomonas spp. or lactic acid bacteria. © 2013 Society of Chemical Industry.

  20. Soil microbial community composition is correlated to soil carbon processing along a boreal wetland formation gradient

    Science.gov (United States)

    Chapman, Eric; Cadillo-Quiroz, Hinsby; Childers, Daniel L.; Turetsky, Merritt R.; Waldrop, Mark P.

    2017-01-01

    Climate change is modifying global biogeochemical cycles. Microbial communities play an integral role in soil biogeochemical cycles; knowledge about microbial composition helps provide a mechanistic understanding of these ecosystem-level phenomena. Next generation sequencing approaches were used to investigate changes in microbial functional groups during ecosystem development, in response to climate change, in northern boreal wetlands. A gradient of wetlands that developed following permafrost degradation was used to characterize changes in the soil microbial communities that mediate C cycling: a bog representing an “undisturbed” system with intact permafrost, and a younger bog and an older bog that formed following the disturbance of permafrost thaw. Reference 16S rRNA databases and several diversity indices were used to assess structural differences among these communities, to assess relationships between soil microbial community composition and various environmental variables including redox potential and pH. Rates of potential CO2 and CH4 gas production were quantified to correlate sequence data with gas flux. The abundance of organic C degraders was highest in the youngest bog, suggesting higher rates of microbial processes, including potential CH4 production. In addition, alpha diversity was also highest in the youngest bog, which seemed to be related to a more neutral pH and a lower redox potential. These results could potentially be driven by increased niche differentiation in anaerobic soils. These results suggest that ecosystem structure, which was largely driven by changes in edaphic and plant community characteristics between the “undisturbed” permafrost bog and the two bogs formed following permafrost thaw, strongly influenced microbial function.

  1. A hybrid clustering approach to recognition of protein families in 114 microbial genomes

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    Gogarten J Peter

    2004-04-01

    Full Text Available Abstract Background Grouping proteins into sequence-based clusters is a fundamental step in many bioinformatic analyses (e.g., homology-based prediction of structure or function. Standard clustering methods such as single-linkage clustering capture a history of cluster topologies as a function of threshold, but in practice their usefulness is limited because unrelated sequences join clusters before biologically meaningful families are fully constituted, e.g. as the result of matches to so-called promiscuous domains. Use of the Markov Cluster algorithm avoids this non-specificity, but does not preserve topological or threshold information about protein families. Results We describe a hybrid approach to sequence-based clustering of proteins that combines the advantages of standard and Markov clustering. We have implemented this hybrid approach over a relational database environment, and describe its application to clustering a large subset of PDB, and to 328577 proteins from 114 fully sequenced microbial genomes. To demonstrate utility with difficult problems, we show that hybrid clustering allows us to constitute the paralogous family of ATP synthase F1 rotary motor subunits into a single, biologically interpretable hierarchical grouping that was not accessible using either single-linkage or Markov clustering alone. We describe validation of this method by hybrid clustering of PDB and mapping SCOP families and domains onto the resulting clusters. Conclusion Hybrid (Markov followed by single-linkage clustering combines the advantages of the Markov Cluster algorithm (avoidance of non-specific clusters resulting from matches to promiscuous domains and single-linkage clustering (preservation of topological information as a function of threshold. Within the individual Markov clusters, single-linkage clustering is a more-precise instrument, discerning sub-clusters of biological relevance. Our hybrid approach thus provides a computationally efficient

  2. Vertical microbial community variability of carbonate-based cones may provide insight into ancient conical stromatolite formation

    Science.gov (United States)

    Bradley, James; Daille, Leslie; Trivedi, Christopher; Bojanowski, Caitlin; Nunn, Heather; Stamps, Blake; Johnson, Hope; Stevenson, Bradley; Berelson, Will; Corsetti, Frank; Spear, John

    2016-04-01

    Stromatolite morphogenesis is poorly understood, and the process by which microbial mats become mineralized is a primary question in microbialite formation. Ancient conical stromatolites are primarily carbonate-based whereas the few modern analogues in hot springs are either non-mineralized or mineralized by silica. A team from the 2015 International GeoBiology Course investigated carbonate-rich microbial cones from near Little Hot Creek (LHC), Long Valley Caldera, California, to investigate how conical stromatolites might form in a hot spring carbonate system. The cones rise up from a layered microbial mat on the east side of a 45° C pool with very low flow that is super-saturated with respect to CaCO3. Cone structures are 8-30 mm in height, are rigid and do not deform when removed from the pool. Morphological characterization through environmental scanning electronic microscopy revealed that the cone structure is maintained by a matrix of intertwining microbial filaments around carbonate grains. This matrix gives rise to cone-filaments that are arranged vertically or horizontally, and provides further stability to the cone. Preliminary 16S rRNA gene analysis indicated variability of community composition between different vertical levels of the cone. The cone tip had comparatively greater abundance of filamentous cyanobacteria including Leptolingbya, Phormidium and Isosphaera and fewer heterotrophs (e.g. Chloroflexi) compared to the cone bottom. This supports the hypothesis that cone formation may depend on the differential abundance of the microbial community and their potential functional roles. Metagenomic analyses of the cones revealed potential genes related to chemotaxis and motility. Specifically, a genomic bin identified as a member of the genus Isosphaera contained an hmp chemotaxis operon implicated in gliding motility in the cyanobacterium Nostoc punctiforme. Isosphaera is a Planctomycete shown to have phototactic capabilities, and may play a role in

  3. Microbial protein synthesis and concentration of urea in dairy heifers fed diets with cactus forage Opuntia

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    Maria do Socorro Mercês Aguiar

    2015-04-01

    Full Text Available The objective was to analyze the influence of increasing levels of forage cactus Opuntia in the diet on the nitrogen balance, the concentrations of urea in urine and plasma and microbial protein synthesis in dairy heifers ¾ Holstein-zebu confined. twenty four heifers were used with initial body weight of 163.00 ± 18 kg, with 8 months old and distributed in a completely randomized design with four treatments and six replications. It was used sorghum silage, concentrate and increasing levels of forage cactus Opuntia in the diet (0, 200, 400 and 600 g kg-1. The nitrogen intake, feces and urine, digested and retained with the addition of forage cactus in the diet showed decreasing linear effect. Nitrogen balance was influenced by the inclusion of forage cactus in the diet of dairy heifers through the values observed for the digested and retained nitrogen, which can be related to similar effects found for the consumption of nitrogen and the nitrogen excretion in feces and urine. Nitrogen digested percentage of intake and nitrogen retention as a percentage of ingested and digested showed no difference with the inclusion of forage cactus in the diet. The concentration of urea nitrogen in the urine of heifers had a quadratic effect point of maximum excretion level of 275.80 g kg-1 of forage cactus in the diet. Consequently, the excretion of urea nitrogen and urea excretion showed similar effect with maximum points excretion levels of 293.75 and 319.00 g kg-1 of forage in the diet. The concentration of ureic nitrogen in plasma showed no difference, with an average value of 13.19 mg dL-1. Synthesis of nitrogen and microbial crude protein adjusted to the quadratic model. The microbial efficiency was not influenced by the inclusion of forage cactus in replacement of sorghum silage and concentrate. The urine volume similar to the treatments, with an average of 5.90 liters of urine per day, proving that the creatinine excretion in urine was not influenced

  4. Rapamycin-induced oligomer formation system of FRB-FKBP fusion proteins.

    Science.gov (United States)

    Inobe, Tomonao; Nukina, Nobuyuki

    2016-07-01

    Most proteins form larger protein complexes and perform multiple functions in the cell. Thus, artificial regulation of protein complex formation controls the cellular functions that involve protein complexes. Although several artificial dimerization systems have already been used for numerous applications in biomedical research, cellular protein complexes form not only simple dimers but also larger oligomers. In this study, we showed that fusion proteins comprising the induced heterodimer formation proteins FRB and FKBP formed various oligomers upon addition of rapamycin. By adjusting the configuration of fusion proteins, we succeeded in generating an inducible tetramer formation system. Proteins of interest also formed tetramers by fusing to the inducible tetramer formation system, which exhibits its utility in a broad range of biological applications. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  5. The stability and formation of native proteins from unfolded monomers is increased through interactions with unrelated proteins.

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    Claudia Rodríguez-Almazán

    Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

  6. Formation of higher plant component microbial community in closed ecological system

    Science.gov (United States)

    Tirranen, L. S.

    2001-07-01

    Closed ecological systems (CES) place at the disposal of a researcher unique possibilities to study the role of microbial communities in individual components and of the entire system. The microbial community of the higher plant component has been found to form depending on specific conditions of the closed ecosystem: length of time the solution is reused, introduction of intrasystem waste water into the nutrient medium, effect of other component of the system, and system closure in terms of gas exchange. The higher plant component formed its own microbial complex different from that formed prior to closure. The microbial complex of vegetable polyculture is more diverse and stable than the monoculture of wheat. The composition of the components' microflora changed, species diversity decreased, individual species of bacteria and fungi whose numbers were not so great before the closure prevailed. Special attention should be paid to phytopathogenic and conditionally pathogenic species of microorganisms potentially hazardous to man or plants and the least controlled in CES. This situation can endanger creation of CES and make conjectural existence of preplanned components, man, specifically, and consequently, of CES as it is.

  7. Modification of the surfaces of medical devices to prevent microbial adhesion and biofilm formation.

    Science.gov (United States)

    Desrousseaux, C; Sautou, V; Descamps, S; Traoré, O

    2013-10-01

    The development of devices with surfaces that have an effect against microbial adhesion or viability is a promising approach to the prevention of device-related infections. To review the strategies used to design devices with surfaces able to limit microbial adhesion and/or growth. A PubMed search of the published literature. One strategy is to design medical devices with a biocidal agent. Biocides can be incorporated into the materials or coated or covalently bonded, resulting either in release of the biocide or in contact killing without release of the biocide. The use of biocides in medical devices is debated because of the risk of bacterial resistance and potential toxicity. Another strategy is to modify the chemical or physical surface properties of the materials to prevent microbial adhesion, a complex phenomenon that also depends directly on microbial biological structure and the environment. Anti-adhesive chemical surface modifications mostly target the hydrophobicity features of the materials. Topographical modifications are focused on roughness and nanostructures, whose size and spatial organization are controlled. The most effective physical parameters to reduce bacterial adhesion remain to be determined and could depend on shape and other bacterial characteristics. A prevention strategy based on reducing microbial attachment rather than on releasing a biocide is promising. Evidence of the clinical efficacy of these surface-modified devices is lacking. Additional studies are needed to determine which physical features have the greatest potential for reducing adhesion and to assess the usefulness of antimicrobial coatings other than antibiotics. Copyright © 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

  8. Study of the effect of presence or absence of protozoa on rumen fermentation and microbial protein contribution to the chyme.

    Science.gov (United States)

    Belanche, A; Abecia, L; Holtrop, G; Guada, J A; Castrillo, C; de la Fuente, G; Balcells, J

    2011-12-01

    The aim of this study was to investigate the effect of presence or absence of protozoa on rumen fermentation and efficiency of microbial protein synthesis under different diets. Of 20 twin paired lambs, 1 lamb of each pair was isolated from the ewe within 24 h after birth and reared in a protozoa-free environment (n = 10), whereas their respective twin-siblings remained with the ewe (faunated, n = 10). When lambs reached 6 mo of age, 5 animals of each group were randomly allocated to 1 of 2 experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain according to a 2 × 2 factorial arrangement of treatments. After 15 d of adaptation to the diet, the animals were euthanized and total rumen and abomasal contents were sampled to estimate rumen microbial synthesis using C(31) alkane as flow marker. Different ((15)N and purine bases) and a novel (recombinant DNA sequences) microbial markers, combined with several microbial reference extracts (rumen protozoa, liquid and solid associated bacteria) were evaluated. Absence of rumen protozoa modified the rumen fermentation pattern and decreased total tract OM and NDF digestibility in 2.0 and 5.1 percentage points, respectively. The effect of defaunation on microbial N flow was weak, however, and was dependent on the microbial marker and microbial reference extract considered. Faunated lambs fed with mixed diet showed the greatest rumen protozoal concentration and the least efficient microbial protein synthesis (29% less than the other treatments), whereas protozoa-free lambs fed with mixed diet presented the smallest ammonia concentration and 34% greater efficiency of N utilization than the other treatments. Although (15)N gave the most precise estimates of microbial synthesis, the use of recombinant DNA sequences represents an alternative that allows separate quantification of the bacteria and protozoa contributions. This marker showed that presence of protozoa decrease the

  9. Evaluation of the Weevil-damaged Sweet Potato as Substrate for Microbial Protein Obtaining

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    Lic. Antonio Montes-de-Oca-Olivares

    2015-11-01

    Full Text Available The production of microbial protein from agricultural and agroindustrial wastes is an important way to supply the demand of this essential nutritional principle. Sweet potato (Ipomea batata tubercles damaged by weevil (Cylas formicarius are considered a waste due to their unpleasant flavor. This research deal in the characterization of sweet potato damaged by weevil, as an alternative substratefor the culture of the fodder yeast Candida utilis. It was found that the damaged tubercle had a similar composition that the healthy one, concerning dry matter, total reducing sugars, nitrogen and minerals; the high content of reducing sugars (30-40 % dry weight recommends the use of this waste as a substrate for single cell protein production. Several fungal strains were assayed to enzymatic degradation of sweet potato polysaccharides; from these ones, Aspergillus oryzae H/28-1 and Neurospora sp. were the more actives to release reducing sugars to the culture medium, being the last one the more prominent. Theyeast Candida utilis showed a satisfactory growth in media formulated in basis to weevil-damaged sweet potato, reaching reducing sugar consumptions over 80 % and biomass yields of 37-58 %; addition of urea as nitrogen source improved both parameters of the growth. The fermentation’s end-product acquired a pleasant flavor, which suggests a better palatability.

  10. Effects of incorporation of whey protein concentrate on physicochemical, texture, and microbial evaluation of developed cookies

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    Safa Hamid Wani

    2015-12-01

    Full Text Available Whey Protein concentrate (WPC was incorporated into cookies at different levels (0, 2, 4, and 6%. Cookies were analyzed for physicochemical, color, textural, microbial, and sensory attributes. Physicochemical analysis revealed that 6% WPC supplemented cookies shows maximum protein content (13.22%, moisture content (11.33%, fat content (23.08%, and ash content (2.02% as compared to control. However, control sample shows significantly different (p ≤ 0.05 value for crude fiber and carbohydrate content. Maximum thickness (9.63 mm, diameter (44.06 mm, and weight (9.10 g were found for control and these decreased significantly (p ≤ 0.05 with increase in WPC supplementation level in cookies. Cookie supplemented with 4% WPC showed maximum overall acceptability (4.76. Texture analysis revealed that 6% WPC supplemented cookie shows maximum cutting force (55.3 N. Lightness (L* value of cookies decreased from 67.32 to 57.94. Where as a* and b* value increased from 0.37 to 3.57 and 25.35 to 27.54, respectively. The total plate count of cookie samples was under acceptable limits.

  11. Evidence for microbial activity in the formation of carbonate-hosted Zn-Pb deposits

    Science.gov (United States)

    Kucha, H.; Raith, J.

    2009-04-01

    *Kucha H **Raith J *University of Mining and Metallurgy, Faculty of Geology, Geophysics and Environmental Protection, Mickiewicza 30, PL-30-059 Krakow, Poland. ** University of Leoben, Department of Applied Geosciences and Geophysics, A-8700 Leoben, Peter Tunner Str. 5, Austria Evidence for microbial activity in the formation of carbonate-hosted Zn-Pb deposits To date evaluation of bacterial processes in the formation of carbonate-hosted Zn-Pb deposits is largely based on sulphur isotope evidence. However, during a past few years, textural criteria, have been established, which support the bacterial origin of many of these deposits. This has received a strong support from micro-, and nano-textures of naturally growing bacterial films in a flooded tunnel within carbonates that host the Piquette Zn-Pb deposit (Druschel et al., 2002). Bacterial textures, micro- and nano textures found in carbonate-hosted Zn-Pb deposits are: i)wavy bacterial films up to a few mm thick to up to a few cm long composed of peloids, ii)semimassive agglomeration of peloids in the carbonate matrix, and iii)solitary peloids dispersed in the carbonate matrix. Peloids are usually composed of a distinct 50-90um core most often made up of Zn-bearing calcite surrounded by 30-60um thick dentate rim composed of ZnS. Etching of Zn-carbonate cores reveals 1 - 2um ZnS filaments, and numerous 15 to 90nm large ZnS nano-spheres (Kucha et al., 2005). In massive ore composite Zn-calcite - sphalerite peloids are entirely replaced by zinc sulphide, and form peloids ghosts within banded sulphide layers. Bacterially derived micro- and nano-textures have been observed in the following carbonate-hosted Zn-Pb deposits: 1)Irish-type Zn-Pb deposits. In the Navan deposit the basic sulphur is isotopically light bacteriogenic S (Fallick at al., 2001). This is corroborated by semimassive agglomerations of composite peloids (Zn-calcite-ZnS corona or ZnS core-melnikovite corona). Etching of Zn-calcite core reveals globular

  12. Development of Equation Based on Urinary Purine Derivatives to Estimate Rumen Microbial Protein Production in Goats

    International Nuclear Information System (INIS)

    Jetana, Thongsuk; Abdullah, Norhani; Liang, Boo Juan; Syed Salim, Syed Jalaludin; Ho, Wan Yin

    2003-06-01

    Three experiments were conducted at the farm of the Universiti Putra Malaysia, Serdang, Selangor, Malaysia, to establish a model as an index for estimating rumen microbial protein production. In Experiment 1, six Ferral male goats (wt. 40.2±4.6 kg) were used to determine the endogenous purine derivatives (PD) excreted in the urine by fasting. In Experiment 2, four Ferral male goats (wt. 39.6±1.8 kg) were used to measure the proportion of plasma PD excreted in the urine by using [ 14 C]-uric acid as a marker at two levels of feed intake (40% and 80% voluntary intake), using an incomplete 2x4 Latin square experimental design. The feed consisted of 40% oil palm frond and 60% concentrate (OPFC). In Experiment 3, four Ferral male goats fed (OPFC)) were slaughtered and rumen contents were taken for measurements of purine and total nitrogen contents of mixed rumen microbes. The results showed that endogenous PD (allantoin, uric acid, xanthine and hypoxanthine) excreted in the urine obtained by the fasting trial was 202±17 μmol/kg BW 0 . 75 d - 1. The average percentage recovery of plasma PD excretion in the urine by using [ 14 C)-uric acid as a marker was 83±2.0% (cv=6.88, ranged 76.3-91.4%, n=8). Percentage recovery was not affected by levels of feed intake. The ratio of purine N: total N in the mixed rumen liquid associated bacteria (LAB) was 0.085. In this study, a preliminary model for goats was established by using the information from the recovery of labeled PD [ 14 C]-uric acid and the fasting PD excretion. The model obtained was Y 0.83X + 0.202 x BW 0 . 75 , where Y = PD excretion in the urine (mmol/d) X PD absorption at small intestine (mmol/d) BW 0 . 75 = Metabolic body weight (kg) Thus the microbial nitrogen based on total PD (MNpd) can be calculated as follows: MNpd = 70 x X = 0.992 x X (g/d) 0.085 x 0.83 x 1000 where 0.085 is the ratio of purine-N: total N in mixed rumen microbes, 0.83 is the average of digestibility of microbial purine from published

  13. Microbial protein and blood parameters of goats fed with licury cake

    Directory of Open Access Journals (Sweden)

    Máikal Souza Borja

    2014-02-01

    Full Text Available The objective of this study was to determine the ideal level of licury cake in the diet of Boer goats through microbial synthesis estimated based on the presence of purine derivatives in the urine and on blood urea and glucose parameters. Twenty uncastrated one-year-old ¾ Boer goats with an average body weight of 18 kg were distributed in a completely randomized design. Each animal was confined to a one m2 suspended stall with access to water ad libitum. The diets were formulated in accordance with the NRC (2007, and the ingredients were: 50% Tifton-85 (Cynodon sp hay, corn meal, soybean meal, premixed vitamin and mineral supplement, and licury cake. The treatments were: 1 0% of the goat’s total diet composed of licury cake (DM basis, 2 15% of the total diet composed of licury cake, 3 30% of the diet composed of licury cake, and 4 45% of the diet composed of licury cake. The experiment lasted for 17 days. The first 10 days were used to adapt the animals to the diets and facilities. The inclusion of the licury cake in the goat’s diets reduced the levels of blood nitrogen and glucose. Urinary excretion decreased linearly with the inclusion of licury cake in the diet. The inclusion of licury cake in the goat’s diets also caused a linear reduction in the excretion of allantoin, xanthine and hypoxanthine and total purine derivative (PD in urine samples. Based on the microbial protein production and blood parameters of goats fed with licury cake, up to 15% of the goat diet may be composed of licury cake.

  14. Nitrogen balance, microbial protein synthesis and blood metabolites in fattening of male Bali cattle fed ration with different protein levels in smallholder farms

    Directory of Open Access Journals (Sweden)

    P. K. Tahuk

    2018-03-01

    Full Text Available Research was aimed to determine nitrogen balance, microbial protein synthesis, and blood metabolites of male Bali cattle fattening fed ration with different protein level in smallholder farms North Central Timor, Province of East Timor Tenggara, Indonesia. The cattle used were 18 heads aged 2 to 2.5 years with initial body weight of 229.86±12.46 kg. The cattle were randomly divided into three treatment groups. The T0 group was given feed the same as traditional fattening cattle practices by farmers,T1 group fed ration containing 12% crude protein (CP and 72% total digestible nutrients (TDN, andT2 group fedration containing 15% CP and 72%TDN. Cattle were fed individually for 90 days and drinkingwater ad libitum. The data were analyzedby analysis of variance.Results of research indicated the nitrogen balance, and blood urea nitrogen between T1 and T2 were relatively similar, but those were higher (P<0.05 than T0 . In contrast, microbial proteins synthesis, and blood glucose at 0, 4, and 6 hours before and after feeding were relatively similar between the groups. Blood glucose of T2 at 2 hours after intake were higher (P <0.05 than T0, but was not different with T1 . It can be concluded, that the fattening maleBali cattle fed ration containing 12% CP and 72% TDNimprovedthe nitrogen balance and blood metabolites, butit was no positive effect on the microbial proteins and N synthesis.

  15. VCP and ATL1 regulate endoplasmic reticulum and protein synthesis for dendritic spine formation.

    Science.gov (United States)

    Shih, Yu-Tzu; Hsueh, Yi-Ping

    2016-03-17

    Imbalanced protein homeostasis, such as excessive protein synthesis and protein aggregation, is a pathogenic hallmark of a range of neurological disorders. Here, using expression of mutant proteins, a knockdown approach and disease mutation knockin mice, we show that VCP (valosin-containing protein), together with its cofactor P47 and the endoplasmic reticulum (ER) morphology regulator ATL1 (Atlastin-1), regulates tubular ER formation and influences the efficiency of protein synthesis to control dendritic spine formation in neurons. Strengthening the significance of protein synthesis in dendritic spinogenesis, the translation blocker cyclohexamide and the mTOR inhibitor rapamycin reduce dendritic spine density, while a leucine supplement that increases protein synthesis ameliorates the dendritic spine defects caused by Vcp and Atl1 deficiencies. Because VCP and ATL1 are the causative genes of several neurodegenerative and neurodevelopmental disorders, we suggest that impaired ER formation and inefficient protein synthesis are significant in the pathogenesis of multiple neurological disorders.

  16. Protein kinase C mediates platelet secretion and thrombus formation through protein kinase D2.

    Science.gov (United States)

    Konopatskaya, Olga; Matthews, Sharon A; Harper, Matthew T; Gilio, Karen; Cosemans, Judith M E M; Williams, Christopher M; Navarro, Maria N; Carter, Deborah A; Heemskerk, Johan W M; Leitges, Michael; Cantrell, Doreen; Poole, Alastair W

    2011-07-14

    Platelets are highly specialized blood cells critically involved in hemostasis and thrombosis. Members of the protein kinase C (PKC) family have established roles in regulating platelet function and thrombosis, but the molecular mechanisms are not clearly understood. In particular, the conventional PKC isoform, PKCα, is a major regulator of platelet granule secretion, but the molecular pathway from PKCα to secretion is not defined. Protein kinase D (PKD) is a family of 3 kinases activated by PKC, which may represent a step in the PKC signaling pathway to secretion. In the present study, we show that PKD2 is the sole PKD member regulated downstream of PKC in platelets, and that the conventional, but not novel, PKC isoforms provide the upstream signal. Platelets from a gene knock-in mouse in which 2 key phosphorylation sites in PKD2 have been mutated (Ser707Ala/Ser711Ala) show a significant reduction in agonist-induced dense granule secretion, but not in α-granule secretion. This deficiency in dense granule release was responsible for a reduced platelet aggregation and a marked reduction in thrombus formation. Our results show that in the molecular pathway to secretion, PKD2 is a key component of the PKC-mediated pathway to platelet activation and thrombus formation through its selective regulation of dense granule secretion.

  17. Development, standardization and validation of nuclear based technologies for estimating microbial protein supply in ruminant livestock for improving productivity

    International Nuclear Information System (INIS)

    Makkar, H.P.S.

    2004-01-01

    The primary constraint to livestock production in developing countries is the scarcity and fluctuating quantity and quality of the year-round feed supply. These countries experience serious shortages of animal feeds and fodders of the conventional type. Natural forages are very variable both in quality and quantity, conventional agro-industrial by-products are scarce and vary seasonal, and grains are required almost exclusively for human consumption. The small farmers in developing countries have limited resources available to them for feeding their ruminant livestock. Poor nutrition results in low rates of reproduction and production as well as increased susceptibility to disease and mortality. Providing adequate good-quality feed to livestock to raise and maintain their productivity is a major challenge to agricultural scientists and policy makers all over the world. Recent advances in ration balancing include manipulation of feed to increase the quantity and quality of protein and energy delivered to the small intestine. Selection of feeds based on high efficiency of microbial protein synthesis in the rumen along with the high dry matter digestibility, and development of feeding strategies based on high efficiency as well as high microbial protein synthesis in the rumen will lead to higher supply of protein post-ruminally. The strategy for improving production has therefore been to maximize the efficiency of utilization of available feed resources in the rumen by providing optimum conditions for microbial growth and thereby supplementing dietary nutrients to complement and balance the products of rumen digestion to the animal's requirement

  18. The complex formation of selected actinides (U, Np, Cm) with microbial ligands

    International Nuclear Information System (INIS)

    Glorius, Maja

    2009-01-01

    One of the urgent tasks in the field of nuclear technology is the final storage of radioactive substances. As a part of the safety requirements the protection of humans and the environment from the danger of radioactive substances in case of the release from the final storage is essential. For performing long-term safety calculations the detailed understanding of the physico-chemical effects and influences which cause the mobilisation and transport of actinides are necessary. The presented work was a discrete part of a project, which was focused on the clarification of the influence of microorganisms on the migration of actinides in case of the release of actinides from a final storage. The influence of microbial produced substances on the mobilisation of selected actinides was studied thereby. The microbial produced substances studied in this project were synthesized by bacteria from the Pseudomonas genus under special conditions. Fluorescent Pseudomonads secrete bacterial pyoverdin-type siderophores with a high potential to complex and transport metals, especially iron(III). The aim of the project was to determine how and under which conditions the bioligands are able to complex also radioactive substances and therefore to transport them. For this work the alpha-emitting actinides uranium, curium and neptunium were chosen because their long-life cycle and their radiotoxicity are a matter of particular interest. This work dealed with the interaction of the actinides U(VI), Np(V) and Cm(III) with model ligands simulating the functionality of the pyoverdins. So, such bioligands can essentially influence the behaviour of actinides in the environment. The results of this work contribute to a better understanding and assessment of the influence of the microbial ligands to the mobilisation and migration of the radionuclides. The outcomes could be used to quantify the actinide-mobilising effect of the bioligands, which are released, for example, in the vicinity of a

  19. Effect of anode polarization on biofilm formation and electron transfer in Shewanella oneidensis/graphite felt microbial fuel cells.

    Science.gov (United States)

    Pinto, David; Coradin, Thibaud; Laberty-Robert, Christel

    2018-04-01

    In microbial fuel cells, electricity generation is assumed by bacterial degradation of low-grade organics generating electrons that are transferred to an electrode. The nature and efficiency of the electron transfer from the bacteria to the electrodes are determined by several chemical, physical and biological parameters. Specifically, the application of a specific potential at the bioanode has been shown to stimulate the formation of an electro-active biofilm, but the underlying mechanisms remain poorly understood. In this study, we have investigated the effect of an applied potential on the formation and electroactivity of biofilms established by Shewanella oneidensis bacteria on graphite felt electrodes in single- and double-chamber reactor configurations in oxic conditions. Using amperometry, cyclic voltammetry, and OCP/Power/Polarization curves techniques, we showed that a potential ranging between -0.3V and +0.5V (vs. Ag/AgCl/KCl sat.) and its converse application to a couple of electrodes leads to different electrochemical behaviors, anodic currents and biofilm architectures. For example, when the bacteria were confined in the anodic compartment of a double-chamber cell, a negative applied potential (-0.3V) at the bioanode favors a mediated electron transfer correlated with the progressive formation of a biofilm that fills the felt porosity and bridges the graphite fibers. In contrast, a positive applied potential (+0.3V) at the bioanode stimulates a direct electron transfer resulting in the fast-bacterial colonization of the fibers only. These results provide significant insight for the understanding of the complex bacteria-electrode interactions in microbial fuel cells. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Formation of Microbial Mats and Salt in Radioactive Paddy Soils in Fukushima, Japan

    Directory of Open Access Journals (Sweden)

    Kazue Tazaki

    2015-12-01

    Full Text Available Coastal areas in Minami-soma City, Fukushima, Japan, were seriously damaged by radioactive contamination from the Fukushima Daiichi Nuclear Power Plant (FDNPP accident that caused multiple pollution by tsunami and radionuclide exposure, after the Great East Japan Earthquake, on 11 March 2011. Some areas will remain no-go zones because radiation levels remain high. In Minami-soma, only 26 percent of decontamination work had been finished by the end of July in 2015. Here, we report the characterization of microbial mats and salt found on flooded paddy fields at Karasuzaki, Minami-soma City, Fukushima Prefecture, Japan which have been heavily contaminated by radionuclides, especially by Cs (134Cs, 137Cs, 40K, Sr (89Sr, 90Sr, and 91 or 95Zr even though it is more than 30 km north of the FDNPP. We document the mineralogy, the chemistry, and the micro-morphology, using a combination of micro techniques. The microbial mats were found to consist of diatoms with mineralized halite and gypsum by using X-ray diffraction (XRD. Particular elements concentrated in microbial mats were detected using scanning electron microscopy equipped with energy dispersive spectroscopy (SEM-EDS and X-ray fluorescence (XRF. The objective of this contribution is to illustrate the ability of various diatoms associated with minerals and microorganisms which are capable of absorbing both radionuclides and stable isotopes from polluted paddy soils in extreme conditions. Ge semiconductor analysis of the microbial mats detected 134Cs, 137Cs, and 40K without 131I in 2012 and in 2013. Quantitative analysis associated with the elemental content maps by SEM-EDS indicated the possibility of absorption of radionuclide and stable isotope elements from polluted paddy soils in Fukushima Prefecture. In addition, radionuclides were detected in solar salts made of contaminated sea water collected from the Karasuzaki ocean bath, Minami-soma, Fukushima in 2015, showing high Zr content associated

  1. Estimation of rumen microbial protein production from urinary purine derivatives in zebu cattle and water buffalo

    International Nuclear Information System (INIS)

    Liang, J.B.; Pimpa, O.; Abdullah, N.; Jelan, Z.A.; Nolan, J.V.

    1999-01-01

    Two experiments were conducted in order to develop equations for predicting rumen microbial protein production for indigenous Kedah-Kelantan (KK) cattle and swamp buffaloes in Malaysia, using urinary purine derivatives (PD) excretion rates. Endogenous PD excretion rates determined by a fasting procedure for KK cattle and swamp buffalo were 275 and 370 μmol/kg W 0.75 /day, respectively. Urinary PD excretion rate per kg digestible organic matter intake (DOMI) for KK cattle was higher than that for swamp buffalo, reconfirming the earlier findings. Glomerular filtration rate, allantoin and uric acid tubular load and PD re-absorption rate for swamp buffalo were generally higher than those for KK cattle. However, due to the large variations among animals within species, these parameters were not significantly different between species. Nevertheless, the higher PD reabsorption in swamp buffalo provides support for the earlier postulation that the lower urinary PD excretion rate of swamp buffalo was due to their higher recycling of plasma PD as compared to KK cattle. Labelled 8- 14 C uric acid was used to estimate the ratio of renal to non-renal PD excretion. The recovery rates of the radioactive tracer via the renal route for both species were much lower than values reported previously for unlabelled PD for European cattle. (author)

  2. Epitopes of microbial and human heat shock protein 60 and their recognition in myalgic encephalomyelitis.

    Directory of Open Access Journals (Sweden)

    Amal Elfaitouri

    Full Text Available Myalgic encephalomyelitis (ME, also called Chronic Fatigue Syndrome, a common disease with chronic fatigability, cognitive dysfunction and myalgia of unknown etiology, often starts with an infection. The chaperonin human heat shock protein 60 (HSP60 occurs in mitochondria and in bacteria, is highly conserved, antigenic and a major autoantigen. The anti-HSP60 humoral (IgG and IgM immune response was studied in 69 ME patients and 76 blood donors (BD (the Training set with recombinant human and E coli HSP60, and 136 30-mer overlapping and targeted peptides from HSP60 of humans, Chlamydia, Mycoplasma and 26 other species in a multiplex suspension array. Peptides from HSP60 helix I had a chaperonin-like activity, but these and other HSP60 peptides also bound IgG and IgM with an ME preference, theoretically indicating a competition between HSP60 function and antibody binding. A HSP60-based panel of 25 antigens was selected. When evaluated with 61 other ME and 399 non-ME samples (331 BD, 20 Multiple Sclerosis and 48 Systemic Lupus Erythematosus patients, a peptide from Chlamydia pneumoniae HSP60 detected IgM in 15 of 61 (24% of ME, and in 1 of 399 non-ME at a high cutoff (p<0.0001. IgM to specific cross-reactive epitopes of human and microbial HSP60 occurs in a subset of ME, compatible with infection-induced autoimmunity.

  3. QUANTIFICATION OF THE EFFICIENCY OF RUMEN MICROBIAL PROTEIN SYNTHESIS IN STEERS FED GREEN TROPICAL GRASS

    Directory of Open Access Journals (Sweden)

    MARTHEN L. MULLIK

    2012-09-01

    Full Text Available ABSTRACT The rate of rumen microbial crude protein (MCP supply to the intestines is a crucial element in the current rumen models to predict respond of ruminants to a certain diet. Data from tropical pastures always below predicted results from the existing rumen models. Thus, quantification of the rumen MCP supply from tropical grass will improve predictive rate under tropical feeding conditions. Four Brahman crossbred steers (457±20.1 kg were used in a metabolism study. Pangola grass (Digitaria erianthe cv. Steudal was harvested every morning and fed to the animals soon after. Parameters measured were EMPS, intake, fractional passage rates, and rumen ammonia concentration. The EMPS was estimated using purine derivative excretion in urine. Crude protein and water soluble carbohydrates content were 6.3 and 7.4% of dry matter (DM respectively. DM intake was 1.6% live weight. Average rumen ammonia concentration was 69 mg/L whilst rumen passage rates were 7.84 and 6.92 %/h for fluid and solids respectively. EMPS was only 72 g MCP/kg digestible organic matter. It might be concluded that EMPS in steers consuming green pangola grass was below the minimum level for forage diets adopted in the current feeding standards. ABSTRAK Tingkat pasokan protein mikroba rumen (MCP ke usus halus merupakan salah satu unsur kunci dalam meramal respon pertumbuhan ruminan terhadap ransum tertentu. Data MCP hijauan tropis selalu berada di bawah nilai prediksi model rumen yang dipakai saat ini. Dengan demikian, kuantifikasi pasokan MCP rumput tropis diharapkan menjadi masukan untuk meningkatkan kemampuan prediksi model rumen untuk pakan daerah tropis. Empat sapi jantan muda Brahman persilangan (457±20,1 kg digunakan dalam sebuah penelitian metabolisme. Rumput pangola (Digitaria erianthe cv. Steudal dipanen setiap pagi dan langsung diberikan kepada ternak dalam kandang metabolis. Parameter yang diukur adalah produksi MCP dan efisiensi sintesis MCP (Emps, konsumsi, laju

  4. Formate as an energy source for microbial metabolism in chemosynthetic zones of hydrothermal ecosystems.

    Science.gov (United States)

    Windman, Todd; Zolotova, Natalya; Schwandner, Florian; Shock, Everett L

    2007-12-01

    Formate, a simple organic acid known to support chemotrophic hyperthermophiles, is found in hot springs of varying temperature and pH. However, it is not yet known how metabolic strategies that use formate could contribute to primary productivity in hydrothermal ecosystems. In an effort to provide a quantitative framework for assessing the role of formate metabolism, concentration data for dissolved formate and many other solutes in samples from Yellowstone hot springs were used, together with data for coexisting gas compositions, to evaluate the overall Gibbs energy for many reactions involving formate oxidation or reduction. The result is the first rigorous thermodynamic assessment of reactions involving formate oxidation to bicarbonate and reduction to methane coupled with various forms of iron, nitrogen, sulfur, hydrogen, and oxygen for hydrothermal ecosystems. We conclude that there are a limited number of reactions that can yield energy through formate reduction, in contrast to numerous formate oxidation reactions that can yield abundant energy for chemosynthetic microorganisms. Because the energy yields are so high, these results challenge the notion that hydrogen is the primary energy source of chemosynthetic microbes in hydrothermal ecosystems.

  5. Exchange Protein Activated by cAMP Enhances Long-Term Memory Formation Independent of Protein Kinase A

    Science.gov (United States)

    Ma, Nan; Abel, Ted; Hernandez, Pepe J.

    2009-01-01

    It is well established that cAMP signaling within neurons plays a major role in the formation of long-term memories--signaling thought to proceed through protein kinase A (PKA). However, here we show that exchange protein activated by cAMP (Epac) is able to enhance the formation of long-term memory in the hippocampus and appears to do so…

  6. Microbial involvement in the formation of Cambrian sea-floor silica-iron oxide deposits, Australia

    Science.gov (United States)

    Duhig, Nathan C.; Davidson, Garry J.; Stolz, Joe

    1992-06-01

    The Cambrian-Ordovician Mount Windsor volcanic belt in northern Australia is host to stratiform lenses of massive ferruginous chert that are spatially associated with volcanogenic massive sulfide occurrences, in particular the Thalanga zinc-lead-copper-silver deposit. The rocks are composed principally of Fe2O3 and SiO2, with very low concentrations of alkalic elements, and lithogenous elements such as Al, Zr, and Ti; they are interpreted as nearly pure chemical sediments. Textural evidence is documented of the integral role of filamentous bacteria (and/or fungi) in depositing iron from hydrothermal fluids, and of the inorganic precipitation of silica-iron-oxyhydroxide gels that subsequently matured to subcrystalline and crystalline silica forms. At least three distinct iron-accumulating microbial forms are distinguished: networks of septate filaments, nonseptate filament networks, and extremely coarse branching filaments that do not reconnect. Values for δ34S in disseminated pyrite are up to 50‰ lighter than those of contemporaneous Cambrian seawater, suggesting postdepositional colonization of some ironstones by sulfur-reducing bacteria. The site not only preserves the textural interplay of biological and inorganic depositional processes in exhalites, but also extends the oldest known instance of microbial mediation in vent-proximal hydrothermal iron precipitation to at least 500 Ma.

  7. Complete genome sequence of Klebsiella pneumoniae J1, a protein-based microbial flocculant-producing bacterium.

    Science.gov (United States)

    Pang, Changlong; Li, Ang; Cui, Di; Yang, Jixian; Ma, Fang; Guo, Haijuan

    2016-02-20

    Klebsiella pneumoniae J1 is a Gram-negative strain, which belongs to a protein-based microbial flocculant-producing bacterium. However, little genetic information is known about this species. Here we carried out a whole-genome sequence analysis of this strain and report the complete genome sequence of this organism and its genetic basis for carbohydrate metabolism, capsule biosynthesis and transport system. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Estimating rumen microbial protein supply for indigenous ruminants using nuclear and purine excretion techniques in Indonesia

    International Nuclear Information System (INIS)

    Soejono, M.; Yusiati, L.M.; Budhi, S.P.S.; Widyobroto, B.P.; Bachrudin, Z.

    1999-01-01

    The microbial protein supply to ruminants can be estimated based on the amount of purine derivatives (PD) excreted in the urine. Four experiments were conducted to evaluate the PD excretion method for Bali and Ongole cattle. In the first experiment, six male, two year old Bali cattle (Bos sondaicus) and six Ongole cattle (Bos indicus) of similar sex and age, were used to quantify the endogenous contribution to total PD excretion in the urine. In the second experiment, four cattle from each breed were used to examine the response of PD excretion to feed intake. 14 C-uric acid was injected in one single dose to define the partitioning ratio of renal:non-renal losses of plasma PD. The third experiment was conducted to examine the ratio of purine N:total N in mixed rumen microbial population. The fourth experiment measured the enzyme activities of blood, liver and intestinal tissues concerned with PD metabolism. The results of the first experiment showed that endogenous PD excretion was 145 ± 42.0 and 132 ± 20.0 μmol/kg W 0.75 /d, for Bali and Ongole cattle, respectively. The second experiment indicated that the proportion of plasma PD excreted in the urine of Bali and Ongole cattle was 0.78 and 0.77 respectively. Hence, the prediction of purine absorbed based on PD excretion can be stated as Y = 0.78 X + 0.145 W 0.75 and Y = 0.77 X + 0.132 W 0.75 for Bali and Ongole cattle, respectively. The third experiment showed that there were no differences in the ratio of purine N:total N in mixed rumen microbes of Bali and Ongole cattle (17% vs 18%). The last experiment, showed that intestinal xanthine oxidase activity of Bali cattle was lower than that of Ongole cattle (0.001 vs 0.015 μmol uric acid produced/min/g tissue) but xanthine oxidase activity in the blood and liver of Bali cattle was higher than that of Ongole cattle (3.48 vs 1.34 μmol/min/L plasma and 0.191 vs 0.131 μmol/min/g liver tissue). Thus, there was no difference in PD excretion between these two breeds

  9. Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation*

    Science.gov (United States)

    Borovok, Natalia; Nesher, Elimelech; Levin, Yishai; Reichenstein, Michal; Pinhasov, Albert

    2016-01-01

    Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein

  10. Dynamics of Hippocampal Protein Expression During Long-term Spatial Memory Formation.

    Science.gov (United States)

    Borovok, Natalia; Nesher, Elimelech; Levin, Yishai; Reichenstein, Michal; Pinhasov, Albert; Michaelevski, Izhak

    2016-02-01

    Spatial memory depends on the hippocampus, which is particularly vulnerable to aging. This vulnerability has implications for the impairment of navigation capacities in older people, who may show a marked drop in performance of spatial tasks with advancing age. Contemporary understanding of long-term memory formation relies on molecular mechanisms underlying long-term synaptic plasticity. With memory acquisition, activity-dependent changes occurring in synapses initiate multiple signal transduction pathways enhancing protein turnover. This enhancement facilitates de novo synthesis of plasticity related proteins, crucial factors for establishing persistent long-term synaptic plasticity and forming memory engrams. Extensive studies have been performed to elucidate molecular mechanisms of memory traces formation; however, the identity of plasticity related proteins is still evasive. In this study, we investigated protein turnover in mouse hippocampus during long-term spatial memory formation using the reference memory version of radial arm maze (RAM) paradigm. We identified 1592 proteins, which exhibited a complex picture of expression changes during spatial memory formation. Variable linear decomposition reduced significantly data dimensionality and enriched three principal factors responsible for variance of memory-related protein levels at (1) the initial phase of memory acquisition (165 proteins), (2) during the steep learning improvement (148 proteins), and (3) the final phase of the learning curve (123 proteins). Gene ontology and signaling pathways analysis revealed a clear correlation between memory improvement and learning phase-curbed expression profiles of proteins belonging to specific functional categories. We found differential enrichment of (1) neurotrophic factors signaling pathways, proteins regulating synaptic transmission, and actin microfilament during the first day of the learning curve; (2) transcription and translation machinery, protein

  11. hPDB – Haskell library for processing atomic biomolecular structures in protein data bank format

    OpenAIRE

    Gajda, Michał Jan

    2013-01-01

    Background Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. Findings I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The propo...

  12. Microbial protein synthesis and nitrogen metabolism in cows bred on tropical pasture and fed on cassava root and corn

    Directory of Open Access Journals (Sweden)

    Ádler Carvalho da Silva

    2014-05-01

    Full Text Available Current experiment evaluated the effect of replacement of full corn meal by dehydrated ground cassava roots at levels 0%, 25%, 50%, 75% and 100% in experimental supplements for lactating cows grazing on irrigated and fertilized tropical pastures. Ten Holstein cows were divided into two 5 x 5 Latin squares, with average initial 150 days of lactation, milk production 22±3.30 kg day-1 at the beginning of experiment and initial body weight of 603±65 kg. Cows were maintained on pasture consisting of elephant grass (Pennisetum purpureum, Schum cultivar Pioneiro, intercropped with Tifton 85 (Cynodon nlemfuensis, fertilized with 600 kg nitrogen per hectare year-1. There was no significant difference (p > 0.05 between the substitution levels of corn meal by ground and dehydrated cassava root in the concentrate on the synthesis of microbial protein with an estimated average of 1,288.49 g day-1 and efficiency in the synthesis of microbial protein per kilogram of TDN with estimated average of 91.30 g kg-1 TDN. Nitrogen equilibrium showed an estimated average of 218.79 g day-1 of retained nitrogen. The microbial protein synthesis and nitrogen balance were not affected by treatments.

  13. Strategies for targeting tetraspanin proteins: potential therapeutic applications in microbial infections.

    Science.gov (United States)

    Hassuna, Noha; Monk, Peter N; Moseley, Gregory W; Partridge, Lynda J

    2009-01-01

    The identification of novel targets and strategies for therapy of microbial infections is an area of intensive research due to the failure of conventional vaccines or antibiotics to combat both newly emerging diseases (e.g. viruses such as severe acute respiratory syndrome (SARS) and new influenza strains, and antibiotic-resistant bacteria) and entrenched, pandemic diseases exemplified by HIV. One clear approach to this problem is to target processes of the host organism rather than the microbe. Recent data have indicated that members of the tetraspanin superfamily, proteins with a widespread distribution in eukaryotic organisms and 33 members in humans, may provide such an approach. Tetraspanins traverse the membrane four times, but are distinguished from other four-pass membrane proteins by the presence of conserved charged residues in the transmembrane domains and a defining 'signature' motif in the larger of the two extracellular domains (the EC2). They characteristically form promiscuous associations with one another and with other membrane proteins and lipids to generate a specialized type of microdomain: the tetraspanin-enriched microdomain (TEM). TEMs are integral to the main role of tetraspanins as 'molecular organizers' involved in functions such as membrane trafficking, cell-cell fusion, motility, and signaling. Increasing evidence demonstrates that tetraspanins are used by intracellular pathogens as a means of entering and replicating within human cells. Although previous investigations focused mainly on viruses such as hepatitis C and HIV, it is now becoming clear that other microbes associate with tetraspanins, using TEMs as a 'gateway' to infection. In this article we review the properties and functions of tetraspanins/TEMs that are relevant to infective processes and discuss the accumulating evidence that shows how different pathogens exploit these properties in infection and in the pathogenesis of disease. We then investigate the novel and exciting

  14. Heat-induced whey protein isolate fibrils: Conversion, hydrolysis, and disulphide bond formation

    NARCIS (Netherlands)

    Bolder, S.G.; Vasbinder, A.; Sagis, L.M.C.; Linden, van der E.

    2007-01-01

    Fibril formation of individual pure whey proteins and whey protein isolate (WPI) was studied. The heat-induced conversion of WPI monomers into fibrils at pH 2 and low ionic strength increased with heating time and protein concentration. Previous studies, using a precipitation method, size-exclusion

  15. Evaluating the influence of process parameters on soluble microbial products formation using response surface methodology coupled with grey relational analysis.

    Science.gov (United States)

    Xu, Juan; Sheng, Guo-Ping; Luo, Hong-Wei; Fang, Fang; Li, Wen-Wei; Zeng, Raymond J; Tong, Zhong-Hua; Yu, Han-Qing

    2011-01-01

    Soluble microbial products (SMPs) present a major part of residual chemical oxygen demand (COD) in the effluents from biological wastewater treatment systems, and the SMP formation is greatly influenced by a variety of process parameters. In this study, response surface methodology (RSM) coupled with grey relational analysis (GRA) method was used to evaluate the effects of substrate concentration, temperature, NH(4)(+)-N concentration and aeration rate on the SMP production in batch activated sludge reactors. Carbohydrates were found to be the major component of SMP, and the influential priorities of these factors were: temperature>substrate concentration > aeration rate > NH(4)(+)-N concentration. On the basis of the RSM results, the interactive effects of these factors on the SMP formation were evaluated, and the optimal operating conditions for a minimum SMP production in such a batch activated sludge system also were identified. These results provide useful information about how to control the SMP formation of activated sludge and ensure the bioreactor high-quality effluent. Copyright © 2010 Elsevier Ltd. All rights reserved.

  16. Influence of microbial composition on foam formation in a manure-based digester

    DEFF Research Database (Denmark)

    Kougias, Panagiotis; Boe, Kanokwan; O-Thong, Sompong

    2012-01-01

    Foaming is one of the major problems that occasionally occur in the biogas plants, affecting negatively the overall digestion process and results in adverse operational, economical and environmental impacts. The most dominant factors contributing to foaming are organic overloading, feedstock...... manure-based digester of Lemvig biogas plant that was facing foaming problem, comparing with three non-foaming digesters. The research was focused on the quantitative and qualitative analysis of Bacteria and Archaea population and on the identification of Gordonia sp. The reactor samples were analysed...... for foaming properties and microbial analysis. The dynamic population of Bacteria and Archaea were studied by PCR-DGGE method. The results obtained from this study showed that the composition of Bacteria in all reactors was not significantly different indicating that foaming was not caused by Bacteria...

  17. Influence of energy concentration and source on the utilization of feed protein and NPN in lambs. 3. Allantoin excretion and microbial protein synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Ulbrich, M; Geissler, C; Bassuny, S M; Borowiec, F; Hoffmann, M

    1989-06-01

    In an N balance experiment with male crossbreeding lambs at an age of 3-4 months four different rations were given differing in energy concentration (high > 700 EFU/sub cattle//kg DM and low < 650 EFU/sub cattle//kg DM) and in the energy source (sugar, starch or crude fibre) with crude protein intake being almost equal. The rations contained 2% urea. Microbial protein synthesis in the rumen was assessed according to Roth and Kichgessner (1978) (1), Rys et al. (1975) (2) and Bickel-Baumann and Landis (1986) (3) on the basis of allantoin excretion in urine. The highest ruminal protein synthesis quotas were 868-921 mg protein N per kg LW/sup 0.75/ in (2). In (3) 723-766 mg protein N/kg LW /sup 0.75/ were synthesized. From the /sup 15/N labelling of the supplemented urea and the excreted allantoin it could be calculated that 26-40% of the microbial protein resulted from the urea-N of the ration. Despite a high crude protein content of the ration of between 16 and 17% in the DM and a relation of NPN: pure protein of 0.95 the utilization of the NPN in the ration was relatively high but slightly lower than the utilization of pure protein. The variants with higher energy concentration showed as a tendency higher allantoin excretion in spite of slightly lower dry matter intake and a slightly higher NPN utilization than the variants with lower energy concentration. (author).

  18. Thermoplastic processing of proteins for film formation--a review.

    Science.gov (United States)

    Hernandez-Izquierdo, V M; Krochta, J M

    2008-03-01

    Increasing interest in high-quality food products with increased shelf life and reduced environmental impact has encouraged the study and development of edible and/or biodegradable polymer films and coatings. Edible films provide the opportunity to effectively control mass transfer among different components in a food or between the food and its surrounding environment. The diversity of proteins that results from an almost limitless number of side-chain amino-acid sequential arrangements allows for a wide range of interactions and chemical reactions to take place as proteins denature and cross-link during heat processing. Proteins such as wheat gluten, corn zein, soy protein, myofibrillar proteins, and whey proteins have been successfully formed into films using thermoplastic processes such as compression molding and extrusion. Thermoplastic processing can result in a highly efficient manufacturing method with commercial potential for large-scale production of edible films due to the low moisture levels, high temperatures, and short times used. Addition of water, glycerol, sorbitol, sucrose, and other plasticizers allows the proteins to undergo the glass transition and facilitates deformation and processability without thermal degradation. Target film variables, important in predicting biopackage performance under various conditions, include mechanical, thermal, barrier, and microstructural properties. Comparisons of film properties should be made with care since results depend on parameters such as film-forming materials, film formulation, fabrication method, operating conditions, testing equipment, and testing conditions. Film applications include their use as wraps, pouches, bags, casings, and sachets to protect foods, reduce waste, and improve package recyclability.

  19. Polarized protein transport and lumen formation during epithelial tissue morphogenesis.

    Science.gov (United States)

    Blasky, Alex J; Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    One of the major challenges in biology is to explain how complex tissues and organs arise from the collective action of individual polarized cells. The best-studied model of this process is the cross talk between individual epithelial cells during their polarization to form the multicellular epithelial lumen during tissue morphogenesis. Multiple mechanisms of apical lumen formation have been proposed. Some epithelial lumens form from preexisting polarized epithelial structures. However, de novo lumen formation from nonpolarized cells has recently emerged as an important driver of epithelial tissue morphogenesis, especially during the formation of small epithelial tubule networks. In this review, we discuss the latest findings regarding the mechanisms and regulation of de novo lumen formation in vitro and in vivo.

  20. Effects of different sources of protein on digestive characteristics, microbial efficiency, and nutrient flow in dairy goats

    Directory of Open Access Journals (Sweden)

    Nivea Regina de Oliveira Felisberto

    2011-10-01

    Full Text Available Diets formulated with protein sources presenting different resistance to ruminal degradation were compared by evaluating ruminal parameters, production and microbial efficiency and nutrients flow to the omasum in goats. Eight rumen cannulated non-lactating, non-pregnant goats were distributed in a 4 × 4 Latin square design with two replicates. Treatments consisted of four diets where different sources of plant protein accounted for the major protein source named soybean meal, source of higher ruminal degradability, and three other sources of higher resistance of degradation: roasted soybean, corn gluten meal, and cottonseed cake. Amounts of rumen protein were similar among rations; however, flows of dry matter, protein and non-fiber carbohydrate to omasum were higher for diets with protein source with reduced rumen degradation rate. Higher values of rumen ammonia were obtained by using ration with soybean meal as major source of protein. Higher values of pH were obtained for rations with roasted soybean e cottonseed cake. Regarding kinetic of transit, similar values were found among rations. Diets with protein sources presenting reduced ruminal degradation increase nutrients flow to the omasum in goats and alter digestive parameters such as pH and ammonia without compromising bacteria growth and efficiency, which grants their use for dairy goats with similar efficiency to rations using more degradable sources of protein.

  1. Microbial pathways and palaeoenvironmental conditions involved in the formation of phosphorite grains, Safaga District, Egypt

    Science.gov (United States)

    Salama, Walid; El-Kammar, Ahmed; Saunders, Martin; Morsy, Rania; Kong, Charlie

    2015-07-01

    Phosphatic grains of the shallow marine phosphorite deposits of Egypt are classified as either phosphatic bioclasts preserving biological structure (e.g. skeletal fragments such as fish bones and teeth) or phosphatic peloids and intraclasts. This study describes the destructive and constructive microbial pathways represented by bioerosion of bones by endolithic cyanobacteria and accretion of phosphatic peloids by bacteria. The palaeoenvironmental conditions and post-depositional/diagenetic history of these grains have also been considered. Scanning and transmission electron microscopy showed that the phosphatic peloids under transmitted light microscopy are composed mainly of microspheres (0.5 to 2.5 μm) similar in shape and size to coccoid-like bacteria. Chemical mapping showed that these microspheres are composed of carbonate-fluorapatite (CFA) and surrounded by degraded carbonaceous matrix. These grains are suggested to be reworked from pre-existing microbial mats during transgressive-regressive cycles affecting the southern Tethyan Campanian-Maastrichtian shallow continental shelf. The bioerosion of phosphatic bones is characterized by a network of meandering microborings that penetrated inward from the bone surface by endolithic cyanobacteria. The bioerosion of bones resulted in a gradual centripetal digestion and conversion of bones into micritic phosphate peloids. The bioerosion mechanism is probably started in the acidic sheath surrounding cyanobacteria followed by supersaturation of PO4 and reprecipitation of crystalline CFA as electron dense remineralized rims. Electron microprobe microanalyses showed that the remineralized microbored areas are higher in CaO, P2O5, and F and depleted in Cl, relative to unaltered bones. A gradual demineralization of remineralized rims followed by dissolution of cyanobacterial cells is probably occurred during diagenesis and meteoric water alteration leaving behind empty microborings. Bone exposed to meteoric water

  2. Physical Principles of Discrete Hierarchies Formation in Protein Macromolecules

    Science.gov (United States)

    Malyshko, E. V.; Tverdislov, V. A.

    2017-11-01

    A model for chiral periodicity with alternating chiral sense in hierarchies of protein and nucleic acid structures is proposed and substantiated. Regular alternation of the chirality sense is revealed in transitions from the lowest to higher levels of structural-functional organization in proteins where it is L-D-L-D. The stratification principle combines the ideas of biomacromolecules folding and molecular biological machines.

  3. Effect of four local vegetable leaves on the protein and microbial ...

    African Journals Online (AJOL)

    5 were run. The processed beef samples were microbially and chemically analysed after treatment and during storage. Beef treated with leaves extracts revealed Staphylococcus aureus after 28 days of storage, while 49 days for those treated ...

  4. Singlet oxygen-mediated formation of protein peroxides within cells

    International Nuclear Information System (INIS)

    Wright, A.; Policarpio, V.

    2003-01-01

    Full text: Singlet oxygen is generated by a number of cellular, enzymatic and chemical reactions as well as by exposure to UV, or visible light in the presence of a sensitizer; as a consequence this oxidant has been proposed as a damaging agent in a number of pathologies including photo-aging and skin cancer. Proteins are major targets for singlet oxygen as a result of their abundance and high rate constants for reaction. In this study it is shown that illumination of viable, sensitizer-loaded, THP-1 (human monocyte-like) cells with visible light gives rise to intra-cellular protein-derived peroxides. The peroxide yield increases with illumination time, requires the presence of the sensitizer, is enhanced in D 2 O, and decreased by azide; these data are consistent with the mediation of singlet oxygen. The concentration of peroxides detected, which is not affected by glucose or ascorbate loading of the cells, corresponds to ca. 1.5 nmoles peroxide per 10 6 cells using rose bengal as sensitizer, or 10 nmoles per mg cell protein and account for up to ca. 15% of the O 2 consumed by the cells. Similar peroxides have been detected on isolated cellular proteins exposed to light in the presence of rose bengal and oxygen. After cessation of illumination, the cellular protein peroxide levels decreases with t 1/2 ca. 4 hrs at 37 deg C, and this is associated with increased cell lysis. Decomposition of protein peroxides formed within cells, or on isolated cellular proteins, by metal ions, gives rise to radicals as detected by EPR spin trapping. These protein peroxides, and radicals derived from them, can inactivate key cellular enzymes (including caspases, GAPDH and glutathione reductase) and induce DNA base oxidation, strand breaks and DNA-protein cross-links. These studies demonstrate that exposure of intact cells to visible light in the presence of a sensitizer gives rise to novel long-lived, but reactive, intra-cellular protein peroxides via singlet oxygen

  5. Conformational Heterogeneity in Antibody-Protein Antigen Recognition IMPLICATIONS FOR HIGH AFFINITY PROTEIN COMPLEX FORMATION

    Czech Academy of Sciences Publication Activity Database

    Addis, P. W.; Hall, c. J.; Bruton, S.; Veverka, Václav; Wilkinson, I. C.; Muskett, F. W.; Renshaw, P. S.; Prosser, C. E.; Carrington, B.; Lawson, A. D. G.; Griffin, R.; Taylor, R. J.; Waters, L. C.; Henry, A. J.; Carr, M. D.

    2014-01-01

    Roč. 289, č. 10 (2014), s. 7200-7210 ISSN 0021-9258 Institutional support: RVO:61388963 Keywords : NMR * antibody * protein-protein interaction * protein conformation Subject RIV: CE - Biochemistry Impact factor: 4.573, year: 2014

  6. Effects of degradable protein and non-fibre carbohydrates on microbial growth and fermentation in the rumen simulating fermenter (Rusitec

    Directory of Open Access Journals (Sweden)

    Xiang H. Zhao

    2015-05-01

    Full Text Available A rumen simulation technique (Rusitec apparatus with eight 800 ml fermentation vessels was used to investigate the effects of rumen degradable protein (RDP level and non-fibre carbohydrate (NFC type on ruminal fermentation, microbial growth, and populations of ruminal cellulolytic bacteria. Treatments consisted of two NFC types (starch and pectin supplemented with 0 g/d (low RDP or 1.56 g/d (high RDP sodium caseinate. Apparent disappearance of dry matter and organic matter was greater for pectin than for starch treatment (P<0.01 with low or high RDP. A NFC × RDP interaction was observed for neutral detergent fibre disappearance (P=0.01, which was lower for pectin than for starch only under low RDP conditions. Compared with starch, pectin treatment increased the copy numbers of Ruminococcus albus (P≤0.01 and Ruminococcus flavefaciens (P≤0.09, the molar proportion of acetate (P<0.01, the acetate:propionate ratio (P<0.01, and methane production (P<0.01, but reduced the propionate proportion (P<0.01. Increasing dietary RDP increased the production of total VFA (P=0.01, methane (P<0.01, ammonia N (P<0.01, and microbial N (P<0.01. Significant NFC × RDP interaction and interaction tendency were observed for ammonia N production (P=0.01 and daily N flow of total microorganisms (P=0.07, which did not differ under low RDP conditions, but pectin produced greater microbial N and less ammonia N than starch with increased RDP. Results showed NFC type, RDP level, and their interaction affected ruminal fermentation and microbial growth, and under sufficient ruminal degradable N pectin had greater advantage in microbial N synthesis than starch in vitro.

  7. SM30 protein function during sea urchin larval spicule formation.

    Science.gov (United States)

    Wilt, Fred; Killian, Christopher E; Croker, Lindsay; Hamilton, Patricia

    2013-08-01

    A central issue in better understanding the process of biomineralization is to elucidate the function of occluded matrix proteins present in mineralized tissues. A potent approach to addressing this issue utilizes specific inhibitors of expression of known genes. Application of antisense oligonucleotides that specifically suppress translation of a given mRNA are capable of causing aberrant biomineralization, thereby revealing, at least in part, a likely function of the protein and gene under investigation. We have applied this approach to study the possible function(s) of the SM30 family of proteins, which are found in spicules, teeth, spines, and tests of Strongylocentrotus purpuratus as well as other euechinoid sea urchins. It is possible using the anti-SM30 morpholino-oligonucleotides (MO's) to reduce the level of these proteins to very low levels, yet the development of skeletal spicules in the embryo shows little or no aberration. This surprising result requires re-thinking about the role of these, and possibly other occluded matrix proteins. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Green fluorescent protein (GFP) leakage from microbial biosensors provides useful information for the evaluation of the scale-down effect

    DEFF Research Database (Denmark)

    Delvigne, Frank; Brognaux, Alison; Francis, Frédéric

    2011-01-01

    Mixing deficiencies can be potentially detected by the use of a dedicated whole cell microbial biosensor. In this work, a csiE promoter induced under carbon-limited conditions was involved in the elaboration of such biosensor. The cisE biosensor exhibited interesting response after up and down......-shift of the dilution rate in chemostat mode. Glucose limitation was accompanied by green fluorescent protein (GFP) leakage to the extracellular medium. In order to test the responsiveness of microbial biosensors to substrate fluctuations in large-scale, a scale-down reactor (SDR) experiment was performed. The glucose...... fluctuations were characterized at the single cell level and tend to decrease the induction of GFP. Simulations run on the basis of a stochastic hydrodynamic model have shown the variability and the frequencies at which biosensors are exposed to glucose gradient in the SDR. GFP leakage was observed to a great...

  9. Relationship between Protein Accumulation Regulation and Yield Formation in Soybean

    Institute of Scientific and Technical Information of China (English)

    CHEN Lihua; LI Jie; LIU Lijun; ZU Wei

    2006-01-01

    Three different genotypes soybeans were adopted in this experiment under three fertilizer levels.The object of this study was to investigate protein accumulation regulation of soybean cultivars under the condition of different nutrient levels, and their effects on soybean yield and quality, and to provide theoretical evidence for breed, cultivation and agricultural production, also man-powered controllable locations. The concentration of N in the leaves declined after seedling stage, then increased again at stage of early flowering, and started to decrease up to leaf senescence, declined rapidly from seed-filling season to stage of yellow ripeness. The concentration of N in the stems and pod walls declined with growth stage. High seed protein genotypes exhibited higher N assimilating and partitioning during whole growth stages. Pod walls were media of N partitioning. Protein was accumulated mainly during the later period of reproductive growth stage up to harvest, so plant growth after stage of yellow ripeness could not be neglected.

  10. Microbial glycoproteomics

    DEFF Research Database (Denmark)

    Halim, Adnan; Anonsen, Jan Haug

    2017-01-01

    Mass spectrometry-based "-omics" technologies are important tools for global and detailed mapping of post-translational modifications. Protein glycosylation is an abundant and important post translational modification widespread throughout all domains of life. Characterization of glycoproteins...... and research in this area is rapidly accelerating. Here, we review recent developments in glycoproteomic technologies with a special focus on microbial protein glycosylation....

  11. Molecular insights into the microbial formation of marine dissolved organic matter: recalcitrant or labile?

    Science.gov (United States)

    Koch, B. P.; Kattner, G.; Witt, M.; Passow, U.

    2014-08-01

    The degradation of marine dissolved organic matter (DOM) is an important control variable in the global carbon cycle. For our understanding of the kinetics of organic matter cycling in the ocean, it is crucial to achieve a mechanistic and molecular understanding of its transformation processes. A long-term microbial experiment was performed to follow the production of non-labile DOM by marine bacteria. Two different glucose concentrations and dissolved algal exudates were used as substrates. We monitored the bacterial abundance, concentrations of dissolved and particulate organic carbon (DOC, POC), nutrients, amino acids and transparent exopolymer particles (TEP) for 2 years. The molecular characterization of extracted DOM was performed by ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) after 70 days and after ∼2 years of incubation. Although glucose quickly degraded, a non-labile DOC background (5-9% of the initial DOC) was generated in the glucose incubations. Only 20% of the organic carbon from the algal exudate degraded within the 2 years of incubation. The degradation rates for the non-labile DOC background in the different treatments varied between 1 and 11 μmol DOC L-1 year-1. Transparent exopolymer particles, which are released by microorganisms, were produced during glucose degradation but decreased back to half of the maximum concentration within less than 3 weeks (degradation rate: 25 μg xanthan gum equivalents L-1 d-1) and were below detection in all treatments after 2 years. Additional glucose was added after 2 years to test whether labile substrate can promote the degradation of background DOC (co-metabolism; priming effect). A priming effect was not observed but the glucose addition led to a slight increase of background DOC. The molecular analysis demonstrated that DOM generated during glucose degradation differed appreciably from DOM transformed during the degradation of the algal exudates. Our

  12. Soluble Microbial Product Characterization of Biofilm Formation in Bench-Scale

    KAUST Repository

    Mines, Paul

    2012-12-01

    The biological process known as activated sludge (AS) in conjunction with membrane separation technology for the treatment of wastewater has been employed for over four decades. While, membrane biological reactors (MBR) are now widely employed, the phenomenon of membrane fouling is still the most significant factor leading to performance decline of MBRs. Although much research has been done on the subject of MBR fouling over the past two decades, many questions remain unanswered, and consensus within the scientific community is rare. However, research has led to one system parameter generally being regarded as a contributor to membrane fouling, extracellular polymeric compounds (EPS). EPS, and more specifically, the soluble fraction of EPS known as soluble microbial products (SMP), must be further investigated in order to better understand membrane fouling. The biological activity and performance of the MBR is affected by myriad operational parameters, which in turn affects the SMP generated. A commonly varied operational parameter is, depending on the specific treatment needs of a MBR, the sludge retention time (SRT). This study aims to characterize the SMP in three bench-scale MBRs as the SRT is gradually lowered. By studying how the SMP change as the operation of the system is altered, greater understanding of how SMP are related to fouling can be achieved. At the onset of the study, a steady state was established in the system with a SRT of 20 days. Upon stabilization of a 20 day SRT, the system was gradually transitioned to a five and a half day SRT, in stepwise adjustments. Initially, both the trans-membrane pressure (TMP) and the SMP concentrations were at relatively low values, indicating the presence of minimal amounts of biofilm on the membrane surfaces. As the system was altered and more activated sludge was wasted from the reactors, the SRT inherently decreased. As the lower SRT was transitioned and established, the data from TMP measurements, as well

  13. Formate-Dependent Microbial Conversion of CO2 and the Dominant Pathways of methanogenesis in production water of high-temperature oil reservoirs amended with bicarbonate

    Directory of Open Access Journals (Sweden)

    Guang-Chao eYang

    2016-03-01

    Full Text Available CO2 sequestration in deep-subsurface formations including oil reservoirs is a potential measure to reduce the CO2 concentration in the atmosphere. However, the fate of the CO2 and the ecological influences in Carbon Dioxide Capture and Storage (CDCS facilities is not understood clearly. In the current study, the fate of CO2 (in bicarbonate form (0~90 mM with 10 mM of formate as electron donor and carbon source was investigated with high-temperature production water from oilfield in China. The isotope data showed that bicarbonate could be reduced to methane by methanogens and major pathway of methanogenesis could be syntrophic formate oxidation coupled with CO2 reduction and formate methanogenesis under the anaerobic conditions. The bicarbonate addition induced the shift of microbial community. Addition of bicarbonate and formate was associated with a decrease of Methanosarcinales, but promotion of Methanobacteriales in all treatments. Thermodesulfovibrio was the major group in all the samples and Thermacetogenium dominated in the high bicarbonate treatments. The results indicated that CO2 from CDCS could be transformed to methane and the possibility of microbial CO2 conversion for enhanced microbial energy recovery in oil reservoirs.

  14. Protein shape and crowding drive domain formation and curvature in biological membranes

    NARCIS (Netherlands)

    Frese, R.N.; Pamies, Josep C.; Olsen, John D.; Bahatyrova, S.; van der Weij-de Wit, Chantal D.; Aartsma, Thijs J.; Otto, Cornelis; Hunter, C. Neil; Frenkel, Daan; van Grondelle, Rienk

    2007-01-01

    Folding, curvature, and domain formation are characteristics of many biological membranes. Yet the mechanisms that drive both curvature and the formation of specialized domains enriched in particular protein complexes are unknown. For this reason, studies in membranes whose shape and organization

  15. Summary of the co-ordinated research project on development, standardization and validation of nuclear based technologies for estimating microbial protein supply in ruminant livestock for improving productivity

    International Nuclear Information System (INIS)

    Jayasuriya, M.C.N.

    1999-01-01

    A major constraint to animal production in developing countries is poor nutrition due to inadequate or fluctuating nutrient supply. This results in low rates of reproduction and production as well as increased susceptibility to disease and mortality. Microbial cells formed as a result of rumen degradation of carbohydrates under anaerobic conditions are a major source of protein for ruminants. They provide the majority of the amino acids that the host animal requires for tissue maintenance, growth and production. In roughage-fed ruminants, micro-organisms are virtually the only source of protein. Therefore, a knowledge of the microbial contribution to the nutrition of the host animal is essential to developing feed supplementation strategies for improving ruminant production. While this factor has been recognized for many years, it has been extremely difficult to determine the microbial protein contribution to ruminant nutrition. The methods generally used for determining microbial protein production depend on the use of natural microbial markers such as RNA (ribonucleic acid) and DAPA (diamino-pimelic acid) or of isotopes 35 S, 15 N or 32 P. However, these methods involve surgical intervention such as post-rumen cannulation and complex procedures that require accurate and quantitative information on both digesta and microbial marker flow. A calorimetric technique using enzymatic procedures was developed for measuring purine derivatives (PD) in urine under a Technical Contract. With knowledge of the amount of PD excreted in the urine, the microbial protein supply to the host animal can be estimated. The principle of the method is that nucleic acids leaving the rumen are essentially of microbial origin. The nucleic acids are extensively digested in the small intestine and the resulting purines are absorbed

  16. New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation.

    Science.gov (United States)

    González-Martínez, Alejandro; Calderón, Kadiya; González-López, Jesús

    2016-05-01

    High concentrations of proteins and amino acids can be found in wastewater and wastewater stream produced in anaerobic digesters, having shown that amino acids could persist over different managements for nitrogen removal affecting the nitrogen removal processes. Nitrogen removal is completely necessary because of their implications and the significant adverse environmental impact of ammonium such as eutrophication and toxicity to aquatic life on the receiving bodies. In the last decade, the treatment of effluents with high ammonium concentration through anammox-based bioprocesses has been enhanced because these biotechnologies are cheaper and more environmentally friendly than conventional technologies. However, it has been shown that the presence of important amounts of proteins and amino acids in the effluents seriously affects the microbial autotrophic consortia leading to important losses in terms of ammonium oxidation efficiency. Particularly the presence of sulfur amino acids such as methionine and cysteine has been reported to drastically decrease the autotrophic denitrification processes as well as affect the microbial community structure promoting the decline of ammonium oxidizing bacteria in favor of other phylotypes. In this context we discuss that new biotechnological processes that improve the degradation of protein and amino acids must be considered as a priority to increase the performance of the autotrophic denitrification biotechnologies.

  17. Urinary excretion of purine derivatives as an index of microbial protein synthesis in the camel (Camelus dromedarius).

    Science.gov (United States)

    Guerouali, Abdelhai; El Gass, Youssef; Balcells, Joaquim; Belenguer, Alvaro; Nolan, John

    2004-08-01

    Five experiments were carried out to extend knowledge of purine metabolism in the camel (Camelus dromedarius) and to establish a model to enable microbial protein outflow from the forestomachs to be estimated from the urinary excretion of purine derivatives (PD; i.e. xanthine, hypoxanthine, uric acid, allantoin). In experiment 1, four camels were fasted for five consecutive days to enable endogenous PD excretion in urine to be determined. Total PD excretion decreased during the fasting period to 267 (SE 41.5) micromol/kg body weight (W)0.75 per d. Allantoin and xanthine + hypoxanthine were consistently 86 and 6.1 % of total urinary PD during this period but uric acid increased from 3.6 % to 7.4 %. Xanthine oxidase activity in tissues (experiment 2) was (micromol/min per g fresh tissue) 0.038 in liver and 0.005 in gut mucosa but was not detected in plasma. In experiment 3, the duodenal supply of yeast containing exogenous purines produced a linear increase in urinary PD excretion rate with the slope indicating that 0.63 was excreted in urine. After taking account of endogenous PD excretion, the relationship can be used to predict purine outflow from the rumen. From the latter prediction, and also the purine:protein ratio in bacteria determined in experiment 5, we predicted the net microbial outflow from the rumen. In experiment 4, with increasing food intake, the rate of PD excretion in the urine increased linearly by about 11.1 mmol PD/kg digestible organic matter intake (DOMI), equivalent to 95 g microbial protein/kg DOMI.

  18. Role of the MAGUK protein family in synapse formation and function.

    Science.gov (United States)

    Oliva, Carlos; Escobedo, Pía; Astorga, César; Molina, Claudia; Sierralta, Jimena

    2012-01-01

    Synaptic function is crucially dependent on the spatial organization of the presynaptic and postsynaptic apparatuses and the juxtaposition of both membrane compartments. This precise arrangement is achieved by a protein network at the submembrane region of each cell that is built around scaffold proteins. The membrane-associated guanylate kinase (MAGUK) family of proteins is a widely expressed and well-conserved group of proteins that plays an essential role in the formation and regulation of this scaffolding. Here, we review general features of this protein family, focusing on the discs large and calcium/calmodulin-dependent serine protein kinase subfamilies of MAGUKs in the formation, function, and plasticity of synapses. Copyright © 2011 Wiley Periodicals, Inc.

  19. 2BC Non-Structural Protein of Enterovirus A71 Interacts with SNARE Proteins to Trigger Autolysosome Formation.

    Science.gov (United States)

    Lai, Jeffrey K F; Sam, I-Ching; Verlhac, Pauline; Baguet, Joël; Eskelinen, Eeva-Liisa; Faure, Mathias; Chan, Yoke Fun

    2017-07-04

    Viruses have evolved unique strategies to evade or subvert autophagy machinery. Enterovirus A71 (EV-A71) induces autophagy during infection in vitro and in vivo. In this study, we report that EV-A71 triggers autolysosome formation during infection in human rhabdomyosarcoma (RD) cells to facilitate its replication. Blocking autophagosome-lysosome fusion with chloroquine inhibited virus RNA replication, resulting in lower viral titres, viral RNA copies and viral proteins. Overexpression of the non-structural protein 2BC of EV-A71 induced autolysosome formation. Yeast 2-hybrid and co-affinity purification assays showed that 2BC physically and specifically interacted with a N -ethylmaleimide-sensitive factor attachment receptor (SNARE) protein, syntaxin-17 (STX17). Co-immunoprecipitation assay further showed that 2BC binds to SNARE proteins, STX17 and synaptosome associated protein 29 (SNAP29). Transient knockdown of STX17, SNAP29, and microtubule-associated protein 1 light chain 3B (LC3B), crucial proteins in the fusion between autophagosomes and lysosomes) as well as the lysosomal-associated membrane protein 1 (LAMP1) impaired production of infectious EV-A71 in RD cells. Collectively, these results demonstrate that the generation of autolysosomes triggered by the 2BC non-structural protein is important for EV-A71 replication, revealing a potential molecular pathway targeted by the virus to exploit autophagy. This study opens the possibility for the development of novel antivirals that specifically target 2BC to inhibit formation of autolysosomes during EV-A71 infection.

  20. Higher accumulation of F1-V fusion recombinant protein in plants after induction of protein body formation.

    Science.gov (United States)

    Alvarez, M Lucrecia; Topal, Emel; Martin, Federico; Cardineau, Guy A

    2010-01-01

    Improving foreign protein accumulation is crucial for enhancing the commercial success of plant-based production systems since product yields have a major influence on process economics. Cereal grain evolved to store large amounts of proteins in tightly organized aggregates. In maize, gamma-Zein is the major storage protein synthesized by the rough endoplasmic reticulum (ER) and stored in specialized organelles called protein bodies (PB). Zera (gamma-Zein ER-accumulating domain) is the N-terminal proline-rich domain of gamma-zein that is sufficient to induce the assembly of PB formation. Fusion of the Zera domain to proteins of interest results in assembly of dense PB-like, ER-derived organelles, containing high concentration of recombinant protein. Our main goal was to increase recombinant protein accumulation in plants in order to enhance the efficiency of orally-delivered plant-made vaccines. It is well known that oral vaccination requires substantially higher doses than parental formulations. As a part of a project to develop a plant-made plague vaccine, we expressed our model antigen, the Yersinia pestis F1-V antigen fusion protein, with and without a fused Zera domain. We demonstrated that Zera-F1-V protein accumulation was at least 3x higher than F1-V alone when expressed in three different host plant systems: Ncotiana benthamiana, Medicago sativa (alfalfa) and Nicotiana tabacum NT1 cells. We confirmed the feasibility of using Zera technology to induce protein body formation in non-seed tissues. Zera expression and accumulation did not affect plant development and growth. These results confirmed the potential exploitation of Zera technology to substantially increase the accumulation of value-added proteins in plants.

  1. Nonfeed application of rendered animal proteins for microbial production of eicosapentaenoic acid by the fungus Pythium irregulare.

    Science.gov (United States)

    Liang, Yi; Garcia, Rafael A; Piazza, George J; Wen, Zhiyou

    2011-11-23

    Rendered animal proteins are well suited for animal nutrition applications, but the market is maturing, and there is a need to develop new uses for these products. The objective of this study is to explore the possibility of using animal proteins as a nutrient source for microbial production of omega-3 polyunsaturated fatty acids by the microalga Schizochytrium limacinum and the fungus Pythium irregulare. To be absorbed by the microorganisms, the proteins needed to be hydrolyzed into small peptides and free amino acids. The utility of the protein hydrolysates for microorganisms depended on the hydrolysis method used and the type of microorganism. The enzymatic hydrolysates supported better cell growth performance than the alkali hydrolysates did. P. irregulare displayed better overall growth performance on the experimental hydrolysates compared to S. limacinum. When P. irregulare was grown in medium containing 10 g/L enzymatic hydrolysate derived from meat and bone meal or feather meal, the performance of cell growth, lipid synthesis, and omega-3 fatty acid production was comparable to the that of culture using commercial yeast extract. The fungal biomass derived from the animal proteins had 26-29% lipid, 32-34% protein, 34-39% carbohydrate, and industrial microorganisms which can produce omega-3 fatty acids for making omega-3-fortified foods or feeds.

  2. Brown pigment formation in heated sugar-protein mixed suspensions containing unmodified and peptically modified whey protein concentrates.

    Science.gov (United States)

    Rongsirikul, Narumol; Hongsprabhas, Parichat

    2016-01-01

    Commercial whey protein concentrate (WPC) was modified by heating the acidified protein suspensions (pH 2.0) at 80 °C for 30 min and treating with pepsin at 37 °C for 60 min. Prior to spray-drying, such modification did not change the molecular weights (MWs) of whey proteins determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After spray-drying the modified whey protein concentrate with trehalose excipient (MWPC-TH), it was found that the α-lactalbumin (α-La) was the major protein that was further hydrolyzed the most. The reconstituted MWPC-TH contained β-lactoglobulin (β-Lg) as the major protein and small molecular weight (MW) peptides of less than 6.5 kDa. The reconstituted MWPC-TH had higher NH2 group, Trolox equivalent antioxidant capacity (TEAC), lower exposed aromatic ring and thiol (SH) contents than did the commercial WPC. Kinetic studies revealed that the addition of MWPC-TH in fructose-glycine solution was able to reduce brown pigment formation in the mixtures heated at 80 to 95 °C by increasing the activation energy (Ea) of brown pigment formation due to the retardation of fluoresced advanced glycation end product (AGEs) formation. The addition of MWPC to reducing sugar-glycine/commercial WPC was also able to lower brown pigment formation in the sterilized (121 °C, 15 min) mixed suspensions containing 0.1 M reducing sugar and 0.5-1.0 % glycine and/or commercial (P < 0.05). It was demonstrated that the modification investigated in this study selectively hydrolyzed α-La and retained β-Lg for the production of antibrowning whey protein concentrate.

  3. mTORC1 Coordinates Protein Synthesis and Immunoproteasome Formation via PRAS40 to Prevent Accumulation of Protein Stress.

    Science.gov (United States)

    Yun, Young Sung; Kim, Kwan Hyun; Tschida, Barbara; Sachs, Zohar; Noble-Orcutt, Klara E; Moriarity, Branden S; Ai, Teng; Ding, Rui; Williams, Jessica; Chen, Liqiang; Largaespada, David; Kim, Do-Hyung

    2016-02-18

    Reduction of translational fidelity often occurs in cells with high rates of protein synthesis, generating defective ribosomal products. If not removed, such aberrant proteins can be a major source of cellular stress causing human diseases. Here, we demonstrate that mTORC1 promotes the formation of immunoproteasomes for efficient turnover of defective proteins and cell survival. mTORC1 sequesters precursors of immunoproteasome β subunits via PRAS40. When activated, mTORC1 phosphorylates PRAS40 to enhance protein synthesis and simultaneously to facilitate the assembly of the β subunits for forming immunoproteasomes. Consequently, the PRAS40 phosphorylations play crucial roles in clearing aberrant proteins that accumulate due to mTORC1 activation. Mutations of RAS, PTEN, and TSC1, which cause mTORC1 hyperactivation, enhance immunoproteasome formation in cells and tissues. Those mutations increase cellular dependence on immunoproteasomes for stress response and survival. These results define a mechanism by which mTORC1 couples elevated protein synthesis with immunoproteasome biogenesis to protect cells against protein stress. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Analysis of substrate degradation, metabolite formation and microbial community responses in sand bioreactors treating winery wastewater: a comparative study.

    Science.gov (United States)

    Welz, P J; Palmer, Z; Isaacs, S; Kirby, B; le Roes-Hill, M

    2014-12-01

    There is a global need for the implementation of more cost-effective green technologies for the treatment of effluent from wineries. However, systems reliant on microbial biodegradation may be adversely affected by the highly seasonal character of cellar waste. In this study, the biodegradation of two different formulations of winery effluent in sand bioreactors was compared. The degradation of organic substrates and formation of metabolites was monitored by physicochemical analyses of pore water and final effluent samples. Changes in the bacterial community structures were detected using molecular fingerprinting. In wastewater with an overall COD of 2027 mg/L, a formulation with a high concentration of acetate (800 mg COD/L) was more recalcitrant to degradation than a formulation with a high concentration of glucose (800 mg COD/L). Ethanol, glucose and phenolics were degraded preferentially in the deeper layers of the sand bioreactors (average Eh 25 mV) than in the superficial layers (average Eh 102 mV). The redox status also played a pivotal role on the bacterial community composition. The study yielded valuable insight that can be utilized in the design (configuration and operation) of full scale sand bioreactors. Copyright © 2014 Elsevier Ltd. All rights reserved.

  5. Polysaccharide charge density regulating protein adsorption to air/water interfaces by protein/polysaccharide complex formation

    NARCIS (Netherlands)

    Ganzevles, R.A.; Kosters, H.; Vliet, T. van; Stuart, M.A.C.; Jongh, H.H.J. de

    2007-01-01

    Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different

  6. Spatial Autocorrelation, Source Water and the Distribution of Total and Viable Microbial Abundances within a Crystalline Formation to a Depth of 800 m

    Directory of Open Access Journals (Sweden)

    E. D. Beaton

    2017-09-01

    Full Text Available Proposed radioactive waste repositories require long residence times within deep geological settings for which we have little knowledge of local or regional subsurface dynamics that could affect the transport of hazardous species over the period of radioactive decay. Given the role of microbial processes on element speciation and transport, knowledge and understanding of local microbial ecology within geological formations being considered as host formations can aid predictions for long term safety. In this relatively unexplored environment, sampling opportunities are few and opportunistic. We combined the data collected for geochemistry and microbial abundances from multiple sampling opportunities from within a proposed host formation and performed multivariate mixing and mass balance (M3 modeling, spatial analysis and generalized linear modeling to address whether recharge can explain how subsurface communities assemble within fracture water obtained from multiple saturated fractures accessed by boreholes drilled into the crystalline formation underlying the Chalk River Laboratories site (Deep River, ON, Canada. We found that three possible source waters, each of meteoric origin, explained 97% of the samples, these are: modern recharge, recharge from the period of the Laurentide ice sheet retreat (ca. ∼12000 years before present and a putative saline source assigned as Champlain Sea (also ca. 12000 years before present. The distributed microbial abundances and geochemistry provide a conceptual model of two distinct regions within the subsurface associated with bicarbonate – used as a proxy for modern recharge – and manganese; these regions occur at depths relevant to a proposed repository within the formation. At the scale of sampling, the associated spatial autocorrelation means that abundances linked with geochemistry were not unambiguously discerned, although fine scale Moran’s eigenvector map (MEM coefficients were correlated with

  7. Evidence of synsedimentary microbial activity and iron deposition in ferruginous crusts of the Late Cenomanian Utrillas Formation (Iberian Basin, central Spain)

    Science.gov (United States)

    García-Hidalgo, José F.; Elorza, Javier; Gil-Gil, Javier; Herrero, José M.; Segura, Manuel

    2018-02-01

    Ferruginous sandstones and crusts are prominent sedimentary features throughout the continental (braided)-coastal siliciclastic (estuarine-tidal) wedges of the Late Cenomanian Utrillas Formation in the Iberian Basin. Crust types recognized are: Ferruginous sandy crusts (Fsc) with oxides-oxyhydroxides (hematite and goethite) concentrated on sandstone tops presenting a fibro-radial internal structure reminding organic structures that penetrate different mineral phases, suggesting the existence of bacterial activity in crust development; Ferruginous muddy crusts (Fmc) consisting of wavy, laminated, microbial mats, being composed mainly of hematite. On the other hand, a more dispersed and broader mineralization included as Ferruginous sandstones with iron oxides and oxyhydroxides (hematite and goethite) representing a limited cement phase on these sediments. The presence of microbial remains, ferruginous minerals, Microbially-induced sedimentary structures, microbial laminites and vertebrate tracks preserved due to the presence of biofilms suggest firstly a direct evidence of syn-depositional microbial activity in these sediments; and, secondly, that iron accumulation and ferruginous crusts development occurred immediately after deposition of the host, still soft sediments. Ferruginous crusts cap sedimentary cycles and they represent the gradual development of hard substrate conditions, and the development of a discontinuity surface at the top of the parasequence sets, related to very low sedimentary rates; the overlying sediments record subsequent flooding of underlying shallower environments; crusts are, consequently, interpreted as boundaries for these higher-order cycles in the Iberian Basin.

  8. Emulsion properties of pork myofibrillar protein in combination with microbial transglutaminase and calcium alginate under various pH conditions.

    Science.gov (United States)

    Hong, Geun Pyo; Min, Sang-Gi; Chin, Koo Bok

    2012-01-01

    In this study, the effects of microbial transglutaminase (MTG) and calcium alginate (CA) systems in combination with soybean oil on the emulsion properties of porcine myofibrillar protein (MP) were evaluated under various pH conditions. MTG was shown to improve emulsifying capacity and creaming stability, which increased with increasing pH values up to 6.5. The CA did not influence emulsifying capacity, but it improved the creaming stability of the MP-stabilized emulsions. Both MTG and CA enhanced the rheological properties, but their effects on the physical characteristics of the protein evidenced an opposite trend in relation to pH, i.e., the MTG system improved both the emulsion and gelling properties with increasing pH, whereas the CA system was effective when the pH was lowered. By combining the two MP gelling systems, a stable and pH-insensible emulsion could be produced. Copyright © 2011 Elsevier Ltd. All rights reserved.

  9. Microbial analysis of in situ biofilm formation in drinking water distribution systems: implications for monitoring and control of drinking water quality.

    Science.gov (United States)

    Douterelo, Isabel; Jackson, M; Solomon, C; Boxall, J

    2016-04-01

    Biofilm formation in drinking water distribution systems (DWDS) is influenced by the source water, the supply infrastructure and the operation of the system. A holistic approach was used to advance knowledge on the development of mixed species biofilms in situ, by using biofilm sampling devices installed in chlorinated networks. Key physico-chemical parameters and conventional microbial indicators for drinking water quality were analysed. Biofilm coverage on pipes was evaluated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The microbial community structure, bacteria and fungi, of water and biofilms was assessed using pyrosequencing. Conventional wisdom leads to an expectation for less microbial diversity in groundwater supplied systems. However, the analysis of bulk water showed higher microbial diversity in groundwater site samples compared with the surface water site. Conversely, higher diversity and richness were detected in biofilms from the surface water site. The average biofilm coverage was similar among sites. Disinfection residual and other key variables were similar between the two sites, other than nitrates, alkalinity and the hydraulic conditions which were extremely low at the groundwater site. Thus, the unexpected result of an exceptionally low diversity with few dominant genera (Pseudomonas and Basidiobolus) in groundwater biofilm samples, despite the more diverse community in the bulk water, is attributed to the low-flow hydraulic conditions. This finding evidences that the local environmental conditions are shaping biofilm formation, composition and amount, and hence managing these is critical for the best operation of DWDS to safeguard water quality.

  10. Generating functional analysis of complex formation and dissociation in large protein interaction networks

    International Nuclear Information System (INIS)

    Coolen, A C C; Rabello, S

    2009-01-01

    We analyze large systems of interacting proteins, using techniques from the non-equilibrium statistical mechanics of disordered many-particle systems. Apart from protein production and removal, the most relevant microscopic processes in the proteome are complex formation and dissociation, and the microscopic degrees of freedom are the evolving concentrations of unbound proteins (in multiple post-translational states) and of protein complexes. Here we only include dimer-complexes, for mathematical simplicity, and we draw the network that describes which proteins are reaction partners from an ensemble of random graphs with an arbitrary degree distribution. We show how generating functional analysis methods can be used successfully to derive closed equations for dynamical order parameters, representing an exact macroscopic description of the complex formation and dissociation dynamics in the infinite system limit. We end this paper with a discussion of the possible routes towards solving the nontrivial order parameter equations, either exactly (in specific limits) or approximately.

  11. Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

    Science.gov (United States)

    Ehrenworth, Amy M; Claiborne, Tauris; Peralta-Yahya, Pamela

    2017-10-17

    Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.

  12. Effect of benzimidazol-derivatives on the DNA-protein binding formation after UV-radiation of chromatin

    International Nuclear Information System (INIS)

    Mil', E.M.; Binyukov, V.I.; Zhil'tsova, V.M.; Stolyarova, L.G.; Kuznetsov, Yu.V.

    1991-01-01

    Effect of benzimidazol-derivatives on the DNA-protein binding formation was studied after UV-radiation of chromatin. These derivatives were shown to protect chromatin from UV-induced DNA-protein binding formation. Structural analog contained two aminomethyl residuals sensibilized additional binding formation in chromatin. Results suggested, that benzimidazol interacted with DNA, while aminomethyl groups interacted with protein and sensibilized binding of DNA, whilt aminomethyl groups interacted with protein and sensibilized binding of DNA with histone H1

  13. Effect of protein supplementation on ruminal parameters and microbial community fingerprint of Nellore steers fed tropical forages.

    Science.gov (United States)

    Bento, C B P; Azevedo, A C; Gomes, D I; Batista, E D; Rufino, L M A; Detmann, E; Mantovani, H C

    2016-01-01

    In tropical regions, protein supplementation is a common practice in dairy and beef farming. However, the effect of highly degradable protein in ruminal fermentation and microbial community composition has not yet been investigated in a systematic manner. In this work, we aimed to investigate the impact of casein supplementation on volatile fatty acids (VFA) production, specific activity of deamination (SAD), ammonia concentration and bacterial and archaeal community composition. The experimental design was a 4×4 Latin square balanced for residual effects, with four animals (average initial weight of 280±10 kg) and four experimental periods, each with duration of 29 days. The diet comprised Tifton 85 (Cynodon sp.) hay with an average CP content of 9.8%, on a dry matter basis. Animals received basal forage (control) or infusions of pure casein (230 g) administered direct into the rumen, abomasum or divided (50 : 50 ratio) in the rumen/abomasum. There was no differences (P>0.05) in ruminal pH and microbial protein concentration between supplemented v. non-supplemented animals. However, in steers receiving ruminal infusion of casein the SAD and ruminal ammonia concentration increased 33% and 76%, respectively, compared with the control. The total concentration of VFA increased (P0.05) in species richness and diversity of γ-proteobacteria, firmicutes and archaea between non-supplemented Nellore steers and steers receiving casein supplementation in the rumen. However, species richness and the Shannon-Wiener index were lower (Pruminal and postruminal protein supplementation on metabolic activities of rumen microbes and the composition of bacterial and archaeal communities in the rumen of steers.

  14. Nitrite promotes protein carbonylation and Strecker aldehyde formation in experimental fermented sausages: are both events connected?

    Science.gov (United States)

    Villaverde, A; Ventanas, J; Estévez, M

    2014-12-01

    The role played by curing agents (nitrite, ascorbate) on protein oxidation and Strecker aldehyde formation is studied. To fulfill this objective, increasing concentrations of nitrite (0, 75 and 150ppm) and ascorbate (0, 250 and 500ppm) were added to sausages subjected to a 54day drying process. The concurrence of intense proteolysis, protein carbonylation and formation of Strecker aldehydes during processing of sausages suggests that α-aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) may be implicated in the formation of Strecker aldehydes. The fact that nitrite (150ppm, ingoing amount) significantly promoted the formation of protein carbonyls at early stages of processing and the subsequent formation of Strecker aldehydes provides strength to this hypothesis. Ascorbate (125 and 250ppm) controlled the overall extent of protein carbonylation in sausages without declining the formation of Strecker aldehydes. These results may contribute to understanding the chemistry fundamentals of the positive influence of nitrite on the flavor and overall acceptability of cured muscle foods. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Biogenic amine formation and microbial spoilage in chilled garfish ( Belone belone belone ) - effect of modified atmosphere packaging and previous frozen storage

    DEFF Research Database (Denmark)

    Dalgaard, Paw; Madsen, H.L.; Samieian, N.

    2006-01-01

    Abstract Aims: To evaluate biogenic amine formation and microbial spoilage in fresh and thawed chilled garfish. Methods and Results: Storage trials were carried out with fresh and thawed garfish fillets at 0 or 5oC in air or in modified atmosphere packaging (MAP: 40% CO2 and 60% N2). During storage...... 1000 ppm of histamine was formed in garfish; thus even when it is chilled this product represents a histamine fish-poisoning risk....

  16. Induction of protein body formation in plant leaves by elastin-like polypeptide fusions

    Directory of Open Access Journals (Sweden)

    Joensuu Jussi J

    2009-08-01

    Full Text Available Abstract Background Elastin-like polypeptides are synthetic biopolymers composed of a repeating pentapeptide 'VPGXG' sequence that are valuable for the simple non-chromatographic purification of recombinant proteins. In addition, elastin-like polypeptide fusions have been shown to enhance the accumulation of a range of different recombinant proteins in plants, thus addressing the major limitation of plant-based expression systems, which is a low production yield. This study's main objectives were to determine the general utility of elastin-like polypeptide protein fusions in various intracellular compartments and to elucidate elastin-like polypeptide's mechanism of action for increasing recombinant protein accumulation in the endoplasmic reticulum of plants. Results The effect of elastin-like polypeptide fusions on the accumulation of green fluorescent protein targeted to the cytoplasm, chloroplasts, apoplast, and endoplasmic reticulum was evaluated. The endoplasmic reticulum was the only intracellular compartment in which an elastin-like polypeptide tag was shown to significantly enhance recombinant protein accumulation. Interestingly, endoplasmic reticulum-targeted elastin-like polypeptide fusions induced the formation of a novel type of protein body, which may be responsible for elastin-like polypeptide's positive effect on recombinant protein accumulation by excluding the heterologous protein from normal physiological turnover. Although expressed in the leaves of plants, these novel protein bodies appeared similar in size and morphology to the prolamin-based protein bodies naturally found in plant seeds. The elastin-like polypeptide-induced protein bodies were highly mobile organelles, exhibiting various dynamic patterns of movement throughout the cells, which were dependent on intact actin microfilaments and a functional actomyosin motility system. Conclusion An endoplasmic reticulum-targeted elastin-like polypeptide fusion approach

  17. Bovine binder-of-sperm protein BSP1 promotes protrusion and nanotube formation from liposomes

    International Nuclear Information System (INIS)

    Lafleur, Michel; Courtemanche, Lesley; Karlsson, Goeran; Edwards, Katarina; Schwartz, Jean-Louis; Manjunath, Puttaswamy

    2010-01-01

    Research highlights: → Binder-of-sperm protein 1 (BSP1) modifies the morphology of lipidic vesicles inducing bead necklace-like and thread-like structures. → In the presence of multilamellar liposomes, BSP1 leads to the formation of long nanotubes. → The insertion of BSP1 in the external lipid leaflet of membranes induces local changes in bilayer curvature. -- Abstract: Binder-of-sperm (BSP) proteins interact with sperm membranes and are proposed to extract selectively phosphatidylcholine and cholesterol from these. This change in lipid composition is a key step in sperm capacitation. The present work demonstrates that the interactions between the protein BSP1 and model membranes composed with phosphatidylcholine lead to drastic changes in the morphology of the lipidic self-assemblies. Using cryo-electron microscopy and fluorescence microscopy, we show that, in the presence of the protein, the lipid vesicles elongate, and form bead necklace-like structures that evolve toward small vesicles or thread-like structures. In the presence of multilamellar vesicles, where a large reservoir of lipid is available, the presence of BSP proteins lead to the formation of long nanotubes. Long spiral-like threads, associated with lipid/protein complexes, are also observed. The local curvature of lipid membranes induced by the BSP proteins may be involved in lipid domain formation and the extraction of some lipids during the sperm maturation process.

  18. Methymercury Formation in Marine and Freshwater Systems: Sediment Characteristics, Microbial Activity and SRB Phylogeny Control Formation Rates and Food-Chain Exposure

    Science.gov (United States)

    King, J. K.; Saunders, F. M.

    2004-05-01

    Mercury research in freshwater and marine systems suggests that sediment characteristics such as organic substrate, mercury speciation, and sulfate/sulfide concentrations influence availability of inorganic mercury for methylation. Similarly, sediment characteristics also influence sulfate-reducing bacterial (SRB) respiration as well as the presence/distribution of phylogenetic groups responsible for mercury methylation. Our work illustrates that the process of methylmercury formation in freshwater and marine systems are not dissimilar. Rather, the same geochemical parameters and SRB phylogenetic groups determine the propensity for methylmercury formation and are applicable in both fresh- and marine-water systems. The presentation will include our integration of sediment geochemical and microbial parameters affecting mercury methylation in specific freshwater and marine systems. Constructed wetlands planted with Schoenoplectus californicus and amended with gypsum (CaSO4) have demonstrated a capacity to remove inorganic mercury from industrial outfalls. However, bioaccumulation studies of periphyton, eastern mosquitofish (Gambusia holbrooki) and lake chubsucker (Erimyzon sucetta) were conducted in order to ascertain the availability of wetland-generated methylmercury to biota. Total mercury concentrations in mosquitofish from non-sulfate treated controls and the reference location were significantly lower than those from the low and high sulfate treatments while mean total mercury concentrations in lake chubsuckers were also significantly elevated in the high sulfate treatment compared to the low sulfate, control and reference populations. Methylmercury concentrations in periphyton also corresponded with mercury levels found in the tissue of the lake chubsuckers, and these findings fit well given the trophic levels identified for both species of fish. Overall, data from this study suggest that the initial use of gypsum to accelerate the maturity of a constructed

  19. Effect of advanced oxidation on N-nitrosodimethylamine (NDMA) formation and microbial ecology during pilot-scale biological activated carbon filtration.

    Science.gov (United States)

    Li, Dong; Stanford, Ben; Dickenson, Eric; Khunjar, Wendell O; Homme, Carissa L; Rosenfeldt, Erik J; Sharp, Jonathan O

    2017-04-15

    Water treatment combining advanced oxidative processes with subsequent exposure to biological activated carbon (BAC) holds promise for the attenuation of recalcitrant pollutants. Here we contrast oxidation and subsequent biofiltration of treated wastewater effluent employing either ozone or UV/H 2 O 2 followed by BAC during pilot-scale implementation. Both treatment trains largely met target water quality goals by facilitating the removal of a suite of trace organics and bulk water parameters. N-nitrosodimethylamine (NDMA) formation was observed in ozone fed BAC columns during biofiltration and to a lesser extent in UV/H 2 O 2 fed columns and was most pronounced at 20 min of empty bed contact time (EBCT) when compared to shorter EBCTs evaluated. While microbial populations were highly similar in the upper reaches, deeper samples revealed a divergence within and between BAC filtration systems where EBCT was identified to be a significant environmental predictor for shifts in microbial populations. The abundance of Nitrospira in the top samples of both columns provides an explanation for the oxidation of nitrite and corresponding increases in nitrate concentrations during BAC transit and support interplay between nitrogen cycling with nitrosamine formation. The results of this study demonstrate that pretreatments using ozone versus UV/H 2 O 2 impart modest differences to the overall BAC microbial population structural and functional attributes, and further highlight the need to evaluate NDMA formation prior to full-scale implementation of BAC in potable reuse applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. A community standard format for the representation of protein affinity reagents

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Orchard, Sandra; Bertinetti, Daniela

    2010-01-01

    Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often...... that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange....... Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of Proteome...

  1. Time of Day Influences Memory Formation and dCREB2 Proteins in Drosophila

    Directory of Open Access Journals (Sweden)

    Robin eFropf

    2014-03-01

    Full Text Available Many biological phenomena oscillate under the control of the circadian system, exhibiting peaks and troughs of activity across the day/night cycle. In most animal models, memory formation also exhibits this property, but the underlying neuronal and molecular mechanisms remain unclear. The dCREB2 transcription factor shows circadian regulated oscillations in its activity, and has been shown to be important for both circadian biology and memory formation. We show that the time-of-day (TOD of behavioral training affects Drosophila memory formation. dCREB2 exhibits complex changes in protein levels across the daytime and nighttime, and these changes in protein abundance are likely to contribute to oscillations in dCREB2 activity and TOD effects on memory formation.

  2. Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

    Science.gov (United States)

    Ryder, Kate; Bekhit, Alaa El-Din; McConnell, Michelle; Carne, Alan

    2016-10-01

    Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Contribution of eukaryotic microbial communities to the formation of Fe-rich accretions in an extreme acidic environment

    Science.gov (United States)

    Rodrigues, L.; Valente, T.; Correia, A.; Alves, A.; Foing, B.; Davies, G. R.

    2012-04-01

    In the acid mine drainage of Valdarcas, northern Portugal, Fe-rich tubular and spherical macroaccretions are directly associated with the presence of eukaryotic microorganisms. This raises the question whether they are biogenically-derived or the result of an abiotic process mediated by microeukaryotic phototrophs. The drainage water at Valdarcas is characterized by very low pH values (pHhigh metal solubility and presence of iron colloids. Mineralogical analysis (XRD and SEM) of the precipitates indicates a mixture of goethite, schwertmannite and jarosite. Euglenophyta and Chlorophyta acidophilic algal were previously identified in this site. The spatial distribution of Euglena mutabilis indicated that it has a preference to grow up on schwertmannite-rich precipitates. Field observations demonstrate the existence of oxygenated microenvironments created by algal activity suggesting that algae influence iron minerals precipitation, especially schwertmannite. The mineral-microorganism interactions are relevant to understanding this unique and extreme environment. Further investigations regarding the mineralogical and chemical characterization of these deposits, and the identification of microorganisms involved in the process could be helpful to enhance our knowledge of past Fe formations throughout Earth's primordial environment. It is expectable that this information will contribute to establish a framework for recognition of biosignatures on other planets and extraterrestrial bodies. In this study, results on the chemical and mineralogical composition of the structures are presented. The biological context is characterised based on observations made by optical microscopy complemented with molecular data on the microbial communities obtained by culture independent methods. The results are discussed within the context of two models: the studied Fe-rich stromatolites are microeukaryotic-mediated as described by previous workers from similar environments or are the

  4. Formation of truncated proteins and high-molecular-mass aggregates upon soft illumination of photosynthetic proteins

    DEFF Research Database (Denmark)

    Rinalducci, Sara; Campostrini, Natascia; Antonioli, Paolo

    2005-01-01

    Different spot profiles were observed in 2D gel electrophoresis of thylakoid membranes performed either under complete darkness or by leaving the sample for a short time to low visible light. In the latter case, a large number of new spots with lower molecular masses, ranging between 15,000 and 25......,000 Da, were observed, and high-molecular-mass aggregates, seen as a smearing in the upper part of the gel, appeared in the region around 250 kDa. Identification of protein(s) contained in these new spots by MS/MS revealed that most of them are simply truncated proteins deriving from native ones...

  5. HPDB-Haskell library for processing atomic biomolecular structures in Protein Data Bank format.

    Science.gov (United States)

    Gajda, Michał Jan

    2013-11-23

    Protein DataBank file format is used for the majority of biomolecular data available today. Haskell is a lazy functional language that enjoys a high-level class-based type system, a growing collection of useful libraries and a reputation for efficiency. I present a fast library for processing biomolecular data in the Protein Data Bank format. I present benchmarks indicating that this library is faster than other frequently used Protein Data Bank parsing programs. The proposed library also features a convenient iterator mechanism, and a simple API modeled after BioPython. I set a new standard for convenience and efficiency of Protein Data Bank processing in a Haskell library, and release it to open source.

  6. HD-GYP domain proteins regulate biofilm formation and virulence in Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ryan, Robert P.; Lucey, Jean; O'Donovan, Karen

    2009-01-01

    residues (YN-GYP). Here we have investigated the role of these proteins in biofilm formation, virulence factor synthesis and virulence of P. aeruginosa. Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP in P. aeruginosa, consistent with the predicted activity of the encoded......2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterized C-terminal domain. Mutation of PA4108, PA4781 and PA2572 had distinct effects on biofilm formation and architecture of P. aeruginosa. All three proteins contributed to virulence of P...

  7. Characterization of tetratricopeptide repeat-containing proteins critical for cilia formation and function.

    Directory of Open Access Journals (Sweden)

    Yanan Xu

    Full Text Available Cilia formation and function require a special set of trafficking machinery termed intraflagellar transport (IFT, consisting mainly of protein complexes IFT-A, IFT-B, BBSome, and microtubule-dependent molecular motors. Tetratricopeptide repeat-containing (TTC proteins are widely involved in protein complex formation. Nine of them are known to serve as components of the IFT or BBSome complexes. How many TTC proteins are cilia-related and how they function, however, remain unclear. Here we show that twenty TTC genes were upregulated by at least 2-fold during the differentiation of cultured mouse tracheal epithelial cells (MTECs into multiciliated cells. Our systematic screen in zebrafish identified four novel TTC genes, ttc4, -9c, -36, and -39c, that are critical for cilia formation and motility. Accordingly, their zebrafish morphants displayed typical ciliopathy-related phenotypes, including curved body, abnormal otolith, hydrocephalus, and defective left-right patterning. The morphants of ttc4 and ttc25, a known cilia-related gene, additionally showed pronephric cyst formation. Immunoprecipitation indicated associations of TTC4, -9c, -25, -36, and -39c with components or entire complexes of IFT-A, IFT-B, or BBSome, implying their participations in IFT or IFT-related activities. Our results provide a global view for the relationship between TTC proteins and cilia.

  8. Nephrin regulates lamellipodia formation by assembling a protein complex that includes Ship2, filamin and lamellipodin.

    Directory of Open Access Journals (Sweden)

    Madhusudan Venkatareddy

    Full Text Available Actin dynamics has emerged at the forefront of podocyte biology. Slit diaphragm junctional adhesion protein Nephrin is necessary for development of the podocyte morphology and transduces phosphorylation-dependent signals that regulate cytoskeletal dynamics. The present study extends our understanding of Nephrin function by showing in cultured podocytes that Nephrin activation induced actin dynamics is necessary for lamellipodia formation. Upon activation Nephrin recruits and regulates a protein complex that includes Ship2 (SH2 domain containing 5' inositol phosphatase, Filamin and Lamellipodin, proteins important in regulation of actin and focal adhesion dynamics, as well as lamellipodia formation. Using the previously described CD16-Nephrin clustering system, Nephrin ligation or activation resulted in phosphorylation of the actin crosslinking protein Filamin in a p21 activated kinase dependent manner. Nephrin activation in cell culture results in formation of lamellipodia, a process that requires specialized actin dynamics at the leading edge of the cell along with focal adhesion turnover. In the CD16-Nephrin clustering model, Nephrin ligation resulted in abnormal morphology of actin tails in human podocytes when Ship2, Filamin or Lamellipodin were individually knocked down. We also observed decreased lamellipodia formation and cell migration in these knock down cells. These data provide evidence that Nephrin not only initiates actin polymerization but also assembles a protein complex that is necessary to regulate the architecture of the generated actin filament network and focal adhesion dynamics.

  9. The effect of starch, inulin, and degradable protein on ruminal fermentation and microbial growth in rumen simulation technique

    Directory of Open Access Journals (Sweden)

    Xiang H. Zhao

    2014-03-01

    Full Text Available A rumen simulation technique apparatus with eight 800 mL fermentation vessels was used to investigate the effects of rumen degradable protein (RDP level and non-fibre carbohydrate (NFC type on ruminal fermentation, microbial growth, and populations of ruminal cellulolytic bacteria. Treatments consisted of two NFC types (starch and inulin supplemented with 0 g/d (low RDP or 1.56 g/d (high RDP sodium caseinate. No significant differences existed among dietary treatments in the apparent disappearance of dietary nutrients except for dietary N, which increased with increased dietary RDP (P<0.001. Compared with starch, inulin treatments reduced the molar proportion of acetate (P<0.001, the acetate:propionate ratio (P<0.001, and methane production (P=0.006, but increased the butyrate proportion (P<0.001. Increased dietary RDP led to increases in production of total volatile fatty acid (P=0.014 and methane (P=0.050, various measures of N (P≤0.046, and 16s rDNA copy numbers of Ruminococcus flavefaciens (P≤0.010. Non-fibre carbohydrate source did not affect daily microbial N flow regardless of dietary RDP, but ammonia N production was lower for inulin than for starch treatments under high RDP conditions (P<0.001. Compared with starch treatments, inulin depressed the copy numbers of Fibrobacter succinogenes in solid fraction (P=0.023 and R. flavefaciens in liquid (P=0.017 and solid fractions (P=0.007, but it increased the carboxymethylcellulase activity in solid fraction (P=0.045. Current results suggest that starch and inulin differ in ruminal volatile fatty acid fermentation but have similar effects on ruminal digestion and microbial synthesis in vitro, although inulin suppressed the growth of partial ruminal cellulolytic bacteria.

  10. Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

    Science.gov (United States)

    Brito-Argáez, Ligia; Tamayo-Sansores, José A; Madera-Piña, Dianeli; García-Villalobos, Francisco J; Moo-Puc, Rosa E; Kú-González, Ángela; Villanueva, Marco A; Islas-Flores, Ignacio

    2016-12-01

    The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an ∼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  11. The Concentrations of Rumen Fluid Volatile Fatty Acids and Ammonia, and Rumen Microbial Protein Production in Sheep Given Feed During the Day and Night Time

    Science.gov (United States)

    Gumilar, D. A. K. W.; Rianto, E.; Arifin, M.

    2018-02-01

    An experimental study was carried out to investigate the concentrations of volatile (VFA), ammonia and microbial protein production of rumen fluid in sheep given fedd during the day and at night. This study used 12 fat-tailed rams aged 12-18 months and weighed 24,12 ± 25 kg (CV = 10,51%). The rams were fed a complete feed containing 16.64% protein and 68,33% total digestible nutrients (TDN). The rams were allocated into a completely randomised design with 3 treatments and 4 replications. The treatments applied were: T1: day time feeding (6.00 hrs - 18.00 hrs); T2: night time feeding (18.00 hrs - 6.00 hrs); and T3: day and night time feedings (6.00 hrs - 6.00 hrs). The parameters observed were dry matter intake (DMI), rumen VFA concentration, rumen ammonia concentration, rumen rmicrobial protein production and the efficiency of rumen microbial protein production. The results showed that feeding time did not significantly affect (P>0.05) all the parameters observed. Dry matter intake, VFA concentration, ammonia concentration, the microbial protein production of rumen fluid and the efficiency of microbial protein production were 1,073g/d, 49.69 mmol; 4.77 mg N/100 ml, 12,111 g/d and 19.96 g per kg digestible organic matter intake (DOMI), respectively. It is concluded that feeding time did not affect DMI, condition of rumen fluid and rumen microbial protein production in sheep.

  12. Rumen microbial protein supply as estimated from purine derivative excretion on sheep receiving faba beans (vicia faba as supplement delivered at different feeding frequencies

    Directory of Open Access Journals (Sweden)

    Asmuddin Natsir

    2008-06-01

    Full Text Available Rapid and extensive degradation of faba beans (Vicia faba by ruminal microbes can result in substantial and undesirable N loss from the rumen. The purpose of this study was to test the hypothesis that offering faba beans as a supplement more than once a day to sheep receiving a combination of oaten chaff and lucerne chaff as a basal diet will increase microbial protein supply to the intestines. The experiment was conducted in a Latin square design (4 x 4 using four mature merino sheep. The treatments were: T0 = basal diet ad libitum + nil supplements, T1 = T0 + faba beans (FB fed once daily, T2 = T0 + FB fed twice daily, T3 = T0 + FB fed 8 times daily. The basal diet was given once per day at 09:00 in the morning while FB were given at the rate of approximately 0.5% of live body weight and delivered according to the treatment protocol. Urinary excretion of purine derivative (PD was used to estimate microbial protein supply. The results indicated that even though treatment statistically had no effects on total urine output, PD excretion in the urine, PD absorbed, estimated microbial N supply, and the efficiency of rumen microbial protein synthesis, provision of supplement to sheep numerically improved microbial N supply by 92% compared to that of control group. However, there were no differences within the supplemented group. Therefore, it is concluded that feeding faba beans more than once a day was unnecessary.

  13. Microbial proteins produced from cannery wastes. II. Different cellulosic wastes used as substrate

    Energy Technology Data Exchange (ETDEWEB)

    Lee, H.C.; Chen, S.H.; Chang, J.S.; Lee, S.C.

    1980-01-01

    The production of protein by the cultivation of Cellulomonas species 34 on cellulosic wastes was studied. Maximum protein production on bamboo shoot husks was 4.77 g/L in 48 h when aeration was 1 vol./min, stirring rate was 1000 rpm, and substrate concentration was 3%. The chemical compounds of asparagus peel and pineapple peel and the conditions for their treatment with NaOH for protein fermentation are given.

  14. The differential role of cortical protein synthesis in taste memory formation and persistence

    Science.gov (United States)

    Levitan, David; Gal-Ben-Ari, Shunit; Heise, Christopher; Rosenberg, Tali; Elkobi, Alina; Inberg, Sharon; Sala, Carlo; Rosenblum, Kobi

    2016-05-01

    The current dogma suggests that the formation of long-term memory (LTM) is dependent on protein synthesis but persistence of the memory trace is not. However, many of the studies examining the effect of protein synthesis inhibitors (PSIs) on LTM persistence were performed in the hippocampus, which is known to have a time-dependent role in memory storage, rather than the cortex, which is considered to be the main structure to store long-term memories. Here we studied the effect of PSIs on LTM formation and persistence in male Wistar Hola (n⩾5) rats by infusing the protein synthesis inhibitor, anisomycin (100 μg, 1 μl), into the gustatory cortex (GC) during LTM formation and persistence in conditioned taste aversion (CTA). We found that local anisomycin infusion to the GC before memory acquisition impaired LTM formation (P=8.9E-5), but had no effect on LTM persistence when infused 3 days post acquisition (P=0.94). However, when we extended the time interval between treatment with anisomycin and testing from 3 days to 14 days, LTM persistence was enhanced (P=0.01). The enhancement was on the background of stable and non-declining memory, and was not recapitulated by another amnesic agent, APV (10 μg, 1 μl), an N-methyl-D-aspartate receptor antagonist (P=0.54). In conclusion, CTA LTM remains sensitive to the action of PSIs in the GC even 3 days following memory acquisition. This sensitivity is differentially expressed between the formation and persistence of LTM, suggesting that increased cortical protein synthesis promotes LTM formation, whereas decreased protein synthesis promotes LTM persistence.

  15. Exploration of freely available web-interfaces for comparative homology modelling of microbial proteins.

    Science.gov (United States)

    Nema, Vijay; Pal, Sudhir Kumar

    2013-01-01

    This study was conducted to find the best suited freely available software for modelling of proteins by taking a few sample proteins. The proteins used were small to big in size with available crystal structures for the purpose of benchmarking. Key players like Phyre2, Swiss-Model, CPHmodels-3.0, Homer, (PS)2, (PS)(2)-V(2), Modweb were used for the comparison and model generation. Benchmarking process was done for four proteins, Icl, InhA, and KatG of Mycobacterium tuberculosis and RpoB of Thermus Thermophilus to get the most suited software. Parameters compared during analysis gave relatively better values for Phyre2 and Swiss-Model. This comparative study gave the information that Phyre2 and Swiss-Model make good models of small and large proteins as compared to other screened software. Other software was also good but is often not very efficient in providing full-length and properly folded structure.

  16. Formation of singlet oxygen by decomposition of protein hydroperoxide in photosystem II.

    Directory of Open Access Journals (Sweden)

    Vinay Pathak

    Full Text Available Singlet oxygen (1O2 is formed by triplet-triplet energy transfer from triplet chlorophyll to O2 via Type II photosensitization reaction in photosystem II (PSII. Formation of triplet chlorophyll is associated with the change in spin state of the excited electron and recombination of triplet radical pair in the PSII antenna complex and reaction center, respectively. Here, we have provided evidence for the formation of 1O2 by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex. Protein hydroperoxide is formed by protein oxidation initiated by highly oxidizing chlorophyll cation radical and hydroxyl radical formed by Type I photosensitization reaction. Under highly oxidizing conditions, protein hydroperoxide is oxidized to protein peroxyl radical which either cyclizes to dioxetane or recombines with another protein peroxyl radical to tetroxide. These highly unstable intermediates decompose to triplet carbonyls which transfer energy to O2 forming 1O2. Data presented in this study show for the first time that 1O2 is formed by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex.

  17. WD40-repeat proteins in plant cell wall formation: current evidence and research prospects

    Directory of Open Access Journals (Sweden)

    Gea eGuerriero

    2015-12-01

    Full Text Available The metabolic complexity of living organisms relies on supramolecular protein structures which ensure vital processes, such as signal transduction, transcription, translation and cell wall synthesis. In eukaryotes WD40-repeat (WDR proteins often function as molecular hubs mediating supramolecular interactions. WDR proteins may display a variety of interacting partners and participate in the assembly of complexes involved in distinct cellular functions. In plants, the formation of lignocellulosic biomass involves extensive synthesis of cell wall polysaccharides, a process that requires the assembly of large transmembrane enzyme complexes, intensive vesicle trafficking, interactions with the cytoskeleton, and coordinated gene expression. Because of their function as supramolecular hubs, WDR proteins could participate in each or any of these steps, although to date only few WDR proteins have been linked to the cell wall by experimental evidence. Nevertheless, several potential cell wall-related WDR proteins were recently identified using in silico aproaches, such as analyses of co-expression, interactome and conserved gene neighbourhood. Notably, some WDR genes are frequently genomic neighbours of genes coding for GT2-family polysaccharide synthases in eukaryotes, and this WDR-GT2 collinear microsynteny is detected in diverse taxa. In angiosperms, two WDR genes are collinear to cellulose synthase genes, CESAs, whereas in ascomycetous fungi several WDR genes are adjacent to chitin synthase genes, chs. In this Perspective we summarize and discuss experimental and in silico studies on the possible involvement of WDR proteins in plant cell wall formation. The prospects of biotechnological engineering for enhanced biomass production are discussed.

  18. Antimicrobial activity of apple cider vinegar against Escherichia coli, Staphylococcus aureus and Candida albicans; downregulating cytokine and microbial protein expression.

    Science.gov (United States)

    Yagnik, Darshna; Serafin, Vlad; J Shah, Ajit

    2018-01-29

    The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.

  19. Interleukin 17 enhances bone morphogenetic protein-2-induced ectopic bone formation

    NARCIS (Netherlands)

    Croes, M.; Kruyt, M. C.; Groen, W. M.; Van Dorenmalen, K. M.A.; Dhert, W. J.A.; Öner, F. C.; Alblas, J.

    2018-01-01

    Interleukin 17 (IL-17) stimulates the osteogenic differentiation of progenitor cells in vitro through a synergy with bone morphogenetic protein (BMP)-2. This study investigates whether the diverse responses mediated by IL-17 in vivo also lead to enhanced BMP-2-induced bone formation. Since IL-17 is

  20. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    KAUST Repository

    Zimaro, Tamara

    2014-04-18

    Background: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system is involved in multicellular processes, such as bacterial biofilm formation has not been elucidated. Here, the phytopathogen Xanthomonas citri subsp. citri (X. citri) was used as a model to gain further insights about the role of the T3SS in biofilm formation. Results: The capacity of biofilm formation of different X. citri T3SS mutants was compared to the wild type strain and it was observed that this secretion system was necessary for this process. Moreover, the T3SS mutants adhered proficiently to leaf surfaces but were impaired in leaf-associated growth. A proteomic study of biofilm cells showed that the lack of the T3SS causes changes in the expression of proteins involved in metabolic processes, energy generation, exopolysaccharide (EPS) production and bacterial motility as well as outer membrane proteins. Furthermore, EPS production and bacterial motility were also altered in the T3SS mutants. Conclusions: Our results indicate a novel role for T3SS in X. citri in the modulation of biofilm formation. Since this process increases X. citri virulence, this study reveals new functions of T3SS in pathogenesis. 2014 Zimaro et al.; licensee BioMed Central Ltd.

  1. CREB binding protein is required for both short-term and long-term memory formation.

    NARCIS (Netherlands)

    Chen, G.; Zou, X.; Watanabe, H.; Deursen, J.M.A. van; Shen, J.

    2010-01-01

    CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated

  2. Cucurbit[8]uril templated supramolecular ring structure formation and protein assembly modulation

    NARCIS (Netherlands)

    Ramaekers, M.; Wijnands, S.P.W.; van Dongen, J.L.J.; Brunsveld, L.; Dankers, P.Y.W.

    2015-01-01

    The interplay of Phe-Gly-Gly (FGG)-tagged proteins and bivalent FGG-tagged penta(ethylene glycol) as guest molecules with cucurbit[8]uril (Q8) hosts is studied to modulate the supramolecular assembly process. Ring structure formation of the bivalent guest molecule with Q8 leads to enhanced binding

  3. The effect of oil type on network formation by protein aggregates into oleogels

    NARCIS (Netherlands)

    Vries, de Auke; Lopez Gomez, Yuly; Linden, van der Erik; Scholten, Elke

    2017-01-01

    The aim of this study was to assess the effect of oil type on the network formation of heat-set protein aggregates in liquid oil. The gelling properties of such aggregates to structure oil into so-called ‘oleogels’ are related to both the particle-particle and particle-solvent interactions. To

  4. Abnormal monocyte recruitment and collateral artery formation in monocyte chemoattractant protein-1 deficient mice

    NARCIS (Netherlands)

    Voskuil, Michiel; Hoefer, Imo E.; van Royen, Niels; Hua, Jing; de Graaf, Stijn; Bode, Christoph; Buschmann, Ivo R.; Piek, Jan J.

    2004-01-01

    Monocyte chemoattractant protein 1 (MCP-1) has been shown to be effective for the stimulation of collateral artery formation in small and large animal models. The availability of a genetic knockout mouse enables evaluation of the importance of the role of MCP-1 in the natural course of collateral

  5. Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

    Science.gov (United States)

    Xiang, Yang; Yan, Chao; Guo, Xiaolong; Zhou, Kaifeng; Li, Sheng'an; Gao, Qian; Wang, Xuan; Zhao, Feng; Liu, Jie; Lee, Wen-Hui; Zhang, Yun

    2014-05-06

    Aerolysins are virulence factors belonging to the bacterial β-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

  6. Elevated temperature triggers human respiratory syncytial virus F protein six-helix bundle formation

    International Nuclear Information System (INIS)

    Yunus, Abdul S.; Jackson, Trent P.; Crisafi, Katherine; Burimski, Irina; Kilgore, Nicole R.; Zoumplis, Dorian; Allaway, Graham P.; Wild, Carl T.; Salzwedel, Karl

    2010-01-01

    Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 deg. C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of

  7. Influence of stage of maturity of grass silages on protein digestion and microbial protein synthesis in the rumen.

    NARCIS (Netherlands)

    Bosch, M.W.; Bruchem, van J.; Bongers, L.J.G.M.; Tamminga, S.

    1994-01-01

    In four change-over experiments, wilted grass silages, differing in growth stage at harvesting and as a consequence in cell wall content, were fed ad libitum to dairy cows in early and late lactation. Ruminal degradation rate of the crude protein fraction of the silages was investigated using nylon

  8. Visualization of red-ox proteins on the gold surface using enzymatic polypyrrole formation

    International Nuclear Information System (INIS)

    Ramanaviciene, A.; Kausaite-Minkstimiene, A.; Voronovic, J.; Ramanavicius, A.; Oztekin, Y.; Carac, G.; German, N.

    2011-01-01

    We describe a new method for the visualization of the activity of red-ox proteins on a gold interface. Glucose oxidase was selected as a model system. Surfaces were modified by adhesion of glucose oxidase on (a) electrochemically cleaned gold; (b) gold films modified with gold nanoparticles, (c) a gold surface modified with self-assembled monolayer, and (d) covalent immobilization of protein on the gold surface modified with a self-assembled monolayer. The simple optical method for the visualization of enzyme on the surfaces is based on the enzymatic formation of polypyrrole. The activity of the enzyme was quantified via enzymatic formation of polypyrrole, which was detected and investigated by quartz microbalance and amperometric techniques. The experimental data suggest that the enzymatic formation of the polymer may serve as a method to indicate the adhesion of active redox enzyme on such surfaces. (author)

  9. Protein Data Bank Japan (PDBj): maintaining a structural data archive and resource description framework format.

    Science.gov (United States)

    Kinjo, Akira R; Suzuki, Hirofumi; Yamashita, Reiko; Ikegawa, Yasuyo; Kudou, Takahiro; Igarashi, Reiko; Kengaku, Yumiko; Cho, Hasumi; Standley, Daron M; Nakagawa, Atsushi; Nakamura, Haruki

    2012-01-01

    The Protein Data Bank Japan (PDBj, http://pdbj.org) is a member of the worldwide Protein Data Bank (wwPDB) and accepts and processes the deposited data of experimentally determined macromolecular structures. While maintaining the archive in collaboration with other wwPDB partners, PDBj also provides a wide range of services and tools for analyzing structures and functions of proteins, which are summarized in this article. To enhance the interoperability of the PDB data, we have recently developed PDB/RDF, PDB data in the Resource Description Framework (RDF) format, along with its ontology in the Web Ontology Language (OWL) based on the PDB mmCIF Exchange Dictionary. Being in the standard format for the Semantic Web, the PDB/RDF data provide a means to integrate the PDB with other biological information resources.

  10. Shedding light on disulfide bond formation: engineering a redox switch in green fluorescent protein

    DEFF Research Database (Denmark)

    Østergaard, H.; Henriksen, A.; Hansen, Flemming G.

    2001-01-01

    To visualize the formation of disulfide bonds in living cells, a pair of redox-active cysteines was introduced into the yellow fluorescent variant of green fluorescent protein. Formation of a disulfide bond between the two cysteines was fully reversible and resulted in a >2-fold decrease...... in the intrinsic fluorescence. Inter conversion between the two redox states could thus be followed in vitro as well as in vivoby non- invasive fluorimetric measurements. The 1.5 Angstrom crystal structure of the oxidized protein revealed a disulfide bond- induced distortion of the beta -barrel, as well...... the physiological range for redox-active cysteines. In the cytoplasm of Escherichia coli, the protein was a sensitive probe for the redox changes that occur upon disruption of the thioredoxin reductive pathway....

  11. Inhibitory effects of flavonoids on biofilm formation by Staphylococcus aureus that overexpresses efflux protein genes.

    Science.gov (United States)

    Lopes, Laênia Angélica Andrade; Dos Santos Rodrigues, Jéssica Bezerra; Magnani, Marciane; de Souza, Evandro Leite; de Siqueira-Júnior, José P

    2017-06-01

    This study evaluated the efficacy of glycone (myricitrin, hesperidin and phloridzin) and aglycone flavonoids (myricetin, hesperetin and phloretin) in inhibiting biofilm formation by Staphylococcus aureus RN4220 and S. aureus SA1199B that overexpress the msrA and norA efflux protein genes, respectively. The minimum inhibitory concentration (MIC) and minimum biofilm inhibitory concentration (MBIC 50 - defined as the lowest concentration that resulted in ≥50% inhibition of biofilm formation) of flavonoids were determined using microdilution in broth procedures. The flavonoids showed MIC >1024 μg/mL against S. aureus RN4220 and S. aureus SA1199B; however, these compounds at lower concentrations (1-256 μg/mL) showed inhibitory effects on biofilm formation by these strains. Aglycone flavonoids showed lower MBIC 50 values than their respective glycone forms. The lowest MBIC 50 values (1 and 4 μg/mL) were observed against S. aureus RN4220. Myricetin, hesperetin and phloretin exhibited biofilm formation inhibition >70% for S. aureus RN4220, and lower biofilm formation inhibition against S. aureus SA1199B. These results indicate that sub-MICs of the tested flavonoids inhibit biofilm formation by S. aureus strains that overexpress efflux protein genes. These effects are more strongly established by aglycone flavonoids. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    Heme proteins, also termed cytochromes, are a widespread class of metalloproteins containing an Fe-protoporphyrin IX cofactor. They perform numerous functions in nature such as oxygen-transport by hemoglobin, monooxygenation reactions catalyzed by Cytochrome P-450, and electron transfer reactions during photosynthesis. The differences between proteincofactor binding characteristics and the cofactor environment greatly influence the extensive range of functions. In this dissertation, proteins from the Mtr pathway of Shewanella oneidensis are characterized. These c-type cytochromes contain multiple heme cofactors per protein molecule that covalently attach to the protein amino acid sequence and are involved in electron transfer to extracellular metal oxides during anaerobic conditions. Successful recombinant expression of pathway components MtrC and MtrA is achieved in Escherichia coli. Heme-dependent gel staining and UV/Vis spectroscopy show characteristic c-type cytochrome characteristics. Mass spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy measurements of MtrA provide intriguing structural information and highlight the strong influence of the heme cofactors within the protein structure. Next, an Arabidopsis thaliana protein is analyzed. It was previously identified via a motif search of the plant genome, based on conserved residues in the H4 NOX pocket. Here, the incorporation of a heme b cofactor is confirmed. UV/Vis spectroscopy under anaerobic conditions demonstrates reversible binding of nitric oxide to the heme iron and depicts the previously published characteristic absorption maxima for other H-NOX proteins.

  13. Formation of protein-birnessite complex: XRD, FTIR, and AFM analysis.

    Science.gov (United States)

    Naidja, A; Liu, C; Huang, P M

    2002-07-01

    Limited information is available on formation chemistry of enzyme-Mn oxide complexes. Adsorption isotherm of protein molecules (tyrosinase) on birnessite (delta-MnO(2)) at pH 6.0 and room temperature (23 degrees C) was of H type, indicating a very high affinity of the enzyme protein molecules to the birnessite mineral surfaces. After thorough washing of the protein-mineral complex with deionized-distilled water, up to 89% of adsorbed protein molecules remained bound to the mineral surfaces. When a high amount of the protein was immobilized, the X-ray diffractogram shows a significant decrease in the intensity of characteristic d-spacings of birnessite. No shift to higher values of the d-spacings of protein-birnessite complex was observed, indicating that the enzyme molecules were not intercalated in the mineral structure but immobilized at the external surfaces and the edges of the mineral oxide. By comparison to the free enzyme, infrared absorption spectra of the protein-birnessite complexes show a shift by up to 11 cm(-1) to lower frequencies in the absorption bands characteristic of amide I and II modes of the polypeptides chains. The mineral surfaces exerted some strain on the protein structure, resulting in an alteration of the protein molecular conformation after binding to the mineral colloid surfaces. In the free state, the globular protein molecules had a spheroid shape with an average cross-sectional diameter of 70+/-6 nm. The unfolding and flattening of the protein molecules after immobilization is clearly shown in atomic force micrographs. Compared to the tyrosinase-birnessite complex, similar FTIR spectra and atomic force micrographs were observed for the pure protein, bovine serum albumin (BSA), after immobilization on birnessite. The information obtained in this study is of fundamental significance for understanding birnessite as an adsorbent of biopolymers and the catalytic role of the enzyme-birnessite complex.

  14. Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress

    Directory of Open Access Journals (Sweden)

    Tyo Keith EJ

    2012-03-01

    Full Text Available Abstract Background The protein secretory pathway must process a wide assortment of native proteins for eukaryotic cells to function. As well, recombinant protein secretion is used extensively to produce many biologics and industrial enzymes. Therefore, secretory pathway dysfunction can be highly detrimental to the cell and can drastically inhibit product titers in biochemical production. Because the secretory pathway is a highly-integrated, multi-organelle system, dysfunction can happen at many levels and dissecting the root cause can be challenging. In this study, we apply a systems biology approach to analyze secretory pathway dysfunctions resulting from heterologous production of a small protein (insulin precursor or a larger protein (α-amylase. Results HAC1-dependent and independent dysfunctions and cellular responses were apparent across multiple datasets. In particular, processes involving (a degradation of protein/recycling amino acids, (b overall transcription/translation repression, and (c oxidative stress were broadly associated with secretory stress. Conclusions Apparent runaway oxidative stress due to radical production observed here and elsewhere can be explained by a futile cycle of disulfide formation and breaking that consumes reduced glutathione and produces reactive oxygen species. The futile cycle is dominating when protein folding rates are low relative to disulfide bond formation rates. While not strictly conclusive with the present data, this insight does provide a molecular interpretation to an, until now, largely empirical understanding of optimizing heterologous protein secretion. This molecular insight has direct implications on engineering a broad range of recombinant proteins for secretion and provides potential hypotheses for the root causes of several secretory-associated diseases.

  15. Estimation of microbial protein supply of lactating dairy cows under smallholder farms in north-east Thailand using urinary purine derivative technique

    International Nuclear Information System (INIS)

    Pimpa, O.; Liang, J.B.

    2004-01-01

    Two experiments were conducted to examine the potential of urinary purine derivative (PD) as a predictive index of microbial protein supply in ruminant livestock under farm conditions. Results of Experiment 1 indicated that diurnal variation in the PDC index ( [mmol/L PD]/[mmol/L creatinine] kgW 0.75 ) in spot urine samples of zebu cattle was small and highly correlated with the daily PD output, suggesting that spot urine samples could be used to derive an index for estimating microbial protein supply of cattle under farm conditions. However, the PDC index for buffaloes was poorly correlated to daily urinary PD output, therefore the use of spot urine samples appeared to be unsuitable for buffaloes. Based on the above results, spot urine samples were used to estimate the microbial protein supply of lactating dairy cows under farm conditions in a follow-up experiment. The study was conducted using 24 lactating cows in 6 smallholder dairy farms situated in Khon Kaen province of Northeast Thailand. The study was conducted over two climatic seasons (raining and dry), where the animals were fed 5 kg of farm-mixed concentrate feed supplemented either with green grass (cut or grazing) or rice straw as roughage source during the raining and dry seasons, respectively. The results indicated that microbial protein supply was not significantly different and therefore, the nutritional status of the lactating cows was not significantly different between the two seasons. The absence of differences in milk yield between seasons seems to support our findings. We conclude that urinary PD technique could be used to estimate rumen microbial protein production for dairy cattle under farm conditions. (author)

  16. Probing the role of intercalating protein sidechains for kink formation in DNA.

    Directory of Open Access Journals (Sweden)

    Achim Sandmann

    Full Text Available Protein binding can induce DNA kinks, which are for example important to enhance the specificity of the interaction and to facilitate the assembly of multi protein complexes. The respective proteins frequently exhibit amino acid sidechains that intercalate between the DNA base steps at the site of the kink. However, on a molecular level there is only little information available about the role of individual sidechains for kink formation. To unravel structural principles of protein-induced DNA kinking we have performed molecular dynamics (MD simulations of five complexes that varied in their architecture, function, and identity of intercalated residues. Simulations were performed for the DNA complexes of wildtype proteins (Sac7d, Sox-4, CcpA, TFAM, TBP and for mutants, in which the intercalating residues were individually or combined replaced by alanine. The work revealed that for systems with multiple intercalated residues, not all of them are necessarily required for kink formation. In some complexes (Sox-4, TBP, one of the residues proved to be essential for kink formation, whereas the second residue has only a very small effect on the magnitude of the kink. In other systems (e.g. Sac7d each of the intercalated residues proved to be individually capable of conferring a strong kink suggesting a partially redundant role of the intercalating residues. Mutation of the key residues responsible for kinking either resulted in stable complexes with reduced kink angles or caused conformational instability as evidenced by a shift of the kink to an adjacent base step. Thus, MD simulations can help to identify the role of individual inserted residues for kinking, which is not readily apparent from an inspection of the static structures. This information might be helpful for understanding protein-DNA interactions in more detail and for designing proteins with altered DNA binding properties in the future.

  17. Formation of protein hydroperoxides in mouse myeloma cell line Sp2/0-Ag14

    International Nuclear Information System (INIS)

    Du, J.; Gebicki, J.

    2000-01-01

    Full text: Free radicals generated by normal cell metabolism or from environmental sources can cause damage to DNA, proteins and lipids-the important components of mammalian cells. As function molecules and cell constituent, the abundant and easily available nature of proteins make them the prime target of free radicals. Previous research in our lab have shown protein hydroperoxides in turn can react with other proteins, result in the lose of enzymatic function of the later, or crosslink with DNA, which may interfere gene transcription if not repaired. The formation of protein hydroperoxides in Sp2/0-Ag14 cells was induced by exposing them to peroxyl radical or gamma radiation. Cells were then washed and precipitated by tichloroacetic acid. Concentration of protein and lipid hydroperoxides were measured by FOX assay. No significant amount of lipid peroxides were detected. The effects of reducing agents dithiothreitol, glutathione, sodium borohydride identified the nature of protein hydroperoxides. The life time of cell protein hydroperoxides is about 2 hours

  18. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation.

    Directory of Open Access Journals (Sweden)

    Liang Zhang

    Full Text Available BACKGROUND: Cell plate formation during plant cytokinesis is facilitated by SNARE complex-mediated vesicle fusion at the cell-division plane. However, our knowledge regarding R-SNARE components of membrane fusion machinery for cell plate formation remains quite limited. METHODOLOGY/PRINCIPAL FINDINGS: We report the in vivo function of Arabidopsis VAMP721 and VAMP722, two closely sequence-related R-SNAREs, in cell plate formation. Double homozygous vamp721vamp722 mutant seedlings showed lethal dwarf phenotypes and were characterized by rudimentary roots, cotyledons and hypocotyls. Furthermore, cell wall stubs and incomplete cytokinesis were frequently observed in vamp721vamp722 seedlings. Confocal images revealed that green fluorescent protein-tagged VAMP721 and VAMP722 were preferentially localized to the expanding cell plates in dividing cells. Drug treatments and co-localization analyses demonstrated that punctuate organelles labeled with VAMP721 and VAMP722 represented early endosomes overlapped with VHA-a1-labeled TGN, which were distinct from Golgi stacks and prevacuolar compartments. In addition, protein traffic to the plasma membrane, but not to the vacuole, was severely disrupted in vamp721vamp722 seedlings by subcellular localization of marker proteins. CONCLUSION/SIGNIFICANCE: These observations suggest that VAMP721 and VAMP722 are involved in secretory trafficking to the plasma membrane via TGN/early endosomal compartment, which contributes substantially to cell plate formation during plant cytokinesis.

  19. RPPAML/RIMS: a metadata format and an information management system for reverse phase protein arrays.

    Science.gov (United States)

    Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

    2008-12-22

    Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1,000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. In this report an RPPA Information Management System (RIMS) is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML). RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.

  20. Amyloid-β Peptide Induces Prion Protein Amyloid Formation: Evidence for Its Widespread Amyloidogenic Effect.

    Science.gov (United States)

    Honda, Ryo

    2018-04-12

    Transmissible spongiform encephalopathy is associated with misfolding of prion protein (PrP) into an amyloid β-rich aggregate. Previous studies have indicated that PrP interacts with Alzheimer's disease amyloid-β peptide (Aβ), but it remains elusive how this interaction impacts on the misfolding of PrP. This study presents the first in vitro evidence that Aβ induces PrP-amyloid formation at submicromolar concentrations. Interestingly, systematic mutagenesis of PrP revealed that Aβ requires no specific amino acid sequences in PrP, and induces the misfolding of other unrelated proteins (insulin and lysozyme) into amyloid fibrils in a manner analogous to PrP. This unanticipated nonspecific amyloidogenic effect of Aβ indicates that this peptide might be involved in widespread protein aggregation, regardless of the amino acid sequences of target proteins, and exacerbate the pathology of many neurodegenerative diseases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Formation of nucleoplasmic protein aggregates impairs nuclear function in response to SiO2 nanoparticles

    International Nuclear Information System (INIS)

    Chen Min; Mikecz, Anna von

    2005-01-01

    Despite of their exponentially growing use, little is known about cell biological effects of nanoparticles. Here, we report uptake of silica (SiO 2 ) nanoparticles to the cell nucleus where they induce aberrant clusters of topoisomerase I (topo I) in the nucleoplasm that additionally contain signature proteins of nuclear domains, and protein aggregation such as ubiquitin, proteasomes, cellular glutamine repeat (polyQ) proteins, and huntingtin. Formation of intranuclear protein aggregates (1) inhibits replication, transcription, and cell proliferation; (2) does not significantly alter proteasomal activity or cell viability; and (3) is reversible by Congo red and trehalose. Since SiO 2 nanoparticles trigger a subnuclear pathology resembling the one occurring in expanded polyglutamine neurodegenerative disorders, we suggest that integrity of the functional architecture of the cell nucleus should be used as a read out for cytotoxicity and considered in the development of safe nanotechnology

  2. Mutations in Three Genes Encoding Proteins Involved in Hair Shaft Formation Cause Uncombable Hair Syndrome

    DEFF Research Database (Denmark)

    Ü Basmanav, F Buket; Cau, Laura; Tafazzoli, Aylar

    2016-01-01

    Uncombable hair syndrome (UHS), also known as "spun glass hair syndrome," "pili trianguli et canaliculi," or "cheveux incoiffables" is a rare anomaly of the hair shaft that occurs in children and improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant...... in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments...... and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic...

  3. Comparison of three 15N methods to correct for microbial contamination when assessing in situ protein degradability of fresh forages.

    Science.gov (United States)

    Kamoun, M; Ammar, H; Théwis, A; Beckers, Y; France, J; López, S

    2014-11-01

    The use of stable (15)N as a marker to determine microbial contamination in nylon bag incubation residues to estimate protein degradability was investigated. Three methods using (15)N were compared: (15)N-labeled forage (dilution method, LF), (15)N enrichment of rumen solids-associated bacteria (SAB), and (15)N enrichment of rumen liquid-associated bacteria (LAB). Herbage from forages differing in protein and fiber contents (early-cut Italian ryegrass, late-cut Italian ryegrass, and red clover) were freeze-dried and ground and then incubated in situ in the rumen of 3 steers for 3, 6, 12, 24, and 48 h using the nylon bag technique. The (15)N-labeled forages were obtained by fertilizing the plots where herbage was grown with (15)NH4 (15)NO3. Unlabeled forages (obtained from plots fertilized with NH4NO3) were incubated at the same time that ((15)NH4)2SO4 was continuously infused into the rumen of the steers, and then pellets of labeled SAB and LAB were isolated by differential centrifugation of samples of ruminal contents. The proportion of bacterial N in the incubation residues increased from 0.09 and 0.45 g bacterial N/g total N at 3 h of incubation to 0.37 and 0.85 g bacterial N/g total N at 48 h of incubation for early-cut and late-cut ryegrass, respectively. There were differences (P forage (late-cut ryegrass) was 0.51, whereas the corrected values were 0.85, 0.84, and 0.77 for the LF, SAB, and LAB methods, respectively. With early-cut ryegrass and red clover, the differences between uncorrected and corrected values ranged between 6% and 13%, with small differences among the labeling methods. Generally, methods using labeled forage or labeled SAB and LAB provided similar corrected degradability values. The accuracy in estimating the extent of degradation of protein in the rumen from in situ disappearance curves is improved when values are corrected for microbial contamination of the bag residue.

  4. 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.

  5. Formation of soluble amyloid oligomers and amyloid fibrils by the multifunctional protein vitronectin

    Directory of Open Access Journals (Sweden)

    Langen Ralf

    2008-10-01

    Full Text Available Abstract Background The multifunctional protein vitronectin is present within the deposits associated with Alzheimer disease (AD, age-related macular degeneration (AMD, atherosclerosis, systemic amyloidoses, and glomerulonephritis. The extent to which vitronectin contributes to amyloid formation within these plaques, which contain misfolded, amyloidogenic proteins, and the role of vitronectin in the pathophysiology of the aforementioned diseases is currently unknown. The investigation of vitronectin aggregation is significant since the formation of oligomeric and fibrillar structures are common features of amyloid proteins. Results We observed vitronectin immunoreactivity in senile plaques of AD brain, which exhibited overlap with the amyloid fibril-specific OC antibody, suggesting that vitronectin is deposited at sites of amyloid formation. Of particular interest is the growing body of evidence indicating that soluble nonfibrillar oligomers may be responsible for the development and progression of amyloid diseases. In this study we demonstrate that both plasma-purified and recombinant human vitronectin readily form spherical oligomers and typical amyloid fibrils. Vitronectin oligomers are toxic to cultured neuroblastoma and retinal pigment epithelium (RPE cells, possibly via a membrane-dependent mechanism, as they cause leakage of synthetic vesicles. Oligomer toxicity was attenuated in RPE cells by the anti-oligomer A11 antibody. Vitronectin fibrils contain a C-terminal protease-resistant fragment, which may approximate the core region of residues essential to amyloid formation. Conclusion These data reveal the propensity of vitronectin to behave as an amyloid protein and put forth the possibilities that accumulation of misfolded vitronectin may contribute to aggregate formation seen in age-related amyloid diseases.

  6. Purification of Microbially Expressed Recombinant Proteins via a Dual ELP Split Intein System.

    Science.gov (United States)

    Shi, Changhua; Han, Tzu-Chiang; Wood, David W

    2017-01-01

    Fusions of elastin-like peptide (ELP) purification tags and self-cleaving inteins provide a powerful platform for purifying tagless recombinant proteins without the need for conventional packed-bed columns. A drawback to this method has been premature cleaving of the ELP tag during expression, before the purification procedure can take place. Here we demonstrate a split-intein method, where the self-cleaving intein is divided into two inactive segments during expression and purification. Spontaneous assembly of the purified intein segments then restores self-cleaving activity to deliver the tagless target protein.

  7. Amyloid fibril formation in vitro from halophilic metal binding protein: Its high solubility and reversibility minimized formation of amorphous protein aggregations

    Science.gov (United States)

    Tokunaga, Yuhei; Matsumoto, Mitsuharu; Tokunaga, Masao; Arakawa, Tsutomu; Sugimoto, Yasushi

    2013-01-01

    Halophilic proteins are characterized by high net negative charges and relatively small fraction of hydrophobic amino acids, rendering them aggregation resistant. These properties are also shared by histidine-rich metal binding protein (HP) from moderate halophile, Chromohalobacter salexigens, used in this study. Here, we examined how halophilic proteins form amyloid fibrils in vitro. His-tagged HP, incubated at pH 2.0 and 58°C, readily formed amyloid fibrils, as observed by thioflavin fluorescence, CD spectra, and transmission or atomic force microscopies. Under these low-pH harsh conditions, however, His-HP was promptly hydrolyzed to smaller peptides most likely responsible for rapid formation of amyloid fibril. Three major acid-hydrolyzed peptides were isolated from fibrils and turned out to readily form fibrils. The synthetic peptides predicted to form fibrils in these peptide sequences by Waltz software also formed fibrils. Amyloid fibril was also readily formed from full-length His-HP when incubated with 10–20% 2,2,2-trifluoroethanol at pH 7.8 and 25°C without peptide bond cleavage. PMID:24038709

  8. [Effects of metal-catalyzed oxidation on the formation of advanced oxidation protein products].

    Science.gov (United States)

    Li, Li; Peng, Ai; Zhu, Kai-Yuan; Yu, Hong; Ll, Xin-Hua; Li, Chang-Bin

    2008-03-11

    To explore the relationship between metal-catalyzed oxidation (MCO) and the formation of advanced oxidation protein products (AOPPs). Specimens of human serum albumin (HSA) and pooled plasma were collected from 3 healthy volunteers and 4 uremia patients were divided into 3 groups: Group A incubated with copper sulfate solution of the concentrations of 0, 0.2, or 0.5 mmol/L, Group B, incubated with hydrogen peroxide 2 mmol/L, and Group C, incubated with copper sulfate 0.2 or 0.5 mmol/L plus hydrogen peroxide 2 mmol/L. 30 min and 24 h later the AOPP level was determined by ultraviolet visible spectrophotometry. High-performance liquid chromatography (HPLC) was used to observe the fragmentation effect on plasma proteins. Ninhydrin method was used to examine the protein fragments. The scavenging capacity of hydroxyl radical by macromolecules was measured so as to estimate the extent of damage for proteins induced by MCO. (1) The AOPP level of the HSA and plasma specimens of the uremia patients increased along with the increase of cupric ion concentration in a dose-dependent manner, especially in the presence of hydrogen peroxide (P < 0.05). (2) Aggregation of proteins was almost negligible in all groups, however, HPLC showed that cupric ion with or without hydrogen peroxide increased the fragments in the HAS specimens (with a relative molecular mass of 5000) and uremia patients' plasma proteins (with the molecular mass 7000). (3) The plasma AOPP level of the healthy volunteers was 68.2 micromol/L +/- 2.4 micromol/L, significantly lower than that of the uremia patients (158.5 micromol/L +/- 8.2 micromol/L). (4) The scavenging ability to clear hydroxyl radical by plasma proteins of the healthy volunteers was 1.38 -9.03 times as higher than that of the uremia patients. MCO contributes to the formation of AOPPs mainly through its fragmentation effect to proteins.

  9. Purine derivatives/creatinine ratio as an index of microbial protein synthesis in lactating Holstein cows

    International Nuclear Information System (INIS)

    Orellana, R.; Pulido, P.; Briones, M.; Sarabia, A.

    2004-01-01

    Three experiments were conducted to study creatinine excretion in milking cows, and its relationship to food intake and purine derivatives (PD) excretion as an index of microbial N synthesized in the rumen. In the first experiment, differences in creatinine determination method were studied: creatinine excretion was 26.8 ± 1.2 and 27.4 ± 2.2 mg/kg live weight, or 1,175.39 ± 74.69 and 1,199.9 ± 103.9 mmol/kgW 0.75 respectively for HPLC and colorimetry analysis. In the second experiment, the effect of concentrate supplement on the PDC index was studied. The inclusion of different levels of concentrate (0, 16, 33 and 50%) increased the PDC index significantly. The PDC index (Y) also showed a significant relationship with DOMI (X, kg/d): Y 16.85 X + 6.23; R 2 =0.84. Total PD excretion was calculated assuming constant daily excretion of creatinine (1.199 mmol/kgW 0.75 ). In the third experiment the effect of different roughage on the PDC index was studied. The substitution of a 23% of rye grass (Lolium sp.) silage by alkali treated wheat straw did not modify DOMI and the PDC index although among groups there was a significant decrease in milk production (1.5 L/cow/d) and in growth rate (-380 g/cow/d). (author)

  10. Changes in microbial populations of WPC34 and WPC80 whey protein during long term storage

    Science.gov (United States)

    The use of whey protein (WPC34 and WPC80) as a food ingredient and as a base for making biodegradable products is increasing. The need to alleviate world hunger in arid and semi-arid regions demands that we investigate the behavior of native bacteria in these products, especially during long term st...

  11. Characterization of Heme Proteins Involved in Microbial Exoelectric Activity and Small Molecule-Sensing

    KAUST Repository

    Vogler, Malvina M.

    2018-01-01

    spectrometry confirms that the correct extensive post-translational modifications were performed and the ten heme groups were incorporated per protein of MtrC and MtrA and the correct lipid-anchor was attached to extracellular MtrC. Raman spectroscopy

  12. Different functional modes of BAR domain proteins in formation and plasticity of mammalian postsynapses.

    Science.gov (United States)

    Kessels, Michael M; Qualmann, Britta

    2015-09-01

    A plethora of cell biological processes involve modulations of cellular membranes. By using extended lipid-binding interfaces, some proteins have the power to shape membranes by attaching to them. Among such membrane shapers, the superfamily of Bin-Amphiphysin-Rvs (BAR) domain proteins has recently taken center stage. Extensive structural work on BAR domains has revealed a common curved fold that can serve as an extended membrane-binding interface to modulate membrane topologies and has allowed the grouping of the BAR domain superfamily into subfamilies with structurally slightly distinct BAR domain subtypes (N-BAR, BAR, F-BAR and I-BAR). Most BAR superfamily members are expressed in the mammalian nervous system. Neurons are elaborately shaped and highly compartmentalized cells. Therefore, analyses of synapse formation and of postsynaptic reorganization processes (synaptic plasticity) - a basis for learning and memory formation - has unveiled important physiological functions of BAR domain superfamily members. These recent advances, furthermore, have revealed that the functions of BAR domain proteins include different aspects. These functions are influenced by the often complex domain organization of BAR domain proteins. In this Commentary, we review these recent insights and propose to classify BAR domain protein functions into (1) membrane shaping, (2) physical integration, (3) action through signaling components, and (4) suppression of other BAR domain functions. © 2015. Published by The Company of Biologists Ltd.

  13. Role of protein-bound carbonyl groups in the formation of advanced glycation endproducts.

    Science.gov (United States)

    Liggins, J; Furth, A J

    1997-08-22

    Several mechanisms have been postulated for the formation of advanced glycation endproducts (AGEs) from glycated proteins; they all feature protein-bound carbonyl intermediates. Using 2,4-dinitrophenylhydrazine (DNPH), we have detected these intermediates on bovine serum albumin, lysozyme and beta-lactoglobulin after in vitro glycation by glucose or fructose. Carbonyls were formed in parallel with AGE-fluorophores, via oxidative Maillard reactions. Neither Amadori nor Heyns products contributed to the DNPH reaction. Fluorophore and carbonyl yields were much enhanced in lipid-associated proteins, but both groups could also be detected in lipid-free proteins. When pre-glycated proteins were incubated in the absence of free sugar, carbonyl groups were rapidly lost in a first-order reaction, while fluorescence continued to develop beyond the 21 days of incubation. Another unexpected finding was that not all carbonyl groups were blocked by aminoguanidine, although there was complete inhibition of reactions leading to AGE-fluorescence. It is suggested that carbonyls acting as fluorophore precursors react readily with aminoguanidine, while others are resistant to this hydrazine, possibly because they are involved in ring closure. Factors influencing the relative rates of acyclisation and hydrazone formation are discussed, together with possible implications for antiglycation therapy.

  14. Entropy Drives the Formation of Salt Bridges in the Protein GB3.

    Science.gov (United States)

    Zhang, Ning; Wang, Yefei; An, Liaoyuan; Song, Xiangfei; Huang, Qingshan; Liu, Zhijun; Yao, Lishan

    2017-06-19

    Salt bridges are very common in proteins. But what drives the formation of protein salt bridges is not clear. In this work, we determined the strength of four salt bridges in the protein GB3 by measuring the ΔpK a values of the basic residues that constitute the salt bridges with a highly accurate NMR titration method at different temperatures. The results show that the ΔpK a values increase with temperature, thus indicating that the salt bridges are stronger at higher temperatures. Fitting of ΔpK a values to the van't Hoff equation yields positive ΔH and ΔS values, thus indicating that entropy drives salt-bridge formation. Molecular dynamics simulations show that the protein and solvent make opposite contributions to ΔH and ΔS. Specifically, the enthalpic gain contributed from the protein is more than offset by the enthalpic loss contributed from the solvent, whereas the entropic gain originates from the desolvation effect. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Alkali solution extraction of rice residue protein isolates: Influence of alkali concentration on protein functional, structural properties and lysinoalanine formation.

    Science.gov (United States)

    Hou, Furong; Ding, Wenhui; Qu, Wenjuan; Oladejo, Ayobami Olayemi; Xiong, Feng; Zhang, Weiwei; He, Ronghai; Ma, Haile

    2017-03-01

    This study evaluated the nutrient property and safety of the rice residue protein isolates (RRPI) product (extracted by different alkali concentrations) by exploring the protein functional, structural properties and lysinoalanine (LAL) formation. The results showed that with the rising of alkali concentration from 0.03M to 0.15M, the solubility, emulsifying and foaming properties of RRPI increased at first and then descended. When the alkali concentration was greater than 0.03M, the RRPI surface hydrophobicity decreased and the content of thiol and disulfide bond, Lys and Cys significantly reduced. By the analysis of HPLC, the content of LAL rose up from 276.08 to 15,198.07mg/kg and decreased to 1340.98mg/kg crude protein when the alkali concentration increased from 0.03 to 0.09M and until to 0.15M. These results indicated that RRPI alkaline extraction concentration above 0.03M may cause severe nutrient or safety problems of protein. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Rumen microbial protein synthesis and nitrogen efficiency as affected by tanniferous and non-tanniferous forage legumes incubated individually or together in Rumen Simulation Technique.

    Science.gov (United States)

    Grosse Brinkhaus, Anja; Bee, Giuseppe; Schwarm, Angela; Kreuzer, Michael; Dohme-Meier, Frigga; Zeitz, Johanna O

    2018-03-01

    A limited availability of microbial protein can impair productivity in ruminants. Ruminal nitrogen efficiency might be optimised by combining high-quality forage legumes such as red clover (RC), which has unfavourably high ruminal protein degradability, with tanniferous legumes like sainfoin (SF) and birdsfoot trefoil (BT). Silages from SF and from BT cultivars [Bull (BB) and Polom (BP)] were incubated singly or in combination with RC using the Rumen Simulation Technique (n = 6). The tanniferous legumes, when compared to RC, changed the total short-chain fatty acid profile by increasing propionate proportions at the expense of butyrate. Silage from SF contained the most condensed tannins (CTs) (136 g CT kg -1 dry matter) and clearly differed in various traits from the BT and RC silages. The apparent nutrient degradability (small with SF), microbial protein synthesis, and calculated content of potentially utilisable crude protein (large with SF) indicated that SF had the greatest efficiency in ruminal protein synthesis. The effects of combining SF with RC were mostly linear. The potential of sainfoin to improve protein supply, demonstrated either individually or in combination with a high-performance forage legume, indicates its potential usefulness in complementing protein-deficient ruminant diets and high-quality forages rich in rumen-degradable protein. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  17. Nuclear techniques to determine microbial protein synthesis and productive performance of barki lambs fed rations containing some medicinal plants

    International Nuclear Information System (INIS)

    Mohamed, M.M.S.

    2009-01-01

    This study included two experiments, the first experiment was carried out in vitro to evaluate the effect of adding two levels of Lemongrass or Rosemary in ruminant rations on microbial protein synthesis using radio active sulfur S 35 . While, the second experiment was to study the effect of Lemongrass (CC) and Rosemary (RO) as feed additives in rations of lambs on feed intake, nutrient digestibility, some parameters of blood and rumen activity. Meanwhile, body weight and economical efficiency were studied. Twenty five of Barki male lambs with average body weight of 19.8 kg ± 1 kg and 3- 4 months of age were divided into 5 similar groups (5 lambs each). The first group (control) (R1) was fed on a concentrate feed mixture (CFM) plus rice straw (RS). While, R2 and R3 were fed as R1 ration supplemented with 100 or 200 mg Lemongrass /kg LBW/d respectively. Meantime, R4 and R5 were fed as R1 ration supplemented with 100 or 200 mg Rosemary /kg LBW/d respectively.The results indicated that more microbial protein synthesis was noticed with 4 mg of Lemongrass followed by 2 mg Rosemary, 2 mg Lemongrass and control which was higher than 4 mg Rosemary/ 0.5 g concentrate mixture. The differences were not statistically significant. The dry matter intake (DMI) was not significantly different for R4 and R3 when compared with R1 (control) and it significantly decreased in R5 and R2 compared with R1. The digestibilities of DM, OM, CP, EE and NFE in the supplemented groups were not significantly differing compared with R1. The digestibility of CF was significantly increased in R2 and R4 compared with R1 and there were no significant differences for R3 and R5 compared with R1. There were no significant differences in nutritive values as TDN, DCP and SV among all supplemented groups compared with R1. Rumen liquor TVFA,s was not significantly differ at zero time, but it decreased at 3 h and 6 h with all additives compared with the control with no significant differences among all

  18. Proteins involved in flor yeast carbon metabolism under biofilm formation conditions.

    Science.gov (United States)

    Moreno-García, Jaime; García-Martínez, Teresa; Moreno, Juan; Mauricio, Juan Carlos

    2015-04-01

    A lack of sugars during the production of biologically aged wines after fermentation of grape must causes flor yeasts to metabolize other carbon molecules formed during fermentation (ethanol and glycerol, mainly). In this work, a proteome analysis involving OFFGEL fractionation prior to LC/MS detection was used to elucidate the carbon metabolism of a flor yeast strain under biofilm formation conditions (BFC). The results were compared with those obtained under non-biofilm formation conditions (NBFC). Proteins associated to processes such as non-fermentable carbon uptake, the glyoxylate and TCA cycles, cellular respiration and inositol metabolism were detected at higher concentrations under BFC than under the reference conditions (NBFC). This study constitutes the first attempt at identifying the flor yeast proteins responsible for the peculiar sensory profile of biologically aged wines. A better metabolic knowledge of flor yeasts might facilitate the development of effective strategies for improved production of these special wines. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Studies on a microbially derived, high molecular weight inhibitor of plasma cholesteryl ester transfer protein

    International Nuclear Information System (INIS)

    Marschke, C.K.; McGee, J.E.; Melchior, G.W.; Castle, C.K.

    1989-01-01

    The authors have isolated an organism which accumulates an inhibitor of Cholesteryl Ester Transfer Protein (CETP). Purification of 100,000-fold was achieved by ammonium sulfate precipitation followed by Hydroxyl Apatite, Agarose AO.5, and Mono Q (Pharmacia) chromatographies. The use of 14 C-labelled protein molecular weight standards followed by SDS-PAGE revealed some proteolytic activity. However, inhibition of the proteases did not affect the inhibitor potency. The inhibitor has an estimated molecular weight of 40 Kd and appears to exist as two forms. One form was eluted from a Mono Q column by 100 mM NaCl while the other was not bound. Our evidence indicated that the bound form was progressively denatured, or proteolyzed, during storage of the fermentation beer, to the unbound form. Importantly though this molecular change did not affect either inhibitory activity or the apparent molecular weight

  20. The Conserved Spore Coat Protein SpoVM Is Largely Dispensable in Clostridium difficile Spore Formation.

    Science.gov (United States)

    Ribis, John W; Ravichandran, Priyanka; Putnam, Emily E; Pishdadian, Keyan; Shen, Aimee

    2017-01-01

    The spore-forming bacterial pathogen Clostridium difficile is a leading cause of health care-associated infections in the United States. In order for this obligate anaerobe to transmit infection, it must form metabolically dormant spores prior to exiting the host. A key step during this process is the assembly of a protective, multilayered proteinaceous coat around the spore. Coat assembly depends on coat morphogenetic proteins recruiting distinct subsets of coat proteins to the developing spore. While 10 coat morphogenetic proteins have been identified in Bacillus subtilis , only two of these morphogenetic proteins have homologs in the Clostridia : SpoIVA and SpoVM. C. difficile SpoIVA is critical for proper coat assembly and functional spore formation, but the requirement for SpoVM during this process was unknown. Here, we show that SpoVM is largely dispensable for C. difficile spore formation, in contrast with B. subtilis . Loss of C. difficile SpoVM resulted in modest decreases (~3-fold) in heat- and chloroform-resistant spore formation, while morphological defects such as coat detachment from the forespore and abnormal cortex thickness were observed in ~30% of spoVM mutant cells. Biochemical analyses revealed that C. difficile SpoIVA and SpoVM directly interact, similarly to their B. subtilis counterparts. However, in contrast with B. subtilis , C. difficile SpoVM was not essential for SpoIVA to encase the forespore. Since C. difficile coat morphogenesis requires SpoIVA-interacting protein L (SipL), which is conserved exclusively in the Clostridia , but not the more broadly conserved SpoVM, our results reveal another key difference between C. difficile and B. subtilis spore assembly pathways. IMPORTANCE The spore-forming obligate anaerobe Clostridium difficile is the leading cause of antibiotic-associated diarrheal disease in the United States. When C. difficile spores are ingested by susceptible individuals, they germinate within the gut and

  1. Effects of Ohmic Heating on Microbial Counts and Denaturatiuon of Proteins in Milk

    OpenAIRE

    SUN, Huixian; KAWAMURA, Shuso; HIMOTO, Jun-ichi; ITOH, Kazuhiko; WADA, Tatsuhiko; KIMURA, Toshinori

    2008-01-01

    The aim of this study was to compare the inactivation effects of ohmic heating (internal heating by electric current) and conventional heating (external heating by hot water) on viable aerobes and Streptococcus thermophilus 2646 in milk under identical temperature history conditions. The effects of the two treatments on quality of milk were also compared by assessing degrees of protein denaturation in raw and sterilized milk (raw milk being sterilized by ohmic heating or conventional heating)...

  2. Effect of diet and absence of protozoa on the rumen microbial community and on the representativeness of bacterial fractions used in the determination of microbial protein synthesis.

    Science.gov (United States)

    Belanche, A; de la Fuente, G; Pinloche, E; Newbold, C J; Balcells, J

    2012-11-01

    Accurate estimates of microbial synthesis in the rumen are vital to optimize ruminant nutrition. Liquid- (LAB) and solid-associated bacterial fractions (SAB) harvested from the rumen are generally considered as microbial references when microbial yield is calculated; however, factors that determine their composition are not completely understood. The aim of this study was to evaluate the effect of diet and absence or presence of rumen protozoa on the rumen microbial community. It was hypothesized that these treatments could modify the composition and representativeness of LAB and SAB. Twenty twin lambs (Ovis aries) were used; one-half of the twins were kept protozoa-free, and each respective twin sibling was faunated. At 6 mo of age, 5 animals from each group were randomly allocated to the experimental diets consisting of either alfalfa hay as the sole diet, or 50:50 mixed with ground barley grain. After 15 d of adaptation to the diet, animals were euthanized, rumen and abomasum contents were sampled, and LAB and SAB isolated. The presence of protozoa buffered the effect of diet on the rumen bacterial population. Faunated animals fed alfalfa hay had a greater abundance of F. succinogenes, anaerobic fungi and methanogens, as well as an enhanced rumen bacterial diversity. Cellulolytic bacteria were more abundant in SAB, whereas the abomasal abundance of most of the microorganisms studied was closer to those values observed in LAB. Rumen and abomasal samples showed similar bacterial DNA concentrations, but the fungal and protozoal DNA concentration in the abomasum was only 69% and 13% of that observed in the rumen, respectively, suggesting fungal and protozoal sequestration in the rumen or possible preferential degradation of fungal and protozoal DNA in the abomasum, or both. In conclusion, absence of protozoa and type of diet extensively modified the chemical composition of LAB and SAB as a consequence of changes in the microbial composition of these fractions.

  3. Evaluation of the Components Released by Wine Yeast Strains on Protein Haze Formation in White Wine

    Directory of Open Access Journals (Sweden)

    Ellen Cristine Giese

    2016-12-01

    Full Text Available Cultures of 23 indigenous yeast strains (22 Saccharomyces cerevisiae and a non-Saccharomyces, Torulaspora delbrueckii, isolated from fermentation tanks at wineries in Castilla-La Mancha (Spain, and were performed under winemaking conditions using a synthetic must. Polysaccharide analysis and turbidity assays were conducted so as to observe the capacity of the released mannoproteins against protein haze formation in white wine, and 3 strains (2 Saccharomyces cerevisiae and T. delbrueckii were chosen for further experiments. The action of a commercial b-glucanolytic enzyme preparation (Lallzyme BETA®, and a β-(1→3-glucanase preparation from Trichoderma harzianum Rifai were evaluated to release polysaccharides from the different yeast strains’ cell walls. Protection against protein haze formation was strain dependent, and only two strains (Sc2 and Sc4 presented >50% stabilization in comparison to controls. Addition of β-glucanases did not increase the concentrations of polysaccharides in the fermentation musts; however, a significant increase of polymeric mannose (mannoproteins was detected using an enzymatic assay following total acid hydrolysis of the soluble polysaccharides. Enzymatic treatment presented positive effects and decreased protein haze formation in white wine. DOI http://dx.doi.org/10.17807/orbital.v8i6.869

  4. Nuclear body formation and PML body remodeling by the human cytomegalovirus protein UL35

    International Nuclear Information System (INIS)

    Salsman, Jayme; Wang Xueqi; Frappier, Lori

    2011-01-01

    The human cytomegalovirus (HCMV) UL35 gene encodes two proteins, UL35 and UL35a. Expression of UL35 in transfected cells results in the formation of UL35 nuclear bodies that associate with promyelocytic leukemia (PML) protein. PML forms the basis for PML nuclear bodies that are important for suppressing viral lytic gene expression. Given the important relationship between PML and viral infection, we have further investigated the association of UL35 with PML bodies. We demonstrate that UL35 bodies form independently of PML and subsequently recruit PML, Sp100 and Daxx. In contrast, UL35a did not form bodies; however, it could bind UL35 and inhibit the formation of UL35 bodies. The HCMV tegument protein pp71 promoted the formation of UL35 bodies and the cytoplasmic localization of UL35a. Similarly, UL35a shifted pp71 to the cytoplasm. These results indicate that the interplay between UL35, UL35a and pp71 affects their subcellular localization and likely their functions throughout infection.

  5. Origins of the Mechanochemical Coupling of Peptide Bond Formation to Protein Synthesis.

    Science.gov (United States)

    Fritch, Benjamin; Kosolapov, Andrey; Hudson, Phillip; Nissley, Daniel A; Woodcock, H Lee; Deutsch, Carol; O'Brien, Edward P

    2018-04-18

    Mechanical forces acting on the ribosome can alter the speed of protein synthesis, indicating that mechanochemistry can contribute to translation control of gene expression. The naturally occurring sources of these mechanical forces, the mechanism by which they are transmitted 10 nm to the ribosome's catalytic core, and how they influence peptide bond formation rates are largely unknown. Here, we identify a new source of mechanical force acting on the ribosome by using in situ experimental measurements of changes in nascent-chain extension in the exit tunnel in conjunction with all-atom and coarse-grained computer simulations. We demonstrate that when the number of residues composing a nascent chain increases, its unstructured segments outside the ribosome exit tunnel generate piconewtons of force that are fully transmitted to the ribosome's P-site. The route of force transmission is shown to be through the nascent polypetide's backbone, not through the wall of the ribosome's exit tunnel. Utilizing quantum mechanical calculations we find that a consequence of such a pulling force is to decrease the transition state free energy barrier to peptide bond formation, indicating that the elongation of a nascent chain can accelerate translation. Since nascent protein segments can start out as largely unfolded structural ensembles, these results suggest a pulling force is present during protein synthesis that can modulate translation speed. The mechanism of force transmission we have identified and its consequences for peptide bond formation should be relevant regardless of the source of the pulling force.

  6. Rumen Microbial Protein Production in Rumen-Simulating-Technique (RUSITEC) Using 15N-Urea Nitrogen, as Influenced By Hay and Barley Ratios in Feed

    International Nuclear Information System (INIS)

    Al-Masri, M. R.; Abel, HJ.; Steinberg, W.

    2004-01-01

    Metabolism of dietary nitrogen using labeled 15 N and the changes in the microbial protein mass and NH3-N were studied in five rumen-simulating-technique(RUSITEC)-fermenters, which were run simultaneously in three identically repeated experiments. Each experiment consisted of a 6-day adaptation period followed directly by a 3-day collection period. The feed of the fermenters (G1, G2, G3, G4 and G5) varied in the ratio of barley. The barley increased by 20% between the fermenters. Grass hay+barley (g/d) in the feed of the fermenters was 10+2 (G1), 8+4 (G2), 6+6 (G3), 4+8 (G4) and 2+10 (G5). The results indicated that there were no significant (P>0.05) changes in the amounts of microbial nitrogen (92-118 mg/d) and microbial mass syntheses which were (mg/d): 1154 (G1), 1063 (G2), 1152 (G3), 1127 (G4) and 1362 (G5). Increasing the proportion of barley in the fermenters (G4 and G5) decreased NH3-N amounts (G2 and G3) significantly (P<0.05). The energy was not efficiently used in G5 having a lower ratio of the microbial nitrogen and microbial mass to the total short chain fatty acids than that other fermenters. (authors)

  7. Sinup, a novel Siaz-interacting nuclear protein, modulates neural plate formation in the zebrafish embryos

    International Nuclear Information System (INIS)

    Ro, Hyunju; Won, Minho; Lee, Su-Ui; Kim, Kyoon E.; Huh, Tae-Lin; Kim, Cheol-Hee; Rhee, Myungchull

    2005-01-01

    Siah, the vertebrate homologue of the Drosophila seven in absentia (sina) gene, is well conserved from Drosophila to mammal and involved in ubiquitination and proteasome-dependent degradation of various target proteins. To identify cellular proteins interacting with Siah, we screened a zebrafish cDNA library with zebrafish Siah (Siaz) as bait in a yeast two-hybrid assay. We identified a cDNA encoding a novel protein composed of 145 amino acids and termed it as Sinup (Siaz-interacting-nuclear-protein). Sinup is a novel nuclear protein that binds to the highly conserved C-terminal protein-interacting domain of Siaz both in vivo and in vitro. During development, sinup transcripts are abundant from the one-cell stage to the early blastula and then markedly diminished, suggesting sinup largely exists as maternal transcripts. sinup overexpression induced lateral expansion of the neural plate and in consequence caused ectopic expression of otx-2 and hoxb1b during the late gastrula stage. In addition, the lateral/paraxial expression of wnt8 at the onset of gastrulation is suppressed by the forced expression of sinup while the expression levels of various dorso-ventral markers are unaffected. In contrast, interfering with sinup functions using sinup morpholino oligonucleotides gradually diminished the anterior neuroectoderm from the posterior region, and resulted in compete loss of hindbrain at the 3-somites stage. Our report suggests that sinup expression should be tightly regulated during early embryonic development for the proper neural plate formation

  8. Porosity and Organic Carbon Controls on Naturally Reduced Zone (NRZ) Formation Creating Microbial ';Hotspots' for Fe, S, and U Cycling in Subsurface Sediments

    Science.gov (United States)

    Jones, M. E.; Janot, N.; Bargar, J.; Fendorf, S. E.

    2013-12-01

    Previous studies have illustrated the importance of Naturally Reduced Zones (NRZs) within saturated sediments for the cycling of metals and redox sensitive contaminants. NRZs can provide a source of reducing equivalents such as reduced organic compounds or hydrogen to stimulate subsurface microbial communities. These NRZ's are typically characterized by low permeability and elevated concentrations of organic carbon and trace metals. However, both the formation of NRZs and their importance to the overall aquifer carbon remineralization is not fully understood. Within NRZs the hydrolysis of particulate organic carbon (POC) and subsequent fermentation of dissolved organic carbon (DOC) to form low molecular weight dissolved organic carbon (LMW-DOC) provides electron donors necessary for the respiration of Fe, S, and in the case of the Rifle aquifer, U. Rates of POC hydrolysis and subsequent fermentation have been poorly constrained and rates in excess and deficit to the rates of subsurface anaerobic respiratory processes have been suggested. In this study, we simulate the development of NRZ sediments in diffusion-limited aggregates to investigate the physical and chemical conditions required for NRZ formation. Effects of sediment porosity and POC loading on Fe, S, and U cycling on molecular and nanoscale are investigated with synchrotron-based Near Edge X-ray Absorption Fine Structure Spectroscopy (NEXAFS). Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR-MS) and Fourier Transform Infrared spectroscopy (FTIR) are used to characterize the transformations in POC and DOC. Sediment aggregates are inoculated with the natural microbial biota from the Rifle aquifer and population dynamics are monitored by 16S RNA analysis. Overall, establishment of low permeability NRZs within the aquifer stimulate microbial respiration beyond the diffusion-limited zones and can limit the transport of U through a contaminated aquifer. However, the long-term stability of

  9. Petrographic Evidence of Microbial Mats in the Upper Cretaceous Fish-Bearing, Organic-Rich Limestone, Agua Nueva Formation, Central Mexico

    Science.gov (United States)

    Blanco, A.; Maurrasse, F. J.; Hernández-Ávila, J.; Ángeles-Trigueros, S. A.; García-Cabrera, M. E.

    2013-05-01

    We document petrographic evidence of microbial mats in the Upper Cretaceous Agua Nueva Formation in the area of Xilitla (San Luis Potosí, Central Mexico), located in the southern part of the Tampico-Misantla basin. The sequence consists predominantly of alternating decimeter-thick beds of fossiliferous dark laminated limestone (C-org > 1.0wt%), and light gray, bioturbated limestone (C-org Duque-Botero and Maurrasse, 2005; 2008). These structures are also analogous to microbial mats in present environments, and Devonian deposits (Kremer, 2006). In addition, the laminae at Xilitla include filamentous bacterial structures, as thin and segmented red elements. In some thin sections, filaments appear to be embedded within the crinkly laminae and shreds showing the same pattern of folding, suggestive of biomorphic elements that represent the main producers of the organic matter associated with the laminae. Thus, exceptional bacterial activity characterizes sedimentation during the accumulation of the Agua Nueva Formation. Oxygen-deficient conditions related to the microbial mats were an important element in the mass mortality and preservation of the fish assemblages. Absence of bioturbation, pervasive framboidal pyrite, and the high concentration of organic matter (TOC ranges from 1.2% to 8wt%) in the dark limestones are consistent with persistent recurring dysoxic/anoxic conditions, and the light-gray bioturbated limestones represent relatively well-oxygenated episodes. Planktonic foraminifera (Rotalipora cushmani) and Inoceramu labiatus indicate a time interval from the latest Cenomanian through the earliest Turonian, thus this long interval of severe oxygen deficiency is coeval with Oceanic Anoxic Event 2 (OAE-2). [Duque-Botero and Maurrasse. 2005. Jour. Iberian Geology (31), 85-98; 2008. Cret. Res., 29, 957-964; Kremer. 2006. Acta Palaeontologica Polonica (51, 1), 143-154

  10. [Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

    Science.gov (United States)

    Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

    1997-01-01

    Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

  11. Stress proteins and phytohormones: their role in formation of plant resistance

    International Nuclear Information System (INIS)

    Kosakivska, I.V.

    2005-01-01

    Full text: Using the disc-electrophoresis methods, we have studied protein biosynthesis of different plants, including 11 species of Orchidaceae, some other tropical and subtropical plants, 9 different fruit plants, and 4 cultivars of Triticum aestivum L. under stresses factors such as high and low temperature, clinostating, radioactive irradiation and osmotic shock. Specific and unspecific reactions of plants protein system on stresses were found. De novo synthesis of 35 and 45 kD polypeptides were observed in total and mitochondrial proteins fractions after heat-shock and radioactive irradiation. This suggests that mitochondries participate in formation of plant resistance. Intensive synthesis of ABA revealed as the universal reaction of all studied plants on action of different kinds of stresses. Specific changes in balance of phytohormones were found under different stresses. We observed the correlation between endogenous ABA, IAA and cytokinin level and plant resistance. We also found the interaction between the process of biosynthesis of proteins and phytohormone balance, as well as their direct participation in formation of plant resistance. (author)

  12. Neuronal response of the hippocampal formation to injury: blood flow, glucose metabolism, and protein synthesis

    International Nuclear Information System (INIS)

    Kameyama, M.; Wasterlain, C.G.; Ackermann, R.F.; Finch, D.; Lear, J.; Kuhl, D.E.

    1983-01-01

    The reaction of the hippocampal formation to entorhinal lesions was studied from the viewpoints of cerebral blood flow ([ 123 I]isopropyl-iodoamphetamine[IMP])-glucose utilization ([ 14 C]2-deoxyglucose), and protein synthesis ([ 14 C]leucine), using single- and double-label autoradiography. Researchers' studies showed decreased glucose utilization in the inner part, and increased glucose utilization in the outer part of the molecular layer of the dentate gyrus, starting 3 days after the lesion; increased uptake of [ 123 I]IMP around the lesion from 1 to 3 days postlesion; and starting 3 days after the lesion, marked decrease in [ 14 C]leucine incorporation into proteins and cell loss in the dorsal CA1 and dorsal subiculum in about one-half of the rats. These changes were present only in animals with lesions which invaded the ventral hippocampal formation in which axons of CA1 cells travel. By contrast, transsection of the 3rd and 4th cranial nerves resulted, 3 to 9 days after injury, in a striking increase in protein synthesis in the oculomotor and trochlear nuclei. These results raise the possibility that in some neurons the failure of central regeneration may result from the cell's inability to increase its rate of protein synthesis in response to axonal injury

  13. Metagenomic analysis of the rumen microbial community following inhibition of methane formation by a halogenated methane analogue

    Directory of Open Access Journals (Sweden)

    Stuart E Denman

    2015-10-01

    Full Text Available Japanese goats fed a diet of 50% Timothy grass and 50% concentrate with increasing levels of the anti-methanogenic compound, bromochloromethane (BCM were investigated with respect to the microbial shifts in the rumen. Microbial ecology methods identified many species that exhibited positive and negative responses to the increasing levels of BCM. The methane-inhibited rumen appeared to adapt to the higher H2 levels by shifting fermentation to propionate which was mediated by an increase in the population of hydrogen-consuming Prevotella and Selenomonas spp. Metagenomic analysis of propionate production pathways was dominated by genomic content from these species. Reductive acetogenic marker gene libraries and metagenomics analysis indicate that reductive acetogenic species do not play a major role in the BCM treated rumen.

  14. Fractal Dimension Analysis of Texture Formation of Whey Protein-Based Foods

    Directory of Open Access Journals (Sweden)

    Robi Andoyo

    2018-01-01

    Full Text Available Whey protein in the form of isolate or concentrate is widely used in food industries due to its functionality to form gel under certain condition and its nutritive value. Controlling or manipulating the formation of gel aggregates is used often to evaluate food texture. Many researchers made use of fractal analysis that provides the quantitative data (i.e., fractal dimension for fundamentally and rationally analyzing and designing whey protein-based food texture. This quantitative analysis is also done to better understand how the texture of whey protein-based food is formed. Two methods for fractal analysis were discussed in this review: image analysis (microscopy and rheology. These methods, however, have several limitations which greatly affect the accuracy of both fractal dimension values and types of aggregation obtained. This review therefore also discussed problem encountered and ways to reduce the potential errors during fractal analysis of each method.

  15. [Decomposition of corpses--a microbial degradation process with special reference to mummification, formation of adipocere and incomplete putrified corpes].

    Science.gov (United States)

    Schoenen, Dirk

    2013-01-01

    Decomposition of the human body is a microbial process. It is influenced by the environmental situation and it depends to a high degree on the exchange of substances between the corpse and the environment. Mummification occurs at low humidity or frost. Adipocere arises from lack of oxygen, incomplete putrified corpses develop when there is no exchange of air or water between the corpse and the environment.

  16. Involvement of microbial mats in early fossilization by decay delay and formation of impressions and replicas of vertebrates and invertebrates

    Science.gov (United States)

    Iniesto, Miguel; Buscalioni, Ángela D.; Carmen Guerrero, M.; Benzerara, Karim; Moreira, David; López-Archilla, Ana I.

    2016-05-01

    Microbial mats have been hypothesized to improve the persistence and the preservation of organic remains during fossilization processes. We test this hypothesis with long-term experiments (up to 5.5 years) using invertebrate and vertebrate corpses. Once placed on mats, the microbial community coats the corpses and forms a three-dimensional sarcophagus composed of microbial cells and exopolymeric substances (EPS). This coverage provides a template for i) moulding superficial features, resulting in negative impressions, and ii) generating replicas. The impressions of fly setulae, fish scales and frog skin verrucae are shaped mainly by small cells in an EPS matrix. Microbes also replicate delicate structures such as the three successive layers that compose a fish eye. The sarcophagus protects the body integrity, allowing the persistence of inner organs such as the ovaries and digestive apparatus in flies, the swim bladder and muscles in fish, and the bone marrow in frog legs. This study brings strong experimental evidence to the idea that mats favour metazoan fossilization by moulding, replicating and delaying decay. Rapid burial has classically been invoked as a mechanism to explain exceptional preservation. However, mats may play a similar role during early fossilization as they can preserve complex features for a long time.

  17. Utilization of solid coffee waste as a substrate for microbial protein production

    Energy Technology Data Exchange (ETDEWEB)

    Arue, C; Bahar, S

    1986-01-01

    The feasibility of using the solid waste from the instant coffee processing industry (NESCAFE) as a substrate for the production of protein from fungi imperfecti in order to be used as an animal feed supplement was studied. Studies on the selected fungi, Paecilomyces elegans, Aspergillus oryzae and Fusarium oxysporum showed that F. oxysporum produces significantly higher protein levels than the other fungi studied. The fungus was grown in batch on the acid hydrolyzed coffee medium. Maximal values for sugar utilization and mycelium production (3-4 mg/ml) were obtained on 0.5% (w/v) acid hydrolyzed substrate (4% w/v) supplemented with 0.05% (w/v) potassium phosphate and 0.2% (w/v) yeast extract. Supplementary nitrogen was not necessary. The fungus was found to require pyridoxine and inositol. Addition of 1.5% (w/v) glucose to the medium increased the biomass production, indicating that the carbon source may be a limiting factor. 37 references.

  18. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb.

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-08-12

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Mechanisms of Host-Pathogen Protein Complex Formation and Bacterial Immune Evasion of Streptococcus suis Protein Fhb*

    Science.gov (United States)

    Li, Xueqin; Liu, Peng; Gan, Shuzhen; Zhang, Chunmao; Zheng, Yuling; Jiang, Yongqiang; Yuan, Yuan

    2016-01-01

    Streptococcus suis serotype 2 (S. suis 2)-induced sepsis and meningitis are often accompanied by bacteremia. The evasion of polymorphonuclear leukocyte-mediated phagocytic clearance is central to the establishment of bacteremia caused by S. suis 2 and is facilitated by the ability of factor H (FH)-binding protein (Fhb) to bind FH on the bacterial surface, thereby impeding alternative pathway complement activation and phagocytic clearance. Here, C3b/C3d was found to bind to Fhb, along with FH, forming a large immune complex. The formation of this immune complex was mediated by domain II of Fhb via electrostatic and hydrophobic interactions, which, to our knowledge, is a new type of interaction. Interestingly, Fhb was found to be associated with the cell envelope and also present in the culture supernatant, where secreted Fhb inhibited complement activation via interactions with domain II, thereby enhancing antiphagocytic clearance by polymorphonuclear leukocytes. Thus, Fhb is a multifunctional bacterial protein, which binds host complement component C3 as well as FH and interferes with innate immune recognition in a secret protein manner. S. suis 2 therefore appears to have developed a new strategy to combat host innate immunity and enhance survival in host blood. PMID:27342778

  20. Biogas Production from Protein-Rich Biomass: Fed-Batch Anaerobic Fermentation of Casein and of Pig Blood and Associated Changes in Microbial Community Composition

    Science.gov (United States)

    Kovács, Etelka; Wirth, Roland; Maróti, Gergely; Bagi, Zoltán; Rákhely, Gábor; Kovács, Kornél L.

    2013-01-01

    It is generally accepted as a fact in the biogas technology that protein-rich biomass substrates should be avoided due to inevitable process inhibition. Substrate compositions with a low C/N ratio are considered difficult to handle and may lead to process failure, though protein-rich industrial waste products have outstanding biogas generation potential. This common belief has been challenged by using protein-rich substrates, i.e. casein and precipitated pig blood protein in laboratory scale continuously stirred mesophilic fed-batch biogas fermenters. Both substrates proved suitable for sustained biogas production (0.447 L CH4/g protein oDM, i.e. organic total solids) in high yield without any additives, following a period of adaptation of the microbial community. The apparent key limiting factors in the anaerobic degradation of these proteinaceous materials were the accumulation of ammonia and hydrogen sulfide. Changes in time in the composition of the microbiological community were determined by next-generation sequencing-based metagenomic analyses. Characteristic rearrangements of the biogas-producing community upon protein feeding and specific differences due to the individual protein substrates were recognized. The results clearly demonstrate that sustained biogas production is readily achievable, provided the system is well-characterized, understood and controlled. Biogas yields (0.45 L CH4/g oDM) significantly exceeding those of the commonly used agricultural substrates (0.25-0.28 L CH4/g oDM) were routinely obtained. The results amply reveal that these high-energy-content waste products can be converted to biogas, a renewable energy carrier with flexible uses that can replace fossil natural gas in its applications. Process control, with appropriate acclimation of the microbial community to the unusual substrate, is necessary. Metagenomic analysis of the microbial community by next-generation sequencing allows a precise determination of the alterations in

  1. Biogas production from protein-rich biomass: fed-batch anaerobic fermentation of casein and of pig blood and associated changes in microbial community composition.

    Directory of Open Access Journals (Sweden)

    Etelka Kovács

    Full Text Available It is generally accepted as a fact in the biogas technology that protein-rich biomass substrates should be avoided due to inevitable process inhibition. Substrate compositions with a low C/N ratio are considered difficult to handle and may lead to process failure, though protein-rich industrial waste products have outstanding biogas generation potential. This common belief has been challenged by using protein-rich substrates, i.e. casein and precipitated pig blood protein in laboratory scale continuously stirred mesophilic fed-batch biogas fermenters. Both substrates proved suitable for sustained biogas production (0.447 L CH4/g protein oDM, i.e. organic total solids in high yield without any additives, following a period of adaptation of the microbial community. The apparent key limiting factors in the anaerobic degradation of these proteinaceous materials were the accumulation of ammonia and hydrogen sulfide. Changes in time in the composition of the microbiological community were determined by next-generation sequencing-based metagenomic analyses. Characteristic rearrangements of the biogas-producing community upon protein feeding and specific differences due to the individual protein substrates were recognized. The results clearly demonstrate that sustained biogas production is readily achievable, provided the system is well-characterized, understood and controlled. Biogas yields (0.45 L CH4/g oDM significantly exceeding those of the commonly used agricultural substrates (0.25-0.28 L CH4/g oDM were routinely obtained. The results amply reveal that these high-energy-content waste products can be converted to biogas, a renewable energy carrier with flexible uses that can replace fossil natural gas in its applications. Process control, with appropriate acclimation of the microbial community to the unusual substrate, is necessary. Metagenomic analysis of the microbial community by next-generation sequencing allows a precise determination of the

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

    Science.gov (United States)

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

    2015-11-20

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

  3. Semi-solid microbial fermentation of rice and wheat straw for protein enrichment and increased digestibility

    Energy Technology Data Exchange (ETDEWEB)

    Balasubramanya, R.H.; Bhatawdekar, S.P.

    1980-12-01

    Rice and wheat straws were hydrolyzed in various concentrations of sulfuric acid at different temperatures and different water: substrate ratios. The maximum amount of sugars of about 30-34% was released when heated at 121 degrees C with 0.5 N H2SO4 at a water: substrate ratio of 3:1. The pH of the hydrolyzed straws was raised to 5.0-5.5 with 5 N NH4OH. Such ammoniated straws were inoculated with the cultures of Penicillium funiculosum Thom. and Candida utilis (Henneb.) Lodder and Kreger-van Rij, and fermentation was carried out on semi-solid substrate for 5-7 days at room temperature. The fermentation resulted in 37-180% increase in crude protein, 23-100% increase in crude fat and 20-30% increase in the digestibility. (Refs. 29).

  4. Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans.

    Science.gov (United States)

    Matsumi, Y; Fujita, K; Takashima, Y; Yanagida, K; Morikawa, Y; Matsumoto-Nakano, M

    2015-06-01

    Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm-forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm-forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1 were elevated compared with MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different compared with that formed by the parental strain. These results suggest that a defect in GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

    Directory of Open Access Journals (Sweden)

    Hennessy Bryan T

    2008-12-01

    Full Text Available Abstract Background Reverse Phase Protein Arrays (RPPA are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of interest. Results In this report an RPPA Information Management System (RIMS is described and made available with open source software. In order to implement the proposed system, we propose a metadata format known as reverse phase protein array markup language (RPPAML. RPPAML would enable researchers to describe, document and disseminate RPPA data. The complexity of the data structure needed to describe the results and the graphic tools necessary to visualize them require a software deployment distributed between a client and a server application. This was achieved without sacrificing interoperability between individual deployments through the use of an open source semantic database, S3DB. This data service backbone is available to multiple client side applications that can also access other server side deployments. The RIMS platform was designed to interoperate with other data analysis and data visualization tools such as Cytoscape. Conclusion The proposed RPPAML data format hopes to standardize RPPA data. Standardization of data would result in diverse client applications being able to operate on the same set of data. Additionally, having data in a standard format would enable data dissemination and data analysis.

  6. Phosphorylation of Staphylococcus aureus Protein-Tyrosine Kinase Affects the Function of Glucokinase and Biofilm Formation.

    Science.gov (United States)

    Vasu, Dudipeta; Kumar, Pasupuleti Santhosh; Prasad, Uppu Venkateswara; Swarupa, Vimjam; Yeswanth, Sthanikam; Srikanth, Lokanathan; Sunitha, Manne Mudhu; Choudhary, Abhijith; Sarma, Potukuchi Venkata Gurunadha Krishna

    2017-03-01

    When Staphylococcus aureus is grown in the presence of high concentration of external glucose, this sugar is phosphorylated by glucokinase (glkA) to form glucose-6-phosphate. This product subsequently enters into anabolic phase, which favors biofilm formation. The presence of ROK (repressor protein, open reading frame, sugar kinase) motif, phosphate-1 and -2 sites, and tyrosine kinase sites in glkA of S. aureus indicates that phosphorylation must regulate the glkA activity. The aim of the present study was to identify the effect of phosphorylation on the function of S. aureus glkA and biofilm formation. Pure glkA and protein-tyrosine kinase (BYK) of S. aureus ATCC 12600 were obtained by fractionating the cytosolic fractions of glkA1 and BYK-1 expressing recombinant clones through nickel metal chelate column. The pure glkA was used as a substrate for BYK and the phosphorylation of glkA was confirmed by treating with reagent A and resolving in SDS-PAGE, as well as staining with reagent A. The kinetic parameters of glkA and phosphorylated glkA were determined spectrophotometrically, and in silico tools were used for validation. S. aureus was grown in brain heart infusion broth, which was supplemented with glucose, and then biofilm units were calculated. Fourfold elevated glkA activity was observed upon the phosphorylation by BYK. Protein-protein docking analysis revealed that glkA structure docked close to the adenosine triphosphate-binding site of BYK structure corroborating the kinetic results. Further, S. aureus grown in the presence of elevated glucose concentration exhibited an increase in the rate of biofilm formation. The elevated function of glkA is an essential requirement for increased biofilm units in S. aureus, a key pathogenic factor that helps its survival and spread the infection.

  7. The effect of polycarboxylate shell of magnetite nanoparticles on protein corona formation in blood plasma

    Energy Technology Data Exchange (ETDEWEB)

    Szekeres, Márta, E-mail: szekeres@chem.u-szeged.hu [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary); Tóth, Ildikó Y. [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary); Turcu, R. [National Institute R& D for Isotopic and Molecular Technology, Cluj-Napoca 400293 (Romania); Tombácz, Etelka [Department of Physical Chemistry and Materials Sciences, University of Szeged, Hungary, 1 Aradi vt, 6720 Szeged (Hungary)

    2017-04-01

    The development of protein corona around nanoparticles upon administration to the human body is responsible in a large part for their biodistribution, cell-internalization and toxicity or biocompatibility. We studied the influence of the chemical composition of polyelectrolyte shells (citric acid (CA) and poly(acrylic-co-maleic acid) (PAM)) of core-shell magnetite nanoparticles (MNPs) on the evolution of protein corona in human plasma (HP). The aggregation state and zeta potential of the particles were measured in the range of HP concentration between 1 and 80 (v/v)% 3 min and 20 h after dispersing the particles in HP diluted with Tris buffered saline. Naked MNPs aggregated in HP solution, but the carboxylated MNPs became stabilized colloidally at higher plasma concentrations. Significant differences were observed at low plasma concentration. CA@MNPs aggregated instantly while the hydrodynamic diameter of PAM@MNP increased only slightly at 1–3 v/v % HP concentrations. The observed differences in protein corona formation can be explained by the differences in the steric effects of the polycarboxylate shells. It is interesting that relatively small but systematic changes in zeta potential alter the aggregation state significantly. - Highlights: • Human plasma protein corona cannot stabilize naked and citrate-coated magnetite nanoparticles. • Polycarboxylic acid (PAM) coated MNPs are well stabilized with HP protein corona. • Stability pattern of naked, CA and PAM-coated MNPs is not predicted by zeta potential.

  8. MARS: A protein family involved in the formation of vertical skeletal elements.

    Science.gov (United States)

    Abehsera, Shai; Peles, Shani; Tynyakov, Jenny; Bentov, Shmuel; Aflalo, Eliahu D; Li, Shihao; Li, Fuhua; Xiang, Jianhai; Sagi, Amir

    2017-05-01

    Vertical organizations of skeletal elements are found in various vertebrate teeth and invertebrate exoskeletons. The molecular mechanism behind the development of such structural organizations is poorly known, although it is generally held that organic matrix proteins play an essential role. While most crustacean cuticular organizations exhibit horizontal chitinous layering, a typical vertical organization is found towards the surface of the teeth in the mandibles of the crayfish Cherax quadricarinatus. Candidate genes encoding for mandible-forming structural proteins were mined in C. quadricarinatus molt-related transcriptomic libraries by using a binary patterning approach. A new protein family, termed the Mandible Alanine Rich Structural (MARS) protein family, with a modular sequence design predicted to form fibers, was found. Investigations of spatial and temporal expression of the different MARS genes suggested specific expression in the mandibular teeth-forming epithelium, particularly during the formation of the chitinous vertical organization. MARS loss-of-function RNAi experiments resulted in the collapse of the organization of the chitin fibers oriented vertically to the surface of the crayfish mandibular incisor tooth. A general search of transcriptomic libraries suggested conservation of MARS proteins across a wide array of crustaceans. Our results provide a first look into the molecular mechanism used to build the complex crustacean mandible and into the specialized vertical structural solution that has evolved in skeletal elements. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Metaproteomics: Harnessing the power of high performance mass spectrometry to identify the suite of proteins that control metabolic activities in microbial communities

    Science.gov (United States)

    Hettich, Robert L.; Pan, Chongle; Chourey, Karuna; Giannone, Richard J.

    2013-01-01

    Summary The availability of extensive genome information for many different microbes, including unculturable species in mixed communities from environmental samples, has enabled systems-biology interrogation by providing a means to access genomic, transcriptomic, and proteomic information. To this end, metaproteomics exploits the power of high performance mass spectrometry for extensive characterization of the complete suite of proteins expressed by a microbial community in an environmental sample. PMID:23469896

  10. The negatively charged regions of lactoferrin binding protein B, an adaptation against anti-microbial peptides.

    Directory of Open Access Journals (Sweden)

    Ari Morgenthau

    Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein's C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides.

  11. New insight on the formation of whey protein microbeads by a microfluidic system

    Science.gov (United States)

    Andoyo, Robi; Guyomarc'h, Fanny; Tabuteau, Hervé; Famelart, Marie-Hélène

    2018-02-01

    The current paper describes the formation of whey protein microbeads (WPM) having a spherical shape and a monodispersed size distribution. A microfluidic flow-focusing geometry was used to control the production of whey protein microdroplets in a hydrophobic phase. The microfluidic system consists of two inlet channels where the WPI solution and the lipophilic phase were separately injected towards the flow-focusing (FF) junction where they eventually meet, then co-flow. A whey protein isolate (WPI) solution of 150 g/kg protein and two types of hydrophobic phases, i.e. sunflower oil and n-dodecane, were tested as the continuous phase. The formation of WPM was observed microscopically. The aim of the present study was to describe the production of stable monodisperse WPM in suspension in milk ultrafiltrate using a microfluidic system. Hints to perform the control of the running parameters, i.e. choice of the hydrophobic phase or fluids flowrates, are provided. The results showed that in the sunflower oil, microdroplets had a large polydisperse size distribution, while in n-dodecane, microdroplets with narrow size distribution were obtained. Stabilization of the whey protein microdroplets through heat-gelation at 75 °C for 20 min in n-dodecane produced WPM and no change in shape nor size is observed. Meanwhile replacing the n-dodecane by MUF using centrifugation and washing caused the swelling of the WPM, but dispersity remained low. From this study, microfluidic system seemed to be a suitable method to be used for producing small quantities of monodisperse WPM.

  12. Development, standardization and validation of purine excretion technique for measuring microbial protein supply for Yerli Kara cross-breed cattle

    International Nuclear Information System (INIS)

    Cetinkaya, N.; Ozdemir, H.; Gucus, A.I.; Ozcan, H.; Sogut, A.; Yaman, S.

    2002-01-01

    constant (C)-the slope as mmol/kg W 0.75 was 0.960. The coefficient of variation (CV) for the uric acid, allantion, PD, creatinine, total-N, PD:CxW 0.75 ratio in spot urine samples of four treatments were less than 5%. Developed banding system can be used for YK and YK-C cattle for the spot urine sample measurement.There were not significant difference for each parameter between the spot urine sampling times of Groups I, II and III. The corresponding microbial-N values to PD:C ratio of Crops I. II and III were 15-25 g/d. Experimentally estimated DOMI was 2.21±0.15 kg/d. By using the equation (DOMI= 344+48.7xPD:C ratio) estimated DOMI (g/d) of Groups I, II, and III were 2.8±0.6, 2.6±0.7 and 2.7±0.7 respectively. CV % of Groups I, II and III were 22, 27 and 26 % respectively. In conclusion, the PD:creatinine ratio in spot urine samples could be used under farm condition as an indicator of microbial protein supply in YK-C cattle, and also DOMI can be estimated from PD:creatinine ratio in spot urine samples under field conditions

  13. Dynamics of protein aggregation and oligomer formation governed by secondary nucleation

    Energy Technology Data Exchange (ETDEWEB)

    Michaels, Thomas C. T., E-mail: tctm3@cam.ac.uk; Lazell, Hamish W.; Arosio, Paolo; Knowles, Tuomas P. J., E-mail: tpjk2@cam.ac.uk [Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW (United Kingdom)

    2015-08-07

    The formation of aggregates in many protein systems can be significantly accelerated by secondary nucleation, a process where existing assemblies catalyse the nucleation of new species. In particular, secondary nucleation has emerged as a central process controlling the proliferation of many filamentous protein structures, including molecular species related to diseases such as sickle cell anemia and a range of neurodegenerative conditions. Increasing evidence suggests that the physical size of protein filaments plays a key role in determining their potential for deleterious interactions with living cells, with smaller aggregates of misfolded proteins, oligomers, being particularly toxic. It is thus crucial to progress towards an understanding of the factors that control the sizes of protein aggregates. However, the influence of secondary nucleation on the time evolution of aggregate size distributions has been challenging to quantify. This difficulty originates in large part from the fact that secondary nucleation couples the dynamics of species distant in size space. Here, we approach this problem by presenting an analytical treatment of the master equation describing the growth kinetics of linear protein structures proliferating through secondary nucleation and provide closed-form expressions for the temporal evolution of the resulting aggregate size distribution. We show how the availability of analytical solutions for the full filament distribution allows us to identify the key physical parameters that control the sizes of growing protein filaments. Furthermore, we use these results to probe the dynamics of the populations of small oligomeric species as they are formed through secondary nucleation and discuss the implications of our work for understanding the factors that promote or curtail the production of these species with a potentially high deleterious biological activity.

  14. Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

    Directory of Open Access Journals (Sweden)

    Masahiro Kawahara

    2011-01-01

    Full Text Available Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP play crucial roles in the pathogenesis of Alzheimer's disease (AD. Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”, and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed.

  15. Protein-adsorption and Ca-phosphate formation on chitosan-bioactive glass composite coatings

    Science.gov (United States)

    Wagener, V.; Boccaccini, A. R.; Virtanen, S.

    2017-09-01

    In the last years, chitosan-bioactive glass (BG) composites have been developed and investigated as bioactive coatings for orthopedic applications. The increase of bioactivity occurs due to the stimulation of calcium-phosphate/hydroxyapatite formation on the surface while the coating is degrading. In the present work, protein adsorption and its influence on calcium-phosphate precipitation was studied for the first time on such composite coatings. The experiments involved coating of 316L stainless steel substrates with chitosan (Ch) and chitosan-bioactive glass (Ch-BG) and immersion of the coated samples in two different bovine serum albumin (BSA) containing solutions, namely DI H2O (with pH adjusted to about 7.2 with diluted NaOH) and simulated body fluid (SBF). In order to investigate the influence of protein adsorption on calcium-phosphate precipitation, samples were also immersed in DI H2O and in SBF without BSA. Samples were analyzed by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Surface analysis revealed that adsorption of BSA takes place on all studied samples and that protein adsorption is influenced by the presence of Ca2+ and PO43- ions. Bioactivity in the form of hydroxyapatite pre-stage formation is significantly increased on Ch-BG composite coating as compared with bare stainless steel surface. However, calcium-phosphate precipitation in SBF is reduced by the presence of BSA.

  16. Formation of Polyphenol-Denatured Protein Flocs in Alcohol Beverages Sweetened with Refined Cane Sugars.

    Science.gov (United States)

    Eggleston, Gillian; Triplett, Alexa

    2017-11-08

    The sporadic appearance of floc from refined, white cane sugars in alcohol beverages remains a technical problem for both beverage manufacturers and sugar refiners. Cane invert sugars mixed with 60% pure alcohol and water increased light scattering by up to ∼1000-fold. Insoluble and soluble starch, fat, inorganic ash, oligosaccharides, Brix, and pH were not involved in the prevailing floc-formation mechanism. Strong polynomial correlations existed between the haze floc and indicator values (IVs) (color at 420 nm pH 9.0/color at pH 4.0-an indirect measure of polyphenolic and flavonoid colorants) (R 2 = 0.815) and protein (R 2 = 0.819) content of the invert sugars. Ethanol-induced denaturation of the protein exposed hydrophobic polyphenol-binding sites that were further exposed when heated to 80 °C. A tentative mechanism for floc formation was advanced by molecular probing with a haze (floc) active protein and polyphenol as well as polar, nonpolar, and ionic solvents.

  17. Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber.

    Science.gov (United States)

    Papanikolopoulou, Katerina; Schoehn, Guy; Forge, Vincent; Forsyth, V Trevor; Riekel, Christian; Hernandez, Jean-François; Ruigrok, Rob W H; Mitraki, Anna

    2005-01-28

    Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.

  18. Matrix Gla Protein is Involved in Crystal Formation in Kidney of Hyperoxaluric Rats

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    Xiuli Lu

    2013-02-01

    Full Text Available Background: Matrix Gla protein (MGP is a molecular determinant regulating vascular calcification of the extracellular matrix. However, it is still unclear how MGP may be invovled in crystal formation in the kidney of hyperoxaluric rats. Methods: Male Sprague-Dawley rats were divided into the hyperoxaluric group and control group. Hyperoxaluric rats were administrated by 0.75% ethylene glycol (EG for up to 8 weeks. Renal MGP expression was detected by the standard avidin-biotin complex (ABC method. Renal crystal deposition was observed by a polarizing microscope. Total RNA and protein from the rat kidney tissue were extracted. The levels of MGP mRNA and protein expression were analyzed by the real-time polymerase chain reaction (RT-PCR and Western blot. Results: Hyperoxaluria was induced successfully in rats. The MGP was polarly distributed, on the apical membrane of renal tubular epithelial cells, and was found in the ascending thick limbs of Henle's loop (cTAL and the distal convoluted tubule (DCT in hyperoxaluric rats, its expression however, was present in the medullary collecting duct (MCD in stone-forming rats. Crystals with multilaminated structure formed in the injurious renal tubules with lack of MGP expression.MGP mRNA expression was significantly upregulated by the crystals' stimulations. Conclusion: Our results suggested that the MGP was involved in crystals formation by the continuous expression, distributing it polarly in the renal tubular cells and binding directly to the crystals.

  19. Formation of DNA-protein crosslinks in gamma-irradiated chromatin

    International Nuclear Information System (INIS)

    Mee, L.K.

    1985-01-01

    Gamma-irradiation of chromatin in vitro and in vivo induces DNA-protein crosslinks which are stable to salt and detergent treatment. The efficiency of crosslink formation is 100 times greater in irradiated isolated chromatin than in chromatin irradiated in cells before isolation. Gamma-irradiation of isolated chromatin in the presence of radical scavengers shows that OH . is the most effective radical for the promotion of crosslinking whereas e/sub aq//sup -/ and O/sub 2//sup -/ are essentially ineffective. For chromatin irradiated in the cell before isolation, fewer crosslinks are formed in air than in an atmosphere of nitrogen; the greatest effect is found in cells irradiated in an atmosphere of nitrous oxide, suggesting that OH . may be involved in the formation of crosslinks in vivo. On the basis of comparing radiation-induced crosslinking in whole chromating (DNA, H1 histone, the core histones - H2A, H2B, H3 and H4 - and non-histone chromosomal proteins) and in a chromatin subunit (DNA and the core histones), the authors identified the core histones as the specific chromosomal proteins predominantly involved in crosslinking to DNA

  20. In vitro microbial protein synthesis, ruminal degradation and post-ruminal digestibility of crude protein of dairy rations containing Quebracho tannin extract.

    Science.gov (United States)

    Castro-Montoya, J; Westreicher-Kristen, E; Henke, A; Diaby, M; Susenbeth, A; Dickhoefer, U

    2018-02-01

    This study evaluated the effects of Quebracho tannin extract (QTE) on in vitro ruminal fermentation, chemical composition of rumen microbes, ruminal degradation and intestinal digestibility of crude protein (iCPd). Three treatments were tested, the control (basal diet without QTE), the basal diet with 15 g QTE/kg dry matter (DM) and the basal diet with 30 g QTE/kg DM. The basal diet contained (g/kg DM): 339 grass silage, 317 maize silage and 344 concentrate. In vitro gas production kinetic was determined using the Hohenheim gas test (Experiment 1). The Ankom RF technique, a batch system with automatic gas pressure recordings, was used to determine in vitro production of short-chain fatty acids (SCFA) and ammonia-nitrogen concentration (NH 3 -N), as well as nitrogen and purine bases content in liquid-associated microbes (LAM) and in a residue of undegraded feed and solid-associated microbes (Feed+SAM) (Experiment 2). Ruminal degradation and iCPd were determined using the nylon bag technique and the mobile nylon bag technique, respectively (Experiment 3). Gas production (Experiment 1), total SCFA and NH 3 -N (Experiment 2) decreased with increasing QTE levels. Microbial mass and composition of LAM were not affected by QTE, but total mass of Feed+SAM linearly increased, likely due to decreased substrate degradation with increasing QTE levels. The total amount of N in microbial mass and undegraded feed after the in vitro incubation increased with increasing QTE levels, suggesting a potential greater N flow from the rumen to the duodenum. In contrast to in vivo studies with the same QTE, no effects were detected on ruminal effective degradability and iCPd, when using the nylon bag techniques. Based on the in vitro procedures, QTE increased the supply of N post-rumen; however, some evidence of a decreased fibre degradation were also observed. Therefore, the benefit of adding QTE to diets of cattle is still questionable. © 2017 Blackwell Verlag GmbH.

  1. Microbial xanthophylls.

    Science.gov (United States)

    Bhosale, Prakash; Bernstein, Paul S

    2005-09-01

    Xanthophylls are oxygenated carotenoids abundant in the human food supply. Lutein, zeaxanthin, and cryptoxanthin are major xanthophyll carotenoids in human plasma. The consumption of these xanthophylls is directly associated with reduction in the risk of cancers, cardiovascular disease, age-related macular degeneration, and cataract formation. Canthaxanthin and astaxanthin also have considerable importance in aquaculture for salmonid and crustacean pigmentation, and are of commercial interest for the pharmaceutical and food industries. Chemical synthesis is a major source for the heavy demand of xanthophylls in the consumer market; however, microbial producers also have potential as commercial sources. In this review, we discuss the biosynthesis, commercial utility, and major microbial sources of xanthophylls. We also present a critical review of current research and technologies involved in promoting microbes as potential commercial sources for mass production.

  2. Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation.

    Science.gov (United States)

    Kowalska, M Anna; Zhao, Guohua; Zhai, Li; David, George; Marcus, Stephen; Krishnaswamy, Sriram; Poncz, Mortimer

    2014-01-01

    Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with marked decrease anticoagulant activity) may modulate this effect. We measured in vitro and in vivo effects of histones, platelet factor 4, and heparinoids on aPC formation, activated partial thromboplastin time, and murine survival. In vitro, histones and platelet factor 4 both affect thrombin/thrombomodulin aPC generation following a bell-shaped curve, with a peak of >5-fold enhancement. Heparinoids shift these curves rightward. Murine aPC generation studies after infusions of histones, platelet factor 4, and heparinoids supported the in vitro data. Importantly, although unfractionated heparin and 2-O, 3-O desulfated heparin both reversed the lethality of high-dose histone infusions, only mice treated with 2-O, 3-O desulfated heparin demonstrated corrected activated partial thromboplastin times and had significant levels of aPC. Our data provide a new contextual model of how histones affect aPC generation, and how heparinoid therapy may be beneficial in sepsis. These studies provide new insights into the complex interactions controlling aPC formation and suggest a novel therapeutic interventional strategy.

  3. Photo-induced formation of nitrous acid (HONO) on protein surfaces

    Science.gov (United States)

    Meusel, Hannah; Elshorbany, Yasin; Bartels-Rausch, Thorsten; Selzle, Kathrin; Lelieveld, Jos; Ammann, Markus; Pöschl, Ulrich; Su, Hang; Cheng, Yafang

    2014-05-01

    The study of nitrous acid (HONO) is of great interest, as the photolysis of HONO leads to the OH radical, which is the most important oxidant in the troposphere. HONO is directly emitted by combustion of fossil fuel and from soil biogenic nitrite (Su et al., 2011), and can also be formed by gas phase reactions of NO and OH and heterogeneous reactions of NO2. Previous atmospheric measurements have shown unexpectedly high HONO concentrations during daytime. Measured mixing ratios were about one order of magnitude higher than model simulations (Kleffmann et al. 2005, Vogel et al. 2003). The additional daytime source of HONO might be attributed to the photolysis of adsorbed nitric acid or heterogeneous photochemistry of NO2 on organic substrates, such as humic acids or polyphenolic compounds (Stemmler et al., 2006), or indirectly through nitration of phenols and subsequent photolysis of nitrophenols (Sosedova et al., 2011, Bejan et al., 2006). An important reactive surface for the heterogeneous formation of HONO could involve proteins, which are ubiquitous in the environment. They are part of coarse biological aerosol particles like pollen grains, fine particles (fragments of pollen, microorganism, plant debris) and dissolved in rainwater, soil and road dust (Miguel et al. 1999). In this project a thin film of bovine serum albumin (BSA), a model protein with 67 kDa and 21 tyrosine residues per molecule, is irradiated and exposed to nitrogen dioxide in humidified nitrogen. The formation of HONO is measured with long path absorption photometry (LOPAP). The generated HONO is in the range of 100 to 1100 ppt depending on light intensity, NO2 concentration and film thickness. Light induced HONO formation on protein surfaces is stable over the 20-hours experiment of irradiation and exposure. On the other hand, light activated proteins reacting with NO2 form nitrated proteins, as detected by liquid chromatography (LC-DAD). Our experiments on tetranitromethane (TNM) nitrated

  4. Microbial-caddisfly bioherm association from the Lower Cretaceous Shinekhudag Formation, Mongolia: Earliest record of plant armoring in fossil caddisfly cases.

    Directory of Open Access Journals (Sweden)

    Tsolmon Adiya

    Full Text Available Caddisfly larvae construct underwater protective cases using surrounding materials, thus providing information on environmental conditions in both modern and ancient systems. Microbial bioherms associated with caddisfly cases are found in the Berriassian-Hauterivian (~140-130 Ma Shinekhudag Formation of Mongolia, and yield new insights into aspects of lacustrine paleoecosystems and paleoenvironments. This formation contains the earliest record of plant-armored caddisfly cases and a rare occurrence of microbial-caddisfly association from the Mesozoic. The bioherms are investigated within the context of stratigraphic correlations, depositional environment interpretations, and basin-evolution models of the sedimentary fill. The bioherms form 0.5-2.0 m diameter mound-shaped bodies and are concentrated within a single, oil shale-bound stratigraphic interval. Each bioherm is composed of up to 40% caddisfly cases along with stromatolites of millimeter-scale, micritic laminations. Petrographic analyses reveal these bioherms are composed of non-systematic associations of columnar and oncoidal microbialites, constructed around colonies of caddisfly cases. The cases are straight to curved, slightly tapered, and tube-shaped, with a progressively increasing length and width trend (7-21 mm by 1.5-2.5 mm. Despite these variations, the case architectures reveal similar construction materials; the particles used for cases are dominated by plant fragments, ostracod valves, carbonate rocks, and rare mica and feldspar grains. Allochems within the bioherms include ooids, ostracods, plant fragments, rare gastropods, feldspar grains bound in micritic matrices, and are consolidated by carbonate dominated cements. The combination of microbial-caddisfly association, plant fragment case particles, and ooids/oncoids are indicative of a shallow, littoral lake setting. Stratigraphic juxtaposition of nearshore bioherms and the bounding distal oil-shale facies suggests that

  5. The Negatively Charged Regions of Lactoferrin Binding Protein B, an Adaptation against Anti-Microbial Peptides

    Science.gov (United States)

    Morgenthau, Ari; Beddek, Amanda; Schryvers, Anthony B.

    2014-01-01

    Lactoferrin binding protein B (LbpB) is a bi-lobed membrane bound lipoprotein that is part of the lactoferrin receptor complex in a variety of Gram-negative pathogens. Despite high sequence diversity among LbpBs from various strains and species, a cluster of negatively charged amino acids is invariably present in the protein’s C-terminal lobe in all species except Moraxella bovis. The function of LbpB in iron acquisition has yet to be experimentally demonstrated, whereas in vitro studies have shown that LbpB confers protection against lactoferricin, a short cationic antimicrobial peptide released from the N- terminus of lactoferrin. In this study we demonstrate that the negatively charged regions can be removed from the Neisseria meningitidis LbpB without compromising stability, and this results in the inability of LbpB to protect against the bactericidal effects of lactoferricin. The release of LbpB from the cell surface by the autotransporter NalP reduces the protection against lactoferricin in the in vitro killing assay, attributed to removal of LbpB during washing steps, but is unlikely to have a similar impact in vivo. The protective effect of the negatively charged polysaccharide capsule in the killing assay was less than the protection conferred by LbpB, suggesting that LbpB plays a major role in protection against cationic antimicrobial peptides in vivo. The selective release of LbpB by NalP has been proposed to be a mechanism for evading the adaptive immune response, by reducing the antibody binding to the cell surface, but may also provide insights into the primary function of LbpB in vivo. Although TbpB and LbpB have been shown to be major targets of the human immune response, the selective release of LbpB suggests that unlike TbpB, LbpB may not be essential for iron acquisition, but important for protection against cationic antimicrobial peptides. PMID:24465982

  6. The role of milk proteins in the structure formation of dairy products

    Directory of Open Access Journals (Sweden)

    Olga Rybak

    2015-04-01

    Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence of fat globules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions. It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

  7. The role of milk proteins in the structure formation of dairy products

    Directory of Open Access Journals (Sweden)

    Olga Rybak

    2014-09-01

    Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence of fat globules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions. It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

  8. The role of milk proteins in the structure formation of dairy products

    Directory of Open Access Journals (Sweden)

    O. Rybak

    2015-05-01

    Full Text Available Introduction. The structure of dairy products is a complex of proteins, fat, minerals and water that determines the texture and sensory properties of the product. Material and methods. The fermented milks (using the example of yogurt, cheese, ice cream, aerated milk and frozen fruit desserts have been researched. Scientific articles, published during 2000 and 2014 years, as well as theses and monographs of dairy science have been analysed too. Methodology of the investigation is based upon the use of the methods of analysis, comparison and synthesis. Results and discussion. The scientific understanding of the milk proteins’ role in the structure formation of dairy product has been summarized. Negligible changes of structure as a result of compositional or technological changes can lead to shifts in the stability, texture and rheology of products, which are closely related to each other. The allowance of these properties has significant influence on the manufacturing. Acid coagulation is a major functional property of milk proteins, which used in the structure formation of cheese and fermented dairy products. However, the form and properties of milk curd depend on the heat treatment of milk before fermentation. Milk proteins exhibit other functional properties (emulsification and partial coalescence o f fatglobules, aeration and foam stability during a churning, viscosity increasing of external phase in the development of structure in the ice cream, aerated milk and frozen fruit desserts. Conclusions.It is expedient to use results into a further study of the structure formation mechanism of dairy products and the development of recommendations in order to an efficient production.

  9. Direct antioxidant properties of methotrexate: Inhibition of malondialdehyde-acetaldehyde-protein adduct formation and superoxide scavenging

    Directory of Open Access Journals (Sweden)

    Matthew C. Zimmerman

    2017-10-01

    Full Text Available Methotrexate (MTX is an immunosuppressant commonly used for the treatment of autoimmune diseases. Recent observations have shown that patients treated with MTX also exhibit a reduced risk for the development of cardiovascular disease (CVD. Although MTX reduces systemic inflammation and tissue damage, the mechanisms by which MTX exerts these beneficial effects are not entirely known. We have previously demonstrated that protein adducts formed by the interaction of malondialdehyde (MDA and acetaldehyde (AA, known as MAA-protein adducts, are present in diseased tissues of individuals with rheumatoid arthritis (RA or CVD. In previously reported studies, MAA-adducts were shown to be highly immunogenic, supporting the concept that MAA-adducts not only serve as markers of oxidative stress but may have a direct role in the pathogenesis of inflammatory diseases. Because MAA-adducts are commonly detected in diseased tissues and are proposed to mitigate disease progression in both RA and CVD, we tested the hypothesis that MTX inhibits the generation of MAA-protein adducts by scavenging reactive oxygen species. Using a cell free system, we found that MTX reduces MAA-adduct formation by approximately 6-fold, and scavenges free radicals produced during MAA-adduct formation. Further investigation revealed that MTX directly scavenges superoxide, but not hydrogen peroxide. Additionally, using the Nrf2/ARE luciferase reporter cell line, which responds to intracellular redox changes, we observed that MTX inhibits the activation of Nrf2 in cells treated with MDA and AA. These studies define previously unrecognized mechanisms by which MTX can reduce inflammation and subsequent tissue damage, namely, scavenging free radicals, reducing oxidative stress, and inhibiting MAA-adduct formation.

  10. The role of plasma proteins in formation of obstructive protamine complexes

    International Nuclear Information System (INIS)

    De Paulis, R.; Mohammad, S.F.; Chiariello, L.; Morea, M.; Olsen, D.B.

    1991-01-01

    Formation of complexes between heparin and protamine (in saline), or heparin, plasma proteins, and protamine (in plasma) was assessed by measurements of light transmission through different test solutions. To examine the formation of these complexes, 125I-labeled protamine was used. Addition of 125I-protamine to plasma or blood resulted in the sedimentation of 125I-protamine in the form of insoluble complexes. This complex formation was not affected by the presence of heparin, suggesting that protamine-plasma protein interaction may be primarily responsible for precipitation of 125I-protamine. To assess the capability of these complexes to obstruct the pulmonary circulation, an in vitro experimental model was developed. Citrated serum, plasma, blood, or saline were allowed to flow through a glass bead column with the help of a peristaltic pump. A pressure transducer positioned before the column allowed pressure measurements at a constant flow rate during the experiment. Mixing of protamine with plasma or blood prior to their passage through the glass bead column resulted in a significant increase in pressure suggesting that the column was being clogged with insoluble complexes. The increase in pressure occurred both in the presence and absence of heparin in plasma or blood. Under identical experimental conditions, the increase in pressure was insignificant when protamine was added to saline or serum regardless of whether heparin was present or absent. This was further confirmed by the use of 125I-protamine. These observations suggest that protamine forms insoluble complexes with certain plasma proteins. Based on these observations, it is hypothesized that following intravenous administration, protamine immediately forms complexes in circulating blood

  11. The accumulation of a platelet protein, thrombospondin, at the site of arterial thrombus formation: Preliminary report

    International Nuclear Information System (INIS)

    Perlman, S.B.; Hammes, R.J.; Besozzi, M.C.; Folts, J.D.; Mosher, D.F.

    1987-01-01

    There are many methods available for the detection of thrombosis, none of which are noninvasive, rapid and accurate. Thrombosponding is a platelet protein that is present in the developing thrombus and may be an effective substance to use for imaging thrombosis. Vascular stenosis and thrombosis were produced in coronary, carotid and femoral arteries in eleven adult mongrel dogs. 131 I labeled thrombosponding was administered to each animal to determine whether the radiotracer accumulated at the site of thrombus formation. The radioactivity per gram of vessels with thrombi was significantly different from the control vessels or whole blood (p=0.0037 and p=0.015, respectively, paired t-test). This preliminary work suggests that iodinated thrombosponding accumulates at the site of thrombus formation. Labeled thrombosponding may be a rapid, safe and accurate method of detecting arterial thrombosis. (orig.)

  12. Synovial fluid lubrication of artificial joints: protein film formation and composition.

    Science.gov (United States)

    Fan, Jingyun; Myant, Connor; Underwood, Richard; Cann, Philippa

    2012-01-01

    Despite design improvements, wear of artificial implants remains a serious health issue particularly for Metal-on-Metal (MoM) hips where the formation of metallic wear debris has been linked to adverse tissue response. Clearly it is important to understand the fundamental lubrication mechanisms which control the wear process. It is usually assumed that MoM hips operate in the ElastoHydrodynamic Lubrication (EHL) regime where film formation is governed by the bulk fluid viscosity; however there is little experimental evidence of this. The current paper critically examines synovial fluid lubrication mechanisms and the effect of synovial fluid chemistry. Two composition parameters were chosen; protein content and pH, both of which are known to change in diseased or post-operative synovial fluid. Film thickness and wear tests were carried out for a series of model synovial fluid solutions. Two distinct film formation mechanisms were identified; an adsorbed surface film and a high-viscosity gel. The entrainment of this gel controls film formation particularly at low speeds. However wear of the femoral head still occurs and this is thought to be due primarily to a tribo-corrosion mechanisms. The implications of this new lubrication mechanism and the effect of different synovial fluid chemistries are examined. One important conclusion is that patient synovial fluid chemistry plays an important role in determining implant wear and the likelihood of failure.

  13. Formation and repair of DNA-protein cross-links (DPCs) in newly replicated DNA

    International Nuclear Information System (INIS)

    Chiu, S.; Friedman, L.R.; Oleinick, N.L.

    1987-01-01

    DPCs preferentially involve proteins of the nuclear matrix, the site of replication and transcription. To elucidate the relationship with replication, the formation and repair of DPCs has been studied in newly replicated DNA. Log-phase V79 cells were pulsed with /sup 3/H-TdR (10-20 μCi/ml) for 30-90 sec at 22 0 followed by up to a 60 min chase at 37 0 . Irradiation (0-100 Gy) immediately after the pulse increases the labeled DNA in DPCs with a dose-dependence that is unaffected by the initial level of labeled DPC or by chase time. When cells are irradiated before the pulse, DNA synthesis is inhibited; however, release of pulse-labeled DPCs appears normal. The data suggest that during replication, DNA is cross-linked to (matrix) protein, contributing to background DPCs

  14. Endogenous contributions to egg protein formation in lesser scaup Aythya affinis

    Science.gov (United States)

    Cutting, Kyle A.; Hobson, Keith A.; Rotella, Jay J.; Warren, Jeffrey M.; Wainwright-de la Cruz, Susan E.; Takekawa, John Y.

    2011-01-01

    Lesser scaup Aythya affinis populations have declined throughout the North American continent for the last three decades. It has been hypothesized that the loss and degradation of staging habitats has resulted in reduced female body condition on the breeding grounds and a concomitant decline in productivity. We explored the importance of body (endogenous) reserves obtained prior to arrival on the breeding ground in egg protein formation in southwestern Montana during 2006–2008 using stable-carbon (δ13C) and nitrogen (δ15N) isotope analyses of scaup egg components, female tissue, and local prey items. From arrival on the breeding grounds through the egg-laying period, δ15N values of scaup red blood cells decreased while δ13C values became less variable; a pattern consistent with endogenous tissues equilibrating with local (freshwater) dietary sources. In 2006 and 2008, isotopic values for egg albumen and yolk protein indicated that most (>90%) protein used to produce these components was obtained on the breeding grounds. However, in 2007, a year with an exceptionally warm and dry spring, endogenous reserves contributed on average 41% of yolk and 29% of albumen. Results from this study suggest that female scaup can meet the protein needs of egg production largely from local dietary food sources. This highlights the importance of providing high-quality breeding habitats for scaup. Whether this pattern holds in areas with similar breeding season lengths but longer migration routes, such as those found in the western boreal forest, should be investigated.

  15. Investigations on the effect of forage source, grinding, and urea supplementation on ruminal fermentation and microbial protein flow in a semi-continuous rumen simulation system.

    Science.gov (United States)

    Hildebrand, Bastian; Boguhn, Jeannette; Rodehutscord, Markus

    2011-10-01

    The objective of the present study was to compare the effect of maize silage and grass silage on microbial fermentation and protein flow in a semi-continuous rumen simulation system (Rusitec) when milling screen size (MSS) during grinding was varied. Oven-dried silages were milled through screens of 1, 4 or 9 mm pore size and incubated for 48 h in a Rusitec system. Furthermore, the effect of N supplementation to maize silage (MSS: 4 mm) was investigated and single dose vs. continuous infusion of urea-N were compared. Degradation of organic matter (OM), crude protein (CP), fibre fractions and non-structural carbohydrates (NSC) as well as short-chain fatty acid production differed significantly between forage sources. Urea-N supplementation improved the degradation of NSC, but not that of fibre fractions in maize silage. The way of urea supply had only marginal effects on fermentation characteristics. An increase in MSS, and consequently in mean feed particle size, led to an improvement in the degradation of OM, CP and NSC, but efficiency of microbial net protein synthesis (EMPS; mg microbial N flow/g degraded OM) and the microbial amino acid profile were less affected. EMPS was higher in grass silage than in maize silage and was improved by urea-N supplementation in maize silage. This study indicates that fermentation of NSC as well as EMPS during incubation of maize silage was limited by availability of NH3-N. Furthermore, an increase in MSS above 1 mm seems to improve fermentation of silages in the Rusitec system.

  16. Effect of sulfur supplements on cellulolytic rumen micro-organisms and microbial protein synthesis in cattle fed a high fibre diet.

    Science.gov (United States)

    McSweeney, C S; Denman, S E

    2007-11-01

    To examine the effect of sulfur-containing compounds on the growth of anaerobic rumen fungi and the fibrolytic rumen bacteria Ruminococcus albus, Ruminococcus flavefaciens and Fibrobacter succinogenes in pure culture and within the cattle rumen. The effect of two reduced sulfur compounds, 3-mercaptopropionic acid (MPA) or 3-mercapto-1-propanesulfonic acid as the sole S source on growth of pure fibroyltic fungal and bacterial cultures showed that these compounds were capable of sustaining growth. An in vivo trial was then conducted to determine the effect of sulfur supplements (MPA and sodium sulfate) on microbial population dynamics in cattle fed the roughage Dichanthium aristatum. Real-time PCR showed significant increases in fibrolytic bacterial and fungal populations when cattle were supplemented with these compounds. Sulfate supplementation leads to an increase in dry matter intake without a change in whole tract dry matter digestibility. Supplementation of low S-containing diets with either sodium sulfate or MPA stimulates microbial growth with an increase in rumen microbial protein supply to the animal. Through the use of real-time PCR monitoring, a better understanding of the effect of S supplementation on discrete microbial populations within the rumen is provided.

  17. Effects of the source of energy and minerals on microbial protein synthesis in rumen using 35S as indicator. Part of a coordinated programme on tracer techniques in studies on the use of non-protein nitrogen in ruminants

    International Nuclear Information System (INIS)

    Durant, M.

    1976-05-01

    Part I. The effect of the nature of carbohydrates and minerals on microbial growth in vitro was studied in vitro to intensify rumen microbial protein synthesis from non-protein nitrogen, using phosphorus incorporation (PR). The nature of starch greatly influences urea utilization. Among the tropical tubers studied, yam Cayenesis and canna Edulis give lower urea utilization than cassava and yam Dumetorum. However, this can be improved by processing. The effects are greatest when the proportion of urea to processed cereal is ca. 4-5%. S-addition as sulfate improves urea utilization with both natural and purified diets. Part II. Results from the Jouy and Ghent laboratories were analyzed statistically, to check the accuracy of the 32 P method for estimating microbial growth, using protein-free substrates. The linear relationship between PR and every other variable was studied. Two multivariate analyses, principal components and multiple regression, were applied. Net NH 3 utilization was predicted using the equations for substrates with proteins likely to be degraded. This method appears more accurate than using the N/P ratio in microflora. Equations should only be used under the specific experiments described

  18. Improving the quality of rice straw by urea and calcium hydroxide on rumen ecology, microbial protein synthesis in beef cattle.

    Science.gov (United States)

    Polyorach, S; Wanapat, M

    2015-06-01

    Four rumen-fistulated beef cattle were randomly assigned to four treatments according to a 4 × 4 Latin square design to study the influence of urea and calcium hydroxide [Ca(OH)2 ] treatment of rice straw to improve the nutritive value of rice straw. Four dietary treatments were as follows: untreated rice straw, 50 g/kg urea-treated rice straw, 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw and 30 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw. All animals were kept in individual pens and fed with concentrate at 0.5 g/kg of BW (DM), rice straw was fed ad libitum. The experiment was conducted for four periods, and each period lasted for 21 days. During the first 14 days, DM feed intake measurements were made while during the last 7 days, all cattle were moved to metabolism crates for total faeces and urine collections. The results revealed that 20 g/kg urea + 20 g/kg calcium hydroxide-treated rice straw improved the nutritive value of rice straw, in terms of dry matter intake, digestibility, ruminal volatile fatty acids, population of bacteria and fungi, nitrogen retention and microbial protein synthesis. Based on this study, it could be concluded that using urea plus calcium hydroxide was one alternative method to improve the nutritive value of rice straw, rumen ecology and fermentation and thus a reduction of treatment cost. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  19. Structural basis of G protein-coupled receptor-Gi protein interaction: formation of the cannabinoid CB2 receptor-Gi protein complex.

    Science.gov (United States)

    Mnpotra, Jagjeet S; Qiao, Zhuanhong; Cai, Jian; Lynch, Diane L; Grossfield, Alan; Leioatts, Nicholas; Hurst, Dow P; Pitman, Michael C; Song, Zhao-Hui; Reggio, Patricia H

    2014-07-18

    In this study, we applied a comprehensive G protein-coupled receptor-Gαi protein chemical cross-linking strategy to map the cannabinoid receptor subtype 2 (CB2)-Gαi interface and then used molecular dynamics simulations to explore the dynamics of complex formation. Three cross-link sites were identified using LC-MS/MS and electrospray ionization-MS/MS as follows: 1) a sulfhydryl cross-link between C3.53(134) in TMH3 and the Gαi C-terminal i-3 residue Cys-351; 2) a lysine cross-link between K6.35(245) in TMH6 and the Gαi C-terminal i-5 residue, Lys-349; and 3) a lysine cross-link between K5.64(215) in TMH5 and the Gαi α4β6 loop residue, Lys-317. To investigate the dynamics and nature of the conformational changes involved in CB2·Gi complex formation, we carried out microsecond-time scale molecular dynamics simulations of the CB2 R*·Gαi1β1γ2 complex embedded in a 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayer, using cross-linking information as validation. Our results show that although molecular dynamics simulations started with the G protein orientation in the β2-AR*·Gαsβ1γ2 complex crystal structure, the Gαi1β1γ2 protein reoriented itself within 300 ns. Two major changes occurred as follows. 1) The Gαi1 α5 helix tilt changed due to the outward movement of TMH5 in CB2 R*. 2) A 25° clockwise rotation of Gαi1β1γ2 underneath CB2 R* occurred, with rotation ceasing when Pro-139 (IC-2 loop) anchors in a hydrophobic pocket on Gαi1 (Val-34, Leu-194, Phe-196, Phe-336, Thr-340, Ile-343, and Ile-344). In this complex, all three experimentally identified cross-links can occur. These findings should be relevant for other class A G protein-coupled receptors that couple to Gi proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Locked chromophore analogs reveal that photoactive yellow protein regulates biofilm formation in the deep sea bacterium Idiomarina loihiensis

    NARCIS (Netherlands)

    van der Horst, M.A.; Stalcup, T.P.; Kaledhonkar, S.; Kumauchi, M.; Hara, M.; Xie, A.; Hellingwerf, K.J.; Hoff, W.D.

    2009-01-01

    Idiomarina loihiensis is a heterotrophic deep sea bacterium with no known photobiology. We show that light suppresses biofilm formation in this organism. The genome of I. loihiensis encodes a single photoreceptor protein: a homologue of photoactive yellow protein (PYP), a blue light receptor with

  1. Preliminary protein corona formation stabilizes gold nanoparticles and improves deposition efficiency

    Science.gov (United States)

    Luby, Alexandra O.; Breitner, Emily K.; Comfort, Kristen K.

    2016-08-01

    Due to their advantageous characteristics, gold nanoparticles (AuNPs) are being increasingly utilized in a vast array of biomedical applications. However, the efficacy of these procedures are highly dependent upon strong interactions between AuNPs and the surrounding environment. While the field of nanotechnology has grown exponentially, there is still much to be discovered with regards to the complex interactions between NPs and biological systems. One area of particular interest is the generation of a protein corona, which instantaneously forms when NPs encounter a protein-rich environment. Currently, the corona is viewed as an obstacle and has been identified as the cause for loss of application efficiency in physiological systems. To date, however, no study has explored if the protein corona could be designed and advantageously utilized to improve both NP behavior and application efficacy. Therefore, we sought to identify if the formation of a preliminary protein corona could modify both AuNP characteristics and association with the HaCaT cell model. In this study, a corona comprised solely of epidermal growth factor (EGF) was successfully formed around 10-nm AuNPs. These EGF-AuNPs demonstrated augmented particle stability, a modified corona composition, and increased deposition over stock AuNPs, while remaining biocompatible. Analysis of AuNP dosimetry was repeated under dynamic conditions, with lateral flow significantly disrupting deposition and the nano-cellular interface. Taken together, this study demonstrated the plausibility and potential of utilizing the protein corona as a means to influence NP behavior; however, fluid dynamics remains a major challenge to progressing NP dosimetry.

  2. Lipoxygenase independent hexanal formation in isolated soy proteins induced by reducing agents.

    Science.gov (United States)

    Lei, Q; Boatright, W L

    2008-08-01

    Compared to corresponding controls, 6.5 mM dithiothreitol (DTT) elevated headspace hexanal level over aqueous slurries of both commercial isolated soy proteins (ISP) and laboratory ISP prepared with 80 degrees C treatment. Further analysis revealed that lipoxygenase (LOX) activity was not detected from these ISP, indicating that LOX is not involved in the observed hexanal increase. Levels of the induced headspace hexanal over the ISP aqueous slurries were proportional to the amount of DTT added in the range of 0 to 65 mM. Subsequent systematic investigations with model systems revealed that iron was required for the reducing agent-induced hexanal formation from linoleic acid. Erythorbate, another reducing agent, can also induce hexanal formation in both ISP and model systems. As a comparison, the LOX activity and hexanal synthesis in defatted soy flour were examined. The corresponding results showed that defatted soy flour maintained high LOX activities and that hexanal synthesis in such sample was significantly inhibited by high concentration DTT (above 130 mM). Data from the current investigation demonstrate the existence of LOX independent hexanal formation induced by reducing agents in ISP and the potential requirement of iron as a catalyst.

  3. CREB binding protein is required for both short-term and long-term memory formation.

    Science.gov (United States)

    Chen, Guiquan; Zou, Xiaoyan; Watanabe, Hirotaka; van Deursen, Jan M; Shen, Jie

    2010-09-29

    CREB binding protein (CBP) is a transcriptional coactivator with histone acetyltransferase activity. Our prior study suggested that CBP might be a key target of presenilins in the regulation of memory formation and neuronal survival. To elucidate the role of CBP in the adult brain, we generated conditional knock-out (cKO) mice in which CBP is completely inactivated in excitatory neurons of the postnatal forebrain. Histological analysis revealed normal neuronal morphology and absence of age-dependent neuronal degeneration in the CBP cKO cerebral cortex. CBP cKO mice exhibited robust impairment in the formation of spatial, associative, and object-recognition memory. In addition to impaired long-term memory, CBP cKO mice also displayed deficits in short-term associative and object-recognition memory. Administration of a histone deacetylase inhibitor, trichostatin A, rescued the reduction of acetylated histones in the CBP cKO cortex but failed to rescue either short- or long-term memory deficits, suggesting that the memory impairment may not be caused by general reduction of histone acetyltransferase activity in CBP cKO mice. Further microarray and Western analysis showed decreased expression of calcium-calmodulin-dependent kinase isoforms and NMDA and AMPA receptor subunits in the cerebral cortex of CBP cKO mice. Collectively, these findings suggest a crucial role for CBP in the formation of both short- and long-term memory.

  4. Functional divergence of platelet protein kinase C (PKC) isoforms in thrombus formation on collagen.

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T; Cosemans, Judith M E M; Konopatskaya, Olga; Munnix, Imke C A; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D; Heemskerk, Johan W M; Poole, Alastair W

    2010-07-23

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent alpha-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCalpha and PKCbeta, whereas the novel isoform, PKC, negatively regulates these events. PKCdelta also negatively regulates thrombus formation but not alpha-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCalpha or PKCbeta showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKC. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

  5. Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen*

    Science.gov (United States)

    Gilio, Karen; Harper, Matthew T.; Cosemans, Judith M. E. M.; Konopatskaya, Olga; Munnix, Imke C. A.; Prinzen, Lenneke; Leitges, Michael; Liu, Qinghang; Molkentin, Jeffery D.; Heemskerk, Johan W. M.; Poole, Alastair W.

    2010-01-01

    Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen. PMID:20479008

  6. G protein-coupled receptor internalization assays in the high-content screening format.

    Science.gov (United States)

    Haasen, Dorothea; Schnapp, Andreas; Valler, Martin J; Heilker, Ralf

    2006-01-01

    High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.

  7. Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein*

    Science.gov (United States)

    Rahman, M. Azizur; Oomori, Tamotsu; Wörheide, Gert

    2011-01-01

    Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature. PMID:21768106

  8. Protein Adsorption and Layer Formation at the Stainless Steel-Solution Interface Mediates Shear-Induced Particle Formation for an IgG1 Monoclonal Antibody.

    Science.gov (United States)

    Kalonia, Cavan K; Heinrich, Frank; Curtis, Joseph E; Raman, Sid; Miller, Maria A; Hudson, Steven D

    2018-03-05

    Passage of specific protein solutions through certain pumps, tubing, and/or filling nozzles can result in the production of unwanted subvisible protein particles (SVPs). In this work, surface-mediated SVP formation was investigated. Specifically, the effects of different solid interface materials, interfacial shear rates, and protein concentrations on SVP formation were measured for the National Institute of Standards and Technology monoclonal antibody (NISTmAb), a reference IgG1 monoclonal antibody (mAb). A stainless steel rotary piston pump was used to identify formulation and process parameters that affect aggregation, and a flow cell (alumina or stainless steel interface) was used to further investigate the effect of different interface materials and/or interfacial shear rates. SVP particles produced were monitored using flow microscopy or flow cytometry. Neutron reflectometry and a quartz crystal microbalance with dissipation monitoring were used to characterize adsorption and properties of NISTmAb at the stainless steel interface. Pump/shear cell experiments showed that the NISTmAb concentration and interface material had a significant effect on SVP formation, while the effects of interfacial shear rate and passage number were less important. At the higher NISTmAb concentrations, the adsorbed protein became structurally altered at the stainless steel interface. The primary adsorbed layer remained largely undisturbed during flow, suggesting that SVP formation at high NISTmAb concentration was caused by the disruption of patches and/or secondary interactions.

  9. Developmental expression of a cell surface protein involved in sea urchin skeleton formation

    International Nuclear Information System (INIS)

    Farach, M.C.; Valdizan, M.; Park, H.R.; Decker, G.L.; Lennarz, W.J.

    1986-01-01

    The authors have previously used a monoclonal antibody (1223) to identify a 130 Kd cell surface protein involved in skeleton formation is sea urchin embryos. In the current study the authors have examined the expression of the 1223 antigen over the course of development of embryos of two species, Strongylocentrotus purpuratus and Lytechinus pictus. The 130 Kd protein is detected in S. purp eggs on immunoblots. Labeling with [ 3 H] leucine and immunoaffinity chromatography show that it also is synthesized shortly after fertilization. Immunofluroescence reveals that at this early stage the 1223 antigen is uniformly distributed on all of the cells. Synthesis decreases to a minimum by the time of hatching (18 h), as does the total amount of antigen present in the embryo. A second period of synthesis commences at the mesenchyme blastula stage, when the spicule-forming primary mesenchyme cells (PMCs) have appeared. During this later stage, synthesis and cell surface expression are restricted to the PMCs. In contrast to S. purp., in L. pictus the 130 Kd protein does not appear until the PMCs are formed. Hybrid embryos demonstrate a pattern of expression of the maternal species. These results suggest that early expression of 1223 antigen in S. purp. is due to utilization of maternal transcripts present in the egg. In both species later expression in PMCs appears to be the result of cell-type specific synthesis, perhaps encoded by embryonic transcripts

  10. Enhanced conformational sampling to visualize a free-energy landscape of protein complex formation.

    Science.gov (United States)

    Iida, Shinji; Nakamura, Haruki; Higo, Junichi

    2016-06-15

    We introduce various, recently developed, generalized ensemble methods, which are useful to sample various molecular configurations emerging in the process of protein-protein or protein-ligand binding. The methods introduced here are those that have been or will be applied to biomolecular binding, where the biomolecules are treated as flexible molecules expressed by an all-atom model in an explicit solvent. Sampling produces an ensemble of conformations (snapshots) that are thermodynamically probable at room temperature. Then, projection of those conformations to an abstract low-dimensional space generates a free-energy landscape. As an example, we show a landscape of homo-dimer formation of an endothelin-1-like molecule computed using a generalized ensemble method. The lowest free-energy cluster at room temperature coincided precisely with the experimentally determined complex structure. Two minor clusters were also found in the landscape, which were largely different from the native complex form. Although those clusters were isolated at room temperature, with rising temperature a pathway emerged linking the lowest and second-lowest free-energy clusters, and a further temperature increment connected all the clusters. This exemplifies that the generalized ensemble method is a powerful tool for computing the free-energy landscape, by which one can discuss the thermodynamic stability of clusters and the temperature dependence of the cluster networks. © 2016 The Author(s).

  11. Ternary complex formation of lanthanides and radiolanthanides with phosphate and serum proteins

    International Nuclear Information System (INIS)

    Neumaier, B.; Roesch, F.

    1999-01-01

    Radioyttrium was recently reported to form ternary complexes with phosphate and serum proteins in blood. In the present work it was investigated whether the trivalent radiolanthanides react in a chemically similar way. In systematic binding studies using gel filtration a ternary complex formation between different lanthanides, phosphate and serum proteins could be identified. The tendency to build a ternary compound of the type Ln III - phosphate - serum protein, however, is dependent on the ionic radii of the lanthanides. Whereas the light and transition lanthanides have a strong inclination to build a ternary complex, this tendency is weaker for the heavier ones. Taking into account the high content of phosphate in human blood, the corresponding ternary complexes of radiolanthanides represent an important transport form of these elements in blood. This finding may contribute to an understanding of the nuclear medical observation on the biodistribution of radiolanthanides. The heavy radiolanthanides can be classified as bone seeking metals, whereas the light and transition lanthanide elements accumulate mainly in the liver and the spleen. For the lighter radiolanthanides the corresponding ternary complexes thus represent an important transport form in blood. This physicochemical form of lanthanides mainly results in reticulo endothelial accumulation; on the other hand, the lower tendency of heavier lanthanides leads to preferential skeletal deposition. (orig.)

  12. Dengue Virus Capsid Protein Binds Core Histones and Inhibits Nucleosome Formation in Human Liver Cells

    Science.gov (United States)

    Colpitts, Tonya M.; Barthel, Sebastian; Wang, Penghua; Fikrig, Erol

    2011-01-01

    Dengue virus (DENV) is a member of the Flaviviridae and a globally (re)emerging pathogen that causes serious human disease. There is no specific antiviral or vaccine for dengue virus infection. Flavivirus capsid (C) is a structural protein responsible for gathering the viral RNA into a nucleocapsid that forms the core of a mature virus particle. Flaviviral replication is known to occur in the cytoplasm yet a large portion of capsid protein localizes to the nucleus during infection. The reasons for the nuclear presences of capsid are not completely understood. Here, we expressed mature DENV C in a tandem affinity purification assay to identify potential binding partners in human liver cells. DENV C targeted the four core histones, H2A, H2B, H3 and H4. DENV C bound recombinant histones in solution and colocalized with histones in the nucleus and cytoplasm of liver cells during DENV infection. We show that DENV C acts as a histone mimic, forming heterodimers with core histones, binding DNA and disrupting nucleosome formation. We also demonstrate that DENV infection increases the amounts of core histones in livers cells, which may be a cellular response to C binding away the histone proteins. Infection with DENV additionally alters levels of H2A phosphorylation in a time-dependent manner. The interactions of C and histones add an interesting new role for the presence of C in the nucleus during DENV infection. PMID:21909430

  13. Nuclear based technologies for estimating microbial protein supply in ruminant livestock. Proceedings of the second research co-ordination meeting of a co-ordinated research project (phase 1)

    International Nuclear Information System (INIS)

    1999-06-01

    The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture through its Co-ordinated Research Projects (CRPs), has been assisting national agricultural research systems in Member States to develop and apply nuclear and related techniques for improving livestock productivity. The programmes have focused on animal nutrition, animal reproduction and more recently on animal nutrition/reproduction interactions with emphasis on smallholder farming systems. The measurement of microbial protein supply to ruminant livestock has been an important area of research in ruminant nutrition. An estimate of microbial protein contribution to the intestinal protein flow is important for estimating the protein requirement of ruminant animals. Understanding the process of microbial protein synthesis has been difficult however, and due to the lack of simple and accurate methods for measuring microbial protein production in vivo, the methods used are based on complex microbial markers which require surgically prepared animals. As a result of a consultants meeting held in May 1995 to advise the Joint FAO/IAEA Division on the feasibility of using nuclear and related techniques for the development and validation of techniques for measuring microbial protein supply in ruminant animals, an FAO/IAEA Co-ordinated Research Project on Development, Standardization and Validation of Nuclear Based Technologies for Measuring Microbial Protein Supply in Ruminant Livestock for Improving Productivity was initiated in 1996, with a view to validating and adapting this technology for use in developing countries. To assist scientists participating in the CRP, a laboratory manual containing experimental protocols and methodologies for standardization and validation of the urine purine derivative technique and the development of models to suit local conditions, was published as IAEA-TECDOC-945. The present publication contains the final reports from participants in Phase 1 of the project

  14. Nuclear based technologies for estimating microbial protein supply in ruminant livestock. Proceedings of the second research co-ordination meeting of a co-ordinated research project (phase 1)

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-06-01

    The Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture through its Co-ordinated Research Projects (CRPs), has been assisting national agricultural research systems in Member States to develop and apply nuclear and related techniques for improving livestock productivity. The programmes have focused on animal nutrition, animal reproduction and more recently on animal nutrition/reproduction interactions with emphasis on smallholder farming systems. The measurement of microbial protein supply to ruminant livestock has been an important area of research in ruminant nutrition. An estimate of microbial protein contribution to the intestinal protein flow is important for estimating the protein requirement of ruminant animals. Understanding the process of microbial protein synthesis has been difficult however, and due to the lack of simple and accurate methods for measuring microbial protein production in vivo, the methods used are based on complex microbial markers which require surgically prepared animals. As a result of a consultants meeting held in May 1995 to advise the Joint FAO/IAEA Division on the feasibility of using nuclear and related techniques for the development and validation of techniques for measuring microbial protein supply in ruminant animals, an FAO/IAEA Co-ordinated Research Project on Development, Standardization and Validation of Nuclear Based Technologies for Measuring Microbial Protein Supply in Ruminant Livestock for Improving Productivity was initiated in 1996, with a view to validating and adapting this technology for use in developing countries. To assist scientists participating in the CRP, a laboratory manual containing experimental protocols and methodologies for standardization and validation of the urine purine derivative technique and the development of models to suit local conditions, was published as IAEA-TECDOC-945. The present publication contains the final reports from participants in Phase 1 of the project

  15. Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

    Directory of Open Access Journals (Sweden)

    Szu-Chia Hsieh

    Full Text Available The envelope (E of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

  16. Evaluating the microbial community and gene regulation involved in crystallization kinetics of ZnS formation in reduced environments

    Science.gov (United States)

    Falk, Nicholas; Chaganti, Subba Rao; Weisener, Christopher G.

    2018-01-01

    In anoxic environments, sulfate-reducing bacteria (SRB) may precipitate sparingly-soluble, fine-grained sulfides as by-products of dissimilatory sulfate reduction. This bio-mechanism lends importance to acid rock drainage (ARD) remediation efforts for its ability to immobilize harmful metals from contaminant pathways, including Zn. However, SRB often coexist alongside multiple bacterial guilds in these environments, and may be sustained or hindered by the activities and metabolic by-products of their cohorts, driven by the commonly available substrates. Thus, the effectiveness of onset sulfate reduction and resultant metal-sulfide generation in ARD treatment can be enhanced by unravelling the complexities associated with these interactions. This research used material sourced from a passive bioreactor system located at the Stockton Coal Mine, New Zealand to investigate SRB activity and associated community function. RNA sequencing showed spore-forming Desulfitobacterium and Desulfotomaculum as the dominant SRB enriched from the reduced zone of the bioreactor. Metatranscriptomic analysis revealed acetogenic bacteria as syntrophic partners in substrate availability and Pseudomonas as metal-resistant community members. ZnS precipitates were observed by scanning electron microscopy (SEM) in short-term batch enrichments as well as long-term raw bioreactor material, with observed differences in mineral arrangement indicative of different nucleation scenarios. Syntrophy, metal response mechanisms, and the capacity for sporulation were observed as key microbial functions in mine waste reclamation settings. Here, Zn and S mass balance calculations coupled with RNA sequence data and microscopy illuminated favourable physicochemical and biological conditions for early metal sulfide precipitation in passive treatment systems for ARD and highlight the advantages of linking both lab and field-scale studies.

  17. Effect of Carbohydrate Sources and Levels of Cotton Seed Meal in Concentrate on Feed Intake, Nutrient Digestibility, Rumen Fermentation and Microbial Protein Synthesis in Young Dairy Bulls

    Directory of Open Access Journals (Sweden)

    M. Wanapat

    2013-04-01

    Full Text Available The objective of this study was to investigate the effect of levels of cottonseed meal with various carbohydrate sources in concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in dairy bulls. Four, 6 months old dairy bulls were randomly assigned to receive four dietary treatments according to a 2×2 factorial arrangement in a 4×4 Latin square design. Factor A was carbohydrate source; cassava chip (CC and cassava chip+rice bran in the ratio of 3:1 (CR3:1, and factor B was cotton seed meal levels in the concentrate; 109 g CP/kg (LCM and 328 g CP/kg (HCM at similar overall CP levels (490 g CP/kg. Bulls received urea-lime treated rice straw ad libitum and were supplemented with 10 g of concentrate/kg BW. It was found that carbohydrate source and level of cotton seed meal did not have significant effects on ruminal pH, ammonia nitrogen concentration, microbial protein synthesis or feed intake. Animals which received CC showed significantly higher BUN concentration, ruminal propionic acid and butyric acid proportions, while dry matter, organic matter digestibility, populations of total viable bacteria and proteolytic bacteria were lower than those in the CR3:1 treatment. The concentration of total volatile fatty acids was higher in HCM than LCM treatments, while the concentration of butyric acid was higher in LCM than HCM treatments. The population of proteolytic bacteria with the LCM treatments was higher than the HCM treatments; however other bacteria groups were similar among the different levels of cotton seed meal. Bulls which received LCM had higher protein digestibility than those receiving HCM. Therefore, using high levels of cassava chip and cotton seed meal might positively impact on energy and nitrogen balance for the microbial population in the rumen of the young dairy bull.

  18. Dietary protein reduction on microbial protein, amino acid digestibility, and body retention in beef cattle: 2. Amino acid intestinal absorption and their efficiency for whole-body deposition.

    Science.gov (United States)

    Mariz, L D S; Amaral, P M; Valadares Filho, S C; Santos, S A; Detmann, E; Marcondes, M I; Pereira, J M V; Silva Júnior, J M; Prados, L F; Faciola, A P

    2018-03-06

    intestinal digestibility of total microbial AA was 80%. The efficiency of utilization of total AA for whole-body protein deposition was 40%. The efficiency of utilization of lysine and methionine was 37% and 58%, respectively. It was concluded that the AA flow to the omasum increases in response to dietary CP content. In addition, there are differences among AA in the efficiency that they are used by beef cattle.

  19. Activity dependent protein degradation is critical for the formation and stability of fear memory in the amygdala.

    Directory of Open Access Journals (Sweden)

    Timothy J Jarome

    Full Text Available Protein degradation through the ubiquitin-proteasome system [UPS] plays a critical role in some forms of synaptic plasticity. However, its role in memory formation in the amygdala, a site critical for the formation of fear memories, currently remains unknown. Here we provide the first evidence that protein degradation through the UPS is critically engaged at amygdala synapses during memory formation and retrieval. Fear conditioning results in NMDA-dependent increases in degradation-specific polyubiquitination in the amygdala, targeting proteins involved in translational control and synaptic structure and blocking the degradation of these proteins significantly impairs long-term memory. Furthermore, retrieval of fear memory results in a second wave of NMDA-dependent polyubiquitination that targets proteins involved in translational silencing and synaptic structure and is critical for memory updating following recall. These results indicate that UPS-mediated protein degradation is a major regulator of synaptic plasticity necessary for the formation and stability of long-term memories at amygdala synapses.

  20. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    International Nuclear Information System (INIS)

    Li Hong; Huang Weiya; Zhang Yuanming; Xue Bo; Wen Xuejun

    2012-01-01

    Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins (EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization, calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a single diffusion system were conducted. The morphology and organization of formed CaP crystals were examined by X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructure can be formed and the aligned HAP assembly tightly bundled by 3–4 rod-like nanocrystals like an enamel prism. However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase was presented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed that the organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystal prism. The study benefits the understanding of biomineralization of tooth enamel. - Highlights: ► An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. ► An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. ► EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  1. Modulation of enamel matrix proteins on the formation and nano-assembly of hydroxyapatite in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Li Hong, E-mail: tlihong@jnu.edu.cn [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States); Huang Weiya [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Department of Materials Science and Engineering, Taizhou, Taizhou University, Zhejiang 317000 (China); Zhang Yuanming [Department of Chemistry, Jinan University, Guangzhou, Guangdong 510630 (China); Xue Bo [Department of Materials Science and Engineering, Jinan University, Guangzhou, Guangdong 510630 (China); Wen Xuejun [Department of Bioengineering, Clemson University, Charleston, SC 29425 (United States)

    2012-05-01

    Natural enamel has a hierarchically nanoassembled architecture that is regulated by enamel matrix proteins (EMPs) during the formation of enamel crystals. To understand the role of EMPs on enamel mineralization, calcium phosphate (CaP) growth experiments in both the presence and absence of native rat EMPs in a single diffusion system were conducted. The morphology and organization of formed CaP crystals were examined by X-Ray Diffraction (XRD), High-Resolution Transmission Microscopy (HRTEM) and Selected Area Electron Diffraction (SAED). In the system containing the EMPs, hydroxyapatite (HAP) with hierarchical lamellar nanostructure can be formed and the aligned HAP assembly tightly bundled by 3-4 rod-like nanocrystals like an enamel prism. However, in the absence of EMPs, only a sheet-like structure of octacalcium phosphate (OCP) phase was presented. EMPs promote HAP formation and inhibit the growth of OCP on the (010) plane. It is discussed that the organized Amelogenin/Amorphous Calcium Phosphate might be the precursor to the bundled HAP crystal prism. The study benefits the understanding of biomineralization of tooth enamel. - Highlights: Black-Right-Pointing-Pointer An aligned hydroxyapatite crystal bundled by rod-like nanosize crystals was obtained. Black-Right-Pointing-Pointer An organized Amel/ACP would be the precursor of the bundled hydroxyapatite crystal prism. Black-Right-Pointing-Pointer EMPs inhibit the growth of octacalcium phosphate in a defined plane.

  2. Genetic Analysis of the Role of Protein Kinase Cθ in Platelet Function and Thrombus Formation

    Science.gov (United States)

    Hall, Kellie J.; Harper, Matthew T.; Gilio, Karen; Cosemans, Judith M.; Heemskerk, Johan W. M.; Poole, Alastair W.

    2008-01-01

    Background PKCθ is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCθ−/− T cells exhibit reduced activation and PKCθ−/− mice are resistant to autoimmune disease, making PKCθ an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCθ positively regulates outside-in signalling through integrin αIIbβ3 in platelets, the role of PKCθ in GPVI-dependent signalling and functional activation of platelets has not been assessed. Methodology/Principal Findings In the present study we assessed static adhesion, cell spreading, granule secretion, integrin αIIbβ3 activation and platelet aggregation in washed mouse platelets lacking PKCθ. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCθ−/− platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCθ positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCθ−/− platelets also exhibited markedly enhanced GPVI-dependent α-granule secretion, although dense granule secretion was unaffected, suggesting that PKCθ differentially regulates these two granules. Inside-out regulation of αIIbβ3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s−1) was enhanced. Conclusions/Significance These data suggest that PKCθ is an important negative regulator of thrombus formation on collagen, potentially mediated by α-granule secretion and αIIbβ3 activation. PKCθ therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCθ inhibitors. PMID:18815612

  3. Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

    Directory of Open Access Journals (Sweden)

    Kellie J Hall

    2008-09-01

    Full Text Available PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/- T cells exhibit reduced activation and PKCtheta(-/- mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIbbeta(3 in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed.In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIbbeta(3 activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/- platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/- platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIbbeta(3 activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1 was enhanced.These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIbbeta(3 activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors.

  4. Epithelial membrane protein-2 promotes endometrial tumor formation through activation of FAK and Src.

    Directory of Open Access Journals (Sweden)

    Maoyong Fu

    Full Text Available Endometrial cancer is the most common gynecologic malignancy diagnosed among women in developed countries. One recent biomarker strongly associated with disease progression and survival is epithelial membrane protein-2 (EMP2, a tetraspan protein known to associate with and modify surface expression of certain integrin isoforms. In this study, we show using a xenograft model system that EMP2 expression is necessary for efficient endometrial tumor formation, and we have started to characterize the mechanism by which EMP2 contributes to this malignant phenotype. In endometrial cancer cells, the focal adhesion kinase (FAK/Src pathway appears to regulate migration as measured through wound healing assays. Manipulation of EMP2 levels in endometrial cancer cells regulates the phosphorylation of FAK and Src, and promotes their distribution into lipid raft domains. Notably, cells with low levels of EMP2 fail to migrate and poorly form tumors in vivo. These findings reveal the pivotal role of EMP2 in endometrial cancer carcinogenesis, and suggest that the association of elevated EMP2 levels with endometrial cancer prognosis may be causally linked to its effect on integrin-mediated signaling.

  5. Distinct activities of Bartonella henselae type IV secretion effector proteins modulate capillary-like sprout formation.

    Science.gov (United States)

    Scheidegger, F; Ellner, Y; Guye, P; Rhomberg, T A; Weber, H; Augustin, H G; Dehio, C

    2009-07-01

    The zoonotic pathogen Bartonella henselae (Bh) can lead to vasoproliferative tumour lesions in the skin and inner organs known as bacillary angiomatosis and bacillary peliosis. The knowledge on the molecular and cellular mechanisms involved in this pathogen-triggered angiogenic process is confined by the lack of a suitable animal model and a physiologically relevant cell culture model of angiogenesis. Here we employed a three-dimensional in vitro angiogenesis assay of collagen gel-embedded endothelial cell (EC) spheroids to study the angiogenic properties of Bh. Spheroids generated from Bh-infected ECs displayed a high capacity to form sprouts, which represent capillary-like projections into the collagen gel. The VirB/VirD4 type IV secretion system and a subset of its translocated Bartonella effector proteins (Beps) were found to profoundly modulate this Bh-induced sprouting activity. BepA, known to protect ECs from apoptosis, strongly promoted sprout formation. In contrast, BepG, triggering cytoskeletal rearrangements, potently inhibited sprouting. Hence, the here established in vitro model of Bartonella- induced angiogenesis revealed distinct and opposing activities of type IV secretion system effector proteins, which together with a VirB/VirD4-independent effect may control the angiogenic activity of Bh during chronic infection of the vasculature.

  6. Estimation of rumen microbial protein production from purine derivatives in urine. A laboratory manual for the FAO/IAEA co-ordinated research programme on development, standardization and validation of nuclear based technologies for measuring microbial protein supply in ruminant livestock for improving productivity

    International Nuclear Information System (INIS)

    1997-05-01

    This laboratory manual contains the methodologies used in the standardization and validation of the urine purine derivative technique for estimating microbial protein supply to the rumen. It includes descriptions of methods that involve both radioactive and stable isotopes as well as non isotopic techniques such as chemical assays, since it has been recognised that while isotopic trace techniques provide a powerful tool for nutrition research they can not and should not be used in isolation. Refs, figs, tabs

  7. Proteomics of plasma membranes from poplar trees reveals tissue distribution of transporters, receptors, and proteins in cell wall formation.

    Science.gov (United States)

    Nilsson, Robert; Bernfur, Katja; Gustavsson, Niklas; Bygdell, Joakim; Wingsle, Gunnar; Larsson, Christer

    2010-02-01

    By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H(+)-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane.

  8. A Maastrichtian microbial reef and associated limestones in the Roca Formation of Patagonia (Neuquén Province, Argentina

    Directory of Open Access Journals (Sweden)

    W. Kiessling

    2006-08-01

    Full Text Available We describe a small microbial reef and associated limestones occurring in a Maastrichtian transgressive succession of mixed carbonate-siliciclastic lithologies at Sierra Huantraico near Chos Malal (Neuquén, Argentina. Strontium isotope data suggest that the reef is of earliest Maastrichtian age. The small reef (0.8 m thick, 2 m wide is mostly composed of peloidal bindstone, dense stromatolite-cement crusts and thrombolite. Except for some ostracods, no metazoan fossils were found in the reef structure, although the majority of peloids are fecal pellets, probably of larger crustaceans. Small foraminifers with calcite tests and probable green algae have also been noted. Sedimentological data and fossils within and immediately above the reef suggest that the reef was formed in a transgressive systems tract under freshwater to brackish-water conditions. Limestones above the reef are serpulid-bryozoan packstones and intraclast-ooid grainstones. These limestones yield a mixture of typical non-tropical (common serpulids and bryozoans and typical tropical aspects (common dasycladaceans and ooids. This mosaic is explained by salinity fluctuations, which in our case dominate over temperature in determining the grain associations. Wir beschreiben ein kleines mikrobielles Riff, das in der Sierra Hunatraico (Neuquén, Argentinien in einer transgressiven, gemischt siliziklastisch-kalkigen Abfolge gefunden wurde. Nach Strontiumisotopen-Datierung ist das Riff in das unterste Maastrichtium zu stellen. Das kleine Riff (0,8 m Mächtigkeit, 2 m Breite besteht überwiegend aus peloidalem Bindstone, dichten Stromatolith-Zement-Krusten und Thrombolith. Mit Ausnahme von Ostrakoden konnten keine Metazoen in der Riffstruktur nachgewiesen werden, obwohl die Mehrzahl der Peloide als Kotpillen zu interpretieren sind, die vermutlich auf größere Krebse zurückgehen. Kleine Foraminiferen und mögliche Grünalgen sind die einzigen zusätzlich nachweisbaren Eukaryoten

  9. The role of microbial-produced extracellular polymeric matrix in the formation and survival of biological soil crusts

    Science.gov (United States)

    Rossi, Federico; Adessi, Alessandra; De Philippis, Roberto

    2016-04-01

    distribution between EPM fractions and in relation to the age of the crusts, hinting at distinct roles of the same fractions within the crust system. References • Belnap, J., Lange, O.L. (Eds.), 2001. Biological soil crusts: structure, function, and management, Ecological studies. Springer, New York. • Rossi, F., De Philippis, R., 2015. Role of Cyanobacterial Exopolysaccharides in Phototrophic Biofilms and in Complex Microbial Mats. Life 5, 1218-1238. doi:10.3390/life5021218

  10. Controlled formation of emulsion gels stabilized by salted myofibrillar protein under malondialdehyde (MDA)-induced oxidative stress.

    Science.gov (United States)

    Zhou, Feibai; Sun, Weizheng; Zhao, Mouming

    2015-04-15

    This study presented the cold-set gelation of emulsions stabilized by salted myofibrillar protein (MP) under oxidative stress originated from malondialdehyde (MDA). Gel properties were compared over a range of MDA/NaCl concentrations including gel viscoelastic properties, strength, water-holding capacity (WHC), amount of protein entrapped, and microstructure. The oxidative stability of emulsion gels as indicated by lipid hydroperoxide was further determined and compared. Results indicated that emulsion stabilized by MP at swollen state under certain ionic strengths (0.2-0.6 M) was the premise of gel formation under MDA. In the presence of intermediate MDA concentrations (2.5-10 mM), the emulsion gels showed an improved elasticity, strength, WHC, and oxidative stability. This improvement should be mainly attributed to the enhanced protein-protein cross-linkings via MDA, which were homogeneously formed among absorbed and/or unabsorbed proteins, entrapping a greater amount and fractions of protein within network. Therefore, the oil droplets were better adherent to the gel matrix. Nevertheless, addition of high MDA concentrations (25-50 mM) led to the formation of excessive covalent bonds, which might break protein-protein bonds and trigger the desorption of protein from the interface. This ultimately caused "oil leak" phenomena as well as the collapse of gel structure and, thus, overall decreased gel properties and oxidative stability.

  11. A single cysteine post-translational oxidation suffices to compromise globular proteins kinetic stability and promote amyloid formation

    Directory of Open Access Journals (Sweden)

    Patrizia Marinelli

    2018-04-01

    Full Text Available Oxidatively modified forms of proteins accumulate during aging. Oxidized protein conformers might act as intermediates in the formation of amyloids in age-related disorders. However, it is not known whether this amyloidogenic conversion requires an extensive protein oxidative damage or it can be promoted just by a discrete, localized post-translational modification of certain residues. Here, we demonstrate that the irreversible oxidation of a single free Cys suffices to severely perturb the folding energy landscape of a stable globular protein, compromise its kinetic stability, and lead to the formation of amyloids under physiological conditions. Experiments and simulations converge to indicate that this specific oxidation-promoted protein aggregation requires only local unfolding. Indeed, a large scale analysis indicates that many cellular proteins are at risk of undergoing this kind of deleterious transition; explaining how oxidative stress can impact cell proteostasis and subsequently lead to the onset of pathological states. Keywords: Protein oxidation, Protein misfolding, Protein aggregation, Oxidative stress, Post-translational modification

  12. Factors Affecting Microbial Contamination of Market Eggs: A Review

    Directory of Open Access Journals (Sweden)

    Svobodová J.

    2015-01-01

    Full Text Available The aim of the review was to analyze the ways of microbial contamination, the protective mechanism of egg, and factors that affect the quantity of contamination and microbial penetration. Eggs can be contaminated during their formation in the infected reproductive organs of hens or after laying, when eggs are exposed to contaminated environment. The eggs are equipped against microbial contamination by several protective mechanisms comprising the presence of cuticle, eggshell, eggshell membranes, occurrence of some antibacterial proteins, and high pH value of albumen. There are several factors that affect the quantity of microbial contamination and penetration such as species of bacteria, the amount of microorganisms, storage conditions, quality of eggshell or number of pores.

  13. Syntrophic interactions and mechanisms underpinning anaerobic methane oxidation: targeted metaproteogenomics, single-cell protein detection and quantitative isotope imaging of microbial consortia

    Energy Technology Data Exchange (ETDEWEB)

    Orphan, Victoria Jeanne [California Inst. of Technology (CalTech), Pasadena, CA (United States). Division of Geological and Planetary Sciences

    2014-11-26

    Syntrophy and mutualism play a central role in carbon and nutrient cycling by microorganisms. Yet, our ability to effectively study symbionts in culture has been hindered by the inherent interdependence of syntrophic associations, their dynamic behavior, and their frequent existence at thermodynamic limits. Now solutions to these challenges are emerging in the form of new methodologies. Developing strategies that establish links between the identity of microorganisms and their metabolic potential, as well as techniques that can probe metabolic networks on a scale that captures individual molecule exchange and processing, is at the forefront of microbial ecology. Understanding the interactions between microorganisms on this level, at a resolution previously intractable, will lead to our greater understanding of carbon turnover and microbial community resilience to environmental perturbations. In this project, we studied an enigmatic syntrophic association between uncultured methane-oxidizing archaea and sulfate-reducing bacteria. This environmental archaeal-bacterial partnership represents a globally important sink for methane in anoxic environments. The specific goals of this project were organized into 3 major tasks designed to address questions relating to the ecophysiology of these syntrophic organisms under changing environmental conditions (e.g. different electron acceptors and nutrients), primarily through the development of microanalytical imaging methods which enable the visualization of the spatial distribution of the partners within aggregates, consumption and exchange of isotopically labeled substrates, and expression of targeted proteins identified via metaproteomics. The advanced tool set developed here to collect, correlate, and analyze these high resolution image and isotope-based datasets from methane-oxidizing consortia has the potential to be widely applicable for studying and modeling patterns of activity and interactions across a broad range of

  14. Effects of Synchronization of Carbohydrate and Protein Supply in Total Mixed Ration with Korean Rice Wine Residue on Ruminal Fermentation, Nitrogen Metabolism and Microbial Protein Synthesis in Holstein Steers

    Directory of Open Access Journals (Sweden)

    Min Yu Piao

    2012-11-01

    Full Text Available Three Holstein steers in the growing phase, each with a ruminal cannula, were used to test the hypothesis that the synchronization of the hourly rate of carbohydrate and nitrogen (N released in the rumen would increase the amount of retained nitrogen for growth and thus improve the efficiency of microbial protein synthesis (EMPS. In Experiment 1, in situ degradability coefficients of carbohydrate and N in feeds including Korean rice wine residue (RWR were determined. In Experiment 2, three total mixed ration (TMR diets having different rates of carbohydrate and N release in the rumen were formulated using the in situ degradability of the feeds. All diets were made to contain similar contents of crude protein (CP and neutral detergent fiber (NDF but varied in their hourly pattern of nutrient release. The synchrony index of the three TMRs was 0.51 (LS, 0.77 (MS and 0.95 (HS, respectively. The diets were fed at a restricted level (2% of the animal’s body weight in a 3×3 Latin-square design. Synchronizing the hourly supply of energy and N in the rumen did not significantly alter the digestibility of dry matter, organic matter, crude protein, NDF or acid detergent fiber (ADF (p>0.05. The ruminal NH3-N content of the LS group at three hours after feeding was significantly higher (p0.05. In addition, the purine derivative (PD excretion in urine and microbial-N production (MN among the three groups were not significantly different (p>0.05. In conclusion, synchronizing dietary energy and N supply to the rumen did not have a major effect on nutrient digestion or microbial protein synthesis (MPS in Holstein steers.

  15. Accelerating anodic biofilms formation and electron transfer in microbial fuel cells: Role of anionic biosurfactants and mechanism.

    Science.gov (United States)

    Zhang, Yunshu; Jiang, Junqiu; Zhao, Qingliang; Gao, YunZhi; Wang, Kun; Ding, Jing; Yu, Hang; Yao, Yue

    2017-10-01

    Anodic electron transfer is the predominant electricity generation process of MFCs. To accelerate anodic biofilms formation and electron transfer, 40mg/L, 80mg/L, and 120mg/L of rhamnolipid biosurfactants were added to the anolyte, resulting in an increased abiotic capacitance from 15.12F/m 2 (control) to 16.54F/m 2 , 18.00F/m 2 , and 19.39F/m 2 , respectively. Anodic biofilm formation was facilitated after dosing 40mg/L of rhamnolipids on the 7th day after inoculation, resulting in an increased anodic biofilm coverage from 0.43% to 42.51%, and an increased maximum power density from 6.92±1.18W/m 3 to 9.93±0.88W/m 3 . Furthermore, the adsorption of rhamnolipids on the anode caused the Frumkin effect, leading to a decrease of equilibrium potential from -0.43V to -0.56V, and an increase of exchange current density from 5.09×10 -3 A/m 2 to 8.72×10 -3 A/m 2 . However, electron transfer was blocked when the rhamnolipid concentration was further increased to 80mg/L, and 120mg/L. Analysis of the anodic bacterial communities revealed that rhamnolipids facilitated the enrichment of exoelectrogen, increasing the total proportion from 65% to 81%. Additionally, biosurfactants were found to have significant impacts on the composition of exoelectrogens. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Heterotrimeric G protein beta1gamma2 subunits change orientation upon complex formation with G protein-coupled receptor kinase 2 (GRK2) on a model membrane.

    Science.gov (United States)

    Boughton, Andrew P; Yang, Pei; Tesmer, Valerie M; Ding, Bei; Tesmer, John J G; Chen, Zhan

    2011-09-13

    Few experimental techniques can assess the orientation of peripheral membrane proteins in their native environment. Sum Frequency Generation (SFG) vibrational spectroscopy was applied to study the formation of the complex between G protein-coupled receptor (GPCR) kinase 2 (GRK2) and heterotrimeric G protein β(1)γ(2) subunits (Gβγ) at a lipid bilayer, without any exogenous labels. The most likely membrane orientation of the GRK2-Gβγ complex differs from that predicted from the known protein crystal structure, and positions the predicted receptor docking site of GRK2 such that it would more optimally interact with GPCRs. Gβγ also appears to change its orientation after binding to GRK2. The developed methodology is widely applicable for the study of other membrane proteins in situ.

  17. Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease

    Directory of Open Access Journals (Sweden)

    Ashutosh Kumar

    2018-05-01

    Full Text Available Exposure to (bisulfite (HSO3– and sulfite (SO32– has been shown to induce a wide range of adverse reactions in sensitive individuals. Studies have shown that peroxidase-catalyzed oxidation of (bisulfite leads to formation of several reactive free radicals, such as sulfur trioxide anion (.SO3–, peroxymonosulfate (–O3SOO., and especially the sulfate (SO4. – anion radicals. One such peroxidase in neutrophils is myeloperoxidase (MPO, which has been shown to form protein radicals. Although formation of (bisulfite-derived protein radicals is documented in isolated neutrophils, its involvement and role in in vivo inflammatory processes, has not been demonstrated. Therefore, we aimed to investigate (bisulfite-derived protein radical formation and its mechanism in LPS aerosol-challenged mice, a model of non-atopic asthma. Using immuno-spin trapping to detect protein radical formation, we show that, in the presence of (bisulfite, neutrophils present in bronchoalveolar lavage and in the lung parenchyma exhibit, MPO-catalyzed oxidation of MPO to a protein radical. The absence of radical formation in LPS-challenged MPO- or NADPH oxidase-knockout mice indicates that sulfite-derived radical formation is dependent on both MPO and NADPH oxidase activity. In addition to its oxidation by the MPO-catalyzed pathway, (bisulfite is efficiently detoxified to sulfate by the sulfite oxidase (SOX pathway, which forms sulfate in a two-electron oxidation reaction. Since SOX activity in rodents is much higher than in humans, to better model sulfite toxicity in humans, we induced SOX deficiency in mice by feeding them a low molybdenum diet with tungstate. We found that mice treated with the SOX deficiency diet prior to exposure to (bisulfite had much higher protein radical formation than mice with normal SOX activity. Altogether, these results demonstrate the role of MPO and NADPH oxidase in (bisulfite-derived protein radical formation and show the involvement of

  18. UVA Light-excited Kynurenines Oxidize Ascorbate and Modify Lens Proteins through the Formation of Advanced Glycation End Products

    Science.gov (United States)

    Linetsky, Mikhail; Raghavan, Cibin T.; Johar, Kaid; Fan, Xingjun; Monnier, Vincent M.; Vasavada, Abhay R.; Nagaraj, Ram H.

    2014-01-01

    Advanced glycation end products (AGEs) contribute to lens protein pigmentation and cross-linking during aging and cataract formation. In vitro experiments have shown that ascorbate (ASC) oxidation products can form AGEs in proteins. However, the mechanisms of ASC oxidation and AGE formation in the human lens are poorly understood. Kynurenines are tryptophan oxidation products produced from the indoleamine 2,3-dioxygenase (IDO)-mediated kynurenine pathway and are present in the human lens. This study investigated the ability of UVA light-excited kynurenines to photooxidize ASC and to form AGEs in lens proteins. UVA light-excited kynurenines in both free and protein-bound forms rapidly oxidized ASC, and such oxidation occurred even in the absence of oxygen. High levels of GSH inhibited but did not completely block ASC oxidation. Upon UVA irradiation, pigmented proteins from human cataractous lenses also oxidized ASC. When exposed to UVA light (320–400 nm, 100 milliwatts/cm2, 45 min to 2 h), young human lenses (20–36 years), which contain high levels of free kynurenines, lost a significant portion of their ASC content and accumulated AGEs. A similar formation of AGEs was observed in UVA-irradiated lenses from human IDO/human sodium-dependent vitamin C transporter-2 mice, which contain high levels of kynurenines and ASC. Our data suggest that kynurenine-mediated ASC oxidation followed by AGE formation may be an important mechanism for lens aging and the development of senile cataracts in humans. PMID:24798334

  19. Evaluation of some feedstuffs with special emphasis on the effect of nitrate and other non-protein nitrogen fractions on ruminal microbial metabolism

    International Nuclear Information System (INIS)

    Nikolic, J.A.

    1988-01-01

    Green herbage grown under widely different conditions was examined for nitrate content. The mean value was 0.14% (range 0.01-0.32%) NO 3 -N in the dry matter (DM). It was concluded that the cut green fodder would induce high levels of blood methaemoglobin when fed to ruminants in mixed diets. Nevertheless, experiments in vitro indicated that relatively small amounts of dietary nitrate induced considerable microbial nitrate reducing activity in rumen contents. Moreover, nitrate reduction to ammonia was accompanied by alterations in microbial sulphur and energy metabolism. A technique for determining organic matter digestibility (OMD) in vitro was standardized and used to evaluate the potential nutritive value of some fruit wastes. Studies on apple pomace silage indicated that added urea did not significantly increase the silage protein content but remained as a potential source of non-protein nitrogen for rumen microorganisms. Apple pomace ensiled alone or preferably with other ingredients was acceptable to sheep and cattle, particularly as a supplement to their winter rations. (author). 27 refs, 2 figs, 5 tabs

  20. Effects of correcting in situ ruminal microbial colonization of feed particles on the relationship between ruminally undegraded and intestinally digested crude protein in concentrate feeds.

    Science.gov (United States)

    González, Javier; Mouhbi, Rabiaa; Guevara-González, Jesús Alberto; Arroyo, José María

    2018-02-01

    In situ estimates of ruminally undegraded protein (RUP) and intestinally digested protein (IDP) of ten concentrates, uncorrected or corrected for the ruminal microbial colonization, were used to examine the effects of this correction on the relationship between IDP and RUP values. Both variables were established for three rumen and duodenum cannulated wethers using 15 N labeling-techniques and considering measured rates of ruminal particle comminution (k c ) and outflow (k p ). A covariance analysis showed that the close relationship found between both variables (IDP = -0.0132 ± 0.00679 + 0.776 ± 0.0002 RUP; n = 60; P content in concentrates and industrial by-products can be predicted from RUP values, thus avoiding the laborious and complex procedure of determining intestinal digestibility; however, a larger sample of feeds is necessary to achieve more accurate predictions. The lack of influence of the correction for microbial contamination on the prediction observed in the present study increases the data available for this prediction. However, only the use of corrected values may provide an accurate evaluation. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  1. Effect of Carbohydrate Source and Cottonseed Meal Level in the Concentrate on Feed Intake, Nutrient Digestibility, Rumen Fermentation and Microbial Protein Synthesis in Swamp Buffaloes

    Directory of Open Access Journals (Sweden)

    M. Wanapat

    2013-07-01

    Full Text Available The objective of this study was to investigate the effect of carbohydrate source and cottonseed meal level in the concentrate on feed intake, nutrient digestibility, rumen fermentation and microbial protein synthesis in swamp buffaloes. Four, 4-yr old rumen fistulated swamp buffaloes were randomly assigned to receive four dietary treatments according to a 2×2 factorial arrangement in a 4×4 Latin square design. Factor A was carbohydrate source; cassava chip (CC and CC+rice bran at a ratio 3:1 (CR3:1, and factor B was level of cottonseed meal (CM; 109 g CP/kg (LCM and 328 g CP/kg (HCM in isonitrogenous diets (490 g CP/kg. Buffaloes received urea-treated rice straw ad libitum and supplemented with 5 g concentrate/kg BW. It was found that carbohydrate source did not affect feed intake, nutrient intake, digested nutrients, nutrient digestibility, ammonia nitrogen concentration, fungi and bacterial populations, or microbial protein synthesis (p>0.05. Ruminal pH at 6 h after feeding and the population of protozoa at 4 h after feeding were higher when buffalo were fed with CC than in the CR3:1 treatment (p0.05. Based on this experiment, concentrate with a low level of cottonseed meal could be fed with cassava chips as an energy source in swamp buffalo receiving rice straw.

  2. A prospective study on evaluation of pathogenesis, biofilm formation, antibiotic susceptibility of microbial community in urinary catheter

    Science.gov (United States)

    Younis, Khansa Mohammed; Usup, Gires; Ahmad, Asmat

    2015-09-01

    This study is aimed to isolate, detect biofilm formation ability and antibiotic susceptibility of urinary catheter adherent microorganisms from elderly hospitalized patient at the Universiti Kebangsaan Malaysia Medical Center. Microorganisms were isolated from three samples of urinary catheters (UC) surface; one of the acute vascular rejection patient (UCB) and two from benign prostate hyperplasia patients (UCC and UCD). A total of 100 isolates was isolated with 35 from UCB, 38 (UCC) and 28 (UCD). Ninety six were identified as Gram-negative bacilli, one Gram-positive bacilli and three yeasts. Results of biofilm forming on sterile foley catheter showed that all the isolates can form biofilm at different degrees; strong biofilm forming: 32% from the 35 isolates (UCB), 25% out of 38 isolates (UCC), 26% out of 28 isolates (UCD). As for moderate biofilm forming; 3% from UCB, 10% from UCC and 2% from UCD. Weak biofilm forming in UCC (3%). The antibiotic susceptibility for (UCB) isolates showed highly resistant to ampicillin, novobiocin and penicillin 100 (%), kanamycin (97%), tetracycline (94%), chloramphenicol (91%), streptomycin (77%) and showed low level of resistance to gentamycin (17%), while all the isolates from (UCC-D) showed high resistant towards ampicillin and penicillin, novobiocin (94%), tetracycline (61%), streptomycin (53%), gentamycin (50%) and low level of resistance to kanamycin (48%), chloramphenicol (47%). The findings indicate that these isolates can spread within the community on urinary catheters surface and produce strong biofilm, therefore, monitoring antibiotic susceptibility of bacteria isolated in the aggregation is recommended.

  3. Inflammation Intensity-dependent Expression of Osteoinductive Wnt Proteins is Critical for Ectopic New Bone Formation in Ankylosing Spondylitis.

    Science.gov (United States)

    Li, Xiang; Wang, Jianru; Zhan, Zhongping; Li, Sibei; Zheng, Zhaomin; Wang, Taiping; Zhang, Kuibo; Pan, Hehai; Li, Zemin; Zhang, Nu; Liu, Hui

    2018-02-26

    To investigate the molecular mechanism underlying the inflammation- related ectopic new bone formation in ankylosing spondylitis (AS). Spinal tissues and sera were collected from patients or normal volunteers to detect the expression of Wnt proteins. An in vitro cell culture system mimicking the local inflammatory microenvironment of bone-forming sites was established to study the relationship between inflammation and Wnt expression, the regulatory mechanism of inflammation-induced Wnt expression and the role of Wnt signaling in new bone formation. A modified collagen-induced arthritis (mCIA) and a proteoglycan -induced spondylitis (PGIS) animal model were used to confirm the key findings in vivo. The levels of osteoinductive Wnt proteins were obviously increased in the sera and spinal ligament tissues of patients with AS. Only constitutive low-intensity TNF-α stimulation, but not short-term or high-intensity TNF-α stimulation, induced persistent expression of osteoinductive Wnt proteins and subsequent bone formation through NF-κB (p65) and JNK/AP-1 (c-Jun) signaling pathways. Furthermore, inhibition of either Wnt/β-catenin or Wnt/PKCδ pathway significantly suppressed new bone formation. The increased expression of Wnt proteins was confirmed in both mCIA and PGIS models. A kyphotic and ankylosing phenotype of the spine was observed during long-term observation in mCIA model. Inhibition of either Wnt/β-catenin or Wnt/PKCδ signaling pathway significantly reduced the incidence and severity of this phenotype. Inflammation intensity-dependent expression of osteoinductive Wnt proteins is a key link between inflammation and ectopic new bone formation in AS. Activation of both canonical Wnt/β-catenin and noncanonical Wnt/PKCδ pathways is required for inflammation-induced new bone formation. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  4. Lysosomal membrane permeability stimulates protein aggregate formation in neurons of a lysosomal disease.

    Science.gov (United States)

    Micsenyi, Matthew C; Sikora, Jakub; Stephney, Gloria; Dobrenis, Kostantin; Walkley, Steven U

    2013-06-26

    Protein aggregates are a common pathological feature of neurodegenerative diseases and several lysosomal diseases, but it is currently unclear what aggregates represent for pathogenesis. Here we report the accumulation of intraneuronal aggregates containing the macroautophagy adapter proteins p62 and NBR1 in the neurodegenerative lysosomal disease late-infantile neuronal ceroid lipofuscinosis (CLN2 disease). CLN2 disease is caused by a deficiency in the lysosomal enzyme tripeptidyl peptidase I, which results in aberrant lysosomal storage of catabolites, including the subunit c of mitochondrial ATP synthase (SCMAS). In an effort to define the role of aggregates in CLN2, we evaluated p62 and NBR1 accumulation in the CNS of Cln2(-/-) mice. Although increases in p62 and NBR1 often suggest compromised degradative mechanisms, we found normal ubiquitin-proteasome system function and only modest inefficiency in macroautophagy late in disease. Importantly, we identified that SCMAS colocalizes with p62 in extra-lysosomal aggregates in Cln2(-/-) neurons in vivo. This finding is consistent with SCMAS being released from lysosomes, an event known as lysosomal membrane permeability (LMP). We predicted that LMP and storage release from lysosomes results in the sequestration of this material as cytosolic aggregates by p62 and NBR1. Notably, LMP induction in primary neuronal cultures generates p62-positive aggregates and promotes p62 localization to lysosomal membranes, supporting our in vivo findings. We conclude that LMP is a previously unrecognized pathogenic event in CLN2 disease that stimulates cytosolic aggregate formation. Furthermore, we offer a novel role for p62 in response to LMP that may be relevant for other diseases exhibiting p62 accumulation.

  5. Glypican-1 mediates both prion protein lipid raft association and disease isoform formation.

    Directory of Open Access Journals (Sweden)

    David R Taylor

    2009-11-01

    Full Text Available In prion diseases, the cellular form of the prion protein, PrP(C, undergoes a conformational conversion to the infectious isoform, PrP(Sc. PrP(C associates with lipid rafts through its glycosyl-phosphatidylinositol (GPI anchor and a region in its N-terminal domain which also binds to heparan sulfate proteoglycans (HSPGs. We show that heparin displaces PrP(C from rafts and promotes its endocytosis, suggesting that heparin competes with an endogenous raft-resident HSPG for binding to PrP(C. We then utilised a transmembrane-anchored form of PrP (PrP-TM, which is targeted to rafts solely by its N-terminal domain, to show that both heparin and phosphatidylinositol-specific phospholipase C can inhibit its association with detergent-resistant rafts, implying that a GPI-anchored HSPG targets PrP(C to rafts. Depletion of the major neuronal GPI-anchored HSPG, glypican-1, significantly reduced the raft association of PrP-TM and displaced PrP(C from rafts, promoting its endocytosis. Glypican-1 and PrP(C colocalised on the cell surface and both PrP(C and PrP(Sc co-immunoprecipitated with glypican-1. Critically, treatment of scrapie-infected N2a cells with glypican-1 siRNA significantly reduced PrP(Sc formation. In contrast, depletion of glypican-1 did not alter the inhibitory effect of PrP(C on the beta-secretase cleavage of the Alzheimer's amyloid precursor protein. These data indicate that glypican-1 is a novel cellular cofactor for prion conversion and we propose that it acts as a scaffold facilitating the interaction of PrP(C and PrP(Sc in lipid rafts.

  6. Multi-component adsorption model for pellicle formation: the influence of salivary proteins and non-salivary phospho proteins on the binding of histatin 5 onto hydroxyapatite.

    Science.gov (United States)

    Yin, A; Margolis, H C; Yao, Y; Grogan, J; Oppenheim, F G

    2006-02-01

    The acquired enamel pellicle formed by selective adsorption of proteins in whole saliva is a protective integument on the tooth surface. The purpose of the present study was to investigate the formation of human acquired enamel pellicle using an in vitro hydroxyapatite (HA) model and 3H-histatin 5 to allow accurate measurement of histatin 5 binding in a multi-component experimental system. A binary system was employed by mixing 3H-histatin 5 with one unlabeled protein prior to incubation with HA or by first incubating 3H-histatin 5 with the HA which had been pre-coated with one of a panel of unlabeled proteins (human albumin, salivary amylase, lysozyme, acidic PIFs, statherin, the N-terminal fragment of statherin, and egg yolk phosvitin). A ternary system was employed by mixing 3H-histatin 5 with HA sequentially pre-coated with two different unlabeled proteins, including recombinant histatin 1. The results showed that only salivary statherin and egg yolk phosvitin promote histatin 5 adsorption significantly. The amount of histatin 5 adsorbed was also found to increase as a function of the amount of phosvitin and statherin used to pre-coat HA up to a maximum level that was two- to four-fold greater than that observed on untreated HA. These data suggest that specific protein-protein interactions may play important roles in pellicle formation in vivo.

  7. How does domain replacement affect fibril formation of the rabbit/human prion proteins.

    Directory of Open Access Journals (Sweden)

    Xu Yan

    Full Text Available It is known that in vivo human prion protein (PrP have the tendency to form fibril deposits and are associated with infectious fatal prion diseases, while the rabbit PrP does not readily form fibrils and is unlikely to cause prion diseases. Although we have previously demonstrated that amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and macromolecular crowding has different effects on fibril formation of the rabbit/human PrPs, we do not know which domains of PrPs cause such differences. In this study, we have constructed two PrP chimeras, rabbit chimera and human chimera, and investigated how domain replacement affects fibril formation of the rabbit/human PrPs.As revealed by thioflavin T binding assays and Sarkosyl-soluble SDS-PAGE, the presence of a strong crowding agent dramatically promotes fibril formation of both chimeras. As evidenced by circular dichroism, Fourier transform infrared spectroscopy, and proteinase K digestion assays, amyloid fibrils formed by human chimera have secondary structures and proteinase K-resistant features similar to those formed by the human PrP. However, amyloid fibrils formed by rabbit chimera have proteinase K-resistant features and secondary structures in crowded physiological environments different from those formed by the rabbit PrP, and secondary structures in dilute solutions similar to the rabbit PrP. The results from transmission electron microscopy show that macromolecular crowding caused human chimera but not rabbit chimera to form short fibrils and non-fibrillar particles.We demonstrate for the first time that the domains beyond PrP-H2H3 (β-strand 1, α-helix 1, and β-strand 2 have a remarkable effect on fibrillization of the rabbit PrP but almost no effect on the human PrP. Our findings can help to explain why amyloid fibrils formed by the rabbit PrP and the human PrP have different secondary structures and why macromolecular crowding has different

  8. Synthetic Electric Microbial Biosensors

    Science.gov (United States)

    2017-06-10

    domains and DNA-binding domains into a single protein for deregulation of down stream genes of have been favored [10]. Initially experiments with... Germany DISTRIBUTION A. Approved for public release: distribution unlimited.   Talk title: “Synthetic biology based microbial biosensors for the...toolbox” in Heidelberg, Germany Poster title: “Anaerobic whole cell microbial biosensors” Link: http://phdsymposium.embl.org/#home   September, 2014

  9. Carotenoid crystal formation in Arabidopsis and carrot roots caused by increased phytoene synthase protein levels.

    Directory of Open Access Journals (Sweden)

    Dirk Maass

    Full Text Available BACKGROUND: As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. METHODOLOGY/PRINCIPAL FINDINGS: In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 microg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to beta-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. CONCLUSIONS: The sequestration of carotenoids into crystals can be driven by the

  10. Expression of bone morphogenetic proteins and Msx genes during root formation.

    Science.gov (United States)

    Yamashiro, T; Tummers, M; Thesleff, I

    2003-03-01

    Like crown development, root formation is also regulated by interactions between epithelial and mesenchymml tissues. Bone morphogenetic proteins (BMPs), together with the transcription factors Msx1 and Msx2, play important roles in these interactions during early tooth morphogenesis. To investigate the involvement of this signaling pathway in root development, we analyzed the expression patterns of Bmp2, Bmp3, Bmp4, and Bmp7 as well as Msx1 and Msx2 in the roots of mouse molars. Bmp4 was expressed in the apical mesenchyme and Msx2 in the root sheath. However, Bmps were not detected in the root sheath epithelium, and Msx transcripts were absent from the underlying mesenchyme. These findings indicate that this Bmp signaling pathway, required for tooth initiation, does not regulate root development, but we suggest that root shape may be regulated by a mechanism similar to that regulating crown shape in cap-stage tooth germs. Msx2 expression continued in the epithelial cell rests of Malassez, and the nearby cementoblasts intensely expressed Bmp3, which may regulate some functions of the fragmented epithelium.

  11. Method-Unifying View of Loop-Formation Kinetics in Peptide and Protein Folding.

    Science.gov (United States)

    Jacob, Maik H; D'Souza, Roy N; Schwarzlose, Thomas; Wang, Xiaojuan; Huang, Fang; Haas, Elisha; Nau, Werner M

    2018-04-26

    Protein folding can be described as a probabilistic succession of events in which the peptide chain forms loops closed by specific amino acid residue contacts, herein referred to as loop nodes. To measure loop rates, several photophysical methods have been introduced where a pair of optically active probes is incorporated at selected chain positions and the excited probe undergoes contact quenching (CQ) upon collision with the second probe. The quenching mechanisms involved triplet-triplet energy transfer, photoinduced electron transfer, and collision-induced fluorescence quenching, where the fluorescence of Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, is quenched by tryptophan. The discrepancy between the loop rates afforded from these three CQ techniques has, however, remained unresolved. In analyzing this discrepancy, we now report two short-distance FRET methods where Dbo acts as an energy acceptor in combination with tryptophan and naphtylalanine, two donors with largely different fluorescence lifetimes of 1.3 and 33 ns, respectively. Despite the different quenching mechanisms, the rates from FRET and CQ methods were, surprisingly, of comparable magnitude. This combination of FRET and CQ data led to a unifying physical model and to the conclusion that the rate of loop formation in folding reactions varies not only with the kind and number of residues that constitute the chain but also in particular with the size and properties of the residues that constitute the loop node.

  12. Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

    Energy Technology Data Exchange (ETDEWEB)

    Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro, E-mail: lidiane.franqui@pos.ft.unicamp.br [Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, SP (Brazil); Coluci, Vitor Rafael [Universidade Estadual de Campinas (UNICAMP), SP (Brazil)

    2016-07-01

    Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

  13. Interaction of single and multi-layer graphene oxide with fetal bovine serum: assessing the protein corona formation

    International Nuclear Information System (INIS)

    Franqui, Lidiane Silva; Farias, Marcelo Alexandre de; Portugal, Rodrigo Villares; Costa, Carlos Alberto; Leme, Adriana Franco Paes; Martinez, Diego Stefani Teodoro; Coluci, Vitor Rafael

    2016-01-01

    Full text: When in contact with biological systems, nanomaterials surface adsorbs biomolecules present in the biological medium, mainly proteins, yielding a molecular coating 'protein corona' which affects the biological response and toxicity of the nanomaterials. Several factors can influence the protein corona formation, such as nanomaterial physicochemical properties and the nature of biological medium. In this work, we have performed a comparative study between the single and multi-layer graphene oxide nanomaterials (SL-GO and ML-GO, respectively) after their interaction with DMEM cell culture medium containing fetal bovine serum (FBS). Bare GOs and FBS protein corona-coated GOs were characterized using dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), cryogenic transmission electron microscopy (Cryo-TEM) and X-ray photoelectron spectroscopy (XPS). The protein corona composition was characterized by gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/MS). Our results showed that, after interaction with FBS, GO particle size increased due to the protein corona formation. Besides, the presence of proteins also has significantly increased the dispersion stability of SLGO and ML-GO over time. Whereas the main proteins have been identified in both SL-GO and ML-GO, SL-GO has adsorbed larger amounts of proteins than ML-GO. Finally, the number of GO layers influences its interactions with FBS proteins and dispersion stability in DMEM medium. These results point out implications for in vitro cytotoxicity assessment and biomedical applications of these promising carbon nanomaterials. (author)

  14. Comparative proteomic analysis in pea treated with microbial consortia of beneficial microbes reveals changes in the protein network to enhance resistance against Sclerotinia sclerotiorum.

    Science.gov (United States)

    Jain, Akansha; Singh, Akanksha; Singh, Surendra; Singh, Vinay; Singh, Harikesh Bahadur

    2015-06-15

    Microbial consortia may provide protection against pathogenic ingress via enhancing plant defense responses. Pseudomonas aeruginosa PJHU15, Trichoderma harzianum TNHU27 and Bacillus subtilis BHHU100 were used either singly or in consortia in the pea rhizosphere to observe proteome level changes upon Sclerotinia sclerotiorum challenge. Thirty proteins were found to increase or decrease differentially in 2-DE gels of pea leaves, out of which 25 were identified by MALDI-TOF MS or MS/MS. These proteins were classified into several functional categories including photosynthesis, respiration, phenylpropanoid metabolism, protein synthesis, stress regulation, carbohydrate and nitrogen metabolism and disease/defense-related processes. The respective homologue of each protein identified was trapped in Pisum sativum and a phylogenetic tree was constructed to check the ancestry. The proteomic view of the defense response to S. sclerotiorum in pea, in the presence of beneficial microbes, highlights the enhanced protection that can be provided by these microbes in challenged plants. Copyright © 2015 Elsevier GmbH. All rights reserved.

  15. The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

    DEFF Research Database (Denmark)

    Yao, Xiao Jie; Vélez Ruiz, Gisselle; Whorton, Matthew R

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs...... of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents...... formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy....

  16. Pulmonary heat shock protein expression after exposure to a metabolically activated Clara cell toxicant: relationship to protein adduct formation

    International Nuclear Information System (INIS)

    Williams, Kurt J.; Cruikshank, Michael K.; Plopper, Charles G.

    2003-01-01

    Heat shock proteins/stress proteins (Hsps) participate in regulation of protein synthesis and degradation and serve as general cytoprotectants, yet their role in lethal Clara cell injury is not clear. To define the pattern of Hsp expression in acute lethal Clara cell injury, mice were treated with the Clara cell-specific toxicant naphthalene (NA), and patterns of expression compared to electrophilic protein adduction and previously established organellar degradation and gluathione (GSH) depletion. In sites of lethal injury (distal bronchiole), prior to organellar degradation (1 h post-NA), protein adduction is detectable and ubiquitin, Hsp 25, Hsp 72, and heme-oxygenase 1 (HO-1) are increased. Maximal Hsp expression, protein adduction, and GSH depletion occur simultaneous (by 2-3 h) with early organelle disruption. Hsp expression is higher later (6-24 h), only in exfoliating cells. In airway sites (proximal bronchiole) with nonlethal Clara cell injury elevation of Hsp 25, 72, and HO-1 expression follows significant GSH depletion (greater than 50% 2 h post-NA). This data build upon our previous studies and we conclude that (1) in lethal (terminal bronchiole) and nonlethal (proximal bronchiole) Clara cell injury, Hsp induction is associated with the loss of GSH and increased protein adduction, and (2) in these same sites, organelle disruption is not a prerequisite for Hsp induction

  17. 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

    Dissection of Molecular Mechanisms Regulating Protein Body Formation in Maize Endosperm - DE-FG03-95-ER20183 Final Technical Report and Patent Summary Dr. Brian A. Larkins, Department of Plant Sciences, University of Arizona, Tucson, AZ 85721 Endosperm texture is an important quality trait in maize, as it influences the shipping characteristics of the grain, its susceptibility to insects, the yield of grits from dry milling, energy costs during wet milling, and the baking and digestibility properties of the flour. There appears to be a causal relationship between kernel hardness and the formation of zein-containing protein bodies, as mutations affecting protein body number and structure are associated with a soft, starchy kernel. In this project we used a variety of approaches to better understand this relationship and investigate the molecular and biochemical changes associated with starchy endosperm mutants. We characterized the distribution of zein mRNAs on endosperm rough endoplasmic reticulum (RER) membranes and the interactions between zein proteins, as each of these could influence the structure of protein bodies. Based on in situ hybridization, mRNAs encoding the 22-kD alpha- and 27-kD gamma-zeins are randomly distributed on RER; hence, mRNA targeting does not appear to influence the formation of protein bodies. Investigation of the interactions between zein proteins (alpha, beta, gamma, delta) with the yeast two-hybrid system showed that interactions between the 19- and 22-alpha-zeins are relatively weak, although each of them interacted strongly with the 10-kD delta-zein. Strong interactions were detected between the alpha- and delta-zeins and the 16-kD gamma- and 15-kD beta-zeins; however, the 50-kD and 27-kD gamma-zeins did not interact detectably with the alpha- and delta-zein proteins. The NH2- and COOH-terminal domains of the 22-kD alpha-zein were found to interact most strongly with the 15-kD beta- and 16-kD gamma-zeins, suggesting the 16-kD and 15

  18. Participatory role of zinc in structural and functional characterization of bioremediase: a unique thermostable microbial silica leaching protein.

    Science.gov (United States)

    Chowdhury, Trinath; Sarkar, Manas; Chaudhuri, Biswadeep; Chattopadhyay, Brajadulal; Halder, Umesh Chandra

    2015-07-01

    A unique protein, bioremediase (UniProt Knowledgebase Accession No.: P86277), isolated from a hot spring bacterium BKH1 (GenBank Accession No.: FJ177512), has shown to exhibit silica leaching activity when incorporated to prepare bio-concrete material. Matrix-assisted laser desorption ionization mass spectrometry analysis suggests that bioremediase is 78% homologous to bovine carbonic anhydrase II though it does not exhibit carbonic anhydrase-like activity. Bioinformatics study is performed for understanding the various physical and chemical parameters of the protein which predicts the involvement of zinc encircled by three histidine residues (His94, His96 and His119) at the active site of the protein. Isothermal titration calorimetric-based thermodynamic study on diethyl pyrocarbonate-modified protein recognizes the presence of Zn(2+) in the enzyme moiety. Exothermic to endothermic transition as observed during titration of the protein with Zn(2+) discloses that there are at least two binding sites for zinc within the protein moiety. Addition of Zn(2+) regains the activity of EDTA chelated bioremediase confirming the presence of extra binding site of Zn(2+) in the protein moiety. Revival of folding pattern of completely unfolded urea-treated protein by Zn(2+) explains the participatory role of zinc in structural stability of the protein. Restoration of the λ max in intrinsic fluorescence emission study of the urea-treated protein by Zn(2+) similarly confirms the involvement of Zn in the refolding of the protein. The utility of bioremediase for silica nanoparticles preparation is observed by field emission scanning electron microscopy.

  19. The type III protein secretion system contributes to Xanthomonas citri subsp. citri biofilm formation

    KAUST Repository

    Zimaro, Tamara; Thomas, Ludivine; Marondedze, Claudius; Sgro, Germá n G; Garofalo, Cecilia G; Ficarra, Florencia A; Gehring, Christoph A; Ottado, Jorgelina; Gottig, Natalia

    2014-01-01

    Background: Several bacterial plant pathogens colonize their hosts through the secretion of effector proteins by a Type III protein secretion system (T3SS). The role of T3SS in bacterial pathogenesis is well established but whether this system

  20. RAE-1, a novel PHR binding protein, is required for axon termination and synapse formation in Caenorhabditis elegans.

    Science.gov (United States)

    Grill, Brock; Chen, Lizhen; Tulgren, Erik D; Baker, Scott T; Bienvenut, Willy; Anderson, Matthew; Quadroni, Manfredo; Jin, Yishi; Garner, Craig C

    2012-02-22

    Previous studies in Caenorhabditis elegans showed that RPM-1 (Regulator of Presynaptic Morphology-1) regulates axon termination and synapse formation. To understand the mechanism of how rpm-1 functions, we have used mass spectrometry to identify RPM-1 binding proteins, and have identified RAE-1 (RNA Export protein-1) as an evolutionarily conserved binding partner. We define a RAE-1 binding region in RPM-1, and show that this binding interaction is conserved and also occurs between Rae1 and the human ortholog of RPM-1 called Pam (protein associated with Myc). rae-1 loss of function causes similar axon and synapse defects, and synergizes genetically with two other RPM-1 binding proteins, GLO-4 and FSN-1. Further, we show that RAE-1 colocalizes with RPM-1 in neurons, and that rae-1 functions downstream of rpm-1. These studies establish a novel postmitotic function for rae-1 in neuronal development.

  1. Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis

    Digital Repository Service at National Institute of Oceanography (India)

    Dusane, D.H; Damare, S.R.; Nancharaiah, Y.V.; Ramaiah, N.; Venugopalan, V.P.; Kumar, A.R.; Zinjarde, S.S.

    -TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75 degrees C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry...

  2. The efficiency of microbial protein production from tropical forages and its measurement using spot samples of urine and CrEDTA clearance from the plasma

    International Nuclear Information System (INIS)

    King, A.; Poppi, D.P.; McLennan, S.R.

    1999-01-01

    The efficiency of microbial crude protein production (MCP) expressed as g MCP/kg digestible organic matter (DOM) was much lower (P 0.75 /d). The fractional disappearance rate of CrEDTA from the plasma was similar for cattle consuming either hay (1.26-1.54%/min). The glomerular filtration rate (GFR) estimated from urinary creatinine excretion was significantly different (289 L/d vs 793 L/d, P vs R respectively). The ratio of predicted allantoin clearance determined by reference to plasma volume and FDR of CrEDTA to the actual urinary excretion was 6.7 and 9.6 for R and P respectively. This difference meant that clearance of CrEDTA could not be used as a technique in association with plasma concentration of allantoin to estimate urinary excretion of allantoin. (author)

  3. Dietary Alfalfa and Calcium Salts of Long-Chain Fatty Acids Alter Protein Utilization, Microbial Populations, and Plasma Fatty Acid Profile in Holstein Freemartin Heifers.

    Science.gov (United States)

    He, Yang; Qiu, Qinghua; Shao, Taoqi; Niu, Wenjing; Xia, Chuanqi; Wang, Haibo; Li, Qianwen; Gao, Zhibiao; Yu, Zhantao; Su, Huawei; Cao, Binghai

    2017-12-20

    This study presented the effects of alfalfa and calcium salts of long-chain fatty acids (CSFA) on feed intake, apparent digestibility, rumen fermentation, microbial community, plasma biochemical parameters, and fatty acid profile in Holstein freemartin heifers. Eight Holstein freemartin heifers were randomly divided into a 4 × 4 Latin Square experiment with 2 × 2 factorial diets, with or without alfalfa or CSFA. Dietary supplementation of CSFA significantly increased the apparent digestibility of dry matter, crude protein, neutral detergent fiber, organic matter, and significantly reduced N retention (P fatty acids in the plasma, which was expressed in reducing saturated fatty acid (ΣSFA) ratio and C14-C17 fatty acids proportion except C16:0 (P fatty acid (ΣPUFA) and unsaturated fatty acid (ΣUFA) (P fatty acids in plasma. Alfalfa and CSFA had mutual interaction effect on fat digestion and plasma triglycerides.

  4. Total replacement of corn by mesquite pod meal considering nutritional value, performance, feeding behavior, nitrogen balance, and microbial protein synthesis of Holstein-Zebu crossbred dairy steers.

    Science.gov (United States)

    de Oliveira Moraes, Gláucia Sabrine; de Souza, Evaristo Jorge Oliveira; Véras, Antonia Sherlânea Chaves; de Paula Almeida, Marina; da Cunha, Márcio Vieira; Torres, Thaysa Rodrigues; da Silva, Camila Sousa; Pereira, Gerfesson Felipe Cavalcanti

    2016-10-01

    The objective of the present study to assess the effects of mesquite pod addition replacing corn (0, 250, 500, 750, and 1000 g/kg in the dry matter basis) on nutrient intake, animal performance, feeding behavior, nutrient digestibility, nitrogen balance, and microbial protein synthesis. Twenty-five Holstein-Zebu crossbred dairy steers at 219 ± 22 kg initial body weight and 18 months of age were used. The experiment lasted 84 days, divided into three periods of 28 days. A completely randomized design was used, and data were submitted to analysis using PROC GLM for analysis of variance and PROC REG for regression analysis using the software Statistical Analysis Systems version 9.1. Experimental diets were composed of Tifton 85 hay, soybean meal, ground corn, mesquite pod meal, and mineral salt. Samples of food offered were collected during the last 3 days of each period, and the leftovers were collected daily, with samples bulked per week. At the end of each 28-day period, the remaining animals were weighed to determine total weight gain and average daily gain. The assessment of behavioral patterns was performed through instantaneous scans in 5-min intervals for three consecutive 12-h days. A single urine sample from each animal was collected on the last day of each collection period at about 4 h after the first feeding. The replacement of corn by mesquite pod meal did not significantly influence treatments regarding nutrients intake, animal performance, and feeding behavior. Retained and consumed nitrogen ratio did not statistically differ between replacement levels. Likewise, there were no statistical differences regarding microbial protein synthesis and efficiency between replacement levels. Mesquite pod meal can be used in Holstein-Zebu crossbred dairy steers' diet with total corn replacement.

  5. Lateral Fluid Percussion Injury Impairs Hippocampal Synaptic Soluble N-Ethylmaleimide Sensitive Factor Attachment Protein Receptor Complex Formation

    Directory of Open Access Journals (Sweden)

    Shaun W. Carlson

    2017-10-01

    Full Text Available Traumatic brain injury (TBI and the activation of secondary injury mechanisms have been linked to impaired cognitive function, which, as observed in TBI patients and animal models, can persist for months and years following the initial injury. Impairments in neurotransmission have been well documented in experimental models of TBI, but the mechanisms underlying this dysfunction are poorly understood. Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE complex facilitates vesicular docking and neurotransmitter release in the synaptic cleft. Published studies highlight a direct link between reduced SNARE complex formation and impairments in neurotransmitter release. While alterations in the SNARE complex have been described following severe focal TBI, it is not known if deficits in SNARE complex formation manifest in a model with reduced severity. We hypothesized that lateral fluid percussion injury (lFPI reduces the abundance of SNARE proteins, impairs SNARE complex formation, and contributes to impaired neurobehavioral function. To this end, rats were subjected to lFPI or sham injury and tested for acute motor performance and cognitive function at 3 weeks post-injury. lFPI resulted in motor impairment between 1 and 5 days post-injury. Spatial acquisition and spatial memory, as assessed by the Morris water maze, were significantly impaired at 3 weeks after lFPI. To examine the effect of lFPI on synaptic SNARE complex formation in the injured hippocampus, a separate cohort of rats was generated and brains processed to evaluate hippocampal synaptosomal-enriched lysates at 1 week post-injury. lFPI resulted in a significant reduction in multiple monomeric SNARE proteins, including VAMP2, and α-synuclein, and SNARE complex abundance. The findings in this study are consistent with our previously published observations suggesting that impairments in hippocampal SNARE complex formation may contribute to

  6. Expression of ErbB3-binding protein-1 (EBP1 during primordial follicle formation: role of estradiol-17ß.

    Directory of Open Access Journals (Sweden)

    Anindit Mukherjee

    Full Text Available The formation of primordial follicles involves the interaction between the oocytes and surrounding somatic cells, which differentiate into granulosa cells. Estradiol-17ß (E promotes primordial follicle formation in vivo and in vitro; however, the underlying mechanisms are poorly understood. The expression of an ERBB3-binding protein 1 (EBP1 is downregulated in 8-day old hamster ovaries concurrent with the increase in serum estradiol levels and the formation of primordial follicles. The objectives of the present study were to determine the spatio-temporal expression and putative E regulation of EBP1 in ovarian cells during perinatal development with respect to primordial follicle formation. Hamster EBP1 nucleic acid and amino acid sequences were more than 93% and 98% similar, respectively, to those of mouse and human, and contained nucleolar localization signal, RNA-binding domain and several phosphorylation sites. EBP1 protein was present in somatic cells and oocytes from E15, and declined in oocytes by P1 and in somatic cells by P5. Thereafter, EBP1 expression increased through P7 with a transient decline on P8 primarily in interstitial cells. EBP1 mRNA levels mirrored protein expression pattern. E treatment on P1 and P4 upregulated EBP1 expression by P8 whereas E treatment on P4 downregulated it by 72 h suggesting a compensatory upregulation due to E pretreatment. Treatment with an FSH-antiserum, which suppressed primordial follicle formation, prevented the decline in EBP1 levels, and the effect was reversed by E treatment. Therefore, the results provide the first evidence that EBP1 may play an important role in mediating the effect of E in the differentiation of somatic cells into granulosa cells during primordial follicle formation.

  7. The streptococcal collagen-like protein-1 (Scl1 is a significant determinant for biofilm formation by group a Streptococcus

    Directory of Open Access Journals (Sweden)

    Oliver-Kozup Heaven A

    2011-12-01

    Full Text Available Abstract Background Group A Streptococcus (GAS is a human-specific pathogen responsible for a number of diseases characterized by a wide range of clinical manifestations. During host colonization GAS-cell aggregates or microcolonies are observed in tissues. GAS biofilm, which is an in vitro equivalent of tissue microcolony, has only recently been studied and little is known about the specific surface determinants that aid biofilm formation. In this study, we demonstrate that surface-associated streptococcal collagen-like protein-1 (Scl1 plays an important role in GAS biofilm formation. Results Biofilm formation by M1-, M3-, M28-, and M41-type GAS strains, representing an intraspecies breadth, were analyzed spectrophotometrically following crystal violet staining, and characterized using confocal and field emission scanning electron microscopy. The M41-type strain formed the most robust biofilm under static conditions, followed by M28- and M1-type strains, while the M3-type strains analyzed here did not form biofilm under the same experimental conditions. Differences in architecture and cell-surface morphology were observed in biofilms formed by the M1- and M41-wild-type strains, accompanied by varying amounts of deposited extracellular matrix and differences in cell-to-cell junctions within each biofilm. Importantly, all Scl1-negative mutants examined showed significantly decreased ability to form biofilm in vitro. Furthermore, the Scl1 protein expressed on the surface of a heterologous host, Lactococcus lactis, was sufficient to induce biofilm formation by this organism. Conclusions Overall, this work (i identifies variations in biofilm formation capacity among pathogenically different GAS strains, (ii identifies GAS surface properties that may aid in biofilm stability and, (iii establishes that the Scl1 surface protein is an important determinant of GAS biofilm, which is sufficient to enable biofilm formation in the heterologous host

  8. Tight junction protein ZO-2 expression and relative function of ZO-1 and ZO-2 during mouse blastocyst formation

    International Nuclear Information System (INIS)

    Sheth, Bhavwanti; Nowak, Rachael L.; Anderson, Rebecca; Kwong, Wing Yee; Papenbrock, Thomas; Fleming, Tom P.

    2008-01-01

    Apicolateral tight junctions (TJs) between epithelial cells are multiprotein complexes regulating membrane polarity and paracellular transport and also contribute to signalling pathways affecting cell proliferation and gene expression. ZO-2 and other ZO family members form a sub-membranous scaffold for binding TJ constituents. We investigated ZO-2 contribution to TJ biogenesis and function during trophectoderm epithelium differentiation in mouse preimplantation embryos. Our data indicate that ZO-2 is expressed from maternal and embryonic genomes with maternal ZO-2 protein associated with nuclei in zygotes and particularly early cleavage stages. Embryonic ZO-2 assembled at outer blastomere apicolateral junctional sites from the late 16-cell stage. Junctional ZO-2 first co-localised with E-cadherin in a transient complex comprising adherens junction and TJ constituents before segregating to TJs after their separation from the blastocyst stage (32-cell onwards). ZO-2 siRNA microinjection into zygotes or 2-cell embryos resulted in specific knockdown of ZO-2 mRNA and protein within blastocysts. Embryos lacking ZO-2 protein at trophectoderm TJs exhibited delayed blastocoel cavity formation but underwent normal cell proliferation and outgrowth morphogenesis. Quantitative analysis of trophectoderm TJs in ZO-2-deficient embryos revealed increased assembly of ZO-1 but not occludin, indicating ZO protein redundancy as a compensatory mechanism contributing to the mild phenotype observed. In contrast, ZO-1 knockdown, or combined ZO-1 and ZO-2 knockdown, generated a more severe inhibition of blastocoel formation indicating distinct roles for ZO proteins in blastocyst morphogenesis

  9. Formation of protein/surfactant adsorption layer at the air/water interface as studied by dilational surface rheology.

    Science.gov (United States)

    Mikhailovskaya, A A; Noskov, B A; Lin, S-Y; Loglio, G; Miller, R

    2011-08-25

    The dynamic dilatational surface elasticity of mixed solutions of globular proteins (β-lactoglobulin (BLG) and bovine serum albumin (BSA)) with cationic (dodecyltrimethylammonium bromide (DTAB)) and anionic (sodium dodecyl sulfate (SDS)) surfactants was measured as a function of the surfactant concentration and surface age. If the cationic surfactant concentration exceeds a certain critical value, the kinetic dependencies of the dynamic surface elasticity of BLG/DTAB and BSA/DTAB solutions become nonmonotonous and resemble those of mixed solutions of proteins with guanidine hydrochloride. This result indicates not only the destruction of the protein tertiary structure in the surface layer of mixed solution but also a strong perturbation of the secondary structure. The corresponding kinetic dependencies for protein solutions with added anionic surfactants are always monotonous, thereby revealing a different mechanism of the adsorption layer formation. One can assume that the secondary structure is destroyed to a lesser extent in the latter case and hinders the formation of loops and tails at the interface. The increase of the solution's ionic strength by the addition of sodium chloride results in stronger changes of the protein conformations in the surface layer and the appearance of a local maximum in the kinetic dependencies of the dynamic surface elasticity in a relatively narrow range of SDS concentration. © 2011 American Chemical Society

  10. Formation of Shc-Grb2 complexes is necessary to induce neoplastic transformation by overexpression of Shc proteins

    DEFF Research Database (Denmark)

    Salcini, A E; McGlade, J; Pelicci, G

    1994-01-01

    The mammalian SHC gene encodes three overlapping proteins which all contain a carboxy-terminal SH2 domain. Shc proteins are phosphorylated on tyrosine by a variety of receptor and cytoplasmic tyrosine kinases. Phosphorylated Shc proteins form a complex with the SH2-SH3 containing Grb2 protein which...... of Grb2 to Shc proteins requires phosphorylation of Shc at Tyr317, which lies within the high affinity binding motif for the Grb2 SH2 domain, pYVNV, where Asn at the +2 position is crucial for complex formation. In vivo, Tyr317 is the major, but not the only, site for Shc phosphorylation, and is the sole...... aminoterminal deletion, but retain the Tyr317 site and the SH2 domain conserve the capacity to be phosphorylated, to bind to Grb2 and to induce cell transformation. These data indicate that the formation of the Shc-Grb2 complex is a crucial event in the transformation induced by overexpression of Shc...

  11. Gut Microbial Glycerol Metabolism as an Endogenous Acrolein Source

    OpenAIRE

    Zhang, Jianbo; Sturla, Shana; Lacroix, Christophe; Schwab, Clarissa

    2018-01-01

    ABSTRACT Acrolein is a highly reactive electrophile causing toxic effects, such as DNA and protein adduction, oxidative stress, endoplasmic reticulum stress, immune dysfunction, and membrane damage. This Opinion/Hypothesis provides an overview of endogenous and exogenous acrolein sources, acrolein’s mode of action, and its metabolic fate. Recent reports underpin the finding that gut microbial glycerol metabolism leading to the formation of reuterin is an additional source of endogenous acrole...

  12. The C-terminal region of A-kinase anchor protein 350 (AKAP350A) enables formation of microtubule-nucleation centers and interacts with pericentriolar proteins.

    Science.gov (United States)

    Kolobova, Elena; Roland, Joseph T; Lapierre, Lynne A; Williams, Janice A; Mason, Twila A; Goldenring, James R

    2017-12-15

    Microtubules in animal cells assemble (nucleate) from both the centrosome and the cis-Golgi cisternae. A-kinase anchor protein 350 kDa (AKAP350A, also called AKAP450/CG-NAP/AKAP9) is a large scaffolding protein located at both the centrosome and Golgi apparatus. Previous findings have suggested that AKAP350 is important for microtubule dynamics at both locations, but how this scaffolding protein assembles microtubule nucleation machinery is unclear. Here, we found that overexpression of the C-terminal third of AKAP350A, enhanced GFP-AKAP350A(2691-3907), induces the formation of multiple microtubule-nucleation centers (MTNCs). Nevertheless, these induced MTNCs lacked "true" centriole proteins, such as Cep135. Mapping analysis with AKAP350A truncations demonstrated that AKAP350A contains discrete regions responsible for promoting or inhibiting the formation of multiple MTNCs. Moreover, GFP-AKAP350A(2691-3907) recruited several pericentriolar proteins to MTNCs, including γ-tubulin, pericentrin, Cep68, Cep170, and Cdk5RAP2. Proteomic analysis indicated that Cdk5RAP2 and Cep170 both interact with the microtubule nucleation-promoting region of AKAP350A, whereas Cep68 interacts with the distal C-terminal AKAP350A region. Yeast two-hybrid assays established a direct interaction of Cep170 with AKAP350A. Super-resolution and deconvolution microscopy analyses were performed to define the association of AKAP350A with centrosomes, and these studies disclosed that AKAP350A spans the bridge between centrioles, co-localizing with rootletin and Cep68 in the linker region. siRNA-mediated depletion of AKAP350A caused displacement of both Cep68 and Cep170 from the centrosome. These results suggest that AKAP350A acts as a scaffold for factors involved in microtubule nucleation at the centrosome and coordinates the assembly of protein complexes associating with the intercentriolar bridge.

  13. Measles virus C protein suppresses gamma-activated factor formation and virus-induced cell growth arrest

    International Nuclear Information System (INIS)

    Yokota, Shin-ichi; Okabayashi, Tamaki; Fujii, Nobuhiro

    2011-01-01

    Measles virus (MeV) produces two accessory proteins, V and C, from the P gene. These accessory proteins have been reported to contribute to efficient virus proliferation through the modulation of host cell events. Our previous paper described that Vero cell-adapted strains of MeV led host cells to growth arrest through the upregulation of interferon regulatory factor 1 (IRF-1), and wild strains did not. In the present study, we found that C protein expression levels varied among MeV strains in infected SiHa cells. C protein levels were inversely correlated with IRF-1 expression levels and with cell growth arrest. Forced expression of C protein released cells from growth arrest. C-deficient recombinant virus efficiently upregulated IRF-1 and caused growth arrest more efficiently than the wild-type virus. C protein preferentially bound to phosphorylated STAT1 and suppressed STAT1 dimer formation. We conclude that MeV C protein suppresses IFN-γ signaling pathway via inhibition of phosphorylated STAT1 dimerization.

  14. Effects of cell culture media on the dynamic formation of protein-nanoparticle complexes and influence on the cellular response.

    Science.gov (United States)

    Maiorano, Gabriele; Sabella, Stefania; Sorce, Barbara; Brunetti, Virgilio; Malvindi, Maria Ada; Cingolani, Roberto; Pompa, Pier Paolo

    2010-12-28

    The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV-visible, plasmon resonance light scattering), that proteins-NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagle's medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.

  15. RPPAML/RIMS: A metadata format and an information management system for reverse phase protein arrays

    OpenAIRE

    Stanislaus, Romesh; Carey, Mark; Deus, Helena F; Coombes, Kevin; Hennessy, Bryan T; Mills, Gordon B; Almeida, Jonas S

    2008-01-01

    Abstract Background Reverse Phase Protein Arrays (RPPA) are convenient assay platforms to investigate the presence of biomarkers in tissue lysates. As with other high-throughput technologies, substantial amounts of analytical data are generated. Over 1000 samples may be printed on a single nitrocellulose slide. Up to 100 different proteins may be assessed using immunoperoxidase or immunoflorescence techniques in order to determine relative amounts of protein expression in the samples of inter...

  16. Dietary Considerations in Autism Spectrum Disorders: The Potential Role of Protein Digestion and Microbial Putrefaction in the Gut-Brain Axis.

    Science.gov (United States)

    Sanctuary, Megan R; Kain, Jennifer N; Angkustsiri, Kathleen; German, J Bruce

    2018-01-01

    Children with autism spectrum disorders (ASD), characterized by a range of behavioral abnormalities and social deficits, display high incidence of gastrointestinal (GI) co-morbidities including chronic constipation and diarrhea. Research is now increasingly able to characterize the "fragile gut" in these children and understand the role that impairment of specific GI functions plays in the GI symptoms associated with ASD. This mechanistic understanding is extending to the interactions between diet and ASD, including food structure and protein digestive capacity in exacerbating autistic symptoms. Children with ASD and gut co-morbidities exhibit low digestive enzyme activity, impaired gut barrier integrity and the presence of antibodies specific for dietary proteins in the peripheral circulation. These findings support the hypothesis that entry of dietary peptides from the gut lumen into the vasculature are associated with an aberrant immune response. Furthermore, a subset of children with ASD exhibit high concentrations of metabolites originating from microbial activity on proteinaceous substrates. Taken together, the combination of specific protein intakes poor digestion, gut barrier integrity, microbiota composition and function all on a background of ASD represents a phenotypic pattern. A potential consequence of this pattern of conditions is that the fragile gut of some children with ASD is at risk for GI symptoms that may be amenable to improvement with specific dietary changes. There is growing evidence that shows an association between gut dysfunction and dysbiosis and ASD symptoms. It is therefore urgent to perform more experimental and clinical research on the "fragile gut" in children with ASD in order to move toward advancements in clinical practice. Identifying those factors that are of clinical value will provide an evidence-based path to individual management and targeted solutions; from real time sensing to the design of diets with personalized

  17. Surface-water interface induces conformational changes critical for protein adsorption: Implications for monolayer formation of EAS hydrophobin

    Directory of Open Access Journals (Sweden)

    Kamron eLey

    2015-11-01

    Full Text Available The class I hydrophobin EAS is part of a family of small, amphiphilic fungal proteins best known for their ability to self-assemble into stable monolayers that modify the hydrophobicity of a surface to facilitate further microbial growth. These proteins have attracted increasing attention for industrial and biomedical applications, with the aim of designing surfaces that have the potential to maintain their clean state by resisting non-specific protein binding. To gain a better understanding of this process, we have employed all-atom molecular dynamics to study initial stages of the spontaneous adsorption of monomeric EAS hydrophobin on fully hydroxylated silica, a commonly used industrial and biomedical substrate. Particular interest has been paid to the Cys3-Cys4 loop, which has been shown to exhibit disruptive behavior in solution, and the Cys7-Cys8 loop, which is believed to be involved in the aggregation of EAS hydrophobin at interfaces. Specific and water mediated interactions with the surface were also analyzed. We have identified two possible binding motifs, one which allows unfolding of the Cys7-Cys8 loop due to the surfactant-like behavior of the Cys3-Cys4 loop, and another which has limited unfolding due to the Cys3-Cys4 loop remaining disordered in solution. We have also identified intermittent interactions with water which mediate the protein adsorption to the surface, as well as longer lasting interactions which control the diffusion of water around the adsorption site. These results have shown that EAS behaves in a similar way at the air-water and surface-water interfaces, and have also highlighted the need for hydrophilic ligand functionalization of the silica surface in order to prevent the adsorption of EAS hydrophobin.

  18. Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity

    Directory of Open Access Journals (Sweden)

    Akiko Ueno

    2018-03-01

    Full Text Available Summary: In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. : Ueno et al. finds that Lrit1 plays an important role in regulating the synaptic connection between cone photoreceptors and cone ON-bipolar cells. The Frmpd2-Lrit1-mGluR6 axis is crucial for selective synapse formation in cone photoreceptors and for development of normal visual function. Keywords: retina, circuit, synapse formation, cone photoreceptor cell, ON-bipolar cell, visual acuity

  19. Analyses of advanced rice anther transcriptomes reveal global tapetum secretory functions and potential proteins for lipid exine formation.

    Science.gov (United States)

    Huang, Ming-Der; Wei, Fu-Jin; Wu, Cheng-Cheih; Hsing, Yue-Ie Caroline; Huang, Anthony H C

    2009-02-01

    The anthers in flowers perform important functions in sexual reproduction. Several recent studies used microarrays to study anther transcriptomes to explore genes controlling anther development. To analyze the secretion and other functions of the tapetum, we produced transcriptomes of anthers of rice (Oryza sativa subsp. japonica) at six progressive developmental stages and pollen with sequencing-by-synthesis technology. The transcriptomes included at least 18,000 unique transcripts, about 25% of which had antisense transcripts. In silico anther-minus-pollen subtraction produced transcripts largely unique to the tapetum; these transcripts include all the reported tapetum-specific transcripts of orthologs in other species. The differential developmental profiles of the transcripts and their antisense transcripts signify extensive regulation of gene expression in the anther, especially the tapetum, during development. The transcriptomes were used to dissect two major cell/biochemical functions of the tapetum. First, we categorized and charted the developmental profiles of all transcripts encoding secretory proteins present in the cellular exterior; these transcripts represent about 12% and 30% of the those transcripts having more than 100 and 1,000 transcripts per million, respectively. Second, we successfully selected from hundreds of transcripts several transcripts encoding potential proteins for lipid exine synthesis during early anther development. These proteins include cytochrome P450, acyltransferases, and lipid transfer proteins in our hypothesized mechanism of exine synthesis in and export from the tapetum. Putative functioning of these proteins in exine formation is consistent with proteins and metabolites detected in the anther locule fluid obtained by micropipetting.

  20. Formation of S-(carboxymethyl)-cysteine in rat liver mitochondrial proteins: effects of caloric and methionine restriction.

    Science.gov (United States)

    Naudí, Alba; Jové, Mariona; Cacabelos, Daniel; Ayala, Victoria; Cabre, Rosanna; Caro, Pilar; Gomez, José; Portero-Otín, Manuel; Barja, Gustavo; Pamplona, Reinald

    2013-02-01

    Maillard reaction contributes to the chemical modification and cross-linking of proteins. This process plays a significant role in the aging process and determination of animal longevity. Oxidative conditions promote the Maillard reaction. Mitochondria are the primary site of oxidants due to the reactive molecular species production. Mitochondrial proteome cysteine residues are targets of oxidative attack due to their specific chemistry and localization. Their chemical, non-enzymatic modification leads to dysfunctional proteins, which entail cellular senescence and organismal aging. Previous studies have consistently shown that caloric and methionine restrictions, nutritional interventions that increase longevity, decrease the rate of mitochondrial oxidant production and the physiological steady-state levels of markers of oxidative damage to macromolecules. In this scenario, we have detected S-(carboxymethyl)-cysteine (CMC) as a new irreversible chemical modification in mitochondrial proteins. CMC content in mitochondrial proteins significantly correlated with that of the lysine-derived analog N (ε)-(carboxymethyl)-lysine. The concentration of CMC is, however, one order of magnitude lower compared with CML likely due in part to the lower content of cysteine with respect to lysine of the mitochondrial proteome. CMC concentrations decreases in liver mitochondrial proteins of rats subjected to 8.5 and 25 % caloric restriction, as well as in 40 and 80 % methionine restriction. This is associated with a concomitant and significant increase in the protein content of sulfhydryl groups. Data presented here evidence that CMC, a marker of Cys-AGE formation, could be candidate as a biomarker of mitochondrial damage during aging.

  1. Formation of poly(butyl 2-cyanoacrylate) microcapsules and the microencapsulation of aqueous solutions of [125I]-labelled proteins

    International Nuclear Information System (INIS)

    Wood, D.A.; Whateley, T.L.; Florence, A.T.

    1981-01-01

    Some featrues of the polymerization reaction of butyl 2-cyanoacrylate at different aqueous/organic solvent interfaces have been investigated. In particular, the effects of pH and the presence of protein on the formation of microcapsules by in situ interfacial polymerization of butyl 2-cyanoacrylate in w/o emulsions have been studied. [ 125 I]-labelled proteins have been used to study the procedure as a method of microencapsulating enzymes or other proteins within potentially biodegradable membranes. Preliminary in vitro degradation studies suggest that degradation of the microcapsules is inhibited by low levels of their breakdown products, thus allowing the storage of the microcapsules as aqueous suspensions for prolonged periods in sealed containers. (Auth.)

  2. Impact of Trichloroethylene Exposure on the Microbial Diversity and Protein Expression in Anaerobic Granular Biomass at 37°C and 15°C

    Directory of Open Access Journals (Sweden)

    Alma Siggins

    2012-01-01

    Full Text Available Granular biomass from a laboratory-scale anaerobic bioreactor trial was analysed to identify changes in microbial community structure and function in response to temperature and trichloroethylene (TCE. Two bioreactors were operated at 37°C, while two were operated at 15°C. At the time of sampling, one of each temperature pair of bioreactors was exposed to process failure-inducing concentrations of TCE (60 mg L−1 while the other served as a TCE-free control. Bacterial community structure was investigated using denaturing gradient gel electrophoresis (DGGE and 16S rRNA gene clone library analysis. Temperature was identified as an important factor for bacterial community composition, while minor differences were associated with trichloroethylene supplementation. Proteobacteria was the dominant phylum in all bioreactors, while clone library analysis revealed a higher proportion of Bacteroidetes-, Chloroflexi-, and Firmicutes-like clones at 15°C than at 37°C. Comparative metaproteomics in the presence and absence of TCE was carried out by two-dimensional gel electrophoresis (2-DGE, and 28 protein spots were identified, with putative functions related to cellular processes, including methanogenesis, glycolysis, the glyoxylate cycle, and the methyl malonyl pathway. A good agreement between metaproteomic species assignment and phylogenetic information was observed, with 10 of the identified proteins associated with members of the phylum Proteobacteria.

  3. Context Memory Formation Requires Activity-Dependent Protein Degradation in the Hippocampus

    Science.gov (United States)

    Cullen, Patrick K.; Ferrara, Nicole C.; Pullins, Shane E.; Helmstetter, Fred J.

    2017-01-01

    Numerous studies have indicated that the consolidation of contextual fear memories supported by an aversive outcome like footshock requires de novo protein synthesis as well as protein degradation mediated by the ubiquitin-proteasome system (UPS). Context memory formed in the absence of an aversive stimulus by simple exposure to a novel…

  4. [The role of Smads and related transcription factors in the signal transduction of bone morphogenetic protein inducing bone formation].

    Science.gov (United States)

    Xu, Xiao-liang; Dai, Ke-rong; Tang, Ting-ting

    2003-09-01

    To clarify the mechanisms of the signal transduction of bone morphogenetic proteins (BMPs) inducing bone formation and to provide theoretical basis for basic and applying research of BMPs. We looked up the literature of the role of Smads and related transcription factors in the signal transduction of BMPs inducing bone formation. The signal transduction processes of BMPs included: 1. BMPs combined with type II and type I receptors; 2. the type I receptor phosphorylated Smads; and 3. Smads entered the cell nucleus, interacted with transcription factors and influenced the transcription of related proteins. Smads could be divided into receptor-regulated Smads (R-Smads: Smad1, Smad2, Smad3, Smad5, Smad8 and Smad9), common-mediator Smad (co-Smad: Smad4), and inhibitory Smads (I-Smads: Smad6 and Smad7). Smad1, Smad5, Smad8, and probable Smad9 were involved in the signal transduction of BMPs. Multiple kinases, such as focal adhesion kinase (FAK), Ras-extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), and Akt serine/threonine kinase were related to Smads signal transduction. Smad1 and Smad5 related with transcription factors included core binding factor A1 (CBFA1), smad-interacting protein 1 (SIP1), ornithine decarboxylase antizyme (OAZ), activating protein-1 (AP-1), xenopus ventralizing homeobox protein-2 (Xvent-2), sandostatin (Ski), antiproliferative proteins (Tob), and homeodomain-containing transcriptian factor-8 (Hoxc-8), et al. CBFA1 could interact with Smad1, Smad2, Smad3, and Smad5, so it was involved in TGF-beta and BMP-2 signal transduction, and played an important role in the bone formation. Cleidocranial dysplasia (CCD) was thought to be caused by heterozygous mutations in CBFA1. The CBFA1 knockout mice showed no osteogenesis and had maturational disturbance of chondrocytes. Smads and related transcription factors, especially Smad1, Smad5, Smad8 and CBFA1, play an important role in the signal transduction of BMPs inducing bone

  5. Behavior of native microbial populations of WPC-34 and WPC-80 whey protein stored at different temperatures

    Science.gov (United States)

    Whey protein (WPC34 and 80) has been used as food ingredients and as a base for making biodegradable product. However, there is limited information on the behavior of native microflora associated with these products. WPC 34 and WPC80 were obtained from the manufacturer, and were stored at 5, 10, 15,...

  6. Protein design and engineering of a de novo pathway for microbial production of 1,3-propanediol from glucose.

    Science.gov (United States)

    Chen, Zhen; Geng, Feng; Zeng, An-Ping

    2015-02-01

    Protein engineering to expand the substrate spectrum of native enzymes opens new possibilities for bioproduction of valuable chemicals from non-natural pathways. No natural microorganism can directly use sugars to produce 1,3-propanediol (PDO). Here, we present a de novo route for the biosynthesis of PDO from sugar, which may overcome the mentioned limitations by expanding the homoserine synthesis pathway. The accomplishment of pathway from homoserine to PDO is achieved by protein engineering of glutamate dehydrogenase (GDH) and pyruvate decarboxylase to sequentially convert homoserine to 4-hydroxy-2-ketobutyrate and 3-hydroxypropionaldehyde. The latter is finally converted to PDO by using a native alcohol dehydrogenase. In this work, we report on experimental accomplishment of this non-natural pathway, especially by protein engineering of GDH for the key step of converting homoserine to 4-hydroxy-2-ketobutyrate. These results show the feasibility and significance of protein engineering for de novo pathway design and overproduction of desired industrial products. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Immobilization-stabilization of proteins on nanofibrillated cellulose derivatives and their bioactive film formation.

    Science.gov (United States)

    Arola, Suvi; Tammelin, Tekla; Setälä, Harri; Tullila, Antti; Linder, Markus B

    2012-03-12

    In a number of different applications for enzymes and specific binding proteins a key technology is the immobilization of these proteins to different types of supports. In this work we describe a concept for protein immobilization that is based on nanofibrillated cellulose (NFC). NFC is a form of cellulose where fibers have been disintegrated into fibrils that are only a few nanometers in diameter and have a very large aspect ratio. Proteins were conjugated through three different strategies using amine, epoxy, and carboxylic acid functionalized NFC. The conjugation chemistries were chosen according to the reactive groups on the NFC derivatives; epoxy amination, heterobifunctional modification of amino groups, and EDC/s-NHS activation of carboxylic acid groups. The conjugation reactions were performed in solution and immobilization was performed by spin coating the protein-NCF conjugates. The structure of NFC was shown to be advantageous for both protein performance and stability. The use of NFC allows all covalent chemistry to be performed in solution, while the immobilization is achieved by a simple spin coating or spreading of the protein-NFC conjugates on a support. This allows more scalable methods and better control of conditions compared to the traditional methods that depend on surface reactions.

  8. Hamster female protein, a pentameric oligomer capable of reassociation and hybrid formation

    International Nuclear Information System (INIS)

    Coe, J.E.; Ross, M.J.

    1987-01-01

    Syrian hamster female protein (SFP), a serum oligomer composed of five identical subunits, was reassociated in vitro monomer subunits. The reconstituted pentamer was genuine by morphologic, antigenic, and structural criteria. Another female protein (FP), a homologue from Armenian hamsters (AFP), also reassociated into a pentamer after dissociation with 5 M guanidine hydrochloride. These two FP's hybridized when a mixture of them was dissociated and then reassociated. Differences between the parent FP's were used to show that the recombinant pentamer contained monomer subunits from both SFP and AFP. Reassociation of both FP's was enhanced by increasing FP concentration and also by adding Ca 2+ during reassembly. The two FP's differed in their reassociation profile in that SFP was especially efficient in reassembly, whereas AFP was more dependent upon Ca 2+ . Female protein is a homologue of C-reactive protein and amyloid P component, and all of these proteins (pentraxins) share a similar structure. The in vitro dissociation-reassociation of female protein described herein may reflect an in vivo dissociation-reassociation which is functionally important and a common metabolic feature within this family of proteins

  9. IL-1 receptor accessory protein-like 1 associated with mental retardation and autism mediates synapse formation by trans-synaptic interaction with protein tyrosine phosphatase δ.

    Science.gov (United States)

    Yoshida, Tomoyuki; Yasumura, Misato; Uemura, Takeshi; Lee, Sung-Jin; Ra, Moonjin; Taguchi, Ryo; Iwakura, Yoichiro; Mishina, Masayoshi

    2011-09-21

    Mental retardation (MR) and autism are highly heterogeneous neurodevelopmental disorders. IL-1-receptor accessory protein-like 1 (IL1RAPL1) is responsible for nonsyndromic MR and is associated with autism. Thus, the elucidation of the functional role of IL1RAPL1 will contribute to our understanding of the pathogenesis of these mental disorders. Here, we showed that knockdown of endogenous IL1RAPL1 in cultured cortical neurons suppressed the accumulation of punctate staining signals for active zone protein Bassoon and decreased the number of dendritic protrusions. Consistently, the expression of IL1RAPL1 in cultured neurons stimulated the accumulation of Bassoon and spinogenesis. The extracellular domain (ECD) of IL1RAPL1 was required and sufficient for the presynaptic differentiation-inducing activity, while both the ECD and cytoplasmic domain were essential for the spinogenic activity. Notably, the synaptogenic activity of IL1RAPL1 was specific for excitatory synapses. Furthermore, we identified presynaptic protein tyrosine phosphatase (PTP) δ as a major IL1RAPL1-ECD interacting protein by affinity chromatography. IL1RAPL1 interacted selectively with certain forms of PTPδ splice variants carrying mini-exon peptides in Ig-like domains. The synaptogenic activity of IL1RAPL1 was abolished in primary neurons from PTPδ knock-out mice. IL1RAPL1 showed robust synaptogenic activity in vivo when transfected into the cortical neurons of wild-type mice but not in PTPδ knock-out mice. These results suggest that IL1RAPL1 mediates synapse formation through trans-synaptic interaction with PTPδ. Our findings raise an intriguing possibility that the impairment of synapse formation may underlie certain forms of MR and autism as a common pathogenic pathway shared by these mental disorders.

  10. Amyloid-like fibril formation by polyQ proteins: a critical balance between the polyQ length and the constraints imposed by the host protein.

    Directory of Open Access Journals (Sweden)

    Natacha Scarafone

    Full Text Available Nine neurodegenerative disorders, called polyglutamine (polyQ diseases, are characterized by the formation of intranuclear amyloid-like aggregates by nine proteins containing a polyQ tract above a threshold length. These insoluble aggregates and/or some of their soluble precursors are thought to play a role in the pathogenesis. The mechanism by which polyQ expansions trigger the aggregation of the relevant proteins remains, however, unclear. In this work, polyQ tracts of different lengths were inserted into a solvent-exposed loop of the β-lactamase BlaP and the effects of these insertions on the properties of BlaP were investigated by a range of biophysical techniques. The insertion of up to 79 glutamines does not modify the structure of BlaP; it does, however, significantly destabilize the enzyme. The extent of destabilization is largely independent of the polyQ length, allowing us to study independently the effects intrinsic to the polyQ length and those related to the structural integrity of BlaP on the aggregating properties of the chimeras. Only chimeras with 55Q and 79Q readily form amyloid-like fibrils; therefore, similarly to the proteins associated with diseases, there is a threshold number of glutamines above which the chimeras aggregate into amyloid-like fibrils. Most importantly, the chimera containing 79Q forms amyloid-like fibrils at the same rate whether BlaP is folded or not, whereas the 55Q chimera aggregates into amyloid-like fibrils only if BlaP is unfolded. The threshold value for amyloid-like fibril formation depends, therefore, on the structural integrity of the β-lactamase moiety and thus on the steric and/or conformational constraints applied to the polyQ tract. These constraints have, however, no significant effect on the propensity of the 79Q tract to trigger fibril formation. These results suggest that the influence of the protein context on the aggregating properties of polyQ disease-associated proteins could be

  11. Formation of a Multiple Protein Complex on the Adenovirus Packaging Sequence by the IVa2 Protein▿

    OpenAIRE

    Tyler, Ryan E.; Ewing, Sean G.; Imperiale, Michael J.

    2007-01-01

    During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first ...

  12. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    Science.gov (United States)

    Sikder, Md. Kabir Uddin; Stone, Kyle A.; Kumar, P. B. Sunil; Laradji, Mohamed

    2014-01-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. PMID:25106608

  13. Microbial expression of proteins containing long repetitive Arg-Gly-Asp cell adhesive motifs created by overlap elongation PCR

    International Nuclear Information System (INIS)

    Kurihara, Hiroyuki; Shinkai, Masashige; Nagamune, Teruyuki

    2004-01-01

    We developed a novel method for creating repetitive DNA libraries using overlap elongation PCR, and prepared a DNA library encoding repetitive Arg-Gly-Asp (RGD) cell adhesive motifs. We obtained various length DNAs encoding repetitive RGD from a short monomer DNA (18 bp) after a thermal cyclic reaction without a DNA template for amplification, and isolated DNAs encoding 2, 21, and 43 repeats of the RGD motif. We cloned these DNAs into a protein expression vector and overexpressed them as thioredoxin fusion proteins: RGD2, RGD21, and RGD43, respectively. The solubility of RGD43 in water was low and it formed a fibrous precipitate in water. Scanning electron microscopy revealed that RGD43 formed a branched 3D-network structure in the solid state. To evaluate the function of the cell adhesive motifs in RGD43, mouse fibroblast cells were cultivated on the RGD43 scaffold. The fibroblast cells adhered to the RGD43 scaffold and extended long filopodia

  14. Kinetics of inclusion body formation and its correlation with the characteristics of protein aggregates in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Arun K Upadhyay

    Full Text Available The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2-3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies.

  15. Kinetics of Inclusion Body Formation and Its Correlation with the Characteristics of Protein Aggregates in Escherichia coli

    Science.gov (United States)

    Upadhyay, Arun K.; Murmu, Aruna; Singh, Anupam; Panda, Amulya K.

    2012-01-01

    The objective of the research was to understand the structural determinants governing protein aggregation into inclusion bodies during expression of recombinant proteins in Escherichia coli. Recombinant human growth hormone (hGH) and asparaginase were expressed as inclusion bodies in E.coli and the kinetics of aggregate formation was analyzed in details. Asparaginase inclusion bodies were of smaller size (200 nm) and the size of the aggregates did not increase with induction time. In contrast, the seeding and growth behavior of hGH inclusion bodies were found to be sequential, kinetically stable and the aggregate size increased from 200 to 800 nm with induction time. Human growth hormone inclusion bodies showed higher resistance to denaturants and proteinase K degradation in comparison to those of asparaginase inclusion bodies. Asparaginase inclusion bodies were completely solubilized at 2–3 M urea concentration and could be refolded into active protein, whereas 7 M urea was required for complete solubilization of hGH inclusion bodies. Both hGH and asparaginase inclusion bodies showed binding with amyloid specific dyes. In spite of its low β-sheet content, binding with dyes was more prominent in case of hGH inclusion bodies than that of asparaginase. Arrangements of protein molecules present in the surface as well as in the core of inclusion bodies were similar. Hydrophobic interactions between partially folded amphiphillic and hydrophobic alpha-helices were found to be one of the main determinants of hGH inclusion body formation. Aggregation behavior of the protein molecules decides the nature and properties of inclusion bodies. PMID:22479486

  16. Dietary protein reduction on microbial protein, amino acids digestibility, and body retention in beef cattle. I. Digestibility sites and ruminal synthesis estimated by purine bases and 15N as markers.

    Science.gov (United States)

    Mariz, Lays Débora Silva; Amaral, Paloma de Melo; Valadares Filho, Sebastião de Campos; Santos, Stefanie Alvarenga; Marcondes, Marcos Inácio; Prados, Laura Franco; Carneiro Pacheco, Marcos Vinícius; Zanetti, Diego; de Castro Menezes, Gustavo Chamon; Faciola, Antonio P

    2018-06-04

    The objectives of this study were to evaluate the effect of reducing dietary CP contents on 1) total and partial nutrient digestion and nitrogen balance and 2) on microbial crude protein (MCP) synthesis and true MCP digestibility in the small intestine obtained with 15N and purine bases (PB) in beef cattle. Eight bulls (4 Nellore and 4 Crossbred Angus × Nellore) cannulated in the rumen and ileum were distributed in duplicated 4 × 4 Latin squares. The diets consisted of increasing CP contents: 100, 120, or 140 g CP/kg DM offered ad libitum, and restricted intake (RI) diet with 120 g CP/kg DM. The experiment lasted four 17-d periods, with 10 d for adaptation to diets and another 7 for data collection. Omasal digesta flow was obtained using Co-EDTA and indigestible NDF (iNDF) as markers, and to estimate ileal digesta flow only iNDF was used. From days 11 to 17 of each experimental period, ruminal infusions of Co-EDTA (5.0 g/d) and 15N (7.03 g of ammonium sulfate enriched with 10% of 15N atoms) were performed. There was no effect of CP contents (linear effect, P = 0.55 and quadratic effect, P = 0.11) on ruminal OM digestibility. Intake of CP linearly increased (P content (P ruminally degradable OM and true ruminally degradable OM) had a quadratic tendency (P = 0.07 and P = 0.08, respectively) to CP increasing and was numerically greatest at 120 g CP/kg DM. The adjusted equations for estimating true intestinal digestibility of MCP (Y1) and total CP (Y2) were, respectively, as follows: Y1 =--16.724(SEM = 40.06) + 0.86X(SEM = 0.05) and Y2 = -43.81(SEM = 49.19) + 0.75X(SEM = 0.05). It was concluded that diets with 120 g/kg of CP optimize the microbial synthesis and efficiency and ruminal ash and protein NDF digestibility, resulting in a better use of N compounds in the rumen. The PB technique can be used as an alternative to the 15N to estimate microbial synthesis.

  17. Urinary excretion of purine derivatives as an indicator of microbial protein supply in Sri Lankan local Zebu cattle and crossbred milking cows

    International Nuclear Information System (INIS)

    Thakshala Seresinhe; Pathirana, K.K.; Jayasuriya, M.C.N.

    2004-01-01

    The objective of the present study was to evaluate the applicability of purine derivative (PD) method to Sri Lankan Zebu cattle and their crosses. In the first experiment four male Zebu cattle (LW 100 kg) were used to determine the response of PD excretion at four levels of intake (95, 80, 60 and 40% of the voluntary intake). Digestibility of dry matter and organic matter were not affected (P > 0.05) but nitrogen retention was increased with increasing levels of feed intake. The PD excretion were 1.91, 1.46, 1.21 and 0.66 mmol/kgW 0.75 /d for 95, 80, 60 and 40% of the voluntary intake levels, respectively. The proportion of allantoin in total PD was 82.6%. The excretion of creatinine was 1.05, 1.04, 0.92 and 0.84 mmol/kgW 0.75 /d, respectively. Daily output of total PD showed a positive response to the level of feed intake, while creatinine excretion was independent of dietary treatments. The correlation between PD excretion and digestible organic matter intake (DOMI) was significant (r 2 0.70). Nevertheless, the PDC index was affected (P > 0.05) by the level of feed intake and the correlation of the PDC index and DOMI was significant as well (r 2 =0.63). Results of spot urine analysis showed that the sampling period had little or no influence on the concentration of total PD or creatinine in urine. The PDC index was affected by the level of feed intake, but not by the time of sampling. In the second experiment, crossbred milking cows showed a higher PD excretion when fed with the experimental ration as compared with the farm ration. The mean PD excretion were 3.45 and 5.21 mmol/d for farm and experimental diets respectively. Allantoin accounted for more than 80% of the total PD, as in the previous experiment. In conclusion, urinary PD excretion appears to be a valid and non-invasive procedure to assess the microbial protein supply in local Zebu cattle and crossbred milking cows in Sri Lanka. Spot urine sampling also appeared to be a satisfactory method for

  18. Biofilm growth on polyvinylchloride surface incubated in suboptimal microbial warm water and effect of sanitizers on biofilm removal post biofilm formation

    Science.gov (United States)

    An in vitro experiment was conducted to understand the nature of biofilm growth on polyvinyl chloride (PVC) surface when exposed to sub optimal quality microbial water (> 4 log10 cfu/ml) obtained from poultry drinking water source mimicking water in waterlines during the first week of poultry broodi...

  19. Conformational co-dependence between Plasmodium berghei LCCL proteins promotes complex formation and stability.

    Science.gov (United States)

    Saeed, Sadia; Tremp, Annie Z; Dessens, Johannes T

    2012-10-01

    Malaria parasites express a conserved family of LCCL-lectin adhesive-like domain proteins (LAPs) that have essential functions in sporozoite transmission. In Plasmodium falciparum all six family members are expressed in gametocytes and form a multi-protein complex. Intriguingly, knockout of P. falciparum LCCL proteins adversely affects expression of other family members at protein, but not at mRNA level, a phenomenon termed co-dependent expression. Here, we investigate this in Plasmodium berghei by crossing a PbLAP1 null mutant parasite with a parasite line expressing GFP-tagged PbLAP3 that displays strong fluorescence in gametocytes. Selected and validated double mutants show normal synthesis and subcellular localization of PbLAP3::GFP. However, GFP-based fluorescence is dramatically reduced without PbLAP1 present, indicating that PbLAP1 and PbLAP3 interact. Moreover, absence of PbLAP1 markedly reduces the half-life of PbLAP3, consistent with a scenario of misfolding. These findings unveil a potential mechanism of conformational interdependence that facilitates assembly and stability of the functional LCCL protein complex. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Radiation induced formation of giant cells in Saccharomyces uvarum. Pt. 4. Macromolecular synthesis and protein patterns

    Energy Technology Data Exchange (ETDEWEB)

    Rink, H; Baumstark-Khan, C; Partke, H J

    1986-08-01

    X-irradiated (1.0 kGy) yeast cells (Saccharomyces uvarum, ATCC 9080), grown in liquid medium stop their mitotic activities and form giant cells by development of several buds which do not separate from mother cells. Depending on the time in culture, wet and dry weights per cell, protein- RNA- and DNA- contents per cell as well as incorporation rates of /sup 14/C-leucine per cell and per hour and patterns (isoelectric focusing) of water soluble proteins were studied. Weights per cell, RNA and protein contents per cell and /sup 14/C-leucine incorporation rates increase markedly in giant cells, whereas DNA content per cell is only duplicated. Protein patterns in isoelectric focusing show one interesting difference. In samples from giant cells one protein band (IP=6.63) decreases after 8 h in culture and later on disappears completely. This finding is not due to primary damage in X-irradiated DNA but seems to be related to the control of cell cycle events.

  1. Studies of the structure and function of Mms6, a bacterial protein that promotes the formation of magnetic nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lijun [Iowa State Univ., Ames, IA (United States)

    2011-01-01

    Here we report structural and functional studies of Mms6, a biomineralization protein that can promote the formation in vitro of magnetic nanoparticles with sizes and morphologies similar to the magnetites synthesized by magnetotactic bacteria. We found the binding pattern of Mms6 to ferric ion to be two-phase and multivalent. We quantatively determined that Mms6 binds one Fe3+ with a very high affinity (Kd = 1016 M). The second phase of iron binding is multivalent and cooperative with respect to iron with a Kd in the μM range and a stoichiometry of about 20 ferric ion per protein molecule. We found that Mms6 exists in large particles of two sizes, one consisting of 20-40 monomeric units and the other of 200 units. From proteolytic digestion, ultracentrifugation and liposome fusion studies, we found that Mms6 forms a large micellar quaternary structure with the N-terminal domain self-assembling into a uniformly sized micelle and the C-terminal domain on the surface. The two-phase iron-binding pattern may be relevant to iron crystal formation. We propose that the first high affinity phase may stabilize a new conformation of the C-terminal domain that allows interaction with other C-terminal domains leading to a structural change in the multimeric protein complex that enables the second low affinity iron binding phase to organize iron and initiate crystal formation. We also observed a dimeric apparent molecular mass of the Mms6 C-terminal peptide (C21Mms6). We speculate that the C-terminal domain may form higher order quaternary arrangements on the surface of the micelle or when anchored to a membrane by the N-terminal domain. The change in fluorescence quenching in the N-terminal domain with iron binding suggests a structural integrity between the C- and N-terminal domains. The slow change in trp fluorescence as a function of time after adding iron suggests a very slow conformational change in the protein that involves

  2. Identification of Chloride Intracellular Channel Protein 3 as a Novel Gene Affecting Human Bone Formation

    DEFF Research Database (Denmark)

    Brum, A M; Leije, M; J, Schreuders-Koedam

    2017-01-01

    is diminished and more adipocytes are seen in the bone marrow, suggesting a shift in MSC lineage commitment. Identification of specific factors that stimulate osteoblast differentiation from human MSCs may deliver therapeutic targets to treat osteoporosis. The aim of this study was to identify novel genes...... an in vivo human bone formation model in which hMSCs lentivirally transduced with the CLIC3 overexpression construct were loaded onto a scaffold (hydroxyapatite-tricalcium-phosphate), implanted under the skin of NOD-SCID mice, and analyzed for bone formation 8 weeks later. CLIC3 overexpression led to a 15...

  3. Utilization of palm oil processing effluents as substrates for microbial protein production by the fungus Aspergillus oryzae

    Energy Technology Data Exchange (ETDEWEB)

    Barker, T.; Worgan, J.T.

    1981-01-01

    The filamentous fungus Aspergillus oryzae grew well on the effluents produced during the extraction of palm oil. Biomass yields of approximately 50 g/100 g organic matter were obtained which contained 40% crude protein and had BOD reductions of 85% and COD reductions of 75-80% in batch culture following optimization of growth conditions. Supplementation with an inorganic N source was necessary. The more resistant substrate constituents to biodegradation were water-soluble carbohydrate and nitrogenous material, possibly Maillard reaction products, and polyphenols.

  4. Inhibition of HCV replication by oxysterol-binding protein-related protein 4 (ORP4 through interaction with HCV NS5B and alteration of lipid droplet formation.

    Directory of Open Access Journals (Sweden)

    In-Woo Park

    Full Text Available Hepatitis C virus (HCV RNA replication involves complex interactions among the 3'x RNA element within the HCV 3' untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3' X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4, a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis.

  5. Formation of protein complex with the aid of polyethylene glycol for deproteinized natural rubber latex

    Science.gov (United States)

    Wei, Lim Keuw; Ing, Wong Kwee; Badri, Khairiah Haji; Ban, Wong Chong

    2013-11-01

    The effect of polyethylene glycol (PEG) as a deproteinizing agent in commercial natural rubber latex (NRL) onto the physicochemical properties of the NRL was investigated. Three types of PEG were used namely PEG200, PEG4000 and PEG20000 (molecular weight of 200, 4000 and 20000 g/mol respectively). The optimum amount of PEG in NRL was determined from viscosity changes, protein content and Fourier Transform Infrared spectroscopy. Level of protein reduction was affected by molecular weight of PEG. The addition of PEG in NRL reduced the protein content of NRL (3.30 %) to the lowest (2.01 %) at 0.40 phr of PEG200 due to more attractive hydrophobic interactions between short chains PEG compared to PEG4000 (2.24%) and PEG20000 (2.15%). This was verified through FTIR spectroscopy analysis by observing the primary and secondary amide peak where PEG4000 has lesser absorption at the region compared to with PEG20000.

  6. Combined effect of cortical cytoskeleton and transmembrane proteins on domain formation in biomembranes

    DEFF Research Database (Denmark)

    Sikder, K. U.; Stone, K. A.; Kumar, P. B. S.

    2014-01-01

    We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find that mic......We investigate the combined effects of transmembrane proteins and the subjacent cytoskeleton on the dynamics of phase separation in multicomponent lipid bilayers using computer simulations of a particle-based implicit solvent model for lipid membranes with soft-core interactions. We find...... that microphase separation can be achieved by the protein confinement by the cytoskeleton. Our results have relevance to the finite size of lipid rafts in the plasma membrane of mammalian cells. (C) 2014 AIP Publishing LLC....

  7. Molecular Stress-inducing Compounds Increase Osteoclast Formation in a Heat Shock Factor 1 Protein-dependent Manner*

    Science.gov (United States)

    Chai, Ryan C.; Kouspou, Michelle M.; Lang, Benjamin J.; Nguyen, Chau H.; van der Kraan, A. Gabrielle J.; Vieusseux, Jessica L.; Lim, Reece C.; Gillespie, Matthew T.; Benjamin, Ivor J.; Quinn, Julian M. W.; Price, John T.

    2014-01-01

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss. PMID:24692538

  8. Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

    Science.gov (United States)

    Chai, Ryan C; Kouspou, Michelle M; Lang, Benjamin J; Nguyen, Chau H; van der Kraan, A Gabrielle J; Vieusseux, Jessica L; Lim, Reece C; Gillespie, Matthew T; Benjamin, Ivor J; Quinn, Julian M W; Price, John T

    2014-05-09

    Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

  9. Consequences of intramolecular dityrosine formation on a DNA-protein complex: a molecular modeling study

    International Nuclear Information System (INIS)

    Gras, Julien; Sy, Denise; Eon, Severine; Charlier, Michel; Spotheim-Maurizot, Melanie

    2005-01-01

    Irradiation of the free lac repressor with γ-rays abolishes protein's ability to specifically bind operator DNA. A possible radiation-induced protein damage is a dityrosine (DTyr) formed by two spatially close radiation-induced tyrosyl radicals. We performed the molecular modeling of complexes between operator DNA and DTyr-bearing parts (headpieces) of the repressor. The presence of DTyr affects the structure and the interactions between partners. A detailed analysis allows to conclude this damage can partially account for the loss of repressor ability to bind DNA

  10. Protein haze formation in wines revisited. The stabilising effect of organic acids

    OpenAIRE

    Batista, L.; Monteiro, L.; Loureiro, V.; Teixeira, A.R.; Ferreira, R.B.

    2010-01-01

    The effect on the wine protein haze potential of five organic acids commonly encountered in wines (L(+)- tartaric, L( )-malic, citric, succinic and gluconic acids) was assessed. All five acids, tested at 20 mM, reduced dramatically the haze potential of proteins, either in wine or dissolved in water, throughout the range of pH values typical of wines (i.e., from 2.8 through 3.8). Subtle differences among the acid effects did not correlate with the number of their carboxyl groups, ...

  11. Perilipin-mediated lipid droplet formation in adipocytes promotes sterol regulatory element-binding protein-1 processing and triacylglyceride accumulation.

    Directory of Open Access Journals (Sweden)

    Yu Takahashi

    Full Text Available Sterol regulatory element-binding protein-1 (SREBP-1 has been thought to be a critical factor that assists adipogenesis. During adipogenesis SREBP-1 stimulates lipogenic gene expression, and peroxisome proliferator-activated receptor γ (PPARγ enhances perilipin (plin gene expression, resulting in generating lipid droplets (LDs to store triacylglycerol (TAG in adipocytes. Plin coats adipocyte LDs and protects them from lipolysis. Here we show in white adipose tissue (WAT of plin-/- mice that nuclear active SREBP-1 and its target gene expression, but not nuclear SREBP-2, significantly decreased on attenuated LD formation. When plin-/- mouse embryonic fibroblasts (MEFs differentiated into adipocytes, attenuated LDs were formed and nuclear SREBP-1 decreased, but enforced plin expression restored them to their original state. Since LDs are largely derived from the endoplasmic reticulum (ER, alterations in the ER cholesterol content were investigated during adipogenesis of 3T3-L1 cells. The ER cholesterol greatly reduced in differentiated adipocytes. The ER cholesterol level in plin-/- WAT was significantly higher than that of wild-type mice, suggesting that increased LD formation caused a change in ER environment along with a decrease in cholesterol. When GFP-SREBP-1 fusion proteins were exogenously expressed in 3T3-L1 cells, a mutant protein lacking the S1P cleavage site was poorly processed during adipogenesis, providing evidence of the increased canonical pathway for SREBP processing in which SREBP-1 is activated by two cleavage enzymes in the Golgi. Therefore, LD biogenesis may create the ER microenvironment favorable for SREBP-1 activation. We describe the novel interplay between LD formation and SREBP-1 activation through a positive feedback loop.

  12. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    Science.gov (United States)

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein. Copyright © 2015. Published by Elsevier Ltd.

  13. Periodontal ligament stem/progenitor cells with protein-releasing scaffolds for cementum formation and integration on dentin surface.

    Science.gov (United States)

    Cho, Hankyu; Tarafder, Solaiman; Fogge, Michael; Kao, Kristy; Lee, Chang H

    2016-11-01

    Purpose/Aim: Cementogenesis is a critical step in periodontal tissue regeneration given the essential role of cementum in anchoring teeth to the alveolar bone. This study is designed to achieve integrated cementum formation on the root surfaces of human teeth using growth factor-releasing scaffolds with periodontal ligament stem/progenitor cells (PDLSCs). Human PDLSCs were sorted by CD146 expression, and characterized using CFU-F assay and induced multi-lineage differentiation. Polycaprolactone scaffolds were fabricated using 3D printing, embedded with poly(lactic-co-glycolic acids) (PLGA) microspheres encapsulating connective tissue growth factor (CTGF), bone morphogenetic protein-2 (BMP-2), or bone morphogenetic protein-7 (BMP-7). After removing cementum on human tooth roots, PDLSC-seeded scaffolds were placed on the exposed dentin surface. After 6-week culture with cementogenic/osteogenic medium, cementum formation and integration were evaluated by histomorphometric analysis, immunofluorescence, and qRT-PCR. Periodontal ligament (PDL) cells sorted by CD146 and single-cell clones show a superior clonogenecity and multipotency as compared with heterogeneous populations. After 6 weeks, all the growth factor-delivered groups showed resurfacing of dentin with a newly formed cementum-like layer as compared with control. BMP-2 and BMP-7 showed de novo formation of tissue layers significantly thicker than all the other groups, whereas CTGF and BMP-7 resulted in significantly improved integration on the dentin surface. The de novo mineralized tissue layer seen in BMP-7-treated samples expressed cementum matrix protein 1 (CEMP1). Consistently, BMP-7 showed a significant increase in CEMP1 mRNA expression. Our findings represent important progress in stem cell-based cementum regeneration as an essential part of periodontium regeneration.

  14. Bone morphogenetic protein-induced heterotopic bone formation: What have we learned from the history of a half century?

    Directory of Open Access Journals (Sweden)

    Takenobu Katagiri, PhD

    2015-05-01

    Full Text Available Bone morphogenetic protein (BMP was originally discovered by Marshall Urist a half century ago following the observation of a unique activity that induced heterotopic bone formation in skeletal muscle tissue. The molecular mechanisms underlying the induction of heterotopic bone formation in skeletal muscle by BMPs were elucidated through the purification and molecular cloning of BMPs and identification of their functional receptors and downstream effectors, as well as from genetic disorders related to BMP activity. BMPs are important regulators of not only skeletal development and regeneration but also the homeostasis of normal skeletal muscle mass. There is still much to learn about the physiology and pathology at the interface of BMPs and skeletal muscle.

  15. Convergence of bone morphogenetic protein and laminin-1 signaling pathways promotes proliferation and colony formation by fetal mouse pancreatic cells

    International Nuclear Information System (INIS)

    Jiang Fangxu; Harrison, Leonard C.

    2005-01-01

    We previously reported that bone morphogenetic proteins (BMPs), members of the transforming growth factor superfamily, together with the basement membrane glycoprotein laminin-1 (Ln-1), promote proliferation of fetal pancreatic cells and formation of colonies containing peripheral insulin-positive cells. Here, we further investigate the cross-talk between BMP and Ln-1 signals. By RT-PCR, receptors for BMP (BMPR) (excepting BMPR-1B) and Ln-1 were expressed in the fetal pancreas between E13.5 and E17.5. Specific blocking antibodies to BMP-4 and -6 and selective BMP antagonists partially inhibited colony formation by fetal pancreas cells. Colony formation induced by BMP-6 and Ln-1 was completely abolished in a dose-dependent manner by blocking Ln-1 binding to its α 6 integrin and α-dystroglycan receptors or by blocking the Ln-1 signaling molecules, phosphatidyl-inositol-3-kinase (P13K) and MAP kinase kinase-1. These results demonstrate a convergence of BMP and Ln-1 signaling through P13K and MAP kinase pathways to induce proliferation and colony formation in E15.5 fetal mouse pancreatic cells

  16. Sulfolobus turreted icosahedral virus c92 protein responsible for the formation of pyramid-like cellular lysis structures.

    Science.gov (United States)

    Snyder, Jamie C; Brumfield, Susan K; Peng, Nan; She, Qunxin; Young, Mark J

    2011-07-01

    Host cells infected by Sulfolobus turreted icosahedral virus (STIV) have been shown to produce unusual pyramid-like structures on the cell surface. These structures represent a virus-induced lysis mechanism that is present in Archaea and appears to be distinct from the holin/endolysin system described for DNA bacteriophages. This study investigated the STIV gene products required for pyramid formation in its host Sulfolobus solfataricus. Overexpression of STIV open reading frame (ORF) c92 in S. solfataricus alone is sufficient to produce the pyramid-like lysis structures in cells. Gene disruption of c92 within STIV demonstrates that c92 is an essential protein for virus replication. Immunolocalization of c92 shows that the protein is localized to the cellular membranes forming the pyramid-like structures.

  17. SSX2 is a novel DNA-binding protein that antagonizes polycomb group body formation and gene repression

    DEFF Research Database (Denmark)

    Gjerstorff, Morten Frier; Relster, Mette Marie; Greve, Katrine Buch Viden

    2014-01-01

    Polycomb group (PcG) complexes regulate cellular identity through epigenetic programming of chromatin. Here, we show that SSX2, a germline-specific protein ectopically expressed in melanoma and other types of human cancers, is a chromatin-associated protein that antagonizes BMI1 and EZH2 PcG body...... formation and derepresses PcG target genes. SSX2 further negatively regulates the level of the PcG-associated histone mark H3K27me3 in melanoma cells, and there is a clear inverse correlation between SSX2/3 expression and H3K27me3 in spermatogenesis. However, SSX2 does not affect the overall composition...

  18. Function of AURKA protein kinase in the formation of vasculogenic mimicry in triple-negative breast cancer stem cells

    Directory of Open Access Journals (Sweden)

    Liu Y

    2016-06-01

    Full Text Available Ying Liu,1,2,* Baocun Sun,1–3,* Tieju Liu,1,2,* Xiulan Zhao,1,2 Xudong Wang,3 Yanlei Li,1,2 Jie Meng,2 Qiang Gu,1,2 Fang Liu,1,2 Xueyi Dong,1,2 Peimei Liu,2 Ran Sun,2 Nan Zhao1 1Department of Pathology, General Hospital of Tianjin Medical University, 2Department of Pathology, Tianjin Medical University, 3Department of Pathology, Cancer Hospital of Tianjin Medical University, Tianjin, People’s Republic of China *These authors contributed equally to this work Abstract: Tumor cell vasculogenic mimicry (VM, a newly defined pattern of tumor blood supply, signifies the functional plasticity of aggressive cancer cells forming vascular networks. VM and cancer stem cells (CSCs have been shown to be associated with tumor growth, local invasion, and distant metastasis. In our previous study, CSCs in triple-negative breast cancer were potential to participate in VM formation. In this study, breast CSCs were isolated from the triple-negative breast cancer cell line MDA-MB-231 by using mammosphere culture. Western blotting and reverse transcription polymerase chain reaction showed that mammosphere cells displayed an increased expression of AURKA protein kinase and stem cell marker c-myc and sox2. The VM formation by mammosphere cells was inhibited by AURKA knockdown or the addition of AURKA inhibitor MLN8237. In the meantime, MLN8237 induced the increased E-cadherin and decreased c-myc, sox2, and β-catenin expressions. The function of AURKA in VM formation was further confirmed using a xenograft-murine model. The results suggested that AURKA protein kinase is involved in VM formation of CSCs and may become a new treatment target in suppressing VM and metastasis of breast cancer. Keywords: AURKA, cancer stem cells, vasculogenic mimicry, breast cancer

  19. Hepatitis C virus (HCV) induces formation of stress granules whose proteins regulate HCV RNA replication and virus assembly and egress.

    Science.gov (United States)

    Garaigorta, Urtzi; Heim, Markus H; Boyd, Bryan; Wieland, Stefan; Chisari, Francis V

    2012-10-01

    Stress granules (SGs) are cytoplasmic structures that are induced in response to environmental stress, including viral infections. Here we report that hepatitis C virus (HCV) triggers the appearance of SGs in a PKR- and interferon (IFN)-dependent manner. Moreover, we show an inverse correlation between the presence of stress granules and the induction of IFN-stimulated proteins, i.e., MxA and USP18, in HCV-infected cells despite high-level expression of the corresponding MxA and USP18 mRNAs, suggesting that interferon-stimulated gene translation is inhibited in stress granule-containing HCV-infected cells. Finally, in short hairpin RNA (shRNA) knockdown experiments, we found that the stress granule proteins T-cell-restricted intracellular antigen 1 (TIA-1), TIA1-related protein (TIAR), and RasGAP-SH3 domain binding protein 1 (G3BP1) are required for efficient HCV RNA and protein accumulation at early time points in the infection and that G3BP1 and TIA-1 are required for intracellular and extracellular infectious virus production late in the infection, suggesting that they are required for virus assembly. In contrast, TIAR downregulation decreases extracellular infectious virus titers with little effect on intracellular RNA content or infectivity late in the infection, suggesting that it is required for infectious particle release. Collectively, these results illustrate that HCV exploits the stress granule machinery at least two ways: by inducing the formation of SGs by triggering PKR phosphorylation, thereby downregulating the translation of antiviral interferon-stimulated genes, and by co-opting SG proteins for its replication, assembly, and egress.

  20. Calcium dependent formation of tubular assemblies by recombinant S-layer proteins in vivo and in vitro

    Science.gov (United States)

    Korkmaz, Nuriye; Ostermann, Kai; Rödel, Gerhard

    2011-03-01

    Surface layer proteins have the appealing property to self-assemble in nanosized arrays in solution and on solid substrates. In this work, we characterize the formation of assembly structures of the recombinant surface layer protein SbsC of Geobacillus stearothermophilus ATTC 12980, which was tagged with enhanced green fluorescent protein and expressed in the yeast Saccharomyces cerevisiae. The tubular structures formed by the protein in vivo are retained upon bursting the cells by osmotic shock; however, their average length is decreased. During dialysis, monomers obtained by treatment with chaotropic chemicals recrystallize again to form tube-like structures. This process is strictly dependent on calcium (Ca2 + ) ions, with an optimal concentration of 10 mM. Further increase of the Ca2 + concentration results in multiple non-productive nucleation points. We further show that the lengths of the S-layer assemblies increase with time and can be controlled by pH. After 48 h, the average length at pH 9.0 is 4.13 µm compared to 2.69 µm at pH 5.5. Successful chemical deposition of platinum indicates the potential of recrystallized mSbsC-eGFP structures for nanobiotechnological applications.

  1. Differential effects of divalent cations on elk prion protein fibril formation and stability

    Science.gov (United States)

    Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, or in this...

  2. Multicompartment lipid cubic nanoparticles with high protein upload: millisecond dynamics of formation

    Czech Academy of Sciences Publication Activity Database

    Angelov, Borislav; Angelova, A.; Filippov, Sergey K.; Drechsler, M.; Štěpánek, Petr; Lesieur, S.

    2014-01-01

    Roč. 8, č. 5 (2014), s. 5216-5226 ISSN 1936-0851 R&D Projects: GA ČR GAP208/10/1600 Institutional support: RVO:61389013 Keywords : lipid- protein nanoassembly * dynamic membrane curvature * amphiphile nanoarchitectonics Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 12.881, year: 2014

  3. Formation and detection of oxidant-generated tryptophan dimers in peptides and proteins

    DEFF Research Database (Denmark)

    Carroll, Luke; Pattison, David I; Davies, Justin B

    2017-01-01

    Free radicals are produced during physiological processes including metabolism and the immune response, as well as on exposure to multiple external stimuli. Many radicals react rapidly with proteins resulting in side-chain modification, backbone fragmentation, aggregation, and changes in structure...

  4. Mechanisms of edema formation in myxedema--increased protein extravasation and relatively slow lymphatic drainage

    DEFF Research Database (Denmark)

    Parving, H H; Hansen, J M; Nielsen, S L

    1979-01-01

    of generalized edema (P less than 0.05). All variables returned to normal during l-thyroxine treatment. The extravascular accumulation of albumin, and presumably of all other plasma proteins, is important in the generalized edema typically found in myxedema. Inadequate lymphatic drainage may also explain...

  5. Microbial protein synthesis, digestion and lactation responses of cows to grass or grass-red clover silage diet supplemented with barley or oats

    Directory of Open Access Journals (Sweden)

    A. VANHATALO

    2008-12-01

    Full Text Available The study was conducted to evaluate effects of silage type (grass-red clover vs. pure grass and grain supplement (oats vs. barley on rumen fermentation, post-ruminal nutrient flows, diet digestion and milk production. Four primiparous Finnish Ayrshire cows fitted with cannulae in the rumen and duodenum were used in a 4 × 4 Latin square experiment with four 28-d experimental periods and 2 × 2 factorial arrangements of treatments. Using red clover-containing (40% silage rather than pure grass silage had minor effects on rumen fermentation or diet digestion but increased non-ammonia nitrogen (N flow in terms of increased flows of microbial and dietary N entering to the small intestine. This was reflected as a reduced ruminal N degradability on grass-red clover diets. Furthermore, grass-red clover diets in comparison to grass silage diets increased milk lactose concentration and yields of milk, protein and lactose. Feeding oats in replacement for barley had minor effects on rumen fermentation or post-ruminal non-ammonia N flows but reduced digestibility of organic matter and neutral detergent fibre in the diet. Using oats rather than barley increased yields of milk and lactose but reduced milk protein concentration. Oats also increased proportions of C18:0 and C18:1 in milk fat and reduced those of C10:0 to C16:0. It is concluded that inclusion of red clover and replacement of barley with oats in grass silage based diets have beneficial effects in dairy cow production.;

  6. Whey protein isolate gel for separation: A formation, characterization, and application study

    Science.gov (United States)

    Teo, Jiunn Yeong

    Novel microporous membranes made of whey protein isolate (WPI) were developed. Aggregates of WPI comprised the bulk of the membrane, the size and packing density of which were varied by changing CaCl2 concentration (0.05--0.3M) and WPI concentration (30--40wt%), respectively. Aggregate sizes of the membranes made with 0.3M, 0.1M, 0.05M CaCl2 were roughly 1.5mum, 1mum, and 0.8mum, respectively. Skin layer of thickness about 0.5mum was found on either side of the membrane, but the thickness could reach 5mum at 0.3M CaCl2. Additionally, the porosity of the skin layer was shown to be modifiable with the addition of surfactant. Membranes were stable in hexane with flux values on the order of 1--1000gal/ft 2·d depending on the morphology of the membrane. The molecular weight cutoffs (MWCOs) of the WPI membranes with skins were evaluated using two different methods: (i) dextran marker method and (ii) protein/vitamin marker method. Membranes were found to have MWCOs of 1,000 or greater with variations when the concentration of salt used to control aggregate size, or surfactant used to modify skin properties were selected. The microporous WPI gel was also used as a cation exchanger and a hydrophobic adsorbent. The WPI cation exchanger has a maximum capacity of 68mg cupric chloride per gram dry WPI gel at neutral pH and can be regenerated effectively by reducing the pH of the solution. The WPI gel has also been found to be an excellent adsorbent for total phenolic compounds from grape extract with a partition coefficient higher than 1000 in aqueous system. The mechanism for total phenolic compounds adsorption is believed to be physical sorption, particularly sorption/condensation of total phenolic compounds in the pores and on all surfaces of WPI gel. The gel has a low extractables of 1ng/ml.g gel, and has an isoelectric point of 5.5. Although WPI gel was made into a monolith for continuous bed chromatography, channeling problems have made it very hard to evaluate the

  7. The synergistic induction of bone formation by the osteogenic proteins of the TGF-β supergene family.

    Science.gov (United States)

    Ripamonti, Ugo; Parak, Ruqayya; Klar, Roland M; Dickens, Caroline; Dix-Peek, Thérèse; Duarte, Raquel

    2016-10-01

    The momentum to compose this Leading Opinion on the synergistic induction of bone formation suddenly arose when a simple question was formulated during a discussion session on how to boost the often limited induction of bone formation seen in clinical contexts. Re-examination of morphological and molecular data available on the rapid induction of bone formation by the recombinant human transforming growth factor-β3 (hTGF-β3) shows that hTGF-β3 replicates the synergistic induction of bone formation as invocated by binary applications of hOP-1:hTGF-β1 at 20:1 by weight when implanted in heterotopic sites of the rectus abdominis muscle of the Chacma baboon, Papio ursinus. The rapid induction of bone formation in primates by hTGF-β3 may stem from bursts of cladistic evolution, now redundant in lower animal species but still activated in primates by relatively high doses of hTGF-β3. Contrary to rodents, lagomorphs and canines, the three mammalian TGF-β isoforms induce rapid and substantial bone formation when implanted in heterotopic rectus abdominis muscle sites of P. ursinus, with unprecedented regeneration of full thickness mandibular defects with rapid mineralization and corticalization. Provocatively, thus providing potential molecular and biological rationales for the apparent redundancy of osteogenic molecular signals in primates, binary applications of recombinant human osteogenic protein-1 (hOP-1) with low doses of hTGF-β1 and -β3, synergize to induce massive ossicles in heterotopic rectus abdominis, orthotopic calvarial and mandibular sites of P. ursinus. The synergistic binary application of homologous but molecularly different soluble molecular signals has indicated that per force several secreted molecular signals are required singly, synchronously and synergistically to induce optimal osteogenesis. The morphological hallmark of the synergistic induction of bone formation is the rapid differentiation of large osteoid seams enveloping

  8. Decomposition of organic matter labelled with 14C, and microbial biomass formation in an acid soil from Piracicaba, Sao Paulo State, Brazil

    International Nuclear Information System (INIS)

    Cerri, C.C.; Volkoff, B.

    1982-01-01

    An experiment was carried out under laboratory conditions (25 0 C, 50% field capacity), using 14 C labelled sugar cane leaves, which were incorporated to a medium texture Dark-Red Latosol. The equivalent of 1 mg C/g was added to soil containing 14 mg native soil carbon. CO 2 release and microbial biomass were periodically determined. After 67 days incubation it was noted that release of native carbon was greater from soil with plant material than the control (724 μg C/g vs 424 μg/g) and that 25% of the incorporated carbon had been mineralized. Addition of plant material doubled the soil microbial biomass, which was formed due to plant material addition rather than to native soil carbon. (Author) [pt

  9. Poly-β-hydroxybutyrate Metabolism Is Unrelated to the Sporulation and Parasporal Crystal Protein Formation in Bacillus thuringiensis.

    Science.gov (United States)

    Wang, Xun; Li, Zhou; Li, Xin; Qian, Hongliang; Cai, Xia; Li, Xinfeng; He, Jin

    2016-01-01

    Poly-3-hydroxybutyrate (PHB) is a natural polymer synthesized by many bacteria as a carbon-energy storage material. It was accumulated maximally prior to the spore formation but was degraded during the process of sporulation in Bacillus thuringiensis. Intriguingly, B. thuringiensis also accumulates large amounts of insecticidal crystal proteins (ICPs) during sporulation, which requires considerable input of carbon and energy sources. How PHB accumulation affects sporulation and ICP formation remains unclear to date. Intuitively, one would imagine that accumulated PHB provides the energy required for ICP formation. Yet our current data indicate that this is not the case. First, growth curves of the deletion mutants of phaC (encoding the PHB synthase) and phaZ (encoding the PHB depolymerase) were found to be similar to the parent strain BMB171; no difference in growth rate could be observed. In addition we further constructed the cry1Ac10 ICP gene overexpression strains of BMB171 (BMB171-cry), as well as its phaC and phaZ deletion mutants ΔphaC-cry and ΔphaZ-cry to compare their spore and ICP production rates. Again, not much change of ICP production was observed among these strains either. In fact, PHB was still degraded in most ΔphaZ-cry cells as observed by transmission electron microscopy. Together these results indicated that there is no direct association between the PHB accumulation and the sporulation and ICP formation in B. thuringiensis. Some other enzymes for PHB degradation or other energy source may be responsible for the sporulation and/or ICP formation in B. thuringiensis.

  10. Elucidation of the Structure Formation of Polymer-Conjugated Proteins in Solution and Block Copolymer Templates

    Science.gov (United States)

    Ferebee, Rachel L.

    The broader technical objective of this work is to contribute to the development of enzyme-functionalized nanoporous membranes that can function as autonomous and target selective dynamic separators. The scientific objective of the research performed within this thesis is to elucidate the parameters that control the mixing of proteins in organic host materials and in block copolymers templates in particular. A "biomimetic" membrane system that uses enzymes to selectively neutralize targets and trigger a change in permeability of nanopores lined with a pH-responsive polymer has been fabricated and characterized. Mechanical and functional stability, as well as scalability, have been demonstrated for this system. Additional research has focused on the role of polymeric ligands on the solubility characteristics of the model protein, Bovine Serum Albumin (BSA). For this purpose BSA was conjugated with poly(ethylene glycol) (PEG) ligands of varied degree of polymerization and grafting density. Combined static and dynamic light scattering was used (in conjunction with MALDI-TOF) to determine the second virial coefficient in PBS solutions. At a given mass fraction PEG or average number of grafts, the solubility of BSA-PEG conjugates is found to increase with the degree of polymerization of conjugated PEG. This result informs the synthesis of protein-conjugate systems that are optimized for the fabrication of block copolymer blend materials with maximum protein loading. Blends of BSA-PEG conjugates and block copolymer (BCP) matrices were fabricated to evaluate the dispersion morphology and solubility limits in a model system. Electron microscopy was used to evaluate the changes in lamellar spacing with increased filling fraction of BSA-PEG conjugates.

  11. Plasma pre beta-HDL formation is decreased by atorvastatin treatment in type 2 diabetes mellitus : Role of phospholipid transfer protein

    NARCIS (Netherlands)

    Dallinga-Thie, G. M.; van Tol, A.; Dullaart, R. P. F.

    Atorvastatin lowers plasma phospholipid transfer protein (PLTP) activity, which stimulates pre-beta-HDL, generation in vitro. We determined the effect of atorvastatin on pre-beta-HDL formation and its relation with PLTP activity in type 2 diabetes. Methods: Plasma pre-beta-HDL formation as well as

  12. Short communication: Effects of increasing protein and energy in the milk replacer with or without direct-fed microbial supplementation on growth and performance of preweaned Holstein calves.

    Science.gov (United States)

    Geiger, A J; Ward, S H; Williams, C C; Rude, B J; Cabrera, C J; Kalestch, K N; Voelz, B E

    2014-11-01

    Forty-four Holstein calves were fed a direct-fed microbial (DFM) and 1 of 2 milk replacers to evaluate calf performance and growth. Treatments were (1) a control milk replacer [22:20; 22% crude protein (CP) and 20% fat], (2) an accelerated milk replacer (27:10; 27% CP and 10% fat), (3) the control milk replacer with added DFM (22:20+D), and (4) the accelerated milk replacer with added DFM (27:10+D). Dry matter intake, rectal temperatures, respiration scores and rates, and fecal scores were collected daily. Body weight, hip and withers height, heart girth, blood, and rumen fluid samples were collected weekly. Effects of treatment, sex, week, and their interactions were analyzed. Calves fed an accelerated milk replacer, regardless of DFM supplementation, consumed more CP and metabolizable energy in the milk replacer. No treatment differences were found for starter intake or intake of neutral detergent fiber or acid detergent fiber in the starter. Calves fed the accelerated milk replacer had greater preweaning and weaning body weight compared with calves fed the control milk replacer. Average daily gain was greater during the preweaning period for calves fed the accelerated milk replacer, but the same pattern did not hold true during the postweaning period. Feed efficiency did not differ among treatments. Hip height tended to be and withers height and heart girth were greater at weaning for calves fed the accelerated milk replacer compared with calves fed the control milk replacer. Fecal scores were greatest in calves fed DFM. Overall acetate, propionate, butyrate, and n-valerate concentrations were lower in calves fed the accelerated milk replacer, but DFM did not have an effect. Rumen pH was not different. Blood metabolites were unaffected by DFM supplementation, but calves fed the accelerated milk replacer had increased partial pressure of CO2, bicarbonate, and total bicarbonate in the blood. Direct-fed microbial supplementation did not appear to benefit the calf

  13. Modulation of Wound Healing and Scar Formation by MG53 Protein-mediated Cell Membrane Repair*

    Science.gov (United States)

    Li, Haichang; Duann, Pu; Lin, Pei-Hui; Zhao, Li; Fan, Zhaobo; Tan, Tao; Zhou, Xinyu; Sun, Mingzhai; Fu, Minghuan; Orange, Matthew; Sermersheim, Matthew; Ma, Hanley; He, Duofen; Steinberg, Steven M.; Higgins, Robert; Zhu, Hua; John, Elizabeth; Zeng, Chunyu; Guan, Jianjun; Ma, Jianjie

    2015-01-01

    Cell membrane repair is an important aspect of physiology, and disruption of this process can result in pathophysiology in a number of different tissues, including wound healing, chronic ulcer and scarring. We have previously identified a novel tripartite motif family protein, MG53, as an essential component of the cell membrane repair machinery. Here we report the functional role of MG53 in the modulation of wound healing and scarring. Although MG53 is absent from keratinocytes and fibroblasts, remarkable defects in skin architecture and collagen overproduction are observed in mg53−/− mice, and these animals display delayed wound healing and abnormal scarring. Recombinant human MG53 (rhMG53) protein, encapsulated in a hydrogel formulation, facilitates wound healing and prevents scarring in rodent models of dermal injuries. An in vitro study shows that rhMG53 protects against acute injury to keratinocytes and facilitates the migration of fibroblasts in response to scratch wounding. During fibrotic remodeling, rhMG53 interferes with TGF-β-dependent activation of myofibroblast differentiation. The resulting down-regulation of α smooth muscle actin and extracellular matrix proteins contributes to reduced scarring. Overall, these studies establish a trifunctional role for MG53 as a facilitator of rapid injury repair, a mediator of cell migration, and a modulator of myofibroblast differentiation during wound healing. Targeting the functional interaction between MG53 and TGF-β signaling may present a potentially effective means for promoting scarless wound healing. PMID:26306047

  14. Dynamic Filament Formation by a Divergent Bacterial Actin-Like ParM Protein.

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    Anthony J Brzoska

    Full Text Available Actin-like proteins (Alps are a diverse family of proteins whose genes are abundant in the chromosomes and mobile genetic elements of many bacteria. The low-copy-number staphylococcal multiresistance plasmid pSK41 encodes ParM, an Alp involved in efficient plasmid partitioning. pSK41 ParM has previously been shown to form filaments in vitro that are structurally dissimilar to those formed by other bacterial Alps. The mechanistic implications of these differences are not known. In order to gain insights into the properties and behavior of the pSK41 ParM Alp in vivo, we reconstituted the parMRC system in the ectopic rod-shaped host, E. coli, which is larger and more genetically amenable than the native host, Staphylococcus aureus. Fluorescence microscopy showed a functional fusion protein, ParM-YFP, formed straight filaments in vivo when expressed in isolation. Strikingly, however, in the presence of ParR and parC, ParM-YFP adopted a dramatically different structure, instead forming axial curved filaments. Time-lapse imaging and selective photobleaching experiments revealed that, in the presence of all components of the parMRC system, ParM-YFP filaments were dynamic in nature. Finally, molecular dissection of the parMRC operon revealed that all components of the system are essential for the generation of dynamic filaments.

  15. Role of the Disulfide Bond in Prion Protein Amyloid Formation: A Thermodynamic and Kinetic Analysis.

    Science.gov (United States)

    Honda, Ryo

    2018-02-27

    Prion diseases are associated with the structural conversion of prion protein (PrP) to a β-sheet-rich aggregate, PrP Sc . Previous studies have indicated that a reduction of the disulfide bond linking C179 and C214 of PrP yields an amyloidlike β-rich aggregate in vitro. To gain mechanistic insights into the reduction-induced aggregation, here I characterized how disulfide bond reduction modulates the protein folding/misfolding landscape of PrP, by examining 1) the equilibrium stabilities of the native (N) and aggregated states relative to the unfolded (U) state, 2) the transition barrier separating the U and aggregated states, and 3) the final structure of amyloidlike misfolded aggregates. Kinetic and thermodynamic experiments revealed that disulfide bond reduction decreases the equilibrium stabilities of both the N and aggregated states by ∼3 kcal/mol, without changing either the amyloidlike aggregate structure, at least at the secondary structural level, or the transition barrier of aggregation. Therefore, disulfide bond reduction modulates the protein folding/misfolding landscape by entropically stabilizing disordered states, including the U and transition state of aggregation. This also indicates that the equilibrium stability of the N state, but not the transition barrier of aggregation, is the dominant factor determining the reduction-induced aggregation of PrP. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Filament formation of the Escherichia coli actin-related protein, MreB, in fission yeast.

    Science.gov (United States)

    Srinivasan, Ramanujam; Mishra, Mithilesh; Murata-Hori, Maki; Balasubramanian, Mohan K

    2007-02-06

    Proteins structurally related to eukaryotic actins have recently been identified in several prokaryotic organisms. These actin-like proteins (MreB and ParM) and the deviant Walker A ATPase (SopA) play a key role in DNA segregation and assemble into polymers in vitro and in vivo. MreB also plays a role in cellular morphogenesis. Whereas the dynamic properties of eukaryotic actins have been extensively characterized, those of bacterial actins are only beginning to emerge. We have established the fission yeast Schizosaccharomyces pombe as a cellular model for the functional analysis of the Escherichia coli actin-related protein MreB. We show that MreB organizes into linear bundles that grow in a symmetrically bidirectional manner at 0.46 +/- 0.03 microm/min, with new monomers and/or oligomers being added along the entire length of the bundle. Organization of linear arrays was dependent on the ATPase activity of MreB, and their alignment along the cellular long axis was achieved by sliding along the cortex of the cylindrical part of the cell. The cell ends appeared to provide a physical barrier for bundle elongation. These experiments provide new insights into the mechanism of assembly and organization of the bacterial actin cytoskeleton.

  17. Formation of W(3)A(1) electron-transferring flavoprotein (ETF) hydroquinone in the trimethylamine dehydrogenase x ETF protein complex.

    Science.gov (United States)

    Jang, M H; Scrutton, N S; Hille, R

    2000-04-28

    The electron-transferring flavoprotein (ETF) from Methylophilus methylotrophus (sp. W(3)A(1)) exhibits unusual oxidation-reduction properties and can only be reduced to the level of the semiquinone under most circumstances (including turnover with its physiological reductant, trimethylamine dehydrogenase (TMADH), or reaction with strong reducing reagents such as sodium dithionite). In the present study, we demonstrate that ETF can be reduced fully to its hydroquinone form both enzymatically and chemically when it is in complex with TMADH. Quantitative titration of the TMADH x ETF protein complex with sodium dithionite shows that a total of five electrons are taken up by the system, indicating that full reduction of ETF occurs within the complex. The results indicate that the oxidation-reduction properties of ETF are perturbed upon binding to TMADH, a conclusion further supported by the observation of a spectral change upon formation of the TMADH x ETF complex that is due to a change in the environment of the FAD of ETF. The results are discussed in the context of ETF undergoing a conformational change during formation of the TMADH x ETF electron transfer complex, which modulates the spectral and oxidation-reduction properties of ETF such that full reduction of the protein can take place.

  18. Introduction of caveolae structural proteins into the protozoan Toxoplasma results in the formation of heterologous caveolae but not caveolar endocytosis.

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    Bao Lige

    Full Text Available Present on the plasma membrane of most metazoans, caveolae are specialized microdomains implicated in several endocytic and trafficking mechanisms. Caveolins and the more recently discovered cavins are the major protein components of caveolae. Previous studies reported that caveolar invaginations can be induced de novo on the surface of caveolae-negative mammalian cells upon heterologous expression of caveolin-1. However, it remains undocumented whether other components in the transfected cells participate in caveolae formation. To address this issue, we have exploited the protozoan Toxoplasma as a heterologous expression system to provide insights into the minimal requirements for caveogenesis and caveolar endocytosis. Upon expression of caveolin-1, Toxoplasma accumulates prototypical exocytic caveolae 'precursors' in the cytoplasm. Toxoplasma expressing caveolin-1 alone, or in conjunction with cavin-1, neither develops surface-located caveolae nor internalizes caveolar ligands. These data suggest that the formation of functional caveolae at the plasma membrane in Toxoplasma and, by inference in all non-mammalian cells, requires effectors other than caveolin-1 and cavin-1. Interestingly, Toxoplasma co-expressing caveolin-1 and cavin-1 displays an impressive spiraled network of membranes containing the two proteins, in the cytoplasm. This suggests a synergistic activity of caveolin-1 and cavin-1 in the morphogenesis and remodeling of membranes, as illustrated for Toxoplasma.

  19. Lrit1, a Retinal Transmembrane Protein, Regulates Selective Synapse Formation in Cone Photoreceptor Cells and Visual Acuity.

    Science.gov (United States)

    Ueno, Akiko; Omori, Yoshihiro; Sugita, Yuko; Watanabe, Satoshi; Chaya, Taro; Kozuka, Takashi; Kon, Tetsuo; Yoshida, Satoyo; Matsushita, Kenji; Kuwahara, Ryusuke; Kajimura, Naoko; Okada, Yasushi; Furukawa, Takahisa

    2018-03-27

    In the vertebrate retina, cone photoreceptors play crucial roles in photopic vision by transmitting light-evoked signals to ON- and/or OFF-bipolar cells. However, the mechanisms underlying selective synapse formation in the cone photoreceptor pathway remain poorly understood. Here, we found that Lrit1, a leucine-rich transmembrane protein, localizes to the photoreceptor synaptic terminal and regulates the synaptic connection between cone photoreceptors and cone ON-bipolar cells. Lrit1-deficient retinas exhibit an aberrant morphology of cone photoreceptor pedicles, as well as an impairment of signal transmission from cone photoreceptors to cone ON-bipolar cells. Furthermore, we demonstrated that Lrit1 interacts with Frmpd2, a photoreceptor scaffold protein, and with mGluR6, an ON-bipolar cell-specific glutamate receptor. Additionally, Lrit1-null mice showed visual acuity impairments in their optokinetic responses. These results suggest that the Frmpd2-Lrit1-mGluR6 axis regulates selective synapse formation in cone photoreceptors and is essential for normal visual function. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

    Energy Technology Data Exchange (ETDEWEB)

    Takanashi, Keisuke; Yamaguchi, Atsushi, E-mail: atsyama@restaff.chiba-u.jp

    2014-09-26

    Highlights: • Aggregation of ALS-linked FUS mutant sequesters ALS-associated RNA-binding proteins (FUS wt, hnRNP A1, and hnRNP A2). • Aggregation of ALS-linked FUS mutant sequesters SMN1 in the detergent-insoluble fraction. • Aggregation of ALS-linked FUS mutant reduced the number of speckles in the nucleus. • Overproduced ALS-linked FUS mutant reduced the number of processing-bodies (PBs). - Abstract: Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS m