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Sample records for psychrophilic alkaline serine

  1. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

    TECHNICAL ABSTRACT A putative protease gene (aprE) from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated the enzyme had...

  2. Growth energetics of an alkaline serine protease-producing strain of Bacillus clausii during continuous cultivation

    DEFF Research Database (Denmark)

    Christiansen, Torben; Nielsen, Jens

    2002-01-01

    the theoretical co-factor and building block requirements needed for biomass formation were calculated. Using the stoichiometric models and data for growth on glucose and citrate the amount of ATP needed for biomass synthesis was estimated to 42.0 mmol ATP/gDW, the P/O ratio to 0.68 and the ATP maintenance to 2......Glucose-limited chemostats were used to determine the growth yields of biomass of Bacillus clausii PP 473-8 producing an alkaline serine protease Savinase (Novozymes A/S, Bagsvaerd, Denmark) and a low yield of biomass on oxygen was observed. The energy metabolism was investigated further by setting...... up simple stoichiometric models for growth on glucose and citrate. In order to determine the parameters in the models, a macromolecular biomass composition was determined based on measured values of protein and RNA combined with literature data. From the macromolecular composition of the biomass...

  3. Purification and characterization of manganese-dependent alkaline serine protease from Bacillus pumilus TMS55.

    Science.gov (United States)

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Karutha Pandian, Shunmugiah

    2011-01-01

    The purification and characterization of a Mn2+-dependent alkaline serine protease produced by Bacillus pumilus TMS55 were investigated. The enzyme was purified in three steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by gel filtration and cation-exchange chromatography. The purified protease had a molecular mass of approximately 35 kDa, was highly active over a broad pH range of 7.0 to 12.0, and remained stable over a pH range of 7.5 to 11.5. The optimum temperature for the enzyme activity was found to be 60 degreesC. PMSF and AEBSF (1 mM) significantly inhibited the protease activity, indicating that the protease is a serine protease. Mn2+ ions enhanced the activity and stability of the enzyme. In addition, the purified protease remained stable with oxidants (H2O2, 2%) and organic solvents (25%), such as benzene, hexane, and toluene. Therefore, these characteristics of the protease and its dehairing ability indicate its potential for a wide range of commercial applications.

  4. A review on production of serine alkaline protease by Bacillus spp

    Directory of Open Access Journals (Sweden)

    Biswanath Bhunia

    2012-04-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 In recent times, protease has gained considerable importance in the world market. Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes. Serine alkaline proteases (SAP are one of the most important groups of industrial enzymes. They account for approximately 35% of the total microbial enzyme sales. Serine protease is produced by various types of fermentation techniques using microorganism. Among the proteases, bacterial proteases are more significant than animal and fungal proteases. Bacillus is the most invigorated species producing extracellular proteases among many bacterial species that have found tremendous application in pharmaceutical, leather, laundry and food processing industry. Mathematical modeling of fermentation process helps understand the relationship between protease production and bacterial growth to provide quantitative information on the behavior of the system. Therefore, high level production of protease in industrial scale should be made feasible. The focus of the present review is to provide an updated overview of fermentative production and the factors that influence production, growth kinetics and downstream processing of serine alkaline protease. /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New Roman"; mso-fareast-theme-font:minor-fareast; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:"Times New Roman"; mso-bidi-theme-font:minor-bidi;}

  5. Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

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    Aysha Kamran

    2015-06-01

    Full Text Available Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80 and other commercial detergents (SDS, Triton X-100.

  6. Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus

    NARCIS (Netherlands)

    van der Laan, J.M.; Teplyakov, A.V.; Kelders, H.; Kalk, K.H.; Misset, O.; Mulleners, L.J.S.M.; Dijkstra, B.W.

    1992-01-01

    The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 Å resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has

  7. Molecular cloning, sequence and structural analysis of dehairing Mn(2+) dependent alkaline serine protease (MASPT) of Bacillus pumilus TMS55.

    Science.gov (United States)

    Ibrahim, Kalibulla Syed; Muniyandi, Jeyaraj; Pandian, Shunmugiah Karutha

    2011-10-01

    Leather industries release a large amount of pollution-causing chemicals which creates one of the major industrial pollutions. The development of enzyme based processes as a potent alternative to pollution-causing chemicals is useful to overcome this issue. Proteases are enzymes which have extensive applications in leather processing and in several bioremediation processes due to their high alkaline protease activity and dehairing efficacy. In the present study, we report cloning, characterization of a Mn2+ dependent alkaline serine protease gene (MASPT) of Bacillus pumilus TMS55. The gene encoding the protease from B. pumilus TMS55 was cloned and its nucleotide sequence was determined. This gene has an open reading frame (ORF) of 1,149 bp that encodes a polypeptide of 383 amino acid residues. Our analysis showed that this polypeptide is composed of 29 residues N-terminal signal peptide, a propeptide of 79 residues and a mature protein of 275 amino acids. We performed bioinformatics analysis to compare MASPT enzyme with other proteases. Homology modeling was employed to model three dimensional structure for MASPT. Structural analysis showed that MASPT structure is composed of nine α-helices and nine β-strands. It has 3 catalytic residues and 14 metal binding residues. Docking analysis showed that residues S223, A260, N263, T328 and S329 interact with Mn2+. This study allows initial inferences about the structure of the protease and will allow the rational design of its derivatives for structure-function studies and also for further improvement of the enzyme.

  8. Alkaline serine protease AprE plays an essential role in poly-γ-glutamate production during natto fermentation.

    Science.gov (United States)

    Kada, Shigeki; Ishikawa, Atsushi; Ohshima, Yoshifumi; Yoshida, Ken-ichi

    2013-01-01

    Natto is a traditional Japanese food made from soybeans fermented by natto starter strains of Bacillus subtilis natto. It has been suggested that extracellular protease activity released by the bacteria are involved in the production of poly-γ-glutamate (γ-PGA) during natto fermentation. One of the natto starters, strain r22, possesses at least seven genes, each of which encoded an extracellular protease orthologous to its counterpart in B. subtilis 168, aprE, bpr, epr, mpr, nprE, vpr, and wprA, but it was found to lack nprB. Inactivating the aprE ortholog alone resulted in a severe decrease in γ-PGA production and in the total extracellular protease activity. The defect in γ-PGA production of the mutant lacking the aprE ortholog was complemented when the medium was supplemented with sufficient glutamate. These results suggest that the alkaline serine protease encoded by aprE plays an indispensable role in supplying materials to produce γ-PGA. On the other hand, simultaneous inactivation of all the protease genes except for aprE did not significantly affect either γ-PGA production or total protease activity.

  9. Comparative proteome analysis of psychrophilic versus mesophilic bacterial species: Insights into the molecular basis of cold adaptation of proteins

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    Reddy Boojala

    2009-01-01

    Full Text Available Abstract Background Cold adapted or psychrophilic organisms grow at low temperatures, where most of other organisms cannot grow. This adaptation requires a vast array of sequence, structural and physiological adjustments. To understand the molecular basis of cold adaptation of proteins, we analyzed proteomes of psychrophilic and mesophilic bacterial species and compared the differences in amino acid composition and substitution patterns to investigate their likely association with growth temperatures. Results In psychrophilic bacteria, serine, aspartic acid, threonine and alanine are overrepresented in the coil regions of secondary structures, whilst glutamic acid and leucine are underrepresented in the helical regions. Compared to mesophiles, psychrophiles comprise a significantly higher proportion of amino acids that contribute to higher protein flexibility in the coil regions of proteins, such as those with tiny/small or neutral side chains. Amino acids with aliphatic, basic, aromatic and hydrophilic side chains are underrepresented in the helical regions of proteins of psychrophiles. The patterns of amino acid substitutions between the orthologous proteins of psychrophiles versus mesophiles are significantly different for several amino acids when compared to their substitutions in orthologous proteins of within the mesophiles or psychrophiles. Conclusion Current results provide quantitative substitution preferences (or avoidance of amino acids that lead to the adaptation of proteins to cold temperatures. These finding would help future efforts in selecting mutations for rational design of proteins with enhanced psychrophilic properties.

  10. Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

    Science.gov (United States)

    Yildirim, Vildan; Baltaci, Mustafa Ozkan; Ozgencli, Ilknur; Sisecioglu, Melda; Adiguzel, Ahmet; Adiguzel, Gulsah

    2017-12-01

    An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 μmol.mL(-)(1).min(-)(1), respectively.

  11. Effect of pH and temperature on stability and kinetics of novel extracellular serine alkaline protease (70 kDa).

    Science.gov (United States)

    Bhunia, Biswanath; Basak, Bikram; Mandal, Tamal; Bhattacharya, Pinaki; Dey, Apurba

    2013-03-01

    A novel extracellular serine protease (70 kDa by SDS-PAGE) was purified and characterized. This enzyme retained more than 93% of its initial activity after preincubation for 30 min at 37 °C in the presence of 25% (v/v) tested organic solvents and showed feather degradation activity. The purified enzyme was deactivated at various combinations of pH and temperature to examine the interactive effect of them on enzyme activity. The deactivation process was modeled as first-order kinetics and the deactivation rate constant (k(d)) was found to be minimum at pH 9 and 37 °C. The kinetic analysis of enzyme over a range of pH values indicated two pK values at 6.21 and at 10.92. The lower pK value was likely due to the catalytic histidine in the free enzyme and higher pK value likely reflected deprotonation of the proline moiety of the substrate but ionization of the active site serine is another possibility. Inhibition kinetic showed that enzyme is serine protease because enzyme was competitively inhibited by antipain and aprotinin as these compounds are known to be competitive inhibitors of serine protease. The organic solvent, thermal and pH tolerances of enzyme suggested that it may have potential for use as a biocatalyst in industry.

  12. Comparative void-volume analysis of psychrophilic and mesophilic enzymes: Structural bioinformatics of psychrophilic enzymes reveals sources of core flexibility

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    Bystroff Christopher

    2011-10-01

    Full Text Available Abstract Background Psychrophiles, cold-adapted organisms, have adapted to live at low temperatures by using a variety of mechanisms. Their enzymes are active at cold temperatures by being structurally more flexible than mesophilic enzymes. Even though, there are some indications of the possible structural mechanisms by which psychrophilic enzymes are catalytic active at cold temperatures, there is not a generalized structural property common to all psychrophilic enzymes. Results We examine twenty homologous enzyme pairs from psychrophiles and mesophiles to investigate flexibility as a key characteristic for cold adaptation. B-factors in protein X-ray structures are one way to measure flexibility. Comparing psychrophilic to mesophilic protein B-factors reveals that psychrophilic enzymes are more flexible in 5-turn and strand secondary structures. Enzyme cavities, identified using CASTp at various probe sizes, indicate that psychrophilic enzymes have larger average cavity sizes at probe radii of 1.4-1.5 Å, sufficient for water molecules. Furthermore, amino acid side chains lining these cavities show an increased frequency of acidic groups in psychrophilic enzymes. Conclusions These findings suggest that embedded water molecules may play a significant role in cavity flexibility, and therefore, overall protein flexibility. Thus, our results point to the important role enzyme flexibility plays in adaptation to cold environments.

  13. Biodiversity and cold adaptive mechanisms of psychrophiles

    Directory of Open Access Journals (Sweden)

    Yuhua Xin

    2013-07-01

    Full Text Available Cold-adapted bacteria and archaea are widely distributed in cold environments on Earth, such as permafrost, cold soils and deserts, glaciers, lakes, sea ice in the Arctic, Antarctic and high mountains, as well as the deep sea, ice caves and the atmospheric stratosphere etc. Cold-adapted organisms inhabiting these environments exhibit rich diversity. Studies on the biogeography of psychrophiles will enable us to understand their biodiversity, distribution and origins. Due to long-term living in cold regions, cold-adapted bacteria and archeae have developed specific physiological mechanisms of adaptation to cold environments. These mechanisms include: regulating the fluidity of the cytoplasmic membrane through adjusting the composition of membrane lipids; achieving low-temperature protection through compatibility solute, antifreeze proteins, ice-binding proteins, ice-nucleation proteins and anti-nucleating proteins; production of heat-shock and coldshock proteins, cold acclimation protein and DEAD-box RNA helicase at low temperatures; production of cold-active enzymes; increasing energy generation and conservation. With the rapid development of sequencing technology, various omics-based approaches have been used to reveal cold-adaptive mechanisms of psychrophiles at the genomic level.

  14. Psychrophilic yeasts and their biotechnological applications - A review

    African Journals Online (AJOL)

    SAM

    2014-05-28

    May 28, 2014 ... Psychrophilic yeast produces cold-active enzymes having numerous applications in .... group of enzymes that hydrolyze the pectic substances present in .... antarctica for the preparation of optically active alcohols has been ...

  15. Psychrophilic and Mesophilic Fungi in Frozen Food Products

    Science.gov (United States)

    Kuehn, Harold H.; Gunderson, Millard F.

    1963-01-01

    The mold flora of certain frozen pastries and chicken pies was investigated. Molds were determined qualitatively or quantitatively, or both, by preparing pour plates of the blended product and incubating the plates at various temperatures. The mesophilic fungal flora developed on plates incubated at 10 and 20 C, whereas psychrophilic fungi were obtained on plates incubated at 0 and 5 C. About 2,000 cultures of fungi, representing about 100 different species, were isolated from various products. Four different brands of blueberry, two brands of cherry pastries, two brands of apple, and one brand of raspberry pastries were examined. In addition, two brands of chicken pies were studied. Blueberry pastries had a much higher total fungal population than the other products, although different brands of blueberry pastries varied considerably. Blueberry pastries had from 347 to 1,586 psychrophilic fungi per g. Cherry pastries had about 70 to 110 psychrophiles per g, and apple pastries had 19 to 92 psychrophiles per g. Chicken pies contained very few psychrophilic fungi, about 15 per g. Aureobasidium pullulans was recovered most frequently. About 90% of the psychrophilic fungi found in blueberry products was A. pullulans. Depending upon the brand of cherry pastry, either Phoma spp. or A. pullulans was the most common fungus present. Apple pastries also displayed brand variation, but were unique in having many mesophilic aspergilli. This genus was generally absent from other products. The Penicillium content of apple pastries was also rather high; 50% of the psychrophilic flora was represented by this genus. The psychrophilic fungal flora of chicken pies was composed primarily of penicillia (50%) and Chrysosporium pannorum (46%). PMID:13927344

  16. Genomics of an extreme psychrophile, Psychromonas ingrahamii

    Directory of Open Access Journals (Sweden)

    Hauser Loren J

    2008-05-01

    Full Text Available Abstract Background The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures. Determination of the whole genome sequence allows comparison with genes of other psychrophiles and mesophiles. Results Correspondence analysis of the composition of all P. ingrahamii proteins showed that (1 there are 6 classes of proteins, at least one more than other bacteria, (2 integral inner membrane proteins are not sharply separated from bulk proteins suggesting that, overall, they may have a lower hydrophobic character, and (3 there is strong opposition between asparagine and the oxygen-sensitive amino acids methionine, arginine, cysteine and histidine and (4 one of the previously unseen clusters of proteins has a high proportion of "orphan" hypothetical proteins, raising the possibility these are cold-specific proteins. Based on annotation of proteins by sequence similarity, (1 P. ingrahamii has a large number (61 of regulators of cyclic GDP, suggesting that this bacterium produces an extracellular polysaccharide that may help sequester water or lower the freezing point in the vicinity of the cell. (2 P. ingrahamii has genes for production of the osmolyte, betaine choline, which may balance the osmotic pressure as sea ice freezes. (3 P. ingrahamii has a large number (11 of three-subunit TRAP systems that may play an important role in the transport of nutrients into the cell at low temperatures. (4 Chaperones and stress proteins may play a critical role in transforming nascent polypeptides into 3-dimensional configurations that permit low temperature growth. (5 Metabolic properties of P. ingrahamii were deduced. Finally, a few small sets of proteins of unknown function which may play a role in psychrophily have been singled out as worthy of future study. Conclusion The results of this genomic analysis

  17. Genomics of an extreme psychrophile, Psychromonas ingrahamii

    Energy Technology Data Exchange (ETDEWEB)

    Riley, Monica [Woods Hole Research Center, Woods Hole, MA; Staley, James T. [University of Washington, Seattle; Danchin, Antoine [Universite Louis Pasteur, France; Wang, T. [University of Washington; Brettin, Tom [Los Alamos National Laboratory (LANL); Hauser, Loren John [ORNL; Land, Miriam L [ORNL; Thompson, Linda S [Los Alamos National Laboratory (LANL)

    2008-05-01

    Background: The genome sequence of the sea-ice bacterium Psychromonas ingrahamii 37, which grows exponentially at -12C, may reveal features that help to explain how this extreme psychrophile is able to grow at such low temperatures. Determination of the whole genome sequence allows comparison with genes of other psychrophiles and mesophiles. Results: Correspondence analysis of the composition of all P. ingrahamii proteins showed that (1) there are 6 classes of proteins, at least one more than other bacteria, (2) integral inner membrane proteins are not sharply separated from bulk proteins suggesting that, overall, they may have a lower hydrophobic character, and (3) there is strong opposition between asparagine and the oxygen-sensitive amino acids methionine, arginine, cysteine and histidine and (4) one of the previously unseen clusters of proteins has a high proportion of "orphan" hypothetical proteins, raising the possibility these are cold-specific proteins. Based on annotation of proteins by sequence similarity, (1) P. ingrahamii has a large number (61) of regulators of cyclic GDP, suggesting that this bacterium produces an extracellular polysaccharide that may help sequester water or lower the freezing point in the vicinity of the cell. (2) P. ingrahamii has genes for production of the osmolyte, betaine choline, which may balance the osmotic pressure as sea ice freezes. (3) P. ingrahamii has a large number (11) of three-subunit TRAP systems that may play an important role in the transport of nutrients into the cell at low temperatures. (4) Chaperones and stress proteins may play a critical role in transforming nascent polypeptides into 3-dimensional configurations that permit low temperature growth. (5) Metabolic properties of P. ingrahamii were deduced. Finally, a few small sets of proteins of unknown function which may play a role in psychrophily have been singled out as worthy of future study. Conclusion: The results of this genomic analysis provide a

  18. Isolation of novel psychrophilic bacteria from Arctic sea ice

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    The phylogenetic diversity of culturable psychrophilic bacteria associated with sea ice from the high latitude regions of Canadian Basin and Chukchi Sea, Arctic, was investigated. A total of 34 psychropilic strains were isolated using three methods of (Ⅰ) dilution plating (at 4 ℃), (Ⅱ) bath culturing (at -1 ℃) and dilution plating, and (Ⅲ) cold shock (-20 ℃ for 24 h), bath culturing and dilution plating under aerobic conditions. Sea-ice samples were exposed to -20 ℃ for 24 h that might reduce the number of common microorganisms and encourage outgrowth of psychrophilic strains. This process might be able to be introduced to isolation psychrophilic bacteria from other environmental samples in future study. 16S rDNA nearly full-length sequence analysis revealed that psychrophilic strains felled in two phylogenetic divisions, γ-proteobacteria (in the genera Colwellia、Marinobacter、Shewanella、Glaciecola、Marinomonas and Pseudoalteromonas) and Cytophaga-Flexibacter-Bacteroides (Flavobacterium and Psychroflexus). Fifteen of bacterial isolates quite likely represented novel species (16S rDNA sequence similarity below 98%). One of strains (BSi20002) from Canadian Basin showed 100% sequence similarity to that of Marinobacter sp. ANT8277 isolated from the Antarctic Weddell sea ice, suggesting bacteria may have a bipolar distribution at the species level.

  19. A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, characterization and l-serine production.

    Science.gov (United States)

    Jiang, Wei; Xia, Bingzhao; Liu, Ziduo

    2013-10-01

    Currently, l-serine is mainly produced by enzymatic conversion, in which serine hydroxymethyltransferase (SHMT) is the key enzyme, suggesting the importance of searching for a SHMT with high activity. Shewanella algae, a methanol-utilizing marine bacterium showing high SHMT activity, was selected based on screening bacterial strains and comparison of the activities of SHMTs. A glyA was isolated from the S. algae through thermal asymmetric interlaced PCR (TAIL-PCR) and it encoded a 417 amino acid polypeptide. The SaSHMT, encoded by the glyA, showed the optimal activity at 50°C and pH 7.0, and retained over 45% of its maximal activity after incubation at 40°C for 3h. The enzyme showed better stability under alkaline environment (pH 6.5-9.0) than Hyphomicrobium methylovorum GM2's SHMT (pH 6.0-7.5). The SaSHMT can produce 77.76mM of l-serine by enzymatic conversion, with the molecular conversion rate in catalyzing glycine to l-serine being 1.41-fold higher than that of Escherichia coli. Therefore, the SaSHMT has the potential for industrial applications due to its tolerance of alkaline environment and a relatively high enzymatic conversion rate. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Serine Hydroxymethyltransferase from the Cold Adapted Microorganism Psychromonas ingrahamii: A Low Temperature Active Enzyme with Broad Substrate Specificity

    Directory of Open Access Journals (Sweden)

    Stefano Pascarella

    2012-01-01

    Full Text Available Serine hydroxymethyltransferase from the psychrophilic microorganism Psychromonas ingrahamii was expressed in Escherichia coli and purified as a His-tag fusion protein. The enzyme was characterized with respect to its spectroscopic, catalytic, and thermodynamic properties. The properties of the psychrophilic enzyme have been contrasted with the characteristics of the homologous counterpart from E. coli, which has been structurally and functionally characterized in depth and with which it shares 75% sequence identity. Spectroscopic measures confirmed that the psychrophilic enzyme displays structural properties almost identical to those of the mesophilic counterpart. At variance, the P. ingrahamii enzyme showed decreased thermostability and high specific activity at low temperature, both of which are typical features of cold adapted enzymes. Furthermore, it was a more efficient biocatalyst compared to E. coli serine hydroxymethyltransferase (SHMT particularly for side reactions. Many β-hydroxy-α-amino acids are SHMT substrates and represent important compounds in the synthesis of pharmaceuticals, agrochemicals and food additives. Thanks to these attractive properties, this enzyme could have a significant potential for biotechnological applications.

  1. Psychrophilic and Mesophilic Fungi in Fruit-Filled Pastries

    Science.gov (United States)

    Kuehn, Harold H.; Gunderson, Millard F.

    1962-01-01

    Surveys of the mold flora of frozen blueberry and cherry pastries were undertaken. Molds were enumerated by preparing pour plates of the blended product and incubating the plates at 0, 5, 10, and 20 C. In this manner, the total fungal content of the product could be ascertained from the 10 and 20 C plates, and the psychrophilic fungal population was represented by those fungi which grew at 0 and 5 C. The pastry portion, or crust, of the blueberry material was sampled separately from the filling portion. Certain differences in fungal flora were apparent. Aureobasidium pullulans was the dominant fungus in crust at all temperatures of isolation. However, Penicillium thomii proved to be the most common mesophilic fungus in the filling portion, and A. pullulans was the most common psychrophile in the filling. Aspergilli were quite common in the crust, but, in general, were absent from the fruit filling. Cherry pastries had a much smaller total fungal flora than did the blueberry product. However, A. pullulans again was the most prevalent fungus in cherry pastries at all temperatures of isolation. Certain differences in fungal flora were apparent in the two fruit products. Phoma spp. were almost completely absent in blueberries, but represented the second most common fungus in cherry pastries. Blueberry filling had 440 psychrophilic fungi per gram of sample (at 0 C), blueberry crust had 65 per gram, and cherry pastries had 77 per gram. Images FIG. 1 PMID:14460237

  2. Anaerobic psychrophiles from Lake Zub and Lake Untersee, Antarctica

    Science.gov (United States)

    Townsend, Alisa; Pikuta, Elena V.; Guisler, Melissa; Stahl, Sarah; Hoover, Richard B.

    2009-08-01

    The study of samples from Antarctica 2008 and 2009 expeditions organized and successfully conducted by Richard Hoover led to the isolation of diverse anaerobic strains with psychrotolerant and psychrophilic physiology. Due to the fact that Lake Untersee has never been subject to microbiological study, this work with the samples has significant and pioneering impact to the knowledge about the biology of this unique ecosystem. Also, the astrobiological significance for the study of these ecosystems is based on new findings of ice covered water systems on other bodies of our solar system. Anaerobic psychrotolerant strain LZ-22 was isolated from a frozen sample of green moss with soils around the rhizosphere collected near Lake Zub in Antarctica. Morphology of strain LZ-22 was observed to be motile, rod shaped and spore-forming cells with sizes 1 x 5-10 μm. This new isolate is a mesophile with the maximum temperature of growth at 40°C. Strain LZ-22 is able to live on media without NaCl and in media with up to 7 % (w/v) NaCl. It is catalase negative and grows only on sugars with the best growth rate being on lactose. The strain is a neutrophile and grows between pH 5 and 9.0 with the optimum at 7.8. Another two strains UL7-96mG and LU-96m7P were isolated from deep water samples of Lake Untersee. Proteolytic strain LU-96m7P had a truly psychrophilic nature and refused to grow at room temperature. Sugarlytic strain UL7-96mG was found to be psychrotolerant, but its rate of growth at 3°C was very high compared with other mesophiles. Two homoacetogenic psychrophilic strains A7AC-96m and AC-DS7 were isolated and purified from samples of Lake Untersee; both of them are able to grow chemolithotrophically on H2+CO2. In the presence of lactate, these strains are able to grow only at 0-18 °C, and growth at 22 °C was observed only with yeast extract stimulation. In this paper, physiological and morphological characteristics of novel psychrophilic and psychrotolerant isolates

  3. Anaerobic Psychrophiles from Lake Zub and Lake Untersee, Antarctica

    Science.gov (United States)

    Townsend, Alisa; Pikuta, Elena V.; Guisler, Melissa; Stahl, Sarah; Hoover, Richard B.

    2009-01-01

    The study of samples from Antarctica 2008 and 2009 expeditions organized and successfully conducted by Richard Hoover led to the isolation of diverse anaerobic strains with psychrotolerant and psychrophilic physiology. Due to the fact that Lake Untersee has never been subject to microbiological study, this work with the samples has significant and pioneering impact to the knowledge about the biology of this unique ecosystem. Also, the astrobiological significance for the study of these ecosystems is based on new findings of ice covered water systems on other bodies of our solar system. Anaerobic psychrotolerant strain LZ-22 was isolated from a frozen sample of green moss with soils around the rhizosphere collected near Lake Zub in Antarctica. Morphology of strain LZ-22 was observed to be motile, rod shaped and spore-forming cells with sizes 1 x 5-10 micron. This new isolate is a mesophile with the maximum temperature of growth at 40C. Strain LZ-22 is able to live on media without NaCl and in media with up to 7% (w/v) NaCl. It is catalase negative and grows only on sugars with the best growth rate being on lactose. The strain is a neutrophile and grows between pH 5 and 9.0 with the optimum at 7.8. Another two strains UL7-96mG and LU-96m7P were isolated from deep water samples of Lake Untersee. Proteolytic strain LU-96m7P had a truly psychrophilic nature and refused to grow at room temperature. Sugarlytic strain UL7-96mG was found to be psychrotolerant, but its rate of growth at 3C was very high compared with other mesophiles. Two homoacetogenic psychrophilic strains A7AC-96m and AC-DS7 were isolated and purified from samples of Lake Untersee; both of them are able to grow chemolithotrophically on H2+CO2. In the presence of lactate, these strains are able to grow only at 0-18C, and growth at 22C was observed only with yeast extract stimulation. In this paper, physiological and morphological characteristics of novel psychrophilic and psychrotolerant isolates from

  4. Psychrophilic Enzymes: From Folding to Function and Biotechnology

    Directory of Open Access Journals (Sweden)

    Georges Feller

    2013-01-01

    Full Text Available Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

  5. Optimization to Low Temperature Activity in Psychrophilic Enzymes

    Directory of Open Access Journals (Sweden)

    Caroline Struvay

    2012-09-01

    Full Text Available Psychrophiles, i.e., organisms thriving permanently at near-zero temperatures, synthesize cold-active enzymes to sustain their cell cycle. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. Considering the subtle structural adjustments required for low temperature activity, directed evolution appears to be the most suitable methodology to engineer cold activity in biological catalysts.

  6. Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments

    DEFF Research Database (Denmark)

    Knoblauch, C.; Jørgensen, BB; Harder, J.

    1999-01-01

    The numbers of sulfate reducers in two Arctic sediments within situ temperatures of 2.6 and -1.7 degrees C were determined. Most-probable-number counts were higher at 10 degrees C than at 20 degrees C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates...... of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than...

  7. Community size and metabolic rates of psychrophilic sulfate-reducing bacteria in Arctic marine sediments

    Energy Technology Data Exchange (ETDEWEB)

    Knoblauch, C.; Joergensen, B.B.; Harder, J.

    1999-09-01

    The numbers of sulfate reducers in two Arctic sediments with in situ temperatures of 2.6 and {minus}1.7C were determined. Most-probable-number counts were higher at 10 C than at 20 C, indicating the predominance of a psychrophilic community. Mean specific sulfate reduction rates of 19 isolated psychrophiles were compared to corresponding rates of 9 marine, mesophilic sulfate-reducing bacteria. The results indicate that, as a physiological adaptation to the permanently cold Arctic environment, psychrophilic sulfate reducers have considerably higher specific metabolic rates than their mesophilic counterparts at similarly low temperatures.

  8. Psychrophilic yeasts from worldwide glacial habitats: diversity, adaptation strategies and biotechnological potential.

    Science.gov (United States)

    Buzzini, Pietro; Branda, Eva; Goretti, Marta; Turchetti, Benedetta

    2012-11-01

    Glacial habitats (cryosphere) include some of the largest unexplored and extreme biospheres on Earth. These habitats harbor a wide diversity of psychrophilic prokaryotic and eukaryotic microorganisms. These highly specialized microorganisms have developed adaptation strategies to overcome the direct and indirect life-endangering influence of low temperatures. For many years Antarctica has been the geographic area preferred by microbiologists for studying the diversity of psychrophilic microorganisms (including yeasts). However, there have been an increasing number of studies on psychrophilic yeasts sharing the non-Antarctic cryosphere. The present paper provides an overview of the distribution and adaptation strategies of psychrophilic yeasts worldwide. Attention is also focused on their biotechnological potential, especially on their exploitation as a source of cold-active enzymes and for bioremediation purposes.

  9. Halophilic-Psychrophilic Bacteria from Tirich Mir Glacier, Pakistan, as Potential Candidate for Astrobiological Studies

    Science.gov (United States)

    Rafiq, M. R.; Anesio, A. M. A.; Hayat, M. H.; Zada, S. Z.; Sajjad, W. S.; Shah, A. A. S.; Hasan, F. H.

    2016-09-01

    Hindu Kush, Karakoram, and Himalaya region is referred to as 'third pole' and could be suitable as a terrestrial analog of Mars and increased possibility of finding polyextremophiles. Study is focused on halophilic psychrophiles.

  10. Psychrophilic and Psychrotolerant Microbial Extremophiles in Polar Environments

    Science.gov (United States)

    Hoover, Richard B.; Pikuta, Elena V.

    2010-01-01

    The microbial extremophiles that inhabit the polar regions of our planet are of tremendous significance. The psychrophilic and psychrotolerant microorganisms, which inhabit all of the cold environments on Earth have important applications to Bioremediation, Medicine, Pharmaceuticals, and many other areas of Biotechnology. Until recently, most of the research on polar microorganisms was confined to studies of polar diatoms, yeast, fungi and cyanobacteria. However, within the past three decades, extensive studies have been conducted to understand the bacteria and archaea that inhabit the Arctic and Antarctic sea-ice, glaciers, ice sheets, permafrost and the cryptoendolithic, cryoconite and ice-bubble environments. These investigations have resulted in the discovery of many new genera and species of anaerobic and aerobic microbial extremophiles. Exotic enzymes, cold-shock proteins and pigments produced by some of the extremophiles from polar environments have the potential to be of great benefit to Mankind. Knowledge about microbial life in the polar regions is crucial to understanding the limitations and biodiversity of life on Earth and may provide valuable clues to the Origin of Life on Earth. The discovery of viable microorganisms in ancient ice from the Fox Tunnel, Alaska and the deep Vostok Ice has shown that microorganisms can remain alive while cryopreserved in ancient ice. The psychrophilic lithoautotrophic homoacetogen isolated from the deep anoxic trough of Lake Untersee is an ideal candidate for life that might inhabit comets or the polar caps of Mars. The spontaneous release of gas from within the Anuchin Glacier above Lake Untersee may provide clues to the ice geysers that erupt from the tiger stripe regions of Saturn s moon Enceladus. The methane productivity in the lower regimes of Lake Untersee may also provide insights into possible mechanisms for the recently discovered methane releases on Mars. Since most of the other water bearing bodies of our

  11. [ATP pool and bioluminescence in psychrophilic bacteria Photobacterium phosphoreum].

    Science.gov (United States)

    Alekserova, L É; Alenina, K A; Efremenko, E N; Mazhul', M M; Piskunova, N F; Ismailov, A D

    2014-01-01

    Bioluminescence activity and ATP pool were investigated in the culture of psychrophilic bacteria Photobacterium phosphoreum collected-from the exponential and stationary growth phases, as well as immobilized in polyvinyl alcohol (PVA) cryogel. In liquid culture, ATP pool remained at an almost a constant level throughout the luminescence cycle (over 100 h). The ATP pool in the stationary-phase and PVA-immobilizedl cells remained constant throughout their incubation in the medium (over 200 h) and in 3% NaCl solution (over 100 h): Quantitative assessment of integral photon yield and ATP pool indicated that bioluminescence decay in growing or stationary cells was not caused by limitation by the energy substrates of the luciferase reaction. Kinetic and quantitative parameters of emission activity and ATP pool excluded the possibility of formation of the aldehyde substrate for luciferase via reduction of the relevant fatty acids in NADPH and ATP-dependent reductase reaction and its oxidation in the monooxygenase reaction. Our results indicate that the aliphatic aldehyde is not utilized in the process of light emission.

  12. Psychrophilic yeasts in glacial environments of Alpine glaciers.

    Science.gov (United States)

    Turchetti, Benedetta; Buzzini, Pietro; Goretti, Marta; Branda, Eva; Diolaiuti, Guglielmina; D'Agata, Carlo; Smiraglia, Claudio; Vaughan-Martini, Ann

    2008-01-01

    The presence of psychrophilic yeasts in supra- and subglacial sediments, ice and meltwater collected from two glaciers of the Italian Alps (Forni and Sforzellina-Ortles-Cevedale group) was investigated. After incubation at 4 degrees C, subglacial sediments contained from 1.3 x 10(3) to 9.6 x 10(3) CFU of yeasts g(-1). The number of yeast cells in supraglacial sediments was c. 10-100-fold lower. A significant proportion of isolated yeasts exhibited one or more extracellular enzymatic activities (starch-degrading, lipolytic, esterolytic, proteolytic and pectinolytic activity) at 4 degrees C. Selected isolates were able to grow at 2 degrees C under laboratory-simulated in situ conditions. In all, 106 isolated yeasts were identified by MSP-PCR fingerprinting and 26S rRNA gene sequencing of the D1/D2 region as belonging to 10 species: Aureobasidium pullulans, Cryptococcus gilvescens (over 50% of the total), Cryptococcus terricolus, Mrakia gelida, Naganishia globosa, Rhodotorula glacialis, Rhodotorula psychrophenolica, Rhodotorula bacarum, Rhodotorula creatinivora and Rhodotorula laryngis. Four strains, all belonging to a new yeast species, yet to be described, were also isolated.

  13. Denitrification characteristics of a marine origin psychrophilic aerobic denitrifying bacterium

    Institute of Scientific and Technical Information of China (English)

    Haiyan Zheng; Ying Liu; Guangdong Sun; Xiyan Gao; Qingling Zhang; Zhipei Liu

    2011-01-01

    A psychrophilic aerobic denitrifying bacterium,strain S1-1,was isolated from a biological aerated filter conducted for treatment of recirculating water in a marine aquaculture system.Strain S1-1 was preliminarily identified as Psychrobacter sp.based on the analysis of its 16S rRNA gene sequence,which showed 100% sequence similarity to that of Psychrobacter sp.TSBY-70.Strain S 1-1 grew well either in high nitrate or high nitrite conditions with a removal of 100% nitrate or 63.50% nitrite,and the total nitrogen removal rates could reach to 46.48% and 31.89%,respectively.The results indicated that nitrate was mainly reduced in its logarithmic growth phase with a very low leve 1 accumulation of nitrite,suggesting that the aerobic denitrification process of strain S l-1 occurred mainly in this phase.The GC-MS results showed that N2O was formed as the major intermediate during the aerobic denitrifying process of strain S1-1.Finally,factors affecting the growth of strain Sl-1 and its aerobic denitrifying ability were also investigated.Results showed that the optimum aerobic denitrification conditions for strain S1-1 were sodium succinate as carbon source,C/N ratio15,salinity 10 g/L NaCl,incubation temperature 20℃ and initial pH 6.5.

  14. The structure of ferricytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H

    Science.gov (United States)

    Harvilla, Paul B.; Wolcott, Holly N.

    2014-01-01

    Approximately 40% of all proteins are metalloproteins, and approximately 80% of Earth’s ecosystems are at temperatures ≤ 5 °C, including 90% of the global ocean. Thus, an essential aspect of marine metallobiochemistry is an understanding of the structure, dynamics, and mechanisms of cold adaptation of metalloproteins from marine microorganisms. Here, the molecular structure of the electron-transfer protein cytochrome c552 from the psychrophilic marine bacterium Colwellia psychrerythraea 34H has been determined by X-ray crystallography (PDB: 4O1W). The structure is highly superimposable with that of the homologous cytochrome from the mesophile Marinobacter hydrocarbonoclasticus. Based on structural analysis and comparison of psychrophilic, psychrotolerant, and mesophilic sequences, a methionine-based ligand-substitution mechanism for psychrophilic protein stabilization is proposed. PMID:24727932

  15. Psychrophilic anaerobic digestion of swine manure slurry in sequencing batch reactors

    Energy Technology Data Exchange (ETDEWEB)

    Masse, D.I. [Agriculture Canada, Ottawa, ON (Canada). Food Research Branch; Droste, R.L. [Ottawa Univ., ON (Canada). Dept. of Civil Engineering

    1993-12-31

    This work presents preliminary results of an ongoing laboratory study to evaluate the feasibility of psychrophilic anaerobic digestion in sequencing batch reactors (SBR) for stabilizing, deodorizing and adding value to swine manure. Preliminary results show that the process is feasible. (author). 14 refs., 7 tabs.

  16. Isolation and Physiological Characterization of Psychrophilic Denitrifying Bacteria from Permanently Cold Arctic Fjord Sediments (Svalbard, Norway)

    Science.gov (United States)

    Canion, Andy; Prakash, Om; Green, Stefan J.; Jahnke, Linda; Kuypers, Marcel M. M.; Kostka, Joel E.

    2013-01-01

    A large proportion of reactive nitrogen loss from polar sediments is mediated by denitrification, but microorganisms mediating denitrification in polar environments remain poorly characterized. A combined approach of most-probable-number (MPN) enumeration, cultivation and physiological characterization was used to describe psychrophilic denitrifying bacterial communities in sediments of three Arctic fjords in Svalbard (Norway). A MPN assay showed the presence of 10(sup 3)-10(sup 6) cells of psychrophilic nitrate-respiring bacteria g(sup -1) of sediment. Fifteen strains within the Proteobacteria were isolated using a systematic enrichment approach with organic acids as electron donors and nitrate as an electron acceptor. Isolates belonged to five genera, including Shewanella, Pseudomonas, Psychromonas (Gammaproteobacteria), Arcobacter (Epsilonproteobacteria) and Herminiimonas (Betaproteobacteria). All isolates were denitrifiers, except Shewanella, which exhibited the capacity for dissimilatory nitrate reduction to ammonium (DNRA). Growth from 0 to 40 degC demonstrated that all genera except Shewanella were psychrophiles with optimal growth below 15 degC, and adaptation to low temperature was demonstrated as a shift from primarily C16:0 saturated fatty acids to C16:1 monounsaturated fatty acids at lower temperatures. This study provides the first targeted enrichment and characterization of psychrophilic denitrifying bacteria from polar sediments, and two genera, Arcobacter and Herminiimonas, are isolated for the first time from permanently cold marine sediments.

  17. Efficient aspartic acid production by a psychrophile-based simple biocatalyst.

    Science.gov (United States)

    Tajima, Takahisa; Hamada, Mai; Nakashimada, Yutaka; Kato, Junichi

    2015-10-01

    We previously constructed a Psychrophile-based Simple bioCatalyst (PSCat) reaction system, in which psychrophilic metabolic enzymes are inactivated by heat treatment, and used it here to study the conversion of aspartic acid from fumaric acid mediated by the activity of aspartate ammonia-lyase (aspartase). In Escherichia coli, the biosynthesis of aspartic acid competes with that of L-malic acid produced from fumaric acid by fumarase. In this study, E. coli aspartase was expressed in psychrophilic Shewanella livingstonensis Ac10 heat treated at 50 °C for 15 min. The resultant PSCat could convert fumaric acid to aspartic acid without the formation of L-malic acid because of heat inactivation of psychrophilic fumarase activity. Furthermore, alginate-immobilized PSCat produced high yields of aspartic acid and could be re-used nine times. The results of our study suggest that PSCat can be applied in biotechnological production as a new approach to increase the yield of target compounds.

  18. Draft Genome Sequence of Sclerotinia borealis, a Psychrophilic Plant Pathogenic Fungus.

    Science.gov (United States)

    Mardanov, Andrey V; Beletsky, Alexey V; Kadnikov, Vitaly V; Ignatov, Alexander N; Ravin, Nikolai V

    2014-01-23

    Sclerotinia borealis is a necrotrophic phytopathogenic fungus notable for its wide host range and environmental persistence. It grows at low temperatures, causing snow mold disease of crop plants. To understand the molecular mechanisms of its pathogenesis and adaptation to the psychrophilic lifestyle, we determined the 39.3-Mb draft genome sequence of S. borealis F-4128.

  19. Effects of alkaline earth metal ion complexation on amino acid zwitterion stability: Results from infrared action spectroscopy

    NARCIS (Netherlands)

    Bush, M. F.; Oomens, J.; Saykally, R. J.; Williams, E. R.

    2008-01-01

    The structures of isolated alkaline earth metal cationized amino acids are investigated using infrared multiple photon dissociation (IRMPD) spectroscopy and theory. These results indicate that arginine, glutamine, proline, serine, and valine all adopt zwitterionic structures when complexed with diva

  20. An update on serine deficiency disorders

    NARCIS (Netherlands)

    van der Crabben, S. N.; Verhoeven-Duif, N. M.; Brilstra, E. H.; Van Maldergem, L.; Coskun, T.; Rubio-Gozalbo, E.; Berger, R.; de Koning, T. J.

    Serine deficiency disorders are caused by a defect in one of the three synthesising enzymes of the L-serine biosynthesis pathway. Serine deficiency disorders give rise to a neurological phenotype with psychomotor retardation, microcephaly and seizures in newborns and children or progressive

  1. [Serine proteinases of lower vertebrates].

    Science.gov (United States)

    Kolodzeĭskaia, M V

    1986-01-01

    Recent data on the effect of serine proteinases of lower vertebrates are generalized. Hydrolysis specificity and kinetics of different synthetic substrates, dependence of the activity of enzymes on pH, their irreversible inhibition by chloromethyl ketones of amino acids and peptides as well as high-molecular proteinase inhibitors are considered in detail. The data testify to the fact that chymotrypsins and trypsins of higher vertebrates and serine proteinases of lower vertebrates act as an acid-base catalysis. Enzymes in the pyloric cacca of fishes are in the state of proenzymes and are transformed into an active form with the aid of their own proteolytic factors. The esterase and proteolytic activity of fish proteinases is concentrated in the same active site and reaches the highest values at pH 7,8. New data are presented on particularities of the lower vertebrate proteinases, on the similarity and differences in their specificity. A distinct difference is shown in the nature of the binding site of the active centre in a number of serine proteinases of fishes as compared to chymotrypsin and trypsin of higher vertebrates.

  2. Thermodynamic Stability of Psychrophilic and Mesophilic Pheromones of the Protozoan Ciliate Euplotes

    Directory of Open Access Journals (Sweden)

    Kurt Wüthrich

    2013-01-01

    Full Text Available Three psychrophilic protein pheromones (En-1, En-2 and En-6 from the polar ciliate, Euplotes nobilii, and six mesophilic pheromones (Er-1, Er-2, Er-10, Er-11, Er-22 and Er-23 from the temperate-water sister species, Euplotes raikovi, were studied in aqueous solution for their thermal unfolding and refolding based on the temperature dependence of their circular dichroism (CD spectra. The three psychrophilic proteins showed thermal unfolding with mid points in the temperature range 55–70 °C. In contrast, no unfolding was observed for any of the six mesophilic proteins and their regular secondary structures were maintained up to 95 °C. Possible causes of these differences are discussed based on comparisons of the NMR structures of the nine proteins.

  3. Cloning and expression of phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica PI12

    Science.gov (United States)

    Jaafar, Nardiah Rizwana; Bakar, Farah Diba Abu; Murad, Abdul Munir Abdul; Mahadi, Nor Muhammad

    2015-09-01

    The conversion of 3-phosphoglycerate to 2-phosphoglycerate during glycolysis and gluconeogenesis is catalyzed by phosphoglycerate mutase (PGM). Better understanding of metabolic reactions performed by this enzyme has been studied extensively in prokaryotes and eukaryotes. Here, we report a phosphoglycerate mutase from the psychrophilic yeast, Glaciozyma antarctica. cDNA encoding for PGM from G. antarctica PI12, a psychrophilic yeast isolated from sea ice at Casey Station, Antarctica was amplified. The gene was then cloned into a cloning vector and sequenced, which verified its identity as the gene putatively encoding for PGM. The recombinant protein was expressed in Escherichia coli BL21 (DE3) as inclusion bodies and this was confirmed by SDS-PAGE and Western blot.

  4. Start-up of a sequential dry anaerobic digestion of paunch under psychrophilic and mesophilic temperatures.

    Science.gov (United States)

    Nkemka, Valentine Nkongndem; Hao, Xiying

    2016-06-21

    The present laboratory study evaluated the sequential leach bed dry anaerobic digestion (DAD) of paunch under psychrophilic (22°C) and mesophilic (40°C) temperatures. Three leach bed reactors were operated under the mesophilic temperature in sequence at a solid retention time (SRT) of 40d with a new batch started 27d into the run of the previous one. A total of six batches were operated for 135d. The results showed that the mesophilic DAD of paunch was efficient, reaching methane yields of 126.9-212.1mLg(-1) volatile solid (VS) and a VS reduction of 32.9-55.5%. The average daily methane production rate increased from 334.3mLd(-1) to 571.4mLd(-1) and 825.7mLd(-1) when one, two and three leach bed reactors were in operation, respectively. The psychrophilic DAD of paunch was operated under a SRT of 100d and a total of three batches were performed in sequence for 300d with each batch starting after completion of the previous one. Improvements in the methane yield from 93.9 to 107.3 and 148.3mLg(-1) VS and VS reductions of 24.8, 30.2 and 38.6% were obtained in the consecutive runs, indicating the adaptation of anaerobic microbes from mesophilic to psychrophilic temperatures. In addition, it took three runs for anaerobic microbes to reduce the volatile fatty acid accumulation observed in the first and second trials. This study demonstrates the potential of renewable energy recovery from paunch under psychrophilic and mesophilic temperatures. Copyright © 2016. Published by Elsevier Ltd.

  5. Endotoxin structures in the psychrophiles Psychromonas marina and Psychrobacter cryohalolentis contain distinctive acyl features.

    Science.gov (United States)

    Sweet, Charles R; Alpuche, Giancarlo M; Landis, Corinne A; Sandman, Benjamin C

    2014-07-09

    Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide), a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME) GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2-) units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

  6. Endotoxin Structures in the Psychrophiles Psychromonas marina and Psychrobacter cryohalolentis Contain Distinctive Acyl Features

    Directory of Open Access Journals (Sweden)

    Charles R. Sweet

    2014-07-01

    Full Text Available Lipid A is the essential component of endotoxin (Gram-negative lipopolysaccharide, a potent immunostimulatory compound. As the outer surface of the outer membrane, the details of lipid A structure are crucial not only to bacterial pathogenesis but also to membrane integrity. This work characterizes the structure of lipid A in two psychrophiles, Psychromonas marina and Psychrobacter cryohalolentis, and also two mesophiles to which they are related using MALDI-TOF MS and fatty acid methyl ester (FAME GC-MS. P. marina lipid A is strikingly similar to that of Escherichia coli in organization and total acyl size, but incorporates an unusual doubly unsaturated tetradecadienoyl acyl residue. P. cryohalolentis also shows structural organization similar to a closely related mesophile, Acinetobacter baumannii, however it has generally shorter acyl constituents and shows many acyl variants differing by single methylene (-CH2- units, a characteristic it shares with the one previously reported psychrotolerant lipid A structure. This work is the first detailed structural characterization of lipid A from an obligate psychrophile and the second from a psychrotolerant species. It reveals distinctive structural features of psychrophilic lipid A in comparison to that of related mesophiles which suggest constitutive adaptations to maintain outer membrane fluidity in cold environments.

  7. Deep-sea fungi as a source of alkaline and cold-tolerant proteases

    Digital Repository Service at National Institute of Oceanography (India)

    Damare, S.; Raghukumar, C.; Muraleedharan, U.; Raghukumar, S.

    and Chang CS. Bleach resistant alkaline protease produced by a Bacillus sp. Isolated from the Korean polychaete, Periserrula leucophryna. Proc Biochem 2004;39:1441-7. [12] Turkiewicz M, Gromek E, Kalinowska H and Zielinska M, Biosynthesis and properties... o C Serine protease [33] Paecilomyces lilacinus Biocontrol agent Azocasein [34] Bacillus sp Korean polychaete Casein 1?g tyrosine min -1 10.0 45-50 o C Serine protease [11] Aspergillus ustus NIOCC #20 Deep-sea sediment Azocasein 1639 ACU m...

  8. Serine recombinases as tools for genome engineering.

    Science.gov (United States)

    Brown, William R A; Lee, Nicholas C O; Xu, Zhengyao; Smith, Margaret C M

    2011-04-01

    The serine recombinases differ mechanistically from the tyrosine recombinases and include proteins such as ϕC31 integrase which, unlike Cre and Flp, promote unidirectional reactions. The serine recombinase family is large and includes many other proteins besides ϕC31 integrase with the potential to be widely used in genome engineering. Here we review the details of the mechanism of the reactions promoted by the serine recombinases and discuss how these not only limit the utility of this class of recombinase but also creates opportunities for the engineering of new enzymes. We discuss the unanswered questions posed by genome engineering experiments in a variety of systems in which the serine recombinases have been used and finally describe more recently discovered serine recombinases that have the potential to be used in genome engineering.

  9. l-Serine Production by a Mutant of Sarcina albida Defective in l-Serine Degradation

    Science.gov (United States)

    Omori, Kenji; Kakimoto, Toshio; Chibata, Ichiro

    1983-01-01

    For improved l-serine production, an l-serine dehydratase-defective mutant of Sarcina albida IAM 1012 was obtained. In the mutant, the activities of the enzymes responsible for l-serine production were as high as those in the parent strain, and, at a low glycine concentration, the mutant accumulated l-serine more efficiently than the parent. Under optimum conditions, 21 mg of l-serine per ml accumulated from 100 mg of glycine per ml. l-Serine was isolated from a reaction mixture as l-serine m-xylene-4-sulfonate, and free amino acid was obtained in high yields by use of an ion-exchange resin. Residual glycine was recovered at a yield of 61%. PMID:16346305

  10. D-serine and serine racemase are associated with PSD-95 and glutamatergic synapse stability

    Directory of Open Access Journals (Sweden)

    Hong eLin

    2016-02-01

    Full Text Available D-serine is an endogenous coagonist at the glycine site of synaptic NMDA receptors (NMDARs, synthesized by serine racemase (SR through conversion of L-serine. It is crucial for synaptic plasticity and is implicated in schizophrenia. Our previous studies demonstrated specific loss of SR, D-serine-responsive synaptic NMDARs, and glutamatergic synapses in cortical neurons lacking alpha7 nicotinic acetylcholine receptors, which promotes glutamatergic synapse formation and maturation during development. We thus hypothesize that D-serine and SR (D-serine/SR are associated with glutamatergic synaptic development. Using morphological and molecular studies in cortical neuronal cultures, we demonstrate that D-serine/SR are associated with PSD-95 and NMDARs in postsynaptic neurons and with glutamatergic synapse stability during synaptic development. Endogenous D-serine and SR colocalize with PSD-95, but not presynaptic vesicular glutamate transporter 1 (VGLUT1, in glutamatergic synapses of cultured cortical neurons. Low-density astrocytes in cortical neuronal cultures lack SR expression but contain enriched D-serine in large vesicle-like structures, suggesting possible synthesis of D-serine in postsynaptic neurons and storage in astrocytes. More interestingly, endogenous D-serine and SR colocalize with PSD-95 in the postsynaptic terminals of glutamatergic synapses during early and late synaptic development, implicating involvement of D-serine/SR in glutamatergic synaptic development. Exogenous application of D-serine enhances the interactions of SR with PSD-95 and NR1, and increases the number of VGLUT1- and PSD-95-positive glutamatergic synapses, suggesting that exogenous D-serine enhances postsynaptic SR/PSD-95 signaling and stabilizes glutamatergic synapses during cortical synaptic development. This is blocked by NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5 and 7-chlorokynurenic acid (7-CK, a specific antagonist at the glycine site of NMDARs

  11. Anodes for alkaline electrolysis

    Science.gov (United States)

    Soloveichik, Grigorii Lev

    2011-02-01

    A method of making an anode for alkaline electrolysis cells includes adsorption of precursor material on a carbonaceous material, conversion of the precursor material to hydroxide form and conversion of precursor material from hydroxide form to oxy-hydroxide form within the alkaline electrolysis cell.

  12. Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

    Science.gov (United States)

    Lonhienne, T; Baise, E; Feller, G; Bouriotis, V; Gerday, C

    2001-02-09

    Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

  13. Psychrophilic proteases dramatically reduce single cell RNA-seq artifacts: A molecular atlas of kidney development.

    Science.gov (United States)

    Adam, Mike; Potter, Andrew S; Potter, S Steven

    2017-08-29

    Single cell RNA-seq is a powerful methodology. Nevertheless there are important limitations, including the technical challenges of breaking down an organ or tissue into a single cell suspension. Invariably this has required enzymatic incubation at 37°C, which can be expected to result in artifact changes in gene expression patterns. We here describe a dissociation method that uses a protease with high activity in the cold, purified from a psychrophilic microorganism. The entire procedure is carried out at 6°C or colder, where mammalian transcriptional machinery is largely inactive, thereby effectively "freezing in" the in vivo gene expression patterns. To test this method we carried out RNA-seq on 20,424 single cells from P1 mouse kidneys, comparing the results of the psychrophilic protease method with procedures using 37°C incubation. We show that the cold protease method provides a great reduction in gene expression artifacts. In addition the results produce a single cell resolution gene expression atlas of the newborn mouse kidney, an interesting time in development when mature nephrons are present yet nephrogenesis remains extremely active. © 2017. Published by The Company of Biologists Ltd.

  14. Purification and characterization of cold-adapted beta-agarase from an Antarctic psychrophilic strain

    Directory of Open Access Journals (Sweden)

    Jiang Li

    2015-09-01

    Full Text Available An extracellular β-agarase was purified from Pseudoalteromonas sp. NJ21, a Psychrophilic agar-degrading bacterium isolated from Antarctic Prydz Bay sediments. The purified agarase (Aga21 revealed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with an apparent molecular weight of 80 kDa. The optimum pH and temperature of the agarase were 8.0 and 30 °C, respectively. However, it maintained as much as 85% of the maximum activities at 10 °C. Significant activation of the agarase was observed in the presence of Mg2+, Mn2+, K+; Ca2+, Na+, Ba2+, Zn2+, Cu2+, Co2+, Fe2+, Sr2+ and EDTA inhibited the enzyme activity. The enzymatic hydrolyzed product of agar was characterized as neoagarobiose. Furthermore, this work is the first evidence of cold-adapted agarase in Antarctic psychrophilic bacteria and these results indicate the potential for the Antarctic agarase as a catalyst in medicine, food and cosmetic industries.

  15. Engineering of a psychrophilic bacterium for the bioremediation of aromatic compounds.

    Science.gov (United States)

    Parrilli, Ermengilda; Papa, Rosanna; Tutino, Maria Luisa; Sannia, Giovanni

    2010-01-01

    Microbial degradation of aromatic hydrocarbons has been studied with the aim of developing applications for the removal of toxic compounds. Efforts have been directed toward the genetic manipulation of mesophilic bacteria to improve their ability to degrade pollutants, even though many pollution problems occur in sea waters and in effluents of industrial processes which are characterized by low temperatures. From these considerations the idea of engineering a psychrophilic microorganism for the oxidation of aromatic compounds was developed.In a previous paper it was demonstrated that the recombinant Antarctic Pseudoalteromonas haloplanktis TAC125 (PhTAC/tou) expressing a toluene-o-xylene monooxygenase (ToMO) is able to convert several aromatic compounds into corresponding catechols. In our work we improved the metabolic capability of PhTAC/tou cells by combining action of recombinant ToMO enzyme with that of the endogenous P. haloplanktis TAC125 laccase-like protein. This strategy allowed conferring new and specific degradative capabilities to a bacterium isolated from an unpolluted environment; indeed engineered PhTAC/tou cells are able to grow on aromatic compounds as sole carbon and energy sources. Our approach demonstrates the possibility to use the engineered psychrophilic bacterium for the bioremediation of chemically contaminated marine environments and/or cold effluents.

  16. Psychrophilic Biomass Producers in the Trophic Chain of the Microbial Community of Lake Untersee, Antarctica

    Science.gov (United States)

    Pikuta, Elena V.; Hoover, Richard B.

    2010-01-01

    The study of photosynthetic microorganisms from the Lake Untersee samples showed dispersed distribution of phototrophs within 80 m water column. Lake Untersee represents a unique ecosystem that experienced complete isolation: sealed by the Anuchin Glacier for many millennia. Consequently, its biocenosis has evolved over a significant period of time without exchange or external interaction with species from other environments. The major producers of organic matter in Lake Untersee are represented by phototrophic and chemolithotrophic microorganisms. This is the traditional trophic scheme for lacustrine ecosystems on Earth. Among the phototrophs, diatoms were not found, which differentiates this lake from other known ecosystems. The dominant species among phototrophs was Chlamydomonas sp. with typical morphostructure: green chloroplasts, bright red round spot, and two polar flagella near the opening. As expected, the physiology of studied phototrophs was limited by low temperature, which defined them as obligate psychrophilic microorganisms. By the quantity estimation of methanogenesis in this lake, the litho-autotrophic production of organic matter is competitive with phototrophic production. However, pure cultures of methanogens have not yet been obtained. We discuss the primary producers of organic matter and the participation of our novel psychrophilic homoacetogen into the litho-autotrophic link of biomass production in Lake Untersee.

  17. Antarctomyces pellizariae sp. nov., a new, endemic, blue, snow resident psychrophilic ascomycete fungus from Antarctica.

    Science.gov (United States)

    de Menezes, Graciéle C A; Godinho, Valéria M; Porto, Bárbara A; Gonçalves, Vívian N; Rosa, Luiz H

    2017-03-01

    In the present study, we have identified and characterised a new snow resident ascomycete blue stain fungus from Antarctica named Antarctomyces pellizariae sp. nov. Menezes, Godinho, Porto, Gonçalves and Rosa, using polyphasic taxonomy techniques. This fungal species was recovered from the seasonal snow of the Antarctic Peninsula. Antarctomyces pellizariae displayed different macro- and micromorphology when compared with A. psychrotrophicus Stchigel and Guarro, the only other Antarctomyces species reported until date. Antarctomyces pellizariae showed psychrophilic behavior and very low growth rate at 22-25 °C, quite different from A. psychrotrophicus that has a higher growth rate at mesophilic temperatures. In addition, micromorphological characteristics and the analysis of the nuclear rDNA internal transcribed spacer, β-tubulin, and RNA polymerase II regions revealed that A. pellizariae is a new species that is related to A. psychrotrophicus and Thelebolus species. Since the Antarctic Peninsula is reported to be one of the main regions of the earth experiencing the effects of global change in climate, species, such as A. pellizariae, might provide information about these effects on the endemic Antarctic biota. In addition, A. pellizariae displayed psychrophilic behavior and might be a source of interesting anti-freeze compounds that might prove useful in biotechnological processes.

  18. Cloning and Overexpression of the Triosephosphate Isomerase Genes from Psychrophilic and Thermophilic Bacteria. Structural Comparison of the Predicted Protein Sequences

    NARCIS (Netherlands)

    Rentier-Delrue, Françoise; Mande, Shekhar C.; Moyens, Sylvianne; Terpstra, Peter; Mainfroid, Véronique; Goraj, Karine; Lion, Michelle; Hol, Wim G.J.; Martial, Joseph A.

    1993-01-01

    We focused on the temperature adaptation of triosephosphate isomerase (TIM; E.C. 5.3.1.1.) by comparing the structure of TIMs isolated from bacterial organisms living in either cold or hot environments. The TIM gene from psychrophilic bacteria Moraxella sp. TA137 was cloned and its nucleotide sequen

  19. Production of gamma-decalactone by a psychrophilic and a mesophilic strain of the yeast Rhodotorula aurantiaca.

    Science.gov (United States)

    Alchihab, Mohamed; Destain, Jacqueline; Aguedo, Mario; Majad, Lamia; Ghalfi, Hakim; Wathelet, Jean-Paul; Thonart, Philippe

    2009-07-01

    Among 18 psychrophilic strains isolated near the Antarctic Station, the psychrophilic strain Rhodotorula aurantiaca A19 was selected for its ability of growth and gamma-decalactone production at low temperatures. The effects of temperature, initial pH, and castor oil concentration on the growth and gamma-decalactone production by a psychrophilic and a mesophilic strain of R. aurantiaca were investigated. The highest gamma-decalactone production in flasks (5.8 g/l) was obtained with the strain A19 at 14 degrees C and initial pH 7.0 in medium containing 20 g/l castor oil. On the other hand, these factors did not affect the production of gamma-decalactone by the mesophilic strain. In fermentor, a gamma-decalactone concentration of 6.6 g/l was reached with the strain A19, whereas a maximum of 0.1 g/l was obtained with the mesophilic strain. Our results suggest that the ability to synthesize gamma-decalactone is a particularity of the strain A19, since the mesophilic strain (no. 30645) produced small amounts, and the other (no. 31354) did not exhibit this property. It is, to our knowledge, the first report of gamma-decalactone production by R. aurantiaca and furthermore by a psychrophilic yeast strain. Moreover, the amount of gamma-decalactone obtained in fermentor with the strain 19 was on the order of concentrations usually described in patents.

  20. Desulfuromonas svalbardensis sp nov and Desulfuromusa ferrireducens sp nov., psychrophilic, Fe(III)-reducing bacteria isolated from Arctic sediments, Svalbard

    DEFF Research Database (Denmark)

    Vandieken, V.; Mussmann, M.; Niemann, Hans Henrik

    2006-01-01

    Two psychrophilic, Gram-negative, rod-shaped, motile bacteria (strains 112(T) and 102(T)) that conserved energy from dissimilatory Fe(III) reduction concomitant with acetate oxidation were isolated from permanently cold Arctic marine sediments. Both strains grew at temperatures down to -2 degrees C...

  1. The vacuolar serine protease, a cross-reactive allergen from Cladosporium herbarum.

    Science.gov (United States)

    Pöll, Verena; Denk, Ursula; Shen, Horng-Der; Panzani, Raphael C; Dissertori, Oliver; Lackner, Peter; Hemmer, Wolfgang; Mari, Adriano; Crameri, Reto; Lottspeich, Friedrich; Rid, Raphaela; Richter, Klaus; Breitenbach, Michael; Simon-Nobbe, Birgit

    2009-04-01

    Subtilisin-like serine proteases make up one of the most important allergen-families regarding the number of individual allergens. Previously, fungal subtilisin-like serine proteases have been identified from Aspergillus-, Penicillium-, and Trichophyton-species having a prevalence of IgE-reactivity between 33% and 80%. Since IgE-cross-reactivity is a common phenomenon within fungal species we wanted to know whether this protein also represents an allergen in Cladosporium herbarum. Hence, a screening of a C. herbarum cDNA library was performed using the coding sequence of the Penicillium oxalicum vacuolar serine protease (Pen o 18) as hybridization probe, ending up with a full-length clone. Biochemical and immunological characterization of this clone revealed that C. herbarum vacuolar serine protease most likely is synthesized as a precursor with an N-terminal pro-enzyme sequence and represents a minor allergen (Cla h 9) with a prevalence of IgE-reactivity of 15.5%. Furthermore Cla h 9 specifically reacted with the two monoclonal antibodies FUM20 and PCM39, as do the vacuolar serine proteases from Aspergillus fumigatus and Penicillium species. Investigation of IgE-cross-reactivity between Cla h 9 and other fungal serine proteases revealed that cross-reactivity is higher between vacuolar than alkaline serine proteases. IgE-epitope mapping of Cla h 9 was done in order to test whether four Cla h 9-peptides having a high sequence homology to previously determined Pen ch 18-IgE-epitopes also harbour IgE-epitopes. Three-dimensional models of the vacuolar serine proteases from C. herbarum and Penicillium chrysogenum were generated for the three-dimensional localization of the Cla h 9- and Pen ch 18- IgE-reactive and -non-reactive peptides. Taken together a new C. herbarum allergen has been identified, which may be useful in a molecule-based approach of C. herbarum allergy-diagnosis and -therapy. Moreover, Cla h 9 represents a further member of the subtilisin-like serine

  2. Alkaline battery operational methodology

    Science.gov (United States)

    Sholklapper, Tal; Gallaway, Joshua; Steingart, Daniel; Ingale, Nilesh; Nyce, Michael

    2016-08-16

    Methods of using specific operational charge and discharge parameters to extend the life of alkaline batteries are disclosed. The methods can be used with any commercial primary or secondary alkaline battery, as well as with newer alkaline battery designs, including batteries with flowing electrolyte. The methods include cycling batteries within a narrow operating voltage window, with minimum and maximum cut-off voltages that are set based on battery characteristics and environmental conditions. The narrow voltage window decreases available capacity but allows the batteries to be cycled for hundreds or thousands of times.

  3. Serine deprivation enhances antineoplastic activity of biguanides.

    Science.gov (United States)

    Gravel, Simon-Pierre; Hulea, Laura; Toban, Nader; Birman, Elena; Blouin, Marie-José; Zakikhani, Mahvash; Zhao, Yunhua; Topisirovic, Ivan; St-Pierre, Julie; Pollak, Michael

    2014-12-15

    Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

  4. Uranium in alkaline rocks

    Energy Technology Data Exchange (ETDEWEB)

    Murphy, M.; Wollenberg, H.; Strisower, B.; Bowman, H.; Flexser, S.; Carmichael, I.

    1978-04-01

    Geologic and geochemical criteria were developed for the occurrence of economic uranium deposits in alkaline igneous rocks. A literature search, a limited chemical analytical program, and visits to three prominent alkaline-rock localities (Ilimaussaq, Greenland; Pocos de Caldas, Brazil; and Powderhorn, Colorado) were made to establish criteria to determine if a site had some uranium resource potential. From the literature, four alkaline-intrusive occurrences of differing character were identified as type-localities for uranium mineralization, and the important aspects of these localities were described. These characteristics were used to categorize and evaluate U.S. occurrences. The literature search disclosed 69 U.S. sites, encompassing nepheline syenite, alkaline granite, and carbonatite. It was possible to compare two-thirds of these sites to the type localities. A ranking system identified ten of the sites as most likely to have uranium resource potential.

  5. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission

    Science.gov (United States)

    Foster, T. L.; Winans, L., Jr.

    1974-01-01

    The ability of psychrophilic microorganisms to grow in some of the environmental conditions suggested for Mars is studied with particular attention given to the effects of moisture and nutrients on growth. Results of growth with the slide culture technique are presented and indicate that this technique can be a rapid and sensitive technique for demonstration of microbial growth under various environmental conditions. Additional soil samples have been obtained from Cape Kennedy, and results of these assays at various low temperatures for psychrophilic populations are presented. The heat resistance of some of the psychrophilic sporeformers have been determined. Psychrophilic organisms were isolated from the teflon ribbons at Cape Kennedy and characterization of these was begun. In addition, heat survivors from the teflon ribbons are being investigated, and partial characterizations of these are presented.

  6. ANAEROBIC DIGESTION MODEL ANALYSIS OF THE FERMENTATION PROCESS IN PSYCHROPHILIC AND MESOPHILIC CHAMBER IN ACCORDANCE WITH THE AMOUNT OF BIOGAS SOURCED

    OpenAIRE

    Dariusz Zdebik; Marcin Głodniok; Paweł Zawartka

    2015-01-01

    The paper presents problems concerning the modelling of anaerobic sludge stabilization, with the additional substrate (waste transported, dairy butchery sewage) in psychrophilic fermentation conditions in the range 10–20 °C and mesophilic at 35 °C. Simulation test was conducted in the two digesters. Results of the study allowed to evaluate the effectiveness of conducting these processes in separate chambers, i.e. the psychrophilic and mesophilic chamber. During the simulations, terms of obtai...

  7. Structural Adaptation of a Thermostable Biotin-binding Protein in a Psychrophilic Environment

    Science.gov (United States)

    Meir, Amit; Bayer, Edward A.; Livnah, Oded

    2012-01-01

    Shwanavidin is an avidin-like protein from the marine proteobactrium Shewanella denitrificans, which exhibits an innate dimeric structure while maintaining high affinity toward biotin. A unique residue (Phe-43) from the L3,4 loop and a distinctive disulfide bridge were shown to account for the high affinity toward biotin. Phe-43 emulates the function and position of the critical intermonomeric Trp that characterizes the tetrameric avidins but is lacking in shwanavidin. The 18 copies of the apo-monomer revealed distinctive snapshots of L3,4 and Phe-43, providing rare insight into loop flexibility, binding site accessibility, and psychrophilic adaptation. Nevertheless, as in all avidins, shwanavidin also displays high thermostability properties. The unique features of shwanavidin may provide a platform for the design of a long sought after monovalent form of avidin, which would be ideal for novel types of biotechnological application. PMID:22493427

  8. Kinetics of psychrophilic anaerobic sequencing batch reactor treating flushed dairy manure.

    Science.gov (United States)

    Ma, Jingwei; Yu, Liang; Frear, Craig; Zhao, Quanbao; Li, Xiujin; Chen, Shulin

    2013-03-01

    In this study, a new strategy, improving biomass retention with fiber material present within the dairy manure as biofilm carriers, was evaluated for treating flushed dairy manure in a psychrophilic anaerobic sequencing batch reactor (ASBR). A kinetic study was carried out for process control and design by comparing four microbial growth kinetic models, i.e. first order, Grau, Monod and Chen and Hashimoto models. A volumetric methane production rate of 0.24L/L/d of and a specific methane productivity of 0.19L/gVSloaded were achieved at 6days HRT. It was proved that an ASBR using manure fiber as support media not only improved methane production but also reduced the necessary HRT and temperature to achieve a similar treating efficiency compared with current technologies. The kinetic model can be used for design and optimization of the process.

  9. Psychrophilic dry anaerobic digestion of dairy cow feces: Long-term operation

    Energy Technology Data Exchange (ETDEWEB)

    Massé, Daniel I., E-mail: Daniel.masse@agr.gc.ca; Cata Saady, Noori M.

    2015-02-15

    Highlights: • Psychrophilic dry anaerobic digestion (PDAD) of cow feces (CF) is feasible. • PDAD of CF is as efficient as mesophilic and thermophilic AD at TCL 21 days. • CF (13–16% TS at OLR 5.0 g TCOD{sub fed} kg{sup −1} inoculum d{sup −1}) yielded 222 ± 27 {sub N}L CH{sub 4} kg{sup −1} VS fed. - Abstract: This paper reports experimental results which demonstrate psychrophilic dry anaerobic digestion of cow feces during long-term operation in sequence batch reactor. Cow feces (13–16% total solids) has been anaerobically digested in 12 successive cycles (252 days) at 21 days treatment cycle length (TCL) and temperature of 20 °C using psychrotrophic anaerobic mixed culture. An average specific methane yield (SMY) of 184.9 ± 24.0, 189.9 ± 27.3, and 222 ± 27.7 {sub N}L CH{sub 4} kg{sup −1} of VS fed has been achieved at an organic loading rate of 3.0, 4.0, and 5.0 g TCOD kg{sup −1} inoculum d{sup −1} and TCL of 21 days, respectively. The corresponding substrate to inoculum ratio (SIR) was 0.39 ± 0.06, 0.48 ± .02, 0.53 ± 0.05, respectively. Average methane production rate of 10 ± 1.4 {sub N}L CH{sub 4} kg{sup −1} VS fed d{sup −1} has been obtained. The low concentration of volatile fatty acids indicated that hydrolysis was the reaction limiting step.

  10. Plasmids of psychrophilic and psychrotolerant bacteria and their role in adaptation to cold environments.

    Science.gov (United States)

    Dziewit, Lukasz; Bartosik, Dariusz

    2014-01-01

    Extremely cold environments are a challenge for all organisms. They are mostly inhabited by psychrophilic and psychrotolerant bacteria, which employ various strategies to cope with the cold. Such harsh environments are often highly vulnerable to the influence of external factors and may undergo frequent dynamic changes. The rapid adjustment of bacteria to changing environmental conditions is crucial for their survival. Such "short-term" evolution is often enabled by plasmids-extrachromosomal replicons that represent major players in horizontal gene transfer. The genomic sequences of thousands of microorganisms, including those of many cold-active bacteria have been obtained over the last decade, but the collected data have yet to be thoroughly analyzed. This report describes the results of a meta-analysis of the NCBI sequence databases to identify and characterize plasmids of psychrophilic and psychrotolerant bacteria. We have performed in-depth analyses of 66 plasmids, almost half of which are cryptic replicons not exceeding 10 kb in size. Our analyses of the larger plasmids revealed the presence of numerous genes, which may increase the phenotypic flexibility of their host strains. These genes encode enzymes possibly involved in (i) protection against cold and ultraviolet radiation, (ii) scavenging of reactive oxygen species, (iii) metabolism of amino acids, carbohydrates, nucleotides and lipids, (iv) energy production and conversion, (v) utilization of toxic organic compounds (e.g., naphthalene), and (vi) resistance to heavy metals, metalloids and antibiotics. Some of the plasmids also contain type II restriction-modification systems, which are involved in both plasmid stabilization and protection against foreign DNA. Moreover, approx. 50% of the analyzed plasmids carry genetic modules responsible for conjugal transfer or mobilization for transfer, which may facilitate the spread of these replicons among various bacteria, including across species boundaries.

  11. Role of fatty acids in cold adaptation of Antarctic psychrophilic Flavobacterium spp.

    Science.gov (United States)

    Králová, Stanislava

    2017-09-01

    Cold-loving microorganisms developed numerous adaptation mechanisms allowing them to survive in extremely cold habitats, such as adaptation of the cell membrane. The focus of this study was on the membrane fatty acids of Antarctic Flavobacterium spp., and their adaptation response to cold-stress. Fatty acids and cold-response of Antarctic flavobacteria was also compared to mesophilic and thermophilic members of the genus Flavobacterium. The results showed that the psychrophiles produced more types of major fatty acids than meso- and thermophilic members of this genus, namely C15:1 iso G, C15:0 iso, C15:0 anteiso, C15:1ω6c, C15:0 iso 3OH, C17:1ω6c, C16:0 iso 3OH and C17:0 iso 3OH, summed features 3 (C16:1ω7cand/or C16:1ω6c) and 9 (C16:0 10-methyl and/or C17:1 iso ω9c). It was shown that the cell membrane of psychrophiles was composed mainly of branched and unsaturated fatty acids. The results also implied that Antarctic flavobacteria mainly used two mechanisms of membrane fluidity alteration in their cold-adaptive response. The first mechanism was based on unsaturation of fatty acids, and the second mechanism on de novo synthesis of branched fatty acids. The alteration of the cell membrane was shown to be similar for all thermotypes of members of the genus Flavobacterium. Copyright © 2017 Elsevier GmbH. All rights reserved.

  12. Progress in Research on Main Psychrophilic Bacteria with Emphasis on Psychrophilic Bacillus in Raw Milk%原料乳中嗜冷菌种类及嗜冷芽孢杆菌研究进展

    Institute of Scientific and Technical Information of China (English)

    徐煜; 任婧

    2015-01-01

    The psychrophilic bacteria present in raw milk can grow under cold storage conditions and secrete protease and lipase which degrade milk protein and fat, resulting in the deterioration of milk quality. The present article summarizes the major species of psychrophilic bacteria in raw milk, with an emphasis on the recent progress that has been made in understanding the hazards of psychrophilic Bacillus including their species, hazard characteristics, proteases and lipases. At last, the future research direction of psychrotrophilic Bacillus is discussed.%嗜冷菌能在原料乳冷藏条件下生长,其分泌蛋白酶和脂肪酶降解牛乳中的蛋白和脂肪,导致牛乳变质。本文归纳了原料乳中嗜冷菌的种类,总结嗜冷芽孢杆菌的危害研究进展,重点包括嗜冷菌常见种型、危害特点、分泌的蛋白酶和脂肪酶。最后探讨了嗜冷芽孢杆菌的研究方向。

  13. Alkaline earth metal thioindates

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov-Ehmin, B.N.; Ivlieva, V.I.; Filatenko, L.A.; Zajtsev, B.E.; Kaziev, G.Z.; Sarabiya, M.G.

    1984-08-01

    Alkaline earth metal thioindates of MIn/sub 2/S/sub 4/ composition were synthesized by interaction of alkaline earth metal oxoindates with hydrogen sulfide during heating. Investigation into the compounds by X-ray analysis showed that calcium compound crystallizes in cubic crystal system and strontium and barium compounds in rhombic crystal system. Lattice parameters and the number of formula units were determined. Thioindates of M/sub 3/In/sub 2/S/sub 6/ composition were synthesized, their individuality was shown.

  14. Significance of the D-serine-deaminase and D-serine metabolism of Staphylococcus saprophyticus for virulence.

    Science.gov (United States)

    Korte-Berwanger, Miriam; Sakinc, Türkan; Kline, Kimberly; Nielsen, Hailyn V; Hultgren, Scott; Gatermann, Sören G

    2013-12-01

    Staphylococcus saprophyticus is the only species of Staphylococcus that is typically uropathogenic and possesses a gene coding for a D-serine-deaminase (DsdA). As D-serine is prevalent in urine and toxic or bacteriostatic to many bacteria, it is not surprising that the D-serine-deaminase gene is found in the genome of uropathogens. It has been suggested that D-serine-deaminase or the ability to respond to or to metabolize D-serine is important for virulence. For uropathogenic Escherichia coli (UPEC), a high intracellular D-serine concentration affects expression of virulence factors. S. saprophyticus is able to grow in the presence of high D-serine concentrations; however, its D-serine metabolism has not been described. The activity of the D-serine-deaminase was verified by analyzing the formation of pyruvate from D-serine in different strains with and without D-serine-deaminase. Cocultivation experiments were performed to show that D-serine-deaminase confers a growth advantage to S. saprophyticus in the presence of D-serine. Furthermore, in vivo coinfection experiments showed a disadvantage for the ΔdsdA mutant during urinary tract infection. Expression analysis of known virulence factors by reverse transcription-quantitative PCR (RT-qPCR) showed that the surface-associated lipase Ssp is upregulated in the presence of D-serine. In addition, we show that S. saprophyticus is able to use D-serine as the sole carbon source, but interestingly, D-serine had a negative effect on growth when glucose was also present. Taken together, D-serine metabolism is associated with virulence in S. saprophyticus, as at least one known virulence factor is upregulated in the presence of D-serine and a ΔdsdA mutant was attenuated in virulence murine model of urinary tract infection.

  15. PURIFICATION AND CHARACTERIZATION OF AN EXTRACELLULAR ALKALINE PROTEASE PRODUCED FROM AN ISOLATED BACILLUS SUBTILIS

    Directory of Open Access Journals (Sweden)

    Vijaya Bundela

    2013-03-01

    Full Text Available This paper describes the studies on the purification and partial characterization of serine alkaline protease produced through submerged fermentation process from a locally isolated Bacillus subtilis. This strain, grown in a highly alkaline medium (pH 10, produces an extracellular proteolytic enzyme. The alkaline protease was purified in a simple two-step procedure involving ammonium sulphate precipitation and gel filtration. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE analysis of the purified alkaline protease indicated an estimated molecular mass of 30KDa. It was more active in the range of 20-60ºC and had an optimum activity at 55ºC with optimum pH of 10.5. Characterization of the protease showed that it required certain cations such as Mg++, Mn++ and Ca++ for maximal activity. The serine nature of the alkaline protease was confirmed by PMSF inhibition. The temperature and pH stability of this Alkaline Protease from Bacillus Subtilismakes it potentially useful forindustrial applications.

  16. Genomic, Transcriptomic, and Proteomic Analysis Provide Insights Into the Cold Adaptation Mechanism of the Obligate Psychrophilic Fungus Mrakia psychrophila

    Directory of Open Access Journals (Sweden)

    Yao Su

    2016-11-01

    Full Text Available Mrakia psychrophila is an obligate psychrophilic fungus. The cold adaptation mechanism of psychrophilic fungi remains unknown. Comparative genomics analysis indicated that M. psychrophila had a specific codon usage preference, especially for codons of Gly and Arg and its major facilitator superfamily (MFS transporter gene family was expanded. Transcriptomic analysis revealed that genes involved in ribosome and energy metabolism were upregulated at 4°, while genes involved in unfolded protein binding, protein processing in the endoplasmic reticulum, proteasome, spliceosome, and mRNA surveillance were upregulated at 20°. In addition, genes related to unfolded protein binding were alternatively spliced. Consistent with other psychrophiles, desaturase and glycerol 3-phosphate dehydrogenase, which are involved in biosynthesis of unsaturated fatty acid and glycerol respectively, were upregulated at 4°. Cold adaptation of M. psychrophila is mediated by synthesizing unsaturated fatty acids to maintain membrane fluidity and accumulating glycerol as a cryoprotectant. The proteomic analysis indicated that the correlations between the dynamic patterns between transcript level changes and protein level changes for some pathways were positive at 4°, but negative at 20°. The death of M. psychrophila above 20° might be caused by an unfolded protein response.

  17. 嗜冷性产甲烷菌的研究进展%Research Progress of Psychrophilic methanogens

    Institute of Scientific and Technical Information of China (English)

    张伟; 万永青; 段开红; 田瑞华

    2013-01-01

    Psychrophilic methanogens play a very important role in global carbon cycle,and attract more and more research interests.Though playing an important role in low-temperature anaerobic digestion,the so-far cultured cold-adaptive methanogenic strains are very few.The research progress of Psychrophilic methanogens was reviewed,with the focus on their classification,isolation and cold-adapted mechanisms,as well as the molecular biology techniques.The application of Psychrophilic methanogens in low-temperature anaerobic digestion was also analyzed.%嗜冷产甲烷菌在全球碳素循环中发挥了非常重要的作用,近年来引起了国内外学者的广泛关注.尽管已知冷适应的产甲烷古菌在低温厌氧消化中发挥着重要作用,但目前获得培养的嗜冷产甲烷古菌却很少.综述了嗜冷产甲烷菌的研究进展,阐述了嗜冷产甲烷古菌的分离、嗜冷机制及分子生物学技术的应用,并对嗜冷产甲烷菌在低温厌氧消化中的应用进行了分析.

  18. [Advances of alkaline amylase production and applications].

    Science.gov (United States)

    Yang, Haiquan; Liu, Long; Li, Jianghua; Du, Guocheng; Chen, Jian

    2012-04-01

    Alkaline amylase is one of alkaline enzymes with optimum pH in the alkaline range, and it could keep stability and efficiently hydrolyze starch under alkaline conditions. Alkaline amylase finds wide applications in textile, detergent, pharmaceutical, food and other fields. Alkaline amylases could be produced by alkaliphilic microorganisms. In this work, the advances of alkaline amylase production and applications were reviewed.

  19. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...

  20. Catabolism of Serine by Pediococcus acidilactici and Pediococcus pentosaceus

    OpenAIRE

    Irmler, Stefan; Bavan, Tharmatha; Oberli, Andrea; Roetschi, Alexandra; Badertscher, René; Guggenbühl, Barbara; Berthoud, Hélène

    2013-01-01

    The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity car...

  1. Increased tolerance towards serine obtained by adaptive laboratory evolution

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna;

    2014-01-01

    by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution...

  2. Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes...... concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

  3. Enhanced Productivity of Serine Alkaline Protease by Bacillus sp. Using Soybean as Substrate

    Directory of Open Access Journals (Sweden)

    Saurabh, S.

    2007-01-01

    Full Text Available The growth and protease production by Bacillus sp. (SBP-29 was examined for poultry processing industries. The maximum protease activity was 3028 U/mL using 1.5% (w/v of soybean meal as substrate. Soybean meal is an inexpensive and readily available, thus it can be used as the cost effective crude material for the production of an extracellular protease. Inorganic nitrogen sources proved to be less favorable, for protease production as strong catabolic repression was observed with ammonium ions. A maximum of 3208 U/mL of protease was produced in 18 h in a 10L bioreactor. The enzyme has temperature and pH optima of 60°C and 9.5 respectively. However, the temperature stability range is from 20-90 °C and pH stability range is from 6.0–12.0. The protease was completely inhibited by phenylmethylsulfonyl fluoride (PMSF and diodopropyl fluorophosphate (DFP, with little increase (10-15% in the production of upon addition of Ca++ and Mg++.

  4. Alkaline broadening in Stars

    CERN Document Server

    De Kertanguy, A

    2015-01-01

    Giving new insight for line broadening theory for atoms with more structure than hydrogen in most stars. Using symbolic software to build precise wave functions corrected for ds;dp quantum defects. The profiles obtained with that approach, have peculiar trends, narrower than hydrogen, all quantum defects used are taken from atomic database topbase. Illustration of stronger effects of ions and electrons on the alkaline profiles, than neutral-neutral collision mechanism. Keywords : Stars: fundamental parameters - Atomic processes - Line: profiles.

  5. Feedback inactivation of D-serine synthesis by NMDA receptor-elicited translocation of serine racemase to the membrane

    DEFF Research Database (Denmark)

    Balan, Livia; Foltyn, Veronika N; Zehl, Martin;

    2009-01-01

    D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDAR-dependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes trans...

  6. Influence of hydraulic retention time on the psychrophilic hydrolysis/acidogenesis of proteins.

    Science.gov (United States)

    Poirrier, Paola; Schiappacasse, María Cristina; Carballa, Marta; Lema, Juan M

    2016-11-01

    The influence of the hydraulic retention time (HRT) on the anaerobic hydrolysis of complex substrates has been studied under psychrophilic conditions. For this purpose, a continuous stirred tank reactor was operated at 15 °C and neutral pH and gelatin was considered as a model protein. Three HRTs have been tested: 12, 21 and 36 h. Gelatin hydrolysis was greatly dependent on HRT, increasing from 40% at 12 h-HRT to a maximum of 65% at 36 h-HRT. Molecular size distribution analyses of the effluent showed that hydrolysation of compounds larger than 10 kDa was poor at 12 h-HRT, whereas the fraction of 1-10 kDa was completely transformed into compounds smaller than 1 kDa. Higher HRT (36 h) improved the degradation of the recalcitrant fraction (>10 kDa), obtaining an effluent with around 95% of soluble molecules (<1 kDa). In that way, the use of membrane bioreactors for the treatment of this type of macromolecules could improve the degradation efficiencies by enabling to increase the residence time of the non-hydrolyzed molecules, with what would be possible to achieve higher organic loading rate operation.

  7. Long-term, high-rate anaerobic biological treatment of whey wastewaters at psychrophilic temperatures.

    Science.gov (United States)

    McHugh, Sharon; Collins, Gavin; O'Flaherty, Vincent

    2006-09-01

    Two laboratory-scale anaerobic hybrid reactors, R1 and R2, treated low- (1 kg COD m-3) and high-strength (10 kg COD m-3) whey-based wastewaters, respectively, in a 500-day trial. The chemical oxygen demand (COD) removal efficiencies of R1 averaged 70-80%, at organic loading rates of 0.5-1.3 kg COD m-3 day-1, between 20 and 12 degrees C. The COD removal efficiencies of R2 exceeded 90%, at organic loading rates up to 13.3 kg COD m-3 day-1, between 20 and 14 degrees C. Lowering the operating temperature of R2 to 12 degrees C resulted in a decrease in COD removal efficiency, to between 50% and 60%, and a disintegration of granular sludge. The decline in performance, and granule disintegration, was reversed by decreasing the organic loading rate of R2 to 6.6 kg m-3 day-1. Specific methanogenic activity profiles revealed mesophilic (37 degrees C) temperature optima for biomass in both reactors, even after 500 days of psychrophilic operation, although the development of psychrotolerance in the biomass was noted.

  8. Rhodoferax antarcticus sp. nov., a moderately psychrophilic purple nonsulfur bacterium isolated from an Antarctic microbial mat

    Science.gov (United States)

    Madigan, M. T.; Jung, D. O.; Woese, C. R.; Achenbach, L. A.

    2000-01-01

    A new species of purple nonsulfur bacteria isolated from an Antarctic microbial mat is described. The organism, designated strain ANT.BR, was mildly psychrophilic, growing optimally at 15-18 degrees C with a growth temperature range of 0-25 degrees C. Cells of strain ANT.BR were highly motile curved rods and spirals, contained bacteriochlorophyll a, and showed a multicomponent in vivo absorption spectrum. A specific phylogenetic relationship was observed between strain ANT.BR and the purple bacterium Rhodoferax fermentans FR2T, and the two organisms shared several physiological and other phenotypic properties, with the notable exception of growth temperature optimum. Tests of genomic DNA hybridization, however, showed Rfx. fermentans FR2T and strain ANT.BR to be genetically distinct bacteria. Because of its unique set of properties, especially its requirement for low growth temperatures, we propose to recognize strain ANT.BR as a new species of the genus Rhodoferax, Rhodoferax antarcticus, named for its known habitat, the Antarctic.

  9. Treatment with amino acids in serine deficiency disorders.

    Science.gov (United States)

    de Koning, T J

    2006-01-01

    Serine deficiency disorders are rare defects in the biosynthesis of the amino acid L-serine. At present two disorders have been reported: 3-phosphoglycerate dehydrogenase deficiency and 3-phosphoserine phosphatase deficiency. These enzyme defects lead to severe neurological symptoms such as congenital microcephaly and severe psychomotor retardation and in addition in patients with 3-phosphoglycerate dehydrogenase deficiency to intractable seizures. These symptoms respond to a variable degree to treatment with L-serine, sometimes combined with glycine. In this paper the current practice of amino acid treatment with L-serine and glycine in serine deficiency is reviewed.

  10. Radiolysis of Serine in High Radiation Field

    Directory of Open Access Journals (Sweden)

    Ellen Y. Aguilar-Ovando

    2016-08-01

    Full Text Available The formation of amino acids under simulated condition suggests that this type of compounds were readily formed on the primitive Earth. Nevertheless, there is no conclusive explanation to the origin of their specific chirality in biological systems. Differences in their stability in the primitive conditions may give some clues about this unsolved problem. Protection mechanisms have been considered, such as the adsorption of the organic compounds onto mineral surfaces. By using HPLC/ELSD to analyze aqueous suspensions of serine adsorbed on clay (sodium montmorillonite and a meteorite (Allende irradiated in different doses with a cobalt-60 gamma source, the aim of this work was to study the possible protector role of these mineral surfaces when an amino acid, serine, is adsorbed onto them and the system is exposed to a high radiation source. The results showed that adsorption is better at acidic pH and desorption from the mineral at basic pH. The irradiation of the free amino acid destroyed it almost completely at a dose of 91 kGy, but the presence of the mineral abruptly decreases the decomposition, acting as a protective agent. At the same time, the results in aqueous solution show no statistically significant differences in adsorption or radiolysis of D and L serine.

  11. A practical total synthesis of the microbial alkaline proteinase inhibitor (MAPI).

    Science.gov (United States)

    Haebich, Dieter; Hillisch, Alexander; El Sheikh, Sherif

    2009-12-01

    Diverse serine and cysteine proteases as well as alkaline proteinases and elastases play a crucial role in numerous biological processes. Natural peptide aldehydes such as the "microbial alkaline proteinase inhibitor" (MAPI, 1) are valuable tools to characterize novel enzymes and to study their function in nature. Within a drug discovery program we wanted to design and explore non-natural MAPI congeners with novel biological profiles. To that end we devised a simple, practical, and scalable synthesis of MAPI 1 from readily available amino acid building blocks. The modular nature of our approach allows convenient structural modification of the MAPI backbone.

  12. Serine proteases, serine protease inhibitors, and protease-activated receptors: roles in synaptic function and behavior.

    Science.gov (United States)

    Almonte, Antoine G; Sweatt, J David

    2011-08-17

    Serine proteases, serine protease inhibitors, and protease-activated receptors have been intensively investigated in the periphery and their roles in a wide range of processes-coagulation, inflammation, and digestion, for example-have been well characterized (see Coughlin, 2000; Macfarlane et al., 2001; Molinari et al., 2003; Wang et al., 2008; Di Cera, 2009 for reviews). A growing number of studies demonstrate that these protein systems are widely expressed in many cell types and regions in mammalian brains. Accumulating lines of evidence suggest that the brain has co-opted the activities of these interesting proteins to regulate various processes underlying synaptic activity and behavior. In this review, we discuss emerging roles for serine proteases in the regulation of mechanisms underlying synaptic plasticity and memory formation. Copyright © 2011 Elsevier B.V. All rights reserved.

  13. Quantitative ecology and dry-heat resistance of psychrophiles. M.S. Thesis; [in soil samples from Viking spacecraft manufacturing areas

    Science.gov (United States)

    Winans, L., Jr.

    1974-01-01

    Microorganisms capable of growth at 7 C were enumerated and isolated from soil samples from the manufacture area (Denver, Colorado) and assembly area (Cape Kennedy, Florida) of the Viking spacecraft. Temperature requirements were determined for these isolates, and those growing at 3 C, but not at 32 C were designated as obligate psychrophiles in this investigation. These were identified to major generic groups, and the population density of obligate psychrophiles from the various groups was determined. Dry heat D-values were found for those spores that demonstrated growth or survival under a simulated Martian environment.

  14. ANAEROBIC DIGESTION MODEL ANALYSIS OF THE FERMENTATION PROCESS IN PSYCHROPHILIC AND MESOPHILIC CHAMBER IN ACCORDANCE WITH THE AMOUNT OF BIOGAS SOURCED

    Directory of Open Access Journals (Sweden)

    Dariusz Zdebik

    2015-03-01

    Full Text Available The paper presents problems concerning the modelling of anaerobic sludge stabilization, with the additional substrate (waste transported, dairy butchery sewage in psychrophilic fermentation conditions in the range 10–20 °C and mesophilic at 35 °C. Simulation test was conducted in the two digesters. Results of the study allowed to evaluate the effectiveness of conducting these processes in separate chambers, i.e. the psychrophilic and mesophilic chamber. During the simulations, terms of obtaining volatile fatty acids and biogas in conjunction with the operating conditions of the chambers indicated.

  15. The Genome Sequence of the psychrophilic archaeon, Methanococcoides burtonii: the Role of Genome Evolution in Cold-adaptation

    Energy Technology Data Exchange (ETDEWEB)

    Allen, Michelle A.; Lauro, Federico M.; Williams, Timothy J.; Burg, Dominic; Siddiqui, Khawar S.; De Francisci, David; Chong, Kevin W.Y.; Pilak, Oliver; Chew, Hwee H.; De Maere, Matthew Z.; Ting, Lily; Katrib, Marilyn; Ng, Charmaine; Sowers, Kevin R.; Galperin, Michael Y.; Anderson, Iain J.; Ivanova, Natalia; Dalin, Eileen; Martinez, Michelle; Lapidus, Alla; Hauser, Loren; Land, Miriam; Thomas, Torsten; Cavicchioli, Ricardo

    2009-04-01

    Psychrophilic archaea are abundant and perform critical roles throughout the Earth's expansive cold biosphere. Here we report the first complete genome sequence for a psychrophilic methanogenic archaeon, Methanococcoides burtonii. The genome sequence was manually annotated including the use of a five tiered Evidence Rating system that ranked annotations from Evidence Rating (ER) 1 (gene product experimentally characterized from the parent organism) to ER5 (hypothetical gene product) to provide a rapid means of assessing the certainty of gene function predictions. The genome is characterized by a higher level of aberrant sequence composition (51%) than any other archaeon. In comparison to hyper/thermophilic archaea which are subject to selection of synonymous codon usage, M. burtonii has evolved cold adaptation through a genomic capacity to accommodate highly skewed amino acid content, while retaining codon usage in common with its mesophilic Methanosarcina cousins. Polysaccharide biosynthesis genes comprise at least 3.3% of protein coding genes in the genome, and Cell wall/membrane/envelope biogenesis COG genes are over-represented. Likewise, signal transduction (COG category T) genes are over-represented and M. burtonii has a high 'IQ' (a measure of adaptive potential) compared to many methanogens. Numerous genes in these two over-represented COG categories appear to have been acquired from {var_epsilon}- and {delta}-proteobacteria, as do specific genes involved in central metabolism such as a novel B form of aconitase. Transposases also distinguish M. burtonii from other archaea, and their genomic characteristics indicate they play an important role in evolving the M. burtonii genome. Our study reveals a capacity for this model psychrophile to evolve through genome plasticity (including nucleotide skew, horizontal gene transfer and transposase activity) that enables adaptation to the cold, and to the biological and physical changes that have

  16. D-serine transporter in Staphylococcus saprophyticus identified.

    Science.gov (United States)

    Marlinghaus, Lennart; Huß, Melanie; Korte-Berwanger, Miriam; Sakinc-Güler, Türkan; Gatermann, Sören G

    2016-07-01

    Among staphylococci Staphylococcus saprophyticus is the only species that is typically uropathogenic and an important cause of urinary tract infections in young women. The amino acid D-serine occurs in relatively high concentrations in human urine and has a bacteriostatic or toxic effect on many bacteria. In uropathogenic Escherichia coli and S. saprophyticus, the amino acid regulates the expression of virulence factors and can be used as a nutrient. The ability of uropathogens to respond to or to metabolize D-serine has been suggested as a factor that enables colonization of the urinary tract. Until now nothing is known about D-serine transport in S. saprophyticus We generated mutants of putative transporter genes in S. saprophyticus 7108 that show homology to the D-serine transporter cycA of E. coli and tested them in a D-serine depletion assay to analyze the D-serine uptake rate of the cells. The mutant of SPP1070 showed a strong decrease in D-serine uptake. Therefore, SSP1070 was identified as a major D-serine transporter in S. saprophyticus 7108 and was named D-serine transporter A (DstA). D-serine caused a prolonged lag phase of S. saprophyticus in a chemically defined medium. This negative effect was dependent on the presence of DstA.

  17. Alkaline fuel cells applications

    Science.gov (United States)

    Kordesch, Karl; Hacker, Viktor; Gsellmann, Josef; Cifrain, Martin; Faleschini, Gottfried; Enzinger, Peter; Fankhauser, Robert; Ortner, Markus; Muhr, Michael; Aronson, Robert R.

    On the world-wide automobile market technical developments are increasingly determined by the dramatic restriction on emissions as well as the regimentation of fuel consumption by legislation. Therefore there is an increasing chance of a completely new technology breakthrough if it offers new opportunities, meeting the requirements of resource preservation and emission restrictions. Fuel cell technology offers the possibility to excel in today's motive power techniques in terms of environmental compatibility, consumer's profit, costs of maintenance and efficiency. The key question is economy. This will be decided by the costs of fuel cell systems if they are to be used as power generators for future electric vehicles. The alkaline hydrogen-air fuel cell system with circulating KOH electrolyte and low-cost catalysed carbon electrodes could be a promising alternative. Based on the experiences of Kordesch [K. Kordesch, Brennstoffbatterien, Springer, Wien, 1984, ISBN 3-387-81819-7; K. Kordesch, City car with H 2-air fuel cell and lead-battery, SAE Paper No. 719015, 6th IECEC, 1971], who operated a city car hybrid vehicle on public roads for 3 years in the early 1970s, improved air electrodes plus new variations of the bipolar stack assembly developed in Graz are investigated. Primary fuel choice will be a major issue until such time as cost-effective, on-board hydrogen storage is developed. Ammonia is an interesting option. The whole system, ammonia dissociator plus alkaline fuel cell (AFC), is characterised by a simple design and high efficiency.

  18. PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats.

    Science.gov (United States)

    Broda, D M; Boerema, J A; Bell, R G

    2003-01-01

    To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of

  19. Impact of Organic Loading Rate on Psychrophilic Anaerobic Digestion of Solid Dairy Manure

    Directory of Open Access Journals (Sweden)

    Noori M. Cata Saady

    2015-03-01

    Full Text Available Increasing the feed total solids to anaerobic digester improves the process economics and decreases the volume of liquid effluent from current wet anaerobic digestion. The objective of this study was to develop a novel psychrophilic (20 °C anaerobic digestion technology of undiluted cow feces (total solids of 11%–16%. Two sets of duplicate laboratory-scale sequence batch bioreactors have been operated at organic loading rates (OLR of 6.0 to 8.0 g total chemical oxygen demand (TCOD kg−1 inoculum day−1 (d−1 during 210 days. The results demonstrated that the process is feasible at treatment cycle length (TCL of 21 days; however, the quality of cow feces rather than the OLR had a direct influence on the specific methane yield (SMY. The SMY ranged between 124.5 ± 1.4 and 227.9 ± 4.8 normalized liter (NL CH4 kg−1 volatile solids (VS fed d−1. Substrate-to-inoculum mass ratio (SIR was 0.63 ± 0.05, 0.90 ± 0.09, and 1.06 ± 0.07 at OLR of 6.0, 7.0, and 8.0 g TCOD kg−1 inoculum d−1, respectively. No volatile fatty acids (VFAs accumulation has been observed which indicated that hydrolysis was the rate limiting step and VFAs have been consumed immediately. Bioreactors performance consistency in terms of the level of SMYs, VFAs concentrations at end of the TCL, pH stability and volatile solids reduction indicates a stable and reproducible process during the entire operation.

  20. Mats of psychrophilic thiotrophic bacteria associated with cold seeps of the Barents Sea

    Directory of Open Access Journals (Sweden)

    S. Grünke

    2012-08-01

    Full Text Available This study investigated the bacterial diversity associated with microbial mats of polar deep-sea cold seeps. The mats were associated with high upward fluxes of sulfide produced by anaerobic oxidation of methane, and grew at temperatures close to the freezing point of seawater. They ranged from small patches of 0.2–5 m in diameter (gray mats to extensive fields covering up to 850 m2 of seafloor (white mats and were formed by diverse sulfide-oxidizing bacteria differing in color and size. Overall, both the dominant mat-forming thiotrophs as well as the associated bacterial communities inhabiting the mats differed in composition for each mat type as determined by microscopy, 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis. While the smaller gray mats were associated with a highly diverse composition of sulfide oxidizers, the larger white mats were composed of only 1–2 types of gliding Beggiatoa filaments. Molecular analyses showed that most of the dominant mat-forming sulfide oxidizers were phylogenetically different from, but still closely related to, thiotrophs known from warmer ocean realms. The psychrophilic nature of the polar mat-forming thiotrophs was tested by visual observation of active mats at in situ temperature compared to their warming to >4 °C. The temperature range of mat habitats and the variation of sulfide and oxygen fluxes appear to be the main factors supporting the diversity of mat-forming thiotrophs in cold seeps at continental margins.

  1. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang;

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending...... of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden....

  2. Fatal cerebral edema associated with serine deficiency in CSF.

    Science.gov (United States)

    Keularts, Irene M L W; Leroy, Piet L J M; Rubio-Gozalbo, Estela M; Spaapen, Leo J M; Weber, Biene; Dorland, Bert; de Koning, Tom J; Verhoeven-Duif, Nanda M

    2010-12-01

    Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and enzyme measurement (in one patient) excluded 3-PGDH deficiency. Deficiencies in other serine biosynthesis enzymes were highly unlikely on clinical grounds. On basis of the fasting state, ketone bodies and lactate in plasma, urine and CSF, we speculate that reduced serine levels were due to its use as gluconeogenic substrate, conversion to pyruvate by brain serine racemase or decreased L-serine production because of a lack of glucose. These are the first strikingly similar cases of patients with a clear secondary serine deficiency associated with a toxic encephalopathy.

  3. An alkaline element

    Energy Technology Data Exchange (ETDEWEB)

    Arita, T.; Murakami, K.; Okha, K.

    1983-04-28

    A cathode with a dual layer active mass is installed in the disk shaped alkaline silver and zinc element. The first layer, which is turned towards the anode, contains 85 parts Ag2O, 5 parts electrolytic MnO2 and 10 parts graphite. The second layer, which contacts the bottom of the element, contains 35 parts Ag2O, 60 parts electrolytic MnO2 and 5 parts graphite. The electrical capacity of the first and second layers is 60 and 40, respectively. The first layer may be discharged with a high current density and the second layer with less current density. The element has high characteristics with comparatively low cost.

  4. Crystal structure of Proteus mirabilis lipase, a novel lipase from the Proteus/psychrophilic subfamily of lipase family I.1.

    Directory of Open Access Journals (Sweden)

    Tyler P Korman

    Full Text Available Bacterial lipases from family I.1 and I.2 catalyze the hydrolysis of triacylglycerol between 25-45°C and are used extensively as biocatalysts. The lipase from Proteus mirabilis belongs to the Proteus/psychrophilic subfamily of lipase family I.1 and is a promising catalyst for biodiesel production because it can tolerate high amounts of water in the reaction. Here we present the crystal structure of the Proteus mirabilis lipase, a member of the Proteus/psychrophilic subfamily of I.1lipases. The structure of the Proteus mirabilis lipase was solved in the absence and presence of a bound phosphonate inhibitor. Unexpectedly, both the apo and inhibitor bound forms of P. mirabilis lipase were found to be in a closed conformation. The structure reveals a unique oxyanion hole and a wide active site that is solvent accessible even in the closed conformation. A distinct mechanism for Ca²⁺ coordination may explain how these lipases can fold without specific chaperones.

  5. Cloning and in-silico analysis of beta-1,3-xylanase from psychrophilic yeast, Glaciozyma antarctica PI12

    Science.gov (United States)

    Nor, Nooraisyah Mohamad; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Murad, Abdul Munir Abdul

    2015-09-01

    A beta-1,3-xylanase (EC 3.2.1.32) gene from psychrophilic yeast, Glaciozyma antarctica has been identified via genome data mining. The enzyme was grouped into GH26 family based on Carbohydrate Active Enzyme (CaZY) database. The molecular weight of this protein was predicted to be 42 kDa and is expected to be soluble for expression. The presence of signal peptide suggested that this enzyme may be released extracellularly into the marine environment of the host's habitat. This supports the theory that such enzymatic activity is required for degradation of nutrients of polysaccharide origins into simpler carbohydrates outside the environment before it could be taken up inside the cell. The sequence for this protein showed very little conservation (< 30%) with other beta-1,3-xylanases from available databases. Based on the phylogenetic analysis, this protein also showed distant relationship to other xylanases from eukaryotic origin. The protein may have undergone major substitution in its gene sequence order to adapt to the cold climate. This is the first report of beta-1,3-xylanase gene isolated from a psychrophilic yeast.

  6. A study of psychrophilic organisms isolated from the manufacture and assembly areas of spacecraft to be used in the Viking mission, 1 January - 30 June 1973

    Science.gov (United States)

    Foster, T. L.; Winans, L., Jr.

    1973-01-01

    Soil samples from the areas associated with the Viking spacecraft were analyzed for major generic groups of microorganisms and the percentage of obligate psychrophiles. Results are presented which show the distribution of organisms isolated at low temperatures and the methods employed for subjecting samples to simulated Martian conditions. Emphasis is placed on application of these results to the objectives of the quarantine program.

  7. Pathways of carbon oxidation in an Arctic fjord sediment (Svalbard) and isolation of psychrophilic and psychrotolerant Fe(III)-reducing bacteria

    DEFF Research Database (Denmark)

    Vandieken, Verona; Finke, Niko; Jørgensen, Bo Barker

    2006-01-01

    , Desulfuromusa, Shewanella and Desulfovibrio were isolated from enrichment cultures of 2 fjord sediments from Svalbard. Strains related to Desulfovibrio reduced Fe(III) without energy generation for growth. All isolates were psychrophilic or psychrotolerant and grew at –2°C, the freezing point of sea water...

  8. Pathways of carbon oxidation in an Arctic fjord sediment (Svalbard) and isolation of psychrophilic and psychrotolerant Fe(III)-reducing bacteria

    DEFF Research Database (Denmark)

    Vandieken, Verona; Finke, Niko; Jørgensen, Bo Barker

    2006-01-01

    Desulfuromonas, Desulfuromusa, Shewanella and Desulfovibrio were isolated from enrichment cultures of 2 fjord sediments from Svalbard. Strains related to Desulfovibrio reduced Fe(III) without energy generation for growth. All isolates were psychrophilic or psychro-tolerant and grew at -2 degrees C, the freezing...

  9. 嗜冷菌对原料奶质量的影响及控制方法%Discussed Psychrophilic Bacteria to Raw Milk Quality Influence and the Control Method

    Institute of Scientific and Technical Information of China (English)

    苏景辉; 于敏艳; 孟凡玉

    2011-01-01

    本文阐述了嗜冷茵对原料奶的危害,并通过对不同牧场嗜冷茵的检测、分析,发现嗜冷茼的来源主要为外源性污染,我们据此提出相应的控制建议.%This paper expounded the hazards of psychrophilic bacteria on the raw milk, and through the inspection and analysis of psychrophilic bacteria in different pasture , found the main source of psychrophilic bacteria for exogenous pollution. Then we put forward the corresponding control Suggestions.

  10. Neonatal disruption of serine racemase causes schizophrenia-like behavioral abnormalities in adulthood: clinical rescue by d-serine.

    Directory of Open Access Journals (Sweden)

    Hiroko Hagiwara

    Full Text Available BACKGROUND: D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA receptor, is synthesized from L-serine by serine racemase (SRR. Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life. METHODOLOGY/PRINCIPAL FINDINGS: Neonatal mice (7-9 days were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day, an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT, and prepulse inhibition (PPI were performed at juvenile (5-6 weeks old and adult (10-12 weeks old stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70 significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure. CONCLUSIONS/SIGNIFICANCE: This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.

  11. Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain

    DEFF Research Database (Denmark)

    Oh, E S; Couchman, J R; Woods, A

    1997-01-01

    Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus seque...

  12. Selective N-hydroxyhydantoin carbamate inhibitors of mammalian serine hydrolases

    Science.gov (United States)

    Cognetta, Armand B.; Niphakis, Micah J.; Lee, Hyeon-Cheol; Martini, Michael L.; Hulce, Jonathan J.; Cravatt, Benjamin F.

    2015-01-01

    Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis and screening of a small library of NHH carbamates by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterease-1 (PPT1), mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis. PMID:26120000

  13. Participation of D-serine in the development and reproduction of the silkworm Bombyx mori.

    Science.gov (United States)

    Tanigawa, Minoru; Suzuki, Chihiro; Niwano, Kimio; Kanekatsu, Rensuke; Tanaka, Hiroyuki; Horiike, Kihachiro; Hamase, Kenji; Nagata, Yoko

    2016-04-01

    The silkworm Bombyx mori contains high concentrations of free D-serine, an optical isomer of L-serine. To elucidate its function, we first investigated the localization of D-serine in various organs of silkworm larvae, pupae, and adult moths. Using immunohistochemical analysis with an anti-D-serine antibody, we found D-serine in the microvilli of midgut goblet and cylindrical cells and in peripheral matrix components of testicular and ovarian cells. By spectrophotometric analysis, D-serine was also found in the hemolymph and fat body. D-Alanine was not detected in the various organs by immunohistochemistry. Serine racemase, which catalyzes the inter-conversion of L- and D-serine, was found to co-localize with D-serine, and D-serine production from L-serine by intrinsic serine racemase was suggested. O-Phospho-L-serine is an inhibitor of serine racemase, and it was administered to the larvae to reduce the D-serine level. This reagent decreased the midgut caspase-3 level and caused a delay in spermatogenesis and oogenesis. The reagent also decreased mature sperm and egg numbers, suggesting D-serine participation in these processes. D-Serine administration induced an increase in pyruvate levels in testis, midgut, and fat body, indicating conversion of D-serine to pyruvate. On the basis of these results, together with our previous investigation of ATP biosynthesis in testis, we consider the possible involvement of D-serine in ATP synthesis for metamorphosis and reproduction.

  14. Effect of Psychrophilic Bacteria on Quality of UHT Milk%嗜冷菌对UHT乳品质的影响研究

    Institute of Scientific and Technical Information of China (English)

    李晓建; 吕阳; 郭慧媛

    2014-01-01

    Raw milk pollution arised by Psychrophilic Bacteria can greatly shorten the shelf-life of UHT milk.The damaging heat-resistant lipase and protease secreted by Psychrophilic Bacteria can still remain after sterilization during UHT production. The distribution of Psychrophilic Bacteria has regional features. In this study, Psychrophilic Bacterium isolated from eight pastures of Mengniu milk industry were investigated on their impact upon UHT milk. Psychrophilic Bacterium screened out were Pseudomonas, Micrococcus and Alkaligenes. Results showed that the number of Psychrophilic Bacteria in raw milk was larger from January to March, compared to the period from June to September, and the more Psychrophilic Bacterium existed in raw milk, the shorter shelf-life of UHT milk seemed to be. Other results showed that, 105CFU/ml of Psychrophilic Bacteria could obviously lead to corruption of UHT milk, simutaneously generating bitter taste(P<0.05). Speciifcally in enzyme activity for lipase of 10IU/mL and protease of 15, 25IU/mL had signiifcantly effect on milk corruption (P<0.05). Also, pH 6.7 and 3~5℃was not suitable for long-term storage of UHT milk, because under this condition the lipase and protease enzyme in UHT milk was still relatively high. In conclusion, excessive Psychrophilic Bacterium in raw milk would largely inlfuence the quality and lfavor of UHT milk, and low temperature may give rise to high-active lipase and protease for the damage of UHT milk, which is not suggested for the UHT milk storage.%原料乳嗜冷菌污染是影响UHT乳保质期的重要原因之一,并且嗜冷菌分布具有地域特征性。本研究对蒙牛8个牧场原料乳中的嗜冷菌进行了分离和鉴定,确定了牧场常见嗜冷菌种类及特性,并分析了其对UHT乳品质的影响。结果表明,从试验样品中分离出的嗜冷菌为假单胞菌属、微球菌属和产碱杆菌属;牧场原料乳的嗜冷菌数量与气温有关,1~3月份数量较高,6

  15. A new alkaline elastase of an alkalophilic bacillus.

    Science.gov (United States)

    Tsai, Y C; Yamasaki, M; Yamamoto-Suzuki, Y; Tamura, G

    1983-11-01

    A new alkaline elastase was purified from the culture broth of an alkalophilic Bacillus sp. Ya-B. This was a serine proteinase. Molecular weight was 25,000. The optimum pH for elastin and casein was 11.75. The enzyme had very high specific activity, 12,400 units/mg protein for casein, and 2,440 units/mg protein for elastin at the optimum pH. It showed marked preference for elastin. The relative activity of elastin/casein of this enzyme was 17 and 6 times higher than those of subtilisin BPN' and subtilisin Carlsberg, respectively. This enzyme also had higher keratin and collagen hydrolyzing activity in comparison with subtilisin.

  16. Catabolism of serine by Pediococcus acidilactici and Pediococcus pentosaceus.

    Science.gov (United States)

    Irmler, Stefan; Bavan, Tharmatha; Oberli, Andrea; Roetschi, Alexandra; Badertscher, René; Guggenbühl, Barbara; Berthoud, Hélène

    2013-02-01

    The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L- and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

  17. Induction of serine racemase expression and D-serine release from microglia by secreted amyloid precursor protein (sAPP).

    Science.gov (United States)

    Wu, Shengzhou; Basile, Anthony S; Barger, Steven W

    2007-07-01

    Alzheimer's disease (AD) involves neuronal loss and reduction of synaptic density in specific brain region. Some of the neuronal deaths are associated with excitotoxicity. We previously reported that amyloid beta-peptide (Abeta) induced release of N-methyl-D-aspartate receptor (NMDA-R) co-agonists, including glutamate and D-serine. The induction of D-serine production by Abeta involves transcriptional and/or translational regulation of serine racemase gene. Similarly, we report here that conditioned medium from microglia treated with secreted amyloid precursor protein (sAPP) contained elevated levels of D-serine. In microglia, sAPP increased the steady-state dimeric protein level of serine racemase. Promoter-reporter and mRNA analyses suggested that serine racemase is transcriptionally induced by sAPP. These data extend the link between excitotoxicity and neuroinflammation. D-serine may cooperate with glutamate to link neuroinflammation with excitotoxicity, suggesting a pathogenic mechanism applicable to neuronal death in AD and other neurodegenerative diseases.

  18. Sugarcane Serine Peptidase Inhibitors, Serine Peptidases, and Clp Protease System Subunits Associated with Sugarcane Borer (Diatraea saccharalis) Herbivory and Wounding

    Science.gov (United States)

    Medeiros, Ane H.; Mingossi, Fabiana B.; Dias, Renata O.; Franco, Flávia P.; Vicentini, Renato; Mello, Marcia O.; Moura, Daniel S.; Silva-Filho, Marcio C.

    2016-01-01

    Sugarcane’s (Saccharum spp.) response to Diatraea saccharalis (F.) (Lepidoptera: (Crambidae) herbivory was investigated using a macroarray spotted with 248 sugarcane Expressed Sequence Tags (ESTs) encoding serine peptidase inhibitors, serine peptidases. and Clp protease system subunits. Our results showed that after nine hours of herbivory, 13 sugarcane genes were upregulated and nine were downregulated. Among the upregulated genes, nine were similar to serine peptidase inhibitors and four were similar to Bowman-Birk Inhibitors (BBIs). Phylogenetic analysis revealed that these sequences belong to a phylogenetic group of sugarcane BBIs that are potentially involved in plant defense against insect predation. The remaining four upregulated genes included serine peptidases and one homolog to the Arabidopsis AAA+ chaperone subunit ClpD, which is a member of the Clp protease system. Among the downregulated genes, five were homologous to serine peptidases and four were homologous to Arabidopsis Clp subunits (three homologous to Clp AAA+ chaperones and one to a ClpP-related ClpR subunit). Although the roles of serine peptidase inhibitors in plant defenses against herbivory have been extensively investigated, the roles of plant serine peptidases and the Clp protease system represent a new and underexplored field of study. The up- and downregulated D. saccharalis genes presented in this study may be candidate genes for the further investigation of the sugarcane response to herbivory. PMID:27598134

  19. Crystallization and preliminary X-ray diffraction studies of tetrameric malate dehydrogenase from the novel Antarctic psychrophile Flavobacterium frigidimaris KUC-1

    Energy Technology Data Exchange (ETDEWEB)

    Fujii, Tomomi [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Oikawa, Tadao; Muraoka, Ikuo [Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Soda, Kenji [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan); Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, Suita, Osaka 564-8680 (Japan); Hata, Yasuo, E-mail: hata@scl.kyoto-u.ac.jp [Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2007-11-01

    A psychrophilic malate dehydrogenase from the novel Antarctic bacterium F. frigidimaris KUC-1 was crystallized using the hanging-drop vapour-diffusion method. The crystals contained one tetrameric molecule per asymmetric unit. The best crystal diffracted to 1.8 Å resolution. Flavobacterium frigidimaris KUC-1 is a novel psychrotolerant bacterium isolated from Antarctic seawater. Malate dehydrogenase (MDH) is an essential metabolic enzyme in the citric acid cycle and has been cloned, overexpressed and purified from F. frigidimaris KUC-1. In contrast to the already known dimeric form of MDH from the psychrophile Aquaspirillium arcticum, F. frigidimaris MDH exists as a tetramer. It was crystallized at 288 K by the hanging-drop vapour-diffusion method using ammonium sulfate as the precipitating agent. The crystal diffracted to a maximum resolution of 1.80 Å. It contains one tetrameric molecule in the asymmetric unit.

  20. Serine one-carbon catabolism with formate overflow

    Science.gov (United States)

    Meiser, Johannes; Tumanov, Sergey; Maddocks, Oliver; Labuschagne, Christiaan Fred; Athineos, Dimitris; Van Den Broek, Niels; Mackay, Gillian M.; Gottlieb, Eyal; Blyth, Karen; Vousden, Karen; Kamphorst, Jurre J.; Vazquez, Alexei

    2016-01-01

    Serine catabolism to glycine and a one-carbon unit has been linked to the anabolic requirements of proliferating mammalian cells. However, genome-scale modeling predicts a catabolic role with one-carbon release as formate. We experimentally prove that in cultured cancer cells and nontransformed fibroblasts, most of the serine-derived one-carbon units are released from cells as formate, and that formate release is dependent on mitochondrial reverse 10-CHO-THF synthetase activity. We also show that in cancer cells, formate release is coupled to mitochondrial complex I activity, whereas in nontransformed fibroblasts, it is partially insensitive to inhibition of complex I activity. We demonstrate that in mice, about 50% of plasma formate is derived from serine and that serine starvation or complex I inhibition reduces formate synthesis in vivo. These observations transform our understanding of one-carbon metabolism and have implications for the treatment of diabetes and cancer with complex I inhibitors.

  1. Identity and physiology of a new psychrophilic eukaryotic green alga, Chlorella sp., strain BI, isolated from a transitory pond near Bratina Island, Antarctica

    Science.gov (United States)

    Morgan-Kiss, R. M.; Ivanov, A.G.; Modla, S.; Czymmek, K.; Huner, N.P.A.; Priscu, J.C.; Lisle, J.T.; Hanson, T.E.

    2008-01-01

    Permanently low temperature environments are one of the most abundant microbial habitats on earth. As in most ecosystems, photosynthetic organisms drive primary production in low temperature food webs. Many of these phototrophic microorganisms are psychrophilic; however, functioning of the photosynthetic processes of these enigmatic psychrophiles (the 'photopsychrophiles') in cold environments is not well understood. Here we describe a new chlorophyte isolated from a low temperature pond, on the Ross Ice Shelf near Bratina Island, Antarctica. Phylogenetic and morphological analyses place this strain in the Chlorella clade, and we have named this new chlorophyte Chlorella BI. Chlorella BI is a psychrophilic species, exhibiting optimum temperature for growth at around 10??C. However, psychrophily in the Antarctic Chlorella was not linked to high levels of membrane-associated poly-unsaturated fatty acids. Unlike the model Antarctic lake alga, Chlamydomonas raudensis UWO241, Chlorella BI has retained the ability for dynamic short term adjustment of light energy distribution between photosystem II (PS II) and photosystem I (PS I). In addition, Chlorella BI can grow under a variety of trophic modes, including heterotrophic growth in the dark. Thus, this newly isolated photopsychrophile has retained a higher versatility in response to environmental change than other well studied cold-adapted chlorophytes. ?? 2008 Springer.

  2. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    Science.gov (United States)

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  3. A metagenomic alkaline protease from saline habitat: cloning, over-expression and functional attributes.

    Science.gov (United States)

    Purohit, Megha K; Singh, Satya P

    2013-02-01

    Metagenomics has opened new horizon to unlock the biotechnological potential for novel enzymes. An alkaline protease gene was obtained from the total environmental DNA extracted from a saline habitat. After cloning and sequencing, it was identified that the protease gene related to uncultivable bacteria (HM219181). The protease was over expressed at 6h of induction with optimum induction at 1mM IPTG and 27°C. The purified enzyme was characterized with respect to various factors; temperature, pH, NaCl and chemical denaturant. The sequence analysis indicated a hydrophobic tendency of the protein, while the predicted 3D structure indicated the enzyme as a serine protease.

  4. 2nd Generation Alkaline Electrolysis

    DEFF Research Database (Denmark)

    Yde, Lars; Kjartansdóttir, Cecilia Kristin; Allebrod, Frank;

    This report provides the results of the 2nd Generation Alkaline Electrolysis project which was initiated in 2008. The project has been conducted from 2009-2012 by a consortium comprising Århus University Business and Social Science – Centre for Energy Technologies (CET (former HIRC)), Technical...

  5. Alkaline resistant ceramics; Alkalimotstaandskraftiga keramer

    Energy Technology Data Exchange (ETDEWEB)

    Westberg, Stig-Bjoern [Vattenfall Utveckling AB, Aelvkarleby (Sweden)

    2001-02-01

    Despite durability in several environments, ceramics and refractories can not endure alkaline environments at high temperature. An example of such an environment is when burning biofuel in modern heat and power plants in which the demand for increasing efficiency results in higher combustion temperatures and content of alkaline substances in the flue gas. Some experiences of these environments has been gained from such vastly different equipment as regenerator chambers in the glass industry and MHD-generators. The grains of a ceramic material are usually bonded together by a glassy phase which despite it frequently being a minor constituent render the materials properties and limits its use at elevated temperature. The damage is usually caused by alkaline containing low-melting phases and the decrease of the viscosity of the bonding glass phase which is caused by the alkaline. The surfaces which are exposed to the flue gas in a modern power plant are not only exposed to the high temperature but also a corroding and eroding, particle containing, gas flow of high velocity. The use of conventional refractory products is limited to 1300-1350 deg C. Higher strength and fracture toughness as well as durability against gases, slag and melts at temperatures exceeding 1700 deg C are expected of the materials of the future. Continuous transport of corrosive compounds to the surface and corrosion products from the surface as well as a suitable environment for the corrosion to occur in are prerequisites for extensive corrosion to come about. The highest corrosion rate is therefore found in a temperature interval between the dew point and the melting point of the alkaline-constituent containing compound. It is therefore important that the corrosion resistance is sufficient in the environment in which alkaline containing melts or slag may appear. In environments such as these, even under normal circumstances durable ceramics, such as alumina and silicon carbide, are attacked

  6. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  7. Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Hsiu-Ying, E-mail: hychung@mail.fcu.edu.tw; Li, Cheng Che

    2013-03-01

    A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3NSS peptide solution, and finally immersed in artificial saliva for 24 h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3NSS peptide compared to samples without peptide treatment. - Highlights: Black-Right-Pointing-Pointer The degree of nanohardness recovery of enamel was 4 times greater with the aid of 3NSS peptide. Black-Right-Pointing-Pointer 3NSS peptide promoted the formation of hydroxyapatites with

  8. Overexpression of serine racemase in retina and overproduction of D-serine in eyes of streptozotocin-induced diabetic retinopathy

    Directory of Open Access Journals (Sweden)

    Yin Guibin

    2011-09-01

    Full Text Available Abstract Background Recent data indicate that inflammatory mechanisms contribute to diabetic retinopathy (DR. We have determined that serine racemase (SR expression is increased by inflammatory stimuli including liposaccharide (LPS, amyloid β-peptide (A-beta, and secreted amyloid precursor protein (sAPP; expression is decreased by the anti-inflammatory drug, dexamethasone. We tested possibility that SR and its product, D-serine, were altered in a rat model of DR. Methods Intraperitoneal injection of streptozotocin (STZ; 70 mg/kg body weight to Sprague-Dawley rats produced type-I diabetic mellitus (fasting blood sugar higher than 300 mg/dL. At 3 and 5 months after STZ or saline injection, retinas from some rats were subjected to cryosectioning for immunofluorescent analysis of SR and TUNEL assay of apoptosis. Retinal homogenates were used to detect SR levels and Jun N-terminal kinase (JNK activation by immunoblotting. Aqueous humor and retina were also collected to assay for neurotransmitters, including glutamate and D-serine, by reverse-phase HPLC. Results Compared to saline-injected rats, STZ-injected (diabetic rats showed elevation of SR protein levels in retinal homogenates, attributed to the inner nuclear layer (INL by immunofluorescence. Aqueous humor fluid from STZ-injected rats contained significantly higher levels of glutamate and D-serine compared to controls; by contrast, D-serine levels in retinas did not differ. Levels of activated JNK were elevated in diabetic retinas compared to controls. Conclusions Increased expression of SR in retina and higher levels of glutamate and D-serine in aqueous humor of STZ-treated rats may result from activation of the JNK pathway in diabetic sequelae. Our data suggest that the inflammatory conditions that prevail during DR result in elevation of D-serine, a neurotransmitter contributing to glutamate toxicity, potentially exacerbating the death of retinal ganglion cells in this condition.

  9. Characterization of NADP(+)-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium, Colwellia psychrerythraea strain 34H.

    Science.gov (United States)

    Suzuki, Kaori; Takada, Yasuhiro

    2016-08-01

    NADP(+)-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.

  10. Purification, characterization, and overexpression of psychrophilic and thermolabile malate dehydrogenase of a novel antarctic psychrotolerant, Flavobacterium frigidimaris KUC-1.

    Science.gov (United States)

    Oikawa, Tadao; Yamamoto, Noriko; Shimoke, Koji; Uesato, Shinichi; Ikeuchi, Toshihiko; Fujioka, Toru

    2005-11-01

    We purified the psychrophilic and thermolabile malate dehydrogenase to homogeneity from a novel psychrotolerant, Flavobacterium frigidimaris KUC-1, isolated from Antarctic seawater. The enzyme was a homotetramer with a molecular weight of about 123 k and that of the subunit was about 32 k. The enzyme required NAD(P)(+) as a coenzyme and catalyzed the oxidation of L-malate and the reduction of oxalacetate specifically. The reaction proceeded through an ordered bi-bi mechanism. The enzyme was highly susceptible to heat treatment, and the half-life time at 40 degrees C was estimated to be 3.0 min. The k(cat)/K(m) (microM(-1).s(-1)) values for L-malate and NAD(+) at 30 degrees C were 289 and 2,790, respectively. The enzyme showed pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of the coenzyme. The enzyme contained 311 amino acid residues and much lower numbers of proline and arginine residues than other malate dehydrogenases.

  11. A novel psychrophilic lipase from Pseudomonas fluorescens with unique property in chiral resolution and biodiesel production via transesterification

    Energy Technology Data Exchange (ETDEWEB)

    Luo Yu; Zheng Yitao; Jiang Zhengbing; Ma Yushu; Wei Dongzhi [East China Univ. of Science and Tech., Shanghai (China). State Key Lab. of Bioreactor Engineering

    2006-11-15

    A lipase-producing bacterium strain B68 screened from soil samples of China was identified as Pseudomonas fluorescens. With GenomeWalker, the open reading frame of lipase gene lipB68, encoding 476 amino acids, was cloned and expressed in Escherichia coli BL21 (DE3). By affinity chromatography, the recombinant LipB68 protein was purified to the purity of 95%. As a member of lipase subfamily I.3, LipB68 has a unique optimum temperature of 20 C, which was the lowest in this subfamily. In chiral resolution, LipB68 effectively catalyzed the transesterification of both a-phenylethanol and a-phenylpropanol at 20 C, achieving E values greater than 100 and 60 after 120 h, respectively. Among all the known catalysts in biodiesel production, LipB68 produced biodiesel with a yield of 92% after 12 h, at the lowest temperature of 20 C, and is the first one of the I.3 lipase subfamily reported to be capable of catalyzing the transesterification reaction of biodiesel production. Since lipasemediated biodiesel production is normally carried out at 35-50 C, the availability of a highly active lipase with a low optimal temperature can provide substantial savings in energy consumption. Thus, this novel psychrophilic lipase (LipB68) may represent a highly competitive energy-saving biocatalyst. (orig.)

  12. Dioszegia antarctica sp. nov. and Dioszegia cryoxerica sp. nov., psychrophilic basidiomycetous yeasts from polar desert soils in Antarctica

    Science.gov (United States)

    Rodriguez, Russell J.; Connell, L.; Redman, R.; Barrett, A.; Iszard, M.; Fonseca, A.

    2010-01-01

    During a survey of the culturable soil fungal population in samples collected in Taylor Valley, South Victoria Land, Antarctica, 13 basidiomycetous yeast strains with orange-coloured colonies were isolated. Phylogenetic analyses of internal transcribed spacer (ITS) and partial LSU rRNA gene sequences showed that the strains belong to the Dioszegia clade of the Tremellales (Tremellomycetes, Agaricomycotina), but did not correspond to any of the hitherto recognized species. Two novel species, Dioszegia antarctica sp. nov. (type strain ANT-03-116T =CBS 10920T =PYCC 5970T) and Dioszegia cryoxerica sp. nov. (type strain ANT-03-071T =CBS 10919T =PYCC 5967T), are described to accommodate ten and three of these strains, respectively. Analysis of ITS sequences demonstrated intrastrain sequence heterogeneity in D. cryoxerica. The latter species is also notable for producing true hyphae with clamp connections and haustoria. However, no sexual structures were observed. The two novel species can be considered obligate psychrophiles, since they failed to grow above 20 °C and grew best between 10 and 15 °C.

  13. Structural Investigation of the Oligosaccharide Portion Isolated from the Lipooligosaccharide of the Permafrost Psychrophile Psychrobacter arcticus 273-4

    Science.gov (United States)

    Casillo, Angela; Parrilli, Ermenegilda; Filomena, Sannino; Lindner, Buko; Lanzetta, Rosa; Parrilli, Michelangelo; Tutino, Maria Luisa; Corsaro, Maria Michela

    2015-01-01

    Psychrophilic microorganisms have successfully colonized all permanently cold environments from the deep sea to mountain and polar regions. The ability of an organism to survive and grow in cryoenviroments depends on a number of adaptive strategies aimed at maintaining vital cellular functions at subzero temperatures, which include the structural modifications of the membrane. To understand the role of the membrane in the adaptation, it is necessary to characterize the cell-wall components, such as the lipopolysaccharides, that represent the major constituent of the outer membrane. The aim of this study was to investigate the structure of the carbohydrate backbone of the lipooligosaccharide (LOS) isolated from the cold-adapted Psychrobacter arcticus 273-4. The strain, isolated from a 20,000-to-30,000-year-old continuously frozen permafrost in Siberia, was cultivated at 4 °C. The LOS was isolated from dry cells and analyzed by means of chemical methods. In particular, it was degraded either by mild acid hydrolysis or by hydrazinolysis and investigated in detail by 1H and 13C NMR spectroscopy and by ESI FT-ICR mass spectrometry. The oligosaccharide was characterized by the substitution of the heptose residue, usually linked to Kdo in the inner core, with a glucose, and for the unusual presence of N-acetylmuramic acid. PMID:26204948

  14. Accelerated evolution of crotalinae snake venom gland serine proteases.

    Science.gov (United States)

    Deshimaru, M; Ogawa, T; Nakashima, K; Nobuhisa, I; Chijiwa, T; Shimohigashi, Y; Fukumaki, Y; Niwa, M; Yamashina, I; Hattori, S; Ohno, M

    1996-11-11

    Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

  15. Positive control of D-serine deaminase synthesis in vitro.

    Science.gov (United States)

    Heincz, M C; Kelker, N E; McFall, E

    1978-04-01

    Efficient constitutive synthesis of D-serine deaminase [D-serine hydro-lyase (deaminating); EC 4.2.1.14] is obtained in vitro by using a slightly modified Zubay system programmed with dsdO6 dsdA+DNA. Synthesis from a dsdO+ dsdA+ template requires active dsdC gene product and 3':5'-cyclic AMP. In vitro synthesis of dsdC product is obtained with a dsdC+ dsdO+ dsdA+ or a dsdCc dsdO+ dsdA+ template; this synthesis is thermosensitive and can be uncoupled from D-serine deaminase synthesis by temperature shift.

  16. Synthesis of o-L-α-glycerylphosphoryl-L-serine

    Institute of Scientific and Technical Information of China (English)

    TANG Rui-ren; YAN Zi-er; LUO Yi-ming

    2005-01-01

    A new efficient method for preparing o-L-α-glycerylphosphoryl-L-serine was presented. D-α, β-isopropylidene glycerol was phosphorylated with phenylphosphoryl dichloride and the resulting o-D-α,β-isopropylidene glycerylphenylphosphoryl chloride was esterified with N-tert-butoxycarbonyl-L-serine ethyl ester in the presence of pyridine to give acetone L-α-glycerylphenylphosphoryl-N-tert-butoxycarbonyl-L-serine ethyl ester. Finally, the protective groups were removed by two-step hydrolysis while strictly controlling pH value. The reaction to produce (1, 2), (5, 6)-diisopropylidene-D-mannitol, which is the key precursor for the preparation of D-α, β-isopropylidene glycerol, was monitored by using gas chromatography-mass spectrometer in order to obtain an optimum yield of 65%. All compounds were characterized through elemental analysis, infrared spectra, 1H, 13C, 31P-nuclear magnetic resonance spectra and mass spectra.

  17. Development of alkaline fuel cells.

    Energy Technology Data Exchange (ETDEWEB)

    Hibbs, Michael R.; Jenkins, Janelle E.; Alam, Todd Michael; Janarthanan, Rajeswari; Horan, James L.; Caire, Benjamin R.; Ziegler, Zachary C.; Herring, Andrew M.; Yang, Yuan; Zuo, Xiaobing; Robson, Michael H.; Artyushkova, Kateryna; Patterson, Wendy; Atanassov, Plamen Borissov

    2013-09-01

    This project focuses on the development and demonstration of anion exchange membrane (AEM) fuel cells for portable power applications. Novel polymeric anion exchange membranes and ionomers with high chemical stabilities were prepared characterized by researchers at Sandia National Laboratories. Durable, non-precious metal catalysts were prepared by Dr. Plamen Atanassovs research group at the University of New Mexico by utilizing an aerosol-based process to prepare templated nano-structures. Dr. Andy Herrings group at the Colorado School of Mines combined all of these materials to fabricate and test membrane electrode assemblies for single cell testing in a methanol-fueled alkaline system. The highest power density achieved in this study was 54 mW/cm2 which was 90% of the project target and the highest reported power density for a direct methanol alkaline fuel cell.

  18. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs: Biogenesis and Function

    Directory of Open Access Journals (Sweden)

    Nathalie Dautin

    2010-05-01

    Full Text Available Serine Protease Autotransporters of Enterobacteriaceae (SPATEs constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

  19. Serine protease identification (in vitro) and molecular structure predictions (in silico) from a phytopathogenic fungus, Alternaria solani.

    Science.gov (United States)

    Chandrasekaran, Murugesan; Chandrasekar, Raman; Sa, Tongmin; Sathiyabama, Muthukrishnan

    2014-07-01

    Serine proteases are involved in an enormous number of biological processes. The present study aims at characterizing three-dimensional (3D) molecular architecture of serine proteases from early blight pathogen, Alternaria solani that are hypothesized to be markers of phytopathogenicity. A serine protease was purified to homogeneity and MALDI-TOF-MS/MS analysis revealed that protease produced by A. solani belongs to alkaline serine proteases (AsP). AsP is made up of 403 amino acid residues with molecular weight of 42.1 kDa (Isoelectric point - 6.51) and its molecular formula was C1859 H2930 N516 O595 S4 . AsP structure model was built based on its comparative homology with serine protease using the program, MODELER. AsP had 16 β-sheets and 10 α-helices, with Ser(350) (G347-G357), Asp(158) (D158-H169), and His(193) (H193-G203) in separate turn/coil structures. Biological metal binding region situated near 6th-helix and His(193) residue is responsible for metal binding site. Also, calcium ion (Ca(2+)) is coordinated by the carboxyl groups of Lys(84), Ile(85), Lys(86), Asp(87), Phe(88), Ala(89), Ala(90) (K84-A90) for first Ca(2+) binding site and carbonyl oxygen atom of Lys(244), Gly(245), Arg(246), Thr(247), Lys(248), Lys(249), and Ala(250) (K244-A250), for second Ca(2+) binding site. Moreover, Ramachandran plot analysis of protein residues falling into most favored secondary structures were determined (83.3%). The predicted molecular 3D structural model was further verified using PROCHECK, ERRAT, and VADAR servers to confirm the geometry and stereo-chemical parameters of the molecular structural design. The functional analysis of AsP 3D molecular structure predictions familiar in the current study may provide a new perspective in the understanding and identification of antifungal protease inhibitor designing. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Structure Prediction of a Novel Exo-β-1,3-Glucanase: Insights into the Cold Adaptation of Psychrophilic Yeast Glaciozyma antarctica PI12.

    Science.gov (United States)

    Mohammadi, Salimeh; Parvizpour, Sepideh; Razmara, Jafar; Abu Bakar, Farah Diba; Illias, Rosli Md; Mahadi, Nor Muhammad; Murad, Abdul MunirAbdul

    2016-07-30

    We report a detailed structural analysis of the psychrophilic exo-β-1,3-glucanase (GaExg55) from Glaciozyma antarctica PI12. This study elucidates the structural basis of exo-1,3-β-1,3-glucanase from this psychrophilic yeast. The structural prediction of GaExg55 remains a challenge because of its low sequence identity (37 %). A 3D model was constructed for GaExg55. Threading approach was employed to determine a suitable template and generate optimal target-template alignment for establishing the model using MODELLER9v15. The primary sequence analysis of GaExg55 with other mesophilic exo-1,3-β-glucanases indicated that an increased flexibility conferred to the enzyme by a set of amino acids substitutions in the surface and loop regions of GaExg55, thereby facilitating its structure to cold adaptation. A comparison of GaExg55 with other mesophilic exo-β-1,3-glucanases proposed that the catalytic activity and structural flexibility at cold environment were attained through a reduced amount of hydrogen bonds and salt bridges, as well as an increased exposure of the hydrophobic side chains to the solvent. A molecular dynamics simulation was also performed using GROMACS software to evaluate the stability of the GaExg55 structure at varying low temperatures. The simulation result confirmed the above findings for cold adaptation of the psychrophilic GaExg55. Furthermore, the structural analysis of GaExg55 with large catalytic cleft and wide active site pocket confirmed the high activity of GaExg55 to hydrolyze polysaccharide substrates.

  1. Intervention with Serine Protease Activity with Small Peptides

    DEFF Research Database (Denmark)

    Xu, Peng

    2015-01-01

    , plasma kallikrein, which contributes to the pathogenesis in hereditary angioedema. According to the X-ray crystal structure analysis, we proposed a principle for designing inhibitors of other serine proteases from mupain-1. In order to be able to evaluate the inhibitory activities of our peptides in vivo...

  2. Intermolecular Phosphoryl Transfer Between Serine and Histidine Residues

    Institute of Scientific and Technical Information of China (English)

    Yu Qian SU; Ming Yu NIU; Shu Xia CAO; Jian Chen ZHANG; Yu Fen ZHAO

    2004-01-01

    A novel intermolecular phosphoryl transfer from O-trimethylsilyl-N-(O, O-diisopropyl) phosphoryl serine trimethylsilyl ester to N, N'-bis(trimethylsilyl) histidine trimethylsilyl ester was studied through electrospray ionization mass spectrometry (ESI-MS). It was proposed that the transfer reaction went through penta-coordinated phosphorus intermediate.

  3. Characterization of the C-Terminal Propeptide Involved in Bacterial Wall Spanning of Alpha-Amylase from the Psychrophile Alteromonas Haloplanctis

    OpenAIRE

    Feller, Georges; D'Amico, Salvino; Benotmane, A. M.; Joly, F.; Van Beeumen, J.; Gerday, Charles

    1998-01-01

    The antarctic psychrophile Alteromonas haloplanctis secretes a Ca2+- and Cl--dependent alpha-amylase. The nucleotide sequence of the amy gene and the amino acid sequences of the gene products indicate that the alpha-amylase precursor is a preproenzyme composed by the signal peptide (24 residues), the mature alpha-amylase (453 residues, 49 kDa), and a long C-terminal propeptide or secretion helper (192 residues, 21 kDa). In cultures of the wild-type strain, the 70-kDa precursor is secreted at ...

  4. Establishment of a cell-based assay system for hepatitis C virus serine protease and its primary applications

    Institute of Scientific and Technical Information of China (English)

    Hong-Xia Mao; Shui-Yun Lan; Yun-Wen Hu; Li Xiang; Zheng-Hong Yuan

    2003-01-01

    AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents.METHOOS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems.RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems,and was apparently decreased after Pefabloc SC treatment.In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a.CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.

  5. Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

    Science.gov (United States)

    Montefusco, David J; Newcomb, Benjamin; Gandy, Jason L; Brice, Sarah E; Matmati, Nabil; Cowart, L Ashley; Hannun, Yusuf A

    2012-03-16

    Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

  6. Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS.

    Directory of Open Access Journals (Sweden)

    Vishnu Chaturvedi

    Full Text Available BACKGROUND: Massive die-offs of little brown bats (Myotis lucifugus have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS. A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals. METHODOLOGY/PRINCIPAL FINDINGS: We have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii G. destructans DNA was directly amplified from infected bat tissues, (iii Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v The fungal isolates showed psychrophilic growth. (vi We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans. CONCLUSIONS/SIGNIFICANCE: Further studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat-fungus relationships, and should aid in the screening of biological and chemical control agents.

  7. UV Radiation and Visible Light Induce hsp70 Gene Expression in the Antarctic Psychrophilic Ciliate Euplotes focardii.

    Science.gov (United States)

    Fulgentini, Lorenzo; Passini, Valerio; Colombetti, Giuliano; Miceli, Cristina; La Terza, Antonietta; Marangoni, Roberto

    2015-08-01

    The psychrophilic ciliate Euplotes focardii inhabits the shallow marine coastal sediments of Antarctica, where, over millions of years of evolution, it has reached a strict molecular adaptation to such a constant-temperature environment (about -2 °C). This long evolution at sub-zero temperatures has made E. focardii unable to respond to heat stress with the activation of its heat shock protein (hsp) 70 genes. These genes can, however, be expressed in response to other stresses, like the oxidative one, thus indicating that the molecular adaptation has exclusively altered the heat stress signaling pathways, while it has preserved hsp70 gene activation in response to other environmental stressors. Since radiative stress has proved to be affine to oxidative stress in several organisms, we investigated the capability of UV radiation to induce hsp70 transcription. E. focardii cell cultures were exposed to several different irradiation regimes, ranging from visible only to a mixture of visible, UV-A and UV-B. The irradiation values of each spectral band have been set to be comparable with those recorded in a typical Antarctic spring. Using Northern blot analysis, we measured the expression level of hsp70 immediately after irradiation (0-h-labeled samples), 1 h, and 2 h from the end of the irradiation. Surprisingly, our results showed that besides UV radiation, the visible light was also able to induce hsp70 expression in E. focardii. Moreover, spectrophotometric measurements have revealed no detectable endogenous pigments in E. focardii, making it difficult to propose a possible explanation for the visible light induction of its hsp70 genes. Further research is needed to conclusively clarify this point.

  8. Expression and characterization of a recombinant psychrophilic Cu/Zn superoxide dismutase from Deschampsia antarctica E. Desv. [Poaceae].

    Science.gov (United States)

    Rojas-Contreras, Juan A; de la Rosa, Ana P Barba; De León-Rodríguez, Antonio

    2015-04-01

    We present here the structural modeling and biochemical characterization of a recombinant superoxide dismutase (SOD) from Deschampsia antarctica E. Desv. [Poaceae] produced in Escherichia coli. The recombinant protein was purified by affinity chromatography nickel-nitrilotriacetic acid (Ni-NTA), and its identity was demonstrated by immunoblotting and inhibition by H2O2 and KCN. Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis confirmed the presence of Cu and Zn. Modeling of the D. antarctica Cu/Zn-SOD (DaSOD) amino acid sequence using the SWISS-MODEL and 2Q2L_B monomer of the psychrophilic Cu/Zu-SOD from Potentilla atrosanguinea (PaSOD) as template produced a structure similar to that of the typical eukaryotic Cu/Zn-SODs. Activity assays using the p-nitro blue tetrazolium chloride (NBT) solution method showed that the purified DaSOD had a specific activity of 5818 U/mg at 25 °C and pH 7.2 and that it was active in a pH interval of 5-8 and a temperature interval of 0-40 °C. Furthermore, DaSOD was still active at -20 °C as observed by a zymogram assay. We found 100 % activity when it was heated at 80 °C for 60 min, indicating a high thermostability. DaSOD properties suggest that this enzyme could be useful for preventing the oxidation of refrigerated or frozen foods, as well as in the preparation of cosmetic and pharmaceutical products.

  9. [Purification and properties of serine proteinases from European catfish Silurus glanis L. pancreas].

    Science.gov (United States)

    Ulitina, N N; Khabliuk, V V; Proskuriakov, M T

    2005-01-01

    Three trypsin isoforms (designated as T1, T2, and T3), three chymotrypsin isoforms (Kh1, Kh2, and Kh3), and two elastase isoforms (E1 and E2) were isolated from the pancreas of European catfish Silurus glanis L. by salting out with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion exchange chromatography on DEAE cellulose. Isoelectric points of the enzymes, determined by isoelectric focusing, amounted to 4.42 for T1, 5.64 for T2, 6.90 for T3, 4.93 for Khl, 5.23 for Kh2, 6.18 for Kh3, 6.17 for E1, and 8.48 for E2. Molecular weights of proteinases within each group were close and amounted to 30100 Da for trypsins, 39800 Da for chymotrypsins, and 24000 Da for elastases. The enzymes isolated displayed maximal activities at alkaline pH values. Inhibitor analysis demonstrated that all the proteinases isolated from European catfish pancreas belonged to the serine type.

  10. Response of maize serine/arginine-rich protein gene family in seedlings to drought stress.

    Science.gov (United States)

    Li, Jiao; Guo, Yuqi; Cui, Weiling; Xu, Aihua; Tian, Zengyuan

    2014-07-01

    Alternative splicing (AS) in eukaryotic organisms is closely related to the gene regulation in plant abiotic stress responses, in which serine/arginine-rich proteins (SR proteins) act as key regulators. The genome sequence of maize inbred line B73 was analyzed, showing that the promoter regions of SR genes possess about three to eight kinds of cis-acting regulatory elements. Twenty-seven SR genes encode alkaline proteins, and 23 of which are divided into five subgroups in terms of the first RNA recognition motif (RRM) at the amino terminal. The expression of SR genes showed tissue-specific and genotype-dependent features under drought stress in the hybrid Zhengdan-958 and its parents, Zheng-58 and Chang-7-2 via bidirectional hierarchical clustering. SR genes were down-regulated in roots while they were up-regulated in shoots under drought stress. However, SR genes were down-regulated in both roots and shoots in three different rehydration stages after severe drought stress. Additionally, a widespread alternative splicing exists in all SR genes although SR genes showed differential expression tendency under drought stress and/or during rehydration stages. Results above will deepen our understanding of the molecular mechanisms of plant response to abiotic stress from the perspective of AS-network.

  11. Development of activity-based probes for trypsin-family serine proteases.

    Science.gov (United States)

    Pan, Zhengying; Jeffery, Douglas A; Chehade, Kareem; Beltman, Jerlyn; Clark, James M; Grothaus, Paul; Bogyo, Matthew; Baruch, Amos

    2006-06-01

    A series of diphenylphosphonate-based probes were developed for the trypsin-like serine proteases. These probes selectively target serine proteases rather than general serine hydrolases that are targets for fluorophosphonate-based probes. This increased selectivity allows detection of low abundance serine proteases in complex proteomes using simple SDS-PAGE methods. We present here the application of multiple probes in enzyme activity profiling of intact mast cells, a type of inflammatory cell implicated in allergy and autoimmune diseases.

  12. Production and Characterization of Alkaline Protease from a High Yielding and Moderately Halophilic Strain of SD11 Marine Bacteria

    Directory of Open Access Journals (Sweden)

    Hongxia Cui

    2015-01-01

    Full Text Available A marine bacterium SD11, which was isolated from sea muds (Geziwo Qinhuangdao Sea area, China, was used to produce thermostable alkaline serine nonmetal protease in the skim milk agar plate medium with 10% NaCl. The optimal temperature about the manufacture of the extracellular protease was ~60°C. The crude enzyme was stable at 20–50°C. The activity was retained to 60% and 45% after heating for 1 h at 60 and 70°C, respectively. The protease was highly active in a wide pH scope (8.0–10.0 and maximum protease activity exhibited at pH 10.0. The activity was restrained by phenylmethylsulfonyl fluoride (PMSF but mildly increased (~107% in the presence of ethylenediaminetetraacetic acid (EDTA, indicating that the production contains serine-protease(s and nonmetal protease(s. Moreover, the crude alkaline protease was active with the 5 mM Ca2+, Mn2+, Zn2+, Cu2+, Na+, and K+ that existed separately. In addition, the protease showed superduper stability when exposed to an anionic surfactant (5 mM SDS, an oxidizing agent (1% H2O2, and several organic solvents (methanol, isopropanol, and acetone. These results suggest that the marine bacterium SD11 is significant in the industry from the prospects of its ability to produce thermally stable alkaline protease.

  13. Alkaline Water and Longevity: A Murine Study.

    Science.gov (United States)

    Magro, Massimiliano; Corain, Livio; Ferro, Silvia; Baratella, Davide; Bonaiuto, Emanuela; Terzo, Milo; Corraducci, Vittorino; Salmaso, Luigi; Vianello, Fabio

    2016-01-01

    The biological effect of alkaline water consumption is object of controversy. The present paper presents a 3-year survival study on a population of 150 mice, and the data were analyzed with accelerated failure time (AFT) model. Starting from the second year of life, nonparametric survival plots suggest that mice watered with alkaline water showed a better survival than control mice. Interestingly, statistical analysis revealed that alkaline water provides higher longevity in terms of "deceleration aging factor" as it increases the survival functions when compared with control group; namely, animals belonging to the population treated with alkaline water resulted in a longer lifespan. Histological examination of mice kidneys, intestine, heart, liver, and brain revealed that no significant differences emerged among the three groups indicating that no specific pathology resulted correlated with the consumption of alkaline water. These results provide an informative and quantitative summary of survival data as a function of watering with alkaline water of long-lived mouse models.

  14. Modeling and structural analysis of PA clan serine proteases

    Directory of Open Access Journals (Sweden)

    Laskar Aparna

    2012-05-01

    Full Text Available Abstract Background Serine proteases account for over a third of all known proteolytic enzymes; they are involved in a variety of physiological processes and are classified into clans sharing structural homology. The PA clan of endopeptidases is the most abundant and over two thirds of this clan is comprised of the S1 family of serine proteases, which bear the archetypal trypsin fold and have a catalytic triad in the order Histidine, Aspartate, Serine. These proteases have been studied in depth and many three dimensional structures have been experimentally determined. However, these structures mostly consist of bacterial and animal proteases, with a small number of plant and fungal proteases and as yet no structures have been determined for protozoa or archaea. The core structure and active site geometry of these proteases is of interest for many applications. This study investigated the structural properties of different S1 family serine proteases from a diverse range of taxa using molecular modeling techniques. Results Our predicted models from protozoa, archaea, fungi and plants were combined with the experimentally determined structures of 16 S1 family members and used for analysis of the catalytic core. Amino acid sequences were submitted to SWISS-MODEL for homology-based structure prediction or the LOOPP server for threading-based structure prediction. Predicted models were refined using INSIGHT II and SCRWL and validated against experimental structures. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. The structural geometry of the catalytic core shows clear deviations between taxa, but the relative positions of the catalytic triad residues were conserved. Some highly conserved residues potentially contributing to the stability of the structural core were identified. Evolutionary divergence was also exhibited by large variation in secondary structure features outside the core

  15. Purification and characterization of alkaline protease from Alcaligenes faecalis.

    Science.gov (United States)

    Thangam, E Berla; Rajkumar, G Suseela

    2002-04-01

    Extracellular alkaline protease from the alkalophilic bacterium Alcaligenes faecalis was purified by a combination of ion-exchange and size-exclusion chromatographic methods, and its properties were examined. The purified enzyme had a specific activity of 563.8 micromol of tyrosine/min per mg of protein and gave a single band on native PAGE and SDS/PAGE with a molecular mass of 67 kDa. Gelatin zymogram also revealed one clear zone of proteolytic activity which corresponded to the band obtained with native PAGE and SDS/PAGE. The enzyme had an optimal pH of 9.0 and exhibited its highest activity at 55 degrees C. The enzyme activity was inhibited by PMSF, suggesting the presence of serine residues at the active site. The enzyme had a K(m) of 1.66 mg/ml and a V(max) of 526 units/min per mg of protein with casein as the substrate.

  16. Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of white nose syndrome.

    Science.gov (United States)

    Johnson, Lynnaun J A N; Miller, Andrew N; McCleery, Robert A; McClanahan, Rod; Kath, Joseph A; Lueschow, Shiloh; Porras-Alfaro, Andrea

    2013-09-01

    Since 2006, Geomyces destructans, the causative agent of white nose syndrome (WNS), has killed over 5.7 million bats in North America. The current hypothesis suggests that this novel fungus is an invasive species from Europe, but little is known about the diversity within the genus Geomyces and its distribution on bats in the United States. We documented the psychrophilic and psychrotolerant fungal flora of hibernating bats prior to the arrival of WNS using culture-based techniques. A total of 149 cultures, which were obtained from 30 bats in five bat hibernacula located in four caves and one mine, were sequenced for the entire internal transcribed spacer (ITS) nuclear ribosomal DNA (nrDNA) region. Approximately 53 operational taxonomic units (OTUs) at 97% similarity were recovered from bat wings, with the community dominated by fungi within the genera Cladosporium, Fusarium, Geomyces, Mortierella, Penicillium, and Trichosporon. Eleven Geomyces isolates were obtained and placed in at least seven distinct Geomyces clades based on maximum-likelihood phylogenetic analyses. Temperature experiments revealed that all Geomyces strains isolated are psychrotolerant, unlike G. destructans, which is a true psychrophile. Our results confirm that a large diversity of fungi, including several Geomyces isolates, occurs on bats prior to the arrival of WNS. Most of these isolates were obtained from damaged wings. Additional studies need to be conducted to determine potential ecological roles of these abundant Geomyces strains isolated from bats.

  17. Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

    Directory of Open Access Journals (Sweden)

    Claudio Laurido

    2012-01-01

    Full Text Available N-methyl-D-aspartic acid receptor (NMDAr activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR. D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS and L-erythro-3-hydroxyaspartate (LEHA, among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL and LEHA (100 μg/10 μL in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

  18. Psychrophilic sulfate-reducing bacteria isolated from permanently cold Arctic marine sediments: description of Desulfofrigrus oceanense gen. nov., sp nov., Desulfofrigus fragile sp nov., Desulfofaba gelida gen. nov., sp nov., Desulfotalea psychrophila gen. nov., sp nov and Desulfotalea arctica sp

    DEFF Research Database (Denmark)

    Knoblauch, C.; Sahm, K.; Jørgensen, BB

    1999-01-01

    Five psychrophilic, Gram-negative, sulfate-reducing bacteria were isolated from marine sediments off the coast of Svalbard. All isolates grew at the in situ temperature of -1.7 degrees C. In batch cultures, strain PSv29(T) had the highest growth rate at 7 degrees C, strains ASv26(T) and LSv54(T...

  19. 杭州地区原料奶中嗜冷菌的分离鉴定%Isolation and identification species of psychrophilic microorganisms from raw milk provided by the pastures in Hangzhou district

    Institute of Scientific and Technical Information of China (English)

    吕元; 叶兴乾

    2012-01-01

    以杭州地区牧场的原料奶为实验对象,对原料奶受嗜冷菌污染情况进行调研,并对其中的嗜冷菌进行分离鉴定,确定革兰氏阴性嗜冷菌占绝大部分,其中荧光假单胞菌、鲁氏不动杆菌、阪崎肠杆菌为优势嗜冷菌种,为进一步研究出一种快速检测原料奶嗜冷菌的方法奠定理论基础.%This paper focused on isolation and identification species of psychrophilic microorganisms from raw* milk provided by the pastures in Hangzhou district. Most of psychrophilic microorganisms in raw milk of Hangzhou district were Gram-negative psychrophilic microorganisms. Pseudomonas fluorescens^Acinetobacter lwofEi and Enterobacter sakazakii were dominant microorganisms. All of the resalts were the basis of set up a new rapid methods for dairy industry to detect the contamination of psychrophilic microorganisms in raw milk.

  20. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna S P; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...... kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1...

  1. Proteolytic profiling and comparative analyses of active trypsin-like serine peptidases in preimaginal stages of Culex quinquefasciatus

    Directory of Open Access Journals (Sweden)

    Borges-Veloso Andre

    2012-06-01

    Full Text Available Abstract Background The mosquito Culex quinquefasciatu s, a widespread insect in tropical and sub-tropical regions of the world, is a vector of multiple arboviruses and parasites, and is considered an important risk to human and veterinary health. Proteolytic enzymes play crucial roles in the insect physiology including the modulation of embryonic development and food digestion. Therefore, these enzymes represent important targets for the development of new control strategies. This study presents zymographic characterization and comparative analysis of the proteolytic activity found in eggs, larval instars and pupae of Culex quinquefasciatus. Methods The proteolytic profiles of eggs, larvae and pupa of Cx. quinquefasciatus were characterized by SDS-PAGE co-polymerized with 0.1% gelatin, according to the pH, temperature and peptidase inhibitor sensitivity. In addition, the proteolytic activities were characterized in solution using 100 μM of the fluorogenic substrate Z-Phe-Arg-AMC. Results Comparison of the proteolytic profiles by substrate-SDS-PAGE from all preimaginal stages of the insect revealed qualitative and quantitative differences in the peptidase expression among eggs, larvae and pupae. Use of specific inhibitors revealed that the proteolytic activity from preimaginal stages is mostly due to trypsin-like serine peptidases that display optimal activity at alkaline pH. In-solution, proteolytic assays of the four larval instars using the fluorogenic substrate Z-Phe-Arg-AMC in the presence or absence of a trypsin-like serine peptidase inhibitor confirmed the results obtained by substrate-SDS-PAGE analysis. The trypsin-like serine peptidases of the four larval instars were functional over a wide range of temperatures, showing activities at 25°C and 65°C, with an optimal activity between 37°C and 50°C. Conclusion The combined use of zymography and in-solution assays, as performed in this study, allowed for a more detailed analysis of the

  2. New Insights into the Regulation of Serine Palmitoyltransferase

    Science.gov (United States)

    2014-02-07

    is catalyzed by the enzyme serine palmitoyltransferase (SPT) which is present in all eukaryotes and even in some prokaryotes . The S. cerevisiae SPT...based on their molecular shapes and chemical properties. They are important components of membranes , predominantly found in the plasma membrane in...contrast to the intracellular membranes which have less sphingolipids (87). The chemical properties of the sphingolipids modulate the physical

  3. Dynamics simulation of the interaction between serine and water

    Science.gov (United States)

    Liu, Yang; Zhang, Peng; Lu, Ying-Bo; Han, Sheng-Hao; Yu, Hui

    2013-05-01

    Using the first principles density functional theory (DFT), we simulated the neutron scattering spectra of the hydration dynamics of serine. Experimental data analyses have shown that dissociative H2O molecules were more likely to form hydrogen bonds (H-bonds) with an -OH group in monohydrated serine and easily shift to a -NH_3 ^ + group at a higher hydration level [P. Zhang, Y. Zhang, S. H. Han, Q. W. Yan, R. C. Ford, and J. C. Li, J. Phys. Chem. A 110, 5000 (2006), 10.1021/jp0569741]. We set the 1:1 ratio hydrated compounds at the two positions and found that the H2O could be optimized to form H-bonds with -OH and -NH3+ separately. When the simulated phonon signals of the -OH…H2O and -NH3+…H2O combinations were summed on a 3:1 scale, the calculating spectra were in good agreement with the experimental results, especially for the peak at 423 cm-1 of the -OH…H2O combination and the peak at 367 cm-1 of the -NH3+…H2O combination, which mutually complemented the real spectrum. We confirm that H2O may break the intermolecular H-bonds of the interlaced binding -OH to form a new structure, and that with the skeleton deformation of serine, H2O forms stronger H-bonds more often with the -NH3+ side indicating the flexible dynamic mechanism of the serine hydration process.

  4. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    Directory of Open Access Journals (Sweden)

    Huan Zhang

    2017-01-01

    Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  5. A serine proteinase inhibitor from frog eggs with bacteriostatic activity.

    Science.gov (United States)

    Han, Yaoping; Yu, Haining; Yang, Xinbo; Rees, Huw H; Liu, Jingze; Lai, Ren

    2008-01-01

    By Sephadex G-50 gel filtration, Resource Q anionic exchange and C4 reversed phase liquid high performance liquid chromatography, a proteinase inhibitor protein (Ranaserpin) was identified and purified from the eggs of the odour frog, Rana grahami. The protein displayed a single band adjacent to the molecular weight marker of 14.4 kDa analyzed by SDS-PAGE. The inhibitor protein homogeneity and its molecular weight were confirmed again by MALDI-TOF mass spectrometry analysis. The MALDI-TOF mass spectrum analysis gave this inhibitor protein an m/z of 14422.26 that was matched well with the result from SDS-PAGE. This protein is a serine proteinase inhibitor targeting multiple proteinases including trypsin, elastase, and subtilisin. Ranaserpin inhibited the proteolytic activities of trypsin, elastase, and subtilisin. It has an inhibitory constant (K(i)) of 6.2 x 10(-8) M, 2.7 x 10(-7) M and 2.2 x 10(-8) M for trypsin, elastase, and subtilisin, respectively. This serine proteinase inhibitor exhibited bacteriostatic effect on Gram-positive bacteria Bacillus subtilis (ATCC 6633). It was suggested that ranaserpin might act as a defensive role in resistance to invasion of pests or pathogens. This is the first report of serine proteinase inhibitor and its direct defensive role from amphibian eggs.

  6. graal: a Drosophila gene coding for several mosaic serine proteases.

    Science.gov (United States)

    Munier, Anne Isabelle; Medzhitov, Ruslan; Janeway, Charles A; Doucet, Daniel; Capovilla, Maria; Lagueux, Marie

    2004-10-01

    Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge.

  7. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    Science.gov (United States)

    Zhang, Huan; Fei, Rui; Xue, Baigong; Yu, Shanshan; Zhang, Zuoming; Zhong, Sheng; Gao, Yuanqi; Zhou, Xiaoli

    2017-01-01

    Serine protease inhibitors (serpins) are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI) of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles. PMID:28067849

  8. Antibacterial activity of silver nanoparticles synthesized from serine

    Energy Technology Data Exchange (ETDEWEB)

    Jayaprakash, N. [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203 (India); Judith Vijaya, J., E-mail: jjvijayaloyola@yahoo.co.in [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); John Kennedy, L. [Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048 (India); Priadharsini, K.; Palani, P. [Department of Center for Advanced Study in Botany, University of Madras, Guindy Campus, Chennai 600 025 (India)

    2015-04-01

    Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV–Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443 nm. The emission spectrum of Ag NPs showed an emission band at 484 nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO{sub 3} against Gram-positive and Gram-negative bacteria. - Highlights: • Microwave irradiation method is used to synthesize silver nanoparticles. • Highly stable silver nanoparticles are produced from serine. • A detailed study of antibacterial activities is discussed. • Formation mechanism of silver microspheres has been proposed.

  9. Serine racemase: a key player in apoptosis and necrosis

    Directory of Open Access Journals (Sweden)

    Nadia eCanu

    2014-04-01

    Full Text Available A fine balance between cell survival and cell death is required to sculpt the nervous system during development. However, an excess of cell death can occur following trauma, exposure to neurotoxins or alcohol, and some developmental and neurodegenerative diseases, such as Alzheimer’s disease (AD. N-Methyl-D-aspartate receptors (NMDARs support synaptic plasticity and survival of many neuronal populations whereas inappropriate activation may promote various forms of cell death, apoptosis and necrosis representing the two extremes of a continuum of cell death processes both in vitro and in vivo. Hence, by identifying the switches controlling pro-survival vs. apoptosis and apoptosis vs. pro-excitotoxic outcome of NMDAR stimulation, NMDAR modulators could be developed that selectively block the cell death enhancing pro-survival signaling or synaptic plasticity mediated by NMDAR. Among these modulators, a role is emerging for the enzyme serine racemase (SR that synthesizes D-serine, a key co-agonist with glutamate at NMDAR. This review summarizes the experimental evidence from in vitro neuronal cultures -- with special emphasis on cerebellar granule neurons (CGNs -- and in vivo models of neurodegeneration, where the dual role of the SR/D-serine pathway as a master regulator of apoptosis and the apoptosis-necrosis shift will be discussed.

  10. Serine racemase expression in mouse cerebral cortex after permanent focal cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Li-zhen WANG; Xing-zu ZHU

    2004-01-01

    AIM: To study the alterations of the expressions of serine racemase in C57BL/6 mouse brain after permanent focal cerebral ischemia. METHODS: The mRNA level and the protein level of serine racemase were assayed by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. The amount of D-serine and L-serine were measured by HPLC. RESULTS: High levels of serine racemase were constitutively expressed in the normal cortex of mouse. At early stage after middle cerebral artery occlusion (MCAO), no significant change in expression of serine racemase was observed in temporoparietal cortex in ipsilateral hemisphere. However,delayed transient decreases of serine racemase in both mRNA and protein levels were detected from d 6 to d 10 after ischemia. Correspondingly, D-serine concentration also declined in the ipsilateral cortex during this period when compared with the D-serine level in the contralateral cortex. CONCLUSION:Delayed decreases in serine racemase expression and D-serine level occurred in the temporoparietal cortex at the late stage after focal cerebral ischemia.

  11. Handbook of Indigenous Foods Involving Alkaline Fermentation

    NARCIS (Netherlands)

    Sarkar, P.K.; Nout, M.J.R.

    2014-01-01

    This book details the basic approaches of alkaline fermentation, provides a brief history, and offers an overview of the subject. The book discusses the diversity of indigenous fermented foods involving an alkaline reaction, as well as the taxonomy, ecology, physiology, and genetics of predominant m

  12. A novel psychrophilic Janthinobacterium lividum MMPP4 isolated from Manimahesh Lake of Chamba District of Himachal Pradesh, India

    Directory of Open Access Journals (Sweden)

    Ranjana Suman

    2015-04-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE MicrosoftInternetExplorer4 We isolated psychrophilic bacterial strains from high altitude (elevation 13,390 ft Manimahesh Lake of Chamba District of Himachal Pradesh. Based on phenotypic characteristics and growth appearance at 4ºC, four bacterial isolates (MMPP2, MMPP4, MMPP5, and MMPP7 were selected and showed optimum growth at 20ºC and no growth was detected at 25ºC.  Qualitative assay showed that cell free medium of MMPP4 prevent inhibition of freezing (antifreeze activity as determined by freezing of cell free medium at -20ºC for seven days. Proteinase K treatment and heat inactivation at 80ºC for 6 h resulted in the loss of antifreeze activity, suggesting a proteinaceous nature of antifreeze activity. MMPP4 produces protease, phytase and lipase enzymes, but did not produce amylase. Moreover, cell free supernatant of MMPP4 showed proteinaceous type of antimicrobial activity against S. aureus as measured by agar well diffusion assay.  To identify the MMPP4 strain, 16s rDNA was amplified using 27F and 1492R standard primers and sequenced on both the strands. Nucleotide BLAST and phylogenetic analysis showed 99% identity to Janthinobacterium lividum strain and deposited in GenBank accession no. as KJ509870. The finding revealed a great potential of the Janthinobacterium lividum strain MMPP4 for biotechnological applications as a source of industrially important enzymes and antimicrobial compound.     /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-qformat:yes; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:11.0pt; font-family:"Calibri","sans-serif"; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-fareast-font-family:"Times New

  13. Multisystemic functions of alkaline phosphatases.

    Science.gov (United States)

    Buchet, René; Millán, José Luis; Magne, David

    2013-01-01

    Human and mouse alkaline phosphatases (AP) are encoded by a multigene family expressed ubiquitously in multiple tissues. Gene knockout (KO) findings have helped define some of the precise exocytic functions of individual isozymes in bone, teeth, the central nervous system, and in the gut. For instance, deficiency in tissue-nonspecific alkaline phosphatase (TNAP) in mice (Alpl (-/-) mice) and humans leads to hypophosphatasia (HPP), an inborn error of metabolism characterized by epileptic seizures in the most severe cases, caused by abnormal metabolism of pyridoxal-5'-phosphate (the predominant form of vitamin B6) and by hypomineralization of the skeleton and teeth featuring rickets and early loss of teeth in children or osteomalacia and dental problems in adults caused by accumulation of inorganic pyrophosphate (PPi). Enzyme replacement therapy with mineral-targeting TNAP prevented all the manifestations of HPP in mice, and clinical trials with this protein therapeutic are showing promising results in rescuing life-threatening HPP in infants. Conversely, TNAP induction in the vasculature during generalized arterial calcification of infancy (GACI), type II diabetes, obesity, and aging can cause medial vascular calcification. TNAP inhibitors, discussed extensively in this book, are in development to prevent pathological arterial calcification. The brush border enzyme intestinal alkaline phosphatase (IAP) plays an important role in fatty acid (FA) absorption, in protecting gut barrier function, and in determining the composition of the gut microbiota via its ability to dephosphorylate lipopolysaccharide (LPS). Knockout mice (Akp3 (-/-)) deficient in duodenal-specific IAP (dIAP) become obese, and develop hyperlipidemia and hepatic steatosis when fed a high-fat diet (HFD). These changes are accompanied by upregulation in the jejunal-ileal expression of the Akp6 IAP isozyme (global IAP, or gIAP) and concomitant upregulation of FAT/CD36, a phosphorylated fatty acid

  14. Association of alkaline phosphatase phenotypes with arthritides

    Directory of Open Access Journals (Sweden)

    Padmini A

    2004-01-01

    Full Text Available Arthritides, a symmetrical polyarticular disease of the bone are a heterogenous group of disorders in which hereditary and environmental factors in combination with an altered immune response appear to play a causative and pathogenic role in its occurrence. Alkaline phosphatase (ALP is an enzyme found in all tissues, with particularly high concentrations of ALP observed in the liver, bile ducts, placenta, and bone.Alkaline phosphatase is an orthophosphoric monoester phosphohydrolase catalyzing the hydrolysis of organic esters at alkaline pH, indicating that alkaline phosphatase is involved in fundamental biological processes.1 The present study envisages on identifying the specific electromorphic association of alkaline phosphatase with arthritides. Phenotyping of serum samples was carried out by PAGE (Polyacrylamide gel electrophoresis following Davies (19642 protocol on 41 juvenile arthritis, 150 rheumatoid arthritis and 100 osteo arthritis apart from, 25 normal children and 100 adult healthy subjects. Phenotyping of alkaline phosphatase revealed an increase in preponderance of p+ and p++ phenotypes in juvenile, rheumatoid and osteo arthritic patients. However a significant association of these phenotypes was observed only with rheumatoid arthritis condition (c2:17.46. Similarly, a significant increase of p+ phenotypes in female rheumatoid arthritis patients was observed (c2:14.973, suggesting that the decrease in p° (tissue non specific synthesis/secretion of alkaline phosphatase could be associated with decreased mineralization and ossification process in arthritis condition.

  15. Alkaline solution neutralization capacity of soil.

    Science.gov (United States)

    Asakura, Hiroshi; Sakanakura, Hirofumi; Matsuto, Toshihiko

    2010-10-01

    Alkaline eluate from municipal solid waste (MSW) incineration residue deposited in landfill alkalizes waste and soil layers. From the viewpoint of accelerating stability and preventing heavy metal elution, pH of the landfill layer (waste and daily cover soil) should be controlled. On the other hand, pH of leachate from existing MSW landfill sites is usually approximately neutral. One of the reasons is that daily cover soil can neutralize alkaline solution containing Ca(2+) as cation. However, in landfill layer where various types of wastes and reactions should be taken into consideration, the ability to neutralize alkaline solutions other than Ca(OH)(2) by soil should be evaluated. In this study, the neutralization capacities of various types of soils were measured using Ca(OH)(2) and NaOH solutions. Each soil used in this study showed approximately the same capacity to neutralize both alkaline solutions of Ca(OH)(2) and NaOH. The cation exchange capacity was less than 30% of the maximum alkali neutralization capacity obtained by the titration test. The mechanism of neutralization by the pH-dependent charge can explain the same neutralization capacities of the soils. Although further investigation on the neutralization capacity of the soils for alkaline substances other than NaOH is required, daily cover soil could serve as a buffer zone for alkaline leachates containing Ca(OH)(2) or other alkaline substances.

  16. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    Directory of Open Access Journals (Sweden)

    Tripathi Lokesh P

    2008-11-01

    Full Text Available Abstract Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc. were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes

  17. An analysis of five serine transfer ribonucleic acids from Drosophila.

    Science.gov (United States)

    White, B N; Dunn, R; Gillam, I; Tener, G M; Armstrong, D J; Skoog, F; Frihart, C R; Leonard, N J

    1975-01-25

    Crude tRNA from adult Drosophila melanogaster was fractionated on bensoylated-diethylaminoethyl cellulose columns. The eluate was assayed for both amino acid acceptance and cytokinin activity. Most of the cytokinin activity was associated with a peak of serine acceptance. The five major serine tRNAs were purified by chromatography on benzoylated-dietyhlaminoethyl cellulose and reversed phase chromatography-5 columns. The major species, tRNA7-Ser was isolated from this tRNA and was shown to be N-6-(delta-2-isopentenyl)adenosine (i-6A) on the basis of ultraviolet and mass spectral data. The nucleoside somposition of all five serine tRNAs was determined directly and by the 3-H derivative method. They all contain pseudouridine, ribothymidine, 1-methyladenosine, 5-methylcytosine, N-2-dimethylguanosine, 5, 6-hydrouridine, and 3-methylcytosine, while two contain an unidentified nucleoside, and one containes 1-methylguanosine. These techniques also confirmed the presence of i-6A in tRNA7-Ser as well as showing its presence in tRNA6-Ser and tRNA4-Ser. These three tRNA-Ser species exhibit marked changes in elution from reversed phase chromatography-5 columns as a function of temperature and this may be related to their minor base composition. The tRNAs-Ser were bound to ribosomes in response to the following triplets: tRNA2-Ser, AGU, AGC; tRNA4-Ser, UCG; tRNA5-Ser, AGU, AGC; tRNA7-Ser, UCG.

  18. Study on low-temperature lipase of psychrophilic bacterium 2-5-10-1 isolated from deep sea of Southern Ocean

    Institute of Scientific and Technical Information of China (English)

    Lin Xuezheng; Yang Xiuxia; Bian Ji; Huang Xiaohang

    2003-01-01

    A strain of psychrophilic bacterium, 2-5-10-1, which produces low-temperature lipase, is isolated from the deep sea of Prydz Bay in Southern Ocean. The highest lipase secretion of this strain is observed at 5 ℃ and this temperature is also for optimal growth. Tween 80 and olive oil enhance secretion of lipase. The optimal temperature and pH for lipase activity are 35 ℃ and 7.5 ℃ respectively. At 0℃, the lipase still has 37% relative enzyme activity. The lipase shows high thermolability, more than 50% activity lost after incubation at 60 ℃ for 15 min. EDTA has no effect on lipase activity, indicating the lipase activity is independent of divalent cation. In contrast, the lipase activity is inhibited drastically by Cu2 + and Zn2 +.

  19. Properties of serine: glyoxylate aminotransferase purified from Arabidopsis thaliana leaves

    Institute of Scientific and Technical Information of China (English)

    Maria Kendziorek; Andrzej Paszkowski

    2008-01-01

    The photorespiratory enzyme L-serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) was purified from Arabidopsis thaliana leaves. The final enzyme was approximately 80% pure as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with silver staining. The identity of the enzyme was confirmed by LC/MS/MS analysis.The molecular mass estimated by gel filtration chromatography on Sephadex G-150 under non-denaturing conditions, mass spectrometry (matrix-assisted laser desorption/ionization/time of flight technique) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 82.4 kDa,42.0 kDa, and 39.8 kDa, respectively, indicating dimer as the active form. The optimum Ph value was 9.2. The enzyme activity was inhibited by aminooxyacetate and β-chloro-L-alanine both compounds reacting with the carbonyl group of pyridoxal phosphate. The enzyme's transaminating activity with L-alanine and glyoxylate as substrates was approximately 55% of that observed with L-serine and glyoxylate, The lower Km value (1.25 Mm) for L-alanine, compared with that of other plant SGATs, and the kcat/Km(Ala) ratio being approximately 2-fold higher than kcat/Km(Ser) suggested that, during photorespiration, Ala and Ser are used by Arabidopsis SGAT with equal efficiency as amino group donors for glyoxylate. The equilibrium constant (Keq), derived from the Haldane relation, for the transamination reaction between L-serine and glyoxylate with the formation of hydroxypyruvate and glycine was 79.1, strongly favoring glycine synthesis. However, it was accompanied by a low Km value of 2.83 Mm for glycine. A comparison of some kinetic properties of the studied enzymes with the recombinant Arabidopsis SGATs previously obtained revealed substantial differences. The ratio of the velocity of the transamination reaction with L-alanine and glyoxylate as substrates versus that with L-serine and glyoxylate was 1:1.8 for the native enzyme, whereas it was 1: 7 for the recombinant SGAT

  20. Structural and functional properties of isocitrate dehydrogenase from the psychrophilic bacterium Desulfotalea psychrophila reveal a cold-active enzyme with an unusual high thermal stability.

    Science.gov (United States)

    Fedøy, Anita-Elin; Yang, Nannan; Martinez, Aurora; Leiros, Hanna-Kirsti S; Steen, Ida Helene

    2007-09-07

    Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)(+)-dependent decarboxylation of isocitrate to alpha-ketoglutarate and CO(2). Here, we present the first crystal structure of IDH from a psychrophilic bacterium, Desulfotalea psychrophila (DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium Desulfitobacterium hafniense (DhIDH), porcine (PcIDH), human cytosolic (HcIDH) and the hyperthermophilic Thermotoga maritima (TmIDH). DpIDH was found to have a higher melting temperature (T(m)=66.9 degrees C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in K(m) for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand, DpIDH was optimized for interacting with NADP(+) and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.

  1. Association between intrinsic disorder and serine/threonine phosphorylation in Mycobacterium tuberculosis

    Directory of Open Access Journals (Sweden)

    Gajinder Pal Singh

    2015-01-01

    Full Text Available Serine/threonine phosphorylation is an important mechanism that is involved in the regulation of protein function. In eukaryotes, phosphorylation occurs predominantly in intrinsically disordered regions of proteins. Though serine/threonine phosphorylation and protein disorder are much less prevalent in prokaryotes, some bacteria have high levels of serine/threonine phosphorylation and disorder, including the medically important M. tuberculosis. Here I show that serine/threonine phosphorylation sites in M. tuberculosis are highly enriched in intrinsically disordered regions, indicating similarity in the substrate recognition mechanisms of eukaryotic and M. tuberculosis kinases. Serine/threonine phosphorylation has been linked to the pathogenicity and survival of M. tuberculosis. Thus, a better understanding of how its kinases recognize their substrates could have important implications in understanding and controlling the biology of this deadly pathogen. These results also indicate that the association between serine/threonine phosphorylation and disorder is not a feature restricted to eukaryotes.

  2. Alkaline tolerant dextranase from streptomyces anulatus

    Science.gov (United States)

    Decker, Stephen R.; Adney, William S.; Vinzant, Todd B.; Himmel, Michael E.

    2003-01-01

    A process for production of an alkaline tolerant dextranase enzyme comprises culturing a dextran-producing microorganism Streptomyces anulatus having accession no. ATCC PTA-3866 to produce an alkaline tolerant dextranase, Dex 1 wherein the protein in said enzyme is characterized by a MW of 63.3 kDa and Dex 2 wherein its protein is characterized by a MW of 81.8 kDa.

  3. Reaction pathway of tryptophanase-catalyzed L-tryptophan synthesis from D-serine

    OpenAIRE

    Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Fujii, Noriko

    2011-01-01

    Tryptophanase, L-tryptophan indole-lyase with extremely absolute stereospecificity, can change the stereospecificity in concentrated diammonium hydrogenphosphate solution. While tryptophanase is not inert to d-serine in the absence of diammonium hydrogenphosphate, it can undergo L-tryptophan synthesis from d-serine along with indole in the presence of it. It has been well known that tryptophanase synthesizes L-tryptophan from l-serine through a β-substitution mechanism of the ping-pong type. ...

  4. Characterization and quantification of biochar alkalinity.

    Science.gov (United States)

    Fidel, Rivka B; Laird, David A; Thompson, Michael L; Lawrinenko, Michael

    2017-01-01

    Lack of knowledge regarding the nature of biochar alkalis has hindered understanding of pH-sensitive biochar-soil interactions. Here we investigate the nature of biochar alkalinity and present a cohesive suite of methods for its quantification. Biochars produced from cellulose, corn stover and wood feedstocks had significant low-pKa organic structural (0.03-0.34 meq g(-1)), other organic (0-0.92 meq g(-1)), carbonate (0.02-1.5 meq g(-1)), and other inorganic (0-0.26 meq g(-1)) alkalinities. All four categories of biochar alkalinity contributed to total biochar alkalinity and are therefore relevant to pH-sensitive soil processes. Total biochar alkalinity was strongly correlated with base cation concentration, but biochar alkalinity was not a simple function of elemental composition, soluble ash, fixed carbon, or volatile matter content. More research is needed to characterize soluble biochar alkalis other than carbonates and to establish predictive relationships among biochar production parameters and the composition of biochar alkalis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Purification and characterization of cloned alkaline protease gene of Geobacillus stearothermophilus.

    Science.gov (United States)

    Iqbal, Irfana; Aftab, Muhammad Nauman; Afzal, Mohammed; Ur-Rehman, Asad; Aftab, Saima; Zafar, Asma; Ud-Din, Zia; Khuharo, Ateeque Rahman; Iqbal, Jawad; Ul-Haq, Ikram

    2015-02-01

    Thermostable alkaline serine protease gene of Geobacillus stearothermophilus B-1172 was cloned and expressed in Escherichia coli BL21 (DE3) using pET-22b(+), as an expression vector. The growth conditions were optimized for maximal production of the protease using variable fermentation parameters, i.e., pH, temperature, and addition of an inducer. Protease, thus produced, was purified by ammonium sulfate precipitation followed by ion exchange chromatography with 13.7-fold purification, with specific activity of 97.5 U mg(-1) , and a recovery of 23.6%. Molecular weight of the purified protease, 39 kDa, was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was stable at 90 °C at pH 9. The enzyme activity was steady in the presence of EDTA indicating that the protease was not a metalloprotease. No significant change in the activity of protease after addition of various metal ions further strengthened this fact. However, an addition of 1% Triton X-100 or SDS surfactants constrained the enzyme specific activity to 34 and 19%, respectively. Among organic solvents, an addition of 1-butanol (20%) augmented the enzyme activity by 29% of the original activity. With casein as a substrate, the enzyme activity under optimized conditions was found to be 73.8 U mg(-1) . The effect of protease expression on the host cells growth was also studied and found to negatively affect E. coli cells to certain extent. Catalytic domains of serine proteases from eight important thermostable organisms were analyzed through WebLogo and found to be conserved in all serine protease sequences suggesting that protease of G. stearothermophilus could be beneficially used as a biocontrol agent and in many industries including detergent industry.

  6. Short hydrogen bonds in the catalytic mechanism of serine proteases

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  7. Purification and characterization of D-serine deaminase activator protein.

    Science.gov (United States)

    Heincz, M C; Bornstein, S M; McFall, E

    1984-10-01

    We purified the dsdC gene product, the specific activator of dsdA (D-serine deaminase) gene expression, to about 25% homogeneity from a strain in which its expression was amplified 100-fold. The purification involved, successively: DNase and high-salt treatment of cell extracts, DNA-cellulose chromatography, and Dyematrex (Amicon Corp.) column chromatography. We identified the protein as a discrete spot on two-dimensional O'Farrell gels after the DNA-cellulose step and quantitated it by densitometry. The active form was found to be a dimer. We estimated that there were eight activator dimers per wild-type cell. The activator is a slightly basic protein, with an experimental Km for its ligand D-serine of about 7 X 10(-6)M. The low concentration of the activator in wild-type cells and its autorepression may explain the previously observed partial dominance of dsdC+ in dsdCc/dsdC+ merodiploids.

  8. Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor.

    Science.gov (United States)

    Odei-Addo, Frank; Frost, Carminita; Smith, Nanette; Ogawa, Tomohisa; Muramoto, Koji; Oliva, Maria Luiza Vilela; Gráf, László; Naude, Ryno

    2014-10-01

    One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

  9. Okadaic acid: the archetypal serine/threonine protein phosphatase inhibitor.

    Science.gov (United States)

    Dounay, A B; Forsyth, C J

    2002-11-01

    As the first recognized member of the "okadaic acid class" of phosphatase inhibitors, the marine natural product okadaic acid is perhaps the most well-known member of a diverse array of secondary metabolites that have emerged as valuable probes for studying the roles of various cellular protein serine/threonine phosphatases. This review provides a historical perspective on the role that okadaic acid has played in stimulating a broad spectrum of modern scientific research as a result of the natural product's ability to bind to and inhibit important classes of protein serine / threonine phosphatases. The relationships between the structure and biological activities of okadaic acid are briefly reviewed, as well as the structural information regarding the particular cellular receptors protein phosphatases 1 (PP1) and 2A. Laboratory syntheses of okadaic acid and its analogs are thoroughly reviewed. Finally, an interpretation of the critical contacts observed between okadaic acid and PP1 by X-ray crystallography is provided, and specific molecular recognition hypotheses that are testable via the synthesis and assay of non-natural analogs of okadaic acid are suggested.

  10. Mechanism of Ribonuclease III Catalytic Regulation by Serine Phosphorylation

    Science.gov (United States)

    Gone, Swapna; Alfonso-Prieto, Mercedes; Paudyal, Samridhdi; Nicholson, Allen W.

    2016-05-01

    Ribonuclease III (RNase III) is a conserved, gene-regulatory bacterial endonuclease that cleaves double-helical structures in diverse coding and noncoding RNAs. RNase III is subject to multiple levels of control, reflective of its global regulatory functions. Escherichia coli (Ec) RNase III catalytic activity is known to increase during bacteriophage T7 infection, reflecting the expression of the phage-encoded protein kinase, T7PK. However, the mechanism of catalytic enhancement is unknown. This study shows that Ec-RNase III is phosphorylated on serine in vitro by purified T7PK, and identifies the targets as Ser33 and Ser34 in the N-terminal catalytic domain. Kinetic experiments reveal a 5-fold increase in kcat and a 1.4-fold decrease in Km following phosphorylation, providing a 7.4–fold increase in catalytic efficiency. Phosphorylation does not change the rate of substrate cleavage under single-turnover conditions, indicating that phosphorylation enhances product release, which also is the rate-limiting step in the steady-state. Molecular dynamics simulations provide a mechanism for facilitated product release, in which the Ser33 phosphomonoester forms a salt bridge with the Arg95 guanidinium group, thereby weakening RNase III engagement of product. The simulations also show why glutamic acid substitution at either serine does not confer enhancement, thus underscoring the specific requirement for a phosphomonoester.

  11. Antibacterial activity of silver nanoparticles synthesized from serine.

    Science.gov (United States)

    Jayaprakash, N; Judith Vijaya, J; John Kennedy, L; Priadharsini, K; Palani, P

    2015-04-01

    Silver nanoparticles (Ag NPs) were synthesized by a simple microwave irradiation method using polyvinyl pyrrolidone (PVP) as a capping agent and serine as a reducing agent. UV-Visible spectra were used to confirm the formation of Ag NPs by observing the surface plasmon resonance (SPR) band at 443nm. The emission spectrum of Ag NPs showed an emission band at 484nm. In the presence of microwave radiation, serine acts as a reducing agent, which was confirmed by Fourier transformed infrared (FT-IR) spectrum. High-resolution transmission electron microscopy (HR-TEM) and high-resolution scanning electron microscopy (HR-SEM) were used to investigate the morphology of the synthesized sample. These images showed the sphere-like morphology. The elemental composition of the sample was determined by the energy dispersive X-ray analysis (EDX). Selected area electron diffraction (SAED) was used to find the crystalline nature of the Ag NPs. The electrochemical behavior of the synthesized Ag NPs was analyzed by the cyclic voltammetry (CV). Antibacterial experiments showed that the prepared Ag NPs showed relatively similar antibacterial activities, when compared with AgNO3 against Gram-positive and Gram-negative bacteria.

  12. Heterogeneity of D-Serine Distribution in the Human Central Nervous System

    Science.gov (United States)

    Suzuki, Masataka; Imanishi, Nobuaki; Mita, Masashi; Hamase, Kenji; Aiso, Sadakazu

    2017-01-01

    D-serine is an endogenous ligand for N-methyl-D-aspartate glutamate receptors. Accumulating evidence including genetic associations of D-serine metabolism with neurological or psychiatric diseases suggest that D-serine is crucial in human neurophysiology. However, distribution and regulation of D-serine in humans are not well understood. Here, we found that D-serine is heterogeneously distributed in the human central nervous system (CNS). The cerebrum contains the highest level of D-serine among the areas in the CNS. There is heterogeneity in its distribution in the cerebrum and even within the cerebral neocortex. The neocortical heterogeneity is associated with Brodmann or functional areas but is unrelated to basic patterns of cortical layer structure or regional expressional variation of metabolic enzymes for D-serine. Such D-serine distribution may reflect functional diversity of glutamatergic neurons in the human CNS, which may serve as a basis for clinical and pharmacological studies on D-serine modulation. PMID:28604057

  13. The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

    DEFF Research Database (Denmark)

    Jürgensen, Vibeke Würtz; Jalkanen, Karl J.

    2006-01-01

    The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory...... the latest methods in experimental and theoretical vibrational spectroscopy are used to gain useful information about the coupling of the nuclear, electronic and magnetic degrees of freedom and structure of tri-L-serine and its capped peptide analog with the environment....

  14. [Alkaline phosphatase in Amoeba proteus].

    Science.gov (United States)

    Sopina, V A

    2005-01-01

    In free-living Amoeba proteus (strain B), 3 phosphatase were found after disc-electrophoresis of 10 microg of protein in PAGE and using 1-naphthyl phosphate as a substrate a pH 9.0. These phosphatases differed in their electrophoretic mobilities - "slow" (1-3 bands), "middle" (one band) and "fast" (one band). In addition to 1-naphthyl phosphate, "slow" phosphatases were able to hydrolyse 2-naphthyl phosphate and p-nitrophenyl phosphate. They were slightly activated by Mg2+, completely inhibited by 3 chelators (EDTA, EGTA and 1,10-phenanthroline), L-cysteine, sodium dodecyl sulfate and Fe2+, Zn2+ and Mn2+ (50 mM), considerably inactivated by orthovanadate, molybdate, phosphatase inhibitor cocktail 1, p-nitrophenyl phosphate, Na2HPO4, DL-dithiothreitol and urea and partly inhibited by H2O2, DL-phenylalanine, 2-mercaptoethanol, phosphatase inhibitor cocktail 2 and Ca2+. Imidazole, L-(+)-tartrate, okadaic acid, NaF and sulfhydryl reagents -p-(hydroxy-mercuri)benzoate and N-ethylmaleimide - had no influence on the activity of "slow" phosphatases. "Middle" and "fast" phosphatases, in contrast to "slow" ones, were not inactivated by 3 chelators. The "middle" phosphatase differed from the "fast" one by smaller resistance to urea, Ca2+, Mn2+, phosphates and H2O2 and greater resistance to dithiothreitol and L-(+)-tartrate. In addition, the "fast" phosphatase was inhibited by L-cysteine but the "middle" one was activated by it. Of 5 tested ions (Mg2+, Cu2+, Mn2+, Ca2+ and Zn2+), only Zn2+ reactivated "slow" phosphatases after their inactivation by EDTA treatment. The reactivation of apoenzyme was only partial (about 35 %). Thus, among phosphatases found in amoebae at pH 9.0, only "slow" ones are Zn-metalloenzymes and may be considered as alkaline phosphatases (EC 3.1.3.1). It still remains uncertain, to which particular phosphatase class "middle" and "fast" phosphatases (pH 9.0) may belong.

  15. Engineering of high yield production of L-serine in Escherichia coli.

    Science.gov (United States)

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre; Nielsen, Alex Toftgaard

    2016-04-01

    L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevented by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very high serine production yield (0.45 g/g glucose) during small-scale batch fermentation in minimal medium. Serine, however, was found to be highly toxic even at low concentrations to this strain, which lead to slow growth and production during fed batch fermentation, resulting in a serine production of 8.3 g/L. The production strain was therefore evolved by random mutagenesis to achieve increased tolerance towards serine. Additionally, overexpression of eamA, a cysteine/homoserine transporter was demonstrated to increase serine tolerance from 1.6 g/L to 25 g/L. During fed batch fermentation, the resulting strain lead to the serine production titer of 11.7 g/L with yield of 0.43 g/g glucose, which is the highest yield reported so far for any organism.

  16. Metabolic engineering and flux analysis of Corynebacterium glutamicum for L-serine production.

    Science.gov (United States)

    Lai, Shujuan; Zhang, Yun; Liu, Shuwen; Liang, Yong; Shang, Xiuling; Chai, Xin; Wen, Tingyi

    2012-04-01

    L-Serine plays a critical role as a building block for cell growth, and thus it is difficult to achieve the direct fermentation of L-serine from glucose. In this study, Corynebacterium glutamicum ATCC 13032 was engineered de novo by blocking and attenuating the conversion of L-serine to pyruvate and glycine, releasing the feedback inhibition by L-serine to 3-phosphoglycerate dehydrogenase (PGDH), in combination with the co-expression of 3-phosphoglycerate kinase (PGK) and feedback-resistant PGDH (PGDH(r)). The resulting strain, SER-8, exhibited a lower specific growth rate and significant differences in L-serine levels from Phase I to Phase V as determined for fed-batch fermentation. The intracellular L-serine pool reached (14.22 ± 1.41) μmol g(CDM) (-1), which was higher than glycine pool, contrary to fermentation with the wild-type strain. Furthermore, metabolic flux analysis demonstrated that the over-expression of PGK directed the flux of the pentose phosphate pathway (PPP) towards the glycolysis pathway (EMP), and the expression of PGDH(r) improved the L-serine biosynthesis pathway. In addition, the flux from L-serine to glycine dropped by 24%, indicating that the deletion of the activator GlyR resulted in down-regulation of serine hydroxymethyltransferase (SHMT) expression. Taken together, our findings imply that L-serine pool management is fundamental for sustaining the viability of C. glutamicum, and improvement of C(1) units generation by introducing the glycine cleavage system (GCV) to degrade the excessive glycine is a promising target for L-serine production in C. glutamicum.

  17. Prophylactic treatment with alkaline phosphatase in cardiac surgery induces endogenous alkaline phosphatase release

    NARCIS (Netherlands)

    Kats, Suzanne; Brands, Ruud; Hamad, Mohamed A. Soliman; Seinen, Willem; Schamhorst, Volkher; Wulkan, Raymond W.; Schoenberger, Jacques P.; van Oeveren, Wim

    2012-01-01

    Introduction: Laboratory and clinical data have implicated endotoxin as an important factor in the inflammatory response to cardiopulmonary bypass. We assessed the effects of the administration of bovine intestinal alkaline phosphatase (bIAP), an endotoxin detoxifier, on alkaline phosphatase levels

  18. Uptake of arsenic by alkaline soils near alkaline coal fly ash disposal facilities.

    Science.gov (United States)

    Khodadoust, Amid P; Theis, Thomas L; Murarka, Ishwar P; Naithani, Pratibha; Babaeivelni, Kamel

    2013-12-01

    The attenuation of arsenic in groundwater near alkaline coal fly ash disposal facilities was evaluated by determining the uptake of arsenic from ash leachates by surrounding alkaline soils. Ten different alkaline soils near a retired coal fly ash impoundment were used in this study with pH ranging from 7.6 to 9.0, while representative coal fly ash samples from two different locations in the coal fly ash impoundment were used to produce two alkaline ash leachates with pH 7.4 and 8.2. The arsenic found in the ash leachates was present as arsenate [As(V)]. Adsorption isotherm experiments were carried out to determine the adsorption parameters required for predicting the uptake of arsenic from the ash leachates. For all soils and leachates, the adsorption of arsenic followed the Langmuir and Freundlich equations, indicative of the favorable adsorption of arsenic from leachates onto all soils. The uptake of arsenic was evaluated as a function of ash leachate characteristics and the soil components. The uptake of arsenic from alkaline ash leachates, which occurred mainly as calcium hydrogen arsenate, increased with increasing clay fraction of soil and with increasing soil organic matter of the alkaline soils. Appreciable uptake of arsenic from alkaline ash leachates with different pH and arsenic concentration was observed for the alkaline soils, thus attenuating the contamination of groundwater downstream of the retired coal fly ash impoundment.

  19. Structural diversity in serine derived homochiral metal organic frameworks

    Indian Academy of Sciences (India)

    Tanay Kundu; Rahul Banerjee

    2014-09-01

    Two new Zn(II) and Cd(II) based homochiral metal-organic frameworks (MOFs) [SerCdOAc and Zn(Ser)2] have been synthesized using pyridyl functionalized amino acid, viz., serine, as an organic linker. The SerCdOAc structure is three dimensional, while that of the Zn(Ser)2 is two dimensional. The polar voids of the corresponding MOFs are filled with solvent molecules (water in the case of SerCdOAc and methanol in the case of Zn(Ser)2). In both cases, metal centres, i.e., Zn(II) and Cd(II), are hexacoordinated. However, with a change in the solvent for synthesis, ligand coordinationmode and incorporation of additional coordinated anion resulted in a great change in the final MOF architecture. Herein, for the first time, we could achieve structural variety and synthesize MOFs composed of only metal ion and pyridyl functionalized amino acid linker.

  20. Serine Proteases an Ab Initio Molecular Dynamics Study

    CERN Document Server

    De Santis, L

    1999-01-01

    In serine proteases (SP's), the H-bond between His-57 and Asp-102, and that between Gly-193 and the transition state intermediate play a crucial role for enzymatic function. To shed light on the nature of these interactions, we have carried out ab initio molecular dynamics simulations on complexes representing adducts between the reaction intermediate and elastase (one protein belonging to the SP family). Our calculations indicate the presence of a low--barrier H-bond between His-57 and Asp-102, in complete agreement with NMR experiments on enzyme--transition state analog complexes. Comparison with an ab initio molecular dynamics simulation on a model of the substrate--enzyme adduct indicates that the Gly-193--induced strong stabilization of the intermediate is accomplished by charge/dipole interactions and not by H-bonding as previously suggested. Inclusion of the protein electric field in the calculations does not affect significantly the charge distribution.

  1. Conformation effects on the molecular orbitals of serine

    Institute of Scientific and Technical Information of China (English)

    Wang Ke-Dong; Ma Peng-Fei; Shan Xu

    2011-01-01

    This paper calculates the five most stable conformers of serine with Hartree-Fock theory, density functional theory (B3LYP), M0ller-Plesset perturbation theory (MP4(SDQ)) and electron propagation theory with the 6-311++G(2d,2p) basis set. The calculated vertical ionization energies for the valence molecular orbitals of each conformer are in agreement with the experimental data, indicating that a range of molecular conformations would coexist in an equilibrium sample. Information of the five outer valence molecular orbitals for each conformer is explored in coordinate and momentum spaces using dual space analysis to investigate the conformational processes, which are generated from the global minimum conformer Serl by rotation of C2-C3 (Ser4), C1-C2 (Ser5) and C1-O2 (Ser2 and Ser3). Orbitals 28a, 27a and 26a are identified as the fingerprint orbitals for all the conformational processes.

  2. Crystallographic Refinement by Incorporation of Molecular Dynamics : Thermostable Serine Protease Thermitase Complexed with Eglin c

    NARCIS (Netherlands)

    Gros, Piet; Fujinaga, Masao; Dijkstra, Bauke W.; Kalk, Kor H.; Hol, W G J

    1989-01-01

    In order to investigate the principles of protein thermostability, the crystal structure of thermitase from Thermoactinomyces vulgaris, a thermostable member of the subtilisin family of serine proteases, has been determined in a complex with eglin c. Eglin c is a serine protease inhibitor from the l

  3. Astrocytes are involved in trigeminal dynamic mechanical allodynia: potential role of D-serine.

    Science.gov (United States)

    Dieb, W; Hafidi, A

    2013-09-01

    Trigeminal neuropathic pain affects millions of people worldwide. Despite decades of study on the neuronal processing of pain, mechanisms underlying enhanced pain states after injury remain unclear. N-methyl-D-aspartate (NMDA) receptor-dependent changes play a critical role in triggering central sensitization in neuropathic pain. These receptors are regulated at the glycine site through a mandatory endogenous co-agonist D-serine, which is synthesized by astrocytes. Therefore, the present study was carried out to determine whether astrocytes are involved, through D-serine secretion, in dynamic mechanical allodynia (DMA) obtained after chronic constriction of the infraorbital nerve (CCI-IoN) in rats. Two weeks after CCI-IoN, an important reaction of astrocytes was present in the medullary dorsal horn (MDH), as revealed by an up-regulation of glial fibrillary acidic protein (GFAP) in allodynic rats. In parallel, an increase in D-serine synthesis, which co-localized with its synthesis enzyme serine racemase, was strictly observed in astrocytes. Blocking astrocyte metabolism by intracisternal delivery of fluorocitrate alleviated DMA. Furthermore, the administration of D-amino-acid oxidase (DAAO), a D-serine-degrading enzyme, or that of L-serine O-sulfate (LSOS), a serine racemase inhibitor, significantly decreased pain behavior in allodynic rats. These results demonstrate that astrocytes are involved in the modulation of orofacial post-traumatic neuropathic pain via the release of the gliotransmitter D-serine.

  4. Engineering of High Yield Production of L-serine in Escherichia coli

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

    2016-01-01

    L-serine is a widely used amino acid that has been proposed as a potential building block biochemical. The high theoretical yield from glucose makes a fermentation based production attractive. In order to achieve this goal, serine degradation to pyruvate and glycine in E. coli MG1655 was prevente...

  5. DMPD: The serine/threonine kinase Pim-1. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15694833 The serine/threonine kinase Pim-1. Bachmann M, Moroy T. Int J Biochem Cell... Biol. 2005 Apr;37(4):726-30. (.png) (.svg) (.html) (.csml) Show The serine/threonine kinase Pim-1. PubmedID 15694833 Title The

  6. Cobalamin deficiency results in severe metabolic disorder of serine and threonine in rats.

    Science.gov (United States)

    Ebara, S; Toyoshima, S; Matsumura, T; Adachi, S; Takenaka, S; Yamaji, R; Watanabe, F; Miyatake, K; Inui, H; Nakano, Y

    2001-12-05

    Dietary cobalamin (vitamin B12; Cbl) deficiency caused significant increases in plasma serine, threonine, glycine, alanine, tyrosine, lysine and histidine levels in rats. In particular, the serine and threonine levels were over five and eight times, respectively, higher in the Cbl-deficient rats than those in the sufficient controls. In addition, some amino acids, including serine and threonine, were excreted into urine at significantly higher levels in the deficient rats. When Cbl was supplemented into the deficient rats for 2 weeks, in coincidence with the disappearance of the urinary excretion of methylmalonic acid (an index of Cbl deficiency), the plasma serine and threonine levels were normalized. These results indicate that Cbl deficiency results in metabolic disorder of certain amino acids, including serine and threonine. The expression level of hepatic serine dehydratase (SDH), which catalyzes the conversion of serine and threonine to pyruvate and 2-oxobutyrate, respectively, was significantly lowered by Cbl deficiency, even though Cbl does not participate directly in the enzyme reaction. The SDH activity in the deficient rats was less than 20% of that in the sufficient controls, and was normalized 2 weeks after the Cbl supplementation. It is thus suggested that the decrease of the SDH expression relates closely with the abnormalities in the plasma and urinary levels of serine and threonine in the Cbl-deficient rats.

  7. Single and chronic L-serine treatments exert antidepressant-like effects in rats possibly by different means.

    Science.gov (United States)

    Nagasawa, Mao; Otsuka, Tsuyoshi; Togo, Yuki; Yamanaga, Masakazu; Yoshida, Junki; Uotsu, Nobuo; Teramoto, Sachiyuki; Yasuo, Shinobu; Furuse, Mitsuhiro

    2017-06-06

    In the present study, the effects of both single (6 mmol L-serine/10 ml/kg orally administrated) and chronic (2% L-serine solution freely given for 28 days) treatments on depression-like behavior were evaluated in Wistar rats, representing the control, and Wistar Kyoto rats, representing an animal model of depression. Both single and chronic L-serine treatments decreased the duration of immobility, which is an index of a depressive-like state, in the forced swimming test in both strains. However, the decreases in the duration of immobility appear to be regulated differently by the different mechanisms involved in single and chronic L-serine treatments. In the prefrontal cortex and hippocampus, single L-serine treatment increased the concentrations of L-serine, but not D-serine, while chronic L-serine treatment increased those of D-serine, but not L-serine. These data suggest that the antidepressant-like effects of single and chronic L-serine treatments may have been induced by the increased L-serine and D-serine concentrations, respectively, in the brain. In addition, chronic L-serine treatment increased cystathionine concentrations in the hippocampus and prefrontal cortex in Wistar rats, but not in Wistar Kyoto rats, suggesting that Wistar Kyoto rats have an abnormality in the serine-cystathionine metabolic pathway. In conclusion, single and chronic L-serine treatments may induce antidepressant-like effects via the different mechanisms related to serine metabolism in the brain.

  8. Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

    Science.gov (United States)

    Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

    2017-04-01

    Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

  9. Biophysicochemical characterization of an alkaline protease from Beauveria sp. MTCC 5184 with multiple applications.

    Science.gov (United States)

    Shankar, Shiv; Laxman, Ryali Seeta

    2015-01-01

    This study illustrates the biophysicochemical properties of an alkaline protease, BAP (Beauveria sp. alkaline protease) from Beauveria sp. MTCC 5184. This protease exhibited maximum activity at 50 °C, pH 9.0, and stability in a broad pH range, in the presence of organic solvents, denaturants, as well as detergents. Wash performance studies revealed that BAP was able to remove blood clots/stains from blood-soaked cloth. Peptide mass fingerprinting results demonstrated partial homology of BAP with subtilisin-like proteinase. BAP showed catalytic activity against natural as well as synthetic substrates. Active site characterization of BAP confirmed the involvement of serine, tryptophan, and aspartic acid in catalytic activity. Detailed kinetic and thermodynamic studies of BAP demonstrated that the activation energy (Ea) for casein hydrolysis was 82.55 kJ/M, the specificity constant (Kcat/K m), and the values of ∆G (change in Gibbs free energy) decreased with increase in temperature, whereas ∆H (change in enthalapy) and ∆S (change in entropy) were constant. The results of the present study indicate that BAP has potential for applications as detergent additive, in peptide synthesis, and in basic research.

  10. Purification and characterization of a novel extracellular alkaline protease from Cellulomonas bogoriensis.

    Science.gov (United States)

    Li, Fan; Yang, Liyuan; Lv, Xue; Liu, Dongbo; Xia, Hongmei; Chen, Shan

    2016-05-01

    An extracellular alkaline protease produced by the alkali-tolerant Cellulomonas bogoriensis was purified by a combination of ammonium sulfate precipitation and cation exchange chromatography. The purity of the protease was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was confirmed to be 18.3 kDa. The enzyme showed optimum activity at 60 °C and pH 11. The stability of the protease was maintained at a wide temperature range of 4-60 °C and pH range of 3-12. Irreversible inhibition of the enzyme activity by phenylmethylsulfonyl fluoride and tosyl-l-phenylalanine chloromethyl ketone demonstrated that the purified enzyme is a chymotrypsin of the serine protease family. The Km and Vmax of the protease activity on casein were 19.2 mg/mL and 25000 μg/min/mg, respectively. The broad substrate specificity and remarkable stability in the presence of organic solvents, salt, and commercial detergents, as well as its excellent stain removal and dehairing capability, make the purified alkaline protease a promising candidate for industrial applications.

  11. Peptidoglycan inducible expression of a serine proteinase homologue from kuruma shrimp (Marsupenaeus japonicus).

    Science.gov (United States)

    Rattanachai, Achara; Hirono, Ikuo; Ohira, Tsuyoshi; Takahashi, Yukinori; Aoki, Takashi

    2005-01-01

    A cDNA encoding a serine proteinase homologue of kuruma shrimp (Marsupenaeus japonicus) was cloned. The 1257 bp cDNA encodes a 339 amino acid putative peptide, with a signal sequence of 16 amino acid residues. The deduced amino acid sequence is 42-67% similar to the immune-related serine proteinases and serine proteinase homologues of arthropods. It contains catalytic triad residues in the putative catalytic domain except for one substitution of Ser by a Gly residue. The six cysteine residues that form three disulphide bridges in most serine proteinases were conserved. The M. japonicus serine proteinase homologue was mainly expressed in haemocytes, in which expression dramatically increased after 3 days feeding with peptidoglycan at 0.2 mg kg(-1) shrimp body weight per day.

  12. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7.

    Science.gov (United States)

    Chapman, Rob D; Heidemann, Martin; Albert, Thomas K; Mailhammer, Reinhard; Flatley, Andrew; Meisterernst, Michael; Kremmer, Elisabeth; Eick, Dirk

    2007-12-14

    RNA polymerase II is distinguished by its large carboxyl-terminal repeat domain (CTD), composed of repeats of the consensus heptapeptide Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Differential phosphorylation of serine-2 and serine-5 at the 5' and 3' regions of genes appears to coordinate the localization of transcription and RNA processing factors to the elongating polymerase complex. Using monoclonal antibodies, we reveal serine-7 phosphorylation on transcribed genes. This position does not appear to be phosphorylated in CTDs of less than 20 consensus repeats. The position of repeats where serine-7 is substituted influenced the appearance of distinct phosphorylated forms, suggesting functional differences between CTD regions. Our results indicate that restriction of serine-7 epitopes to the Linker-proximal region limits CTD phosphorylation patterns and is a requirement for optimal gene expression.

  13. Some durability aspects of hybrid alkaline cements

    Directory of Open Access Journals (Sweden)

    Donatello S.

    2014-04-01

    Full Text Available Blended cements that contain a high content of fly ash and a low content of Portland cement typically suffer from low early strength development and long setting times. Recently, one method of overcoming these problems has been to use an alkali activator to enhance the reactivity of fly ash particles at early ages. Such cements can be grouped under the generic term “hybrid alkaline cements”, where both cement clinker and fly ash, encouraged by the presence of alkalis, are expected to contribute to cementitious gel formation. The work presented here examines some of the durability aspects of high fly ash content hybrid alkaline cement. Specifically, the aspects investigated were: exposure at high temperatures (up to 1000°C, resistance to immersion in aggressive solutions and susceptibility to the alkali aggregate reaction. All tests were repeated with a commercially available sulfate resistant Portland cement for comparison. When exposed to high temperatures, the hybrid alkaline cement showed strikingly different behaviour compared to the control Portland cement, showing fewer micro-cracks and maintaining residual compressive strengths at least equal to original strengths. Beyond 700°C, the hybrid alkaline cement began to sinter, which resulted in shrinkage of around 5% and a 100% increase in residual compressive strengths. No such sintering event was noted in the control Portland cement, which showed a drastic loss in residual compressive strengths upon heating. In immersion tests, the hybrid alkaline cement possessed excellent resistance to sulfate and seawater attack, similar to the control sulfate resistant cement. Both cements were however severely degraded by immersion in 0.1M HCl for 90 days. Both binders complied with the accelerated alkali-aggregate test but when this test was extended, the hybrid alkaline binder showed much greater dimensional stability. Possible reasons for the differences in durability behaviour in both cements

  14. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Science.gov (United States)

    Rendón-Ramírez, Adela; Shukla, Manish; Oda, Masataka; Chakraborty, Sandeep; Minda, Renu; Dandekar, Abhaya M; Ásgeirsson, Bjarni; Goñi, Félix M; Rao, Basuthkar J

    2013-01-01

    Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP). CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP) and a scaffold recognizing a β-lactam (imipenem) in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV), we sought to query other OMV proteins, like phospholipase C (PLC), using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM). Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  15. A computational module assembled from different protease family motifs identifies PI PLC from Bacillus cereus as a putative prolyl peptidase with a serine protease scaffold.

    Directory of Open Access Journals (Sweden)

    Adela Rendón-Ramírez

    Full Text Available Proteolytic enzymes have evolved several mechanisms to cleave peptide bonds. These distinct types have been systematically categorized in the MEROPS database. While a BLAST search on these proteases identifies homologous proteins, sequence alignment methods often fail to identify relationships arising from convergent evolution, exon shuffling, and modular reuse of catalytic units. We have previously established a computational method to detect functions in proteins based on the spatial and electrostatic properties of the catalytic residues (CLASP. CLASP identified a promiscuous serine protease scaffold in alkaline phosphatases (AP and a scaffold recognizing a β-lactam (imipenem in a cold-active Vibrio AP. Subsequently, we defined a methodology to quantify promiscuous activities in a wide range of proteins. Here, we assemble a module which encapsulates the multifarious motifs used by protease families listed in the MEROPS database. Since APs and proteases are an integral component of outer membrane vesicles (OMV, we sought to query other OMV proteins, like phospholipase C (PLC, using this search module. Our analysis indicated that phosphoinositide-specific PLC from Bacillus cereus is a serine protease. This was validated by protease assays, mass spectrometry and by inhibition of the native phospholipase activity of PI-PLC by the well-known serine protease inhibitor AEBSF (IC50 = 0.018 mM. Edman degradation analysis linked the specificity of the protease activity to a proline in the amino terminal, suggesting that the PI-PLC is a prolyl peptidase. Thus, we propose a computational method of extending protein families based on the spatial and electrostatic congruence of active site residues.

  16. Electrochemical behaviour of alkaline copper complexes

    Indian Academy of Sciences (India)

    C L Aravinda; S M Mayanna; V R Muralidharan

    2000-10-01

    A search for non-cyanide plating baths for copper resulted in the development of alkaline copper complex baths containing trisodium citrate [TSC] and triethanolamine [TEA]. Voltammetric studies were carried out on platinum to understand the electrochemical behaviour of these complexes. In TSC solutions, the deposition of copper involves the slow formation of a monovalent species. Adsorption of this species obeys Langmuir isotherm. In TEA solutions the deposition involves the formation of monovalent ions obeying the non-activated Temkin isotherm. Conversion of divalent to monovalent copper is also slow. In TEA and TSC alkaline copper solutions, the predominant species that undergo stepwise reduction contain only TEA ligands

  17. Alkaline Capacitors Based on Nitride Nanoparticles

    Science.gov (United States)

    Aldissi, Matt

    2003-01-01

    High-energy-density alkaline electrochemical capacitors based on electrodes made of transition-metal nitride nanoparticles are undergoing development. Transition- metal nitrides (in particular, Fe3N and TiN) offer a desirable combination of high electrical conductivity and electrochemical stability in aqueous alkaline electrolytes like KOH. The high energy densities of these capacitors are attributable mainly to their high capacitance densities, which, in turn, are attributable mainly to the large specific surface areas of the electrode nanoparticles. Capacitors of this type could be useful as energy-storage components in such diverse equipment as digital communication systems, implanted medical devices, computers, portable consumer electronic devices, and electric vehicles.

  18. Differential gene expression for suicide-substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley

    DEFF Research Database (Denmark)

    Roberts, T.H.; Marttila, S.; Rasmussen, S.K.;

    2003-01-01

    Proteins of the serpin superfamily (similar to43 kDa) from mature cereal grains are in vitro suicide-substrate inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (

  19. Structural and chemical modification of Fe-rich smectite associated with microbial Fe-respiration by psychrophilic bacteria in King George Island, West Antarctica

    Science.gov (United States)

    Jung, J.; Kim, J. Y.; Lim, H. S.; Lee, Y. K.; Kim, O. S.; Park, K.; Lee, J.; Yoon, H.; Kim, J. W.

    2015-12-01

    Biotic/abiotic redox reaction is a ubiquitous process in a mineral alteration and an elemental cycling in the sediments/ocean. The role of psychrophiles in clay mineral alteration was tested in the soil for the seven sites from the coast to the inland at Barton Peninsula. Batch experiments of microbe-mineral interaction under the various temperatures (4 ℃, 15 ℃) that mimics the Antarctic condition were performed to understand the mechanism of biogeochemical alteration of clay minerals. After 12 months incubation of the bulk surface soil samples in the M1 minimal medium, the extent of Fe reduction was reached up to 49 and 42 % at 4 ℃ and 15 ℃. The increase in CEC corresponds to the extent of Fe reduction. Moreover, precipitations of secondary phase mineral such as vivianite were observed only in 12 months enrichment samples at 4 ℃ and 15 ℃. Sulfate reducing bacteria and Fe-reducing bacteria capable of reducing Fe were identified by 16S rRNA pyrosequencing. The Fe reduction coupled to oxidation of organic matter might be enhanced by cooperation of a consortium of Sulfate reducing bacteria and Fe-reducing bacteria. Moreover, Nitrate reducing bacteria which have an ability to oxidize ferrous iron anaerobically with nitrate reduction were identified at 15 ℃. The lower values observed in the extent of Fe reduction at 15 ℃ may be associated with Fe-oxidation induced by nitrate reduction.In order to verify the mechanism of microbial Fe reduction in clay minerals at low temperatures (4 and 15 ℃), Fe-rich Nontronite (NAu-1) and Psychrophilic bacteria were incubated for 4 months in anaerobic condition. Total structural Fe in NAu-1 is 16.4 % and 99.6 % of the total Fe is ferric. The extent of Fe reduction in nontronite was reached up to 11.5 % and 11 % at 4 ℃ and 15 ℃, respectively. The structural modification of biologically Fe-reduced nontronite was observed in the (001) peak shift to the lower 2 theta indicating the layer collapse associated with K

  20. Intraperitoneal injection of d-serine inhibits high-fat diet intake and preference in male mice.

    Science.gov (United States)

    Sasaki, Tsutomu; Yasoshima, Yasunobu; Matsui, Sho; Yokota-Hashimoto, Hiromi; Kobayashi, Masaki; Kitamura, Tadahiro

    2017-11-01

    d-serine is a co-agonist of the N-methyl d-aspartate (NMDA) receptor, an important modulator of glutamatergic excitatory synaptic transmission. We previously reported that oral d-serine ingestion inhibited the intake of highly preferred food and promoted the intake of less preferred food in mice. Here, we analyzed the effects of intraperitoneal (IP) d-serine injections on feeding behavior in mice. We assessed the effects of d-serine during both the acquisition and maintenance of a preference for high-fat diets (HFDs). Aversiveness of IP d-serine was analyzed in the conditioned taste aversion paradigm. The effects on food intake were assessed by providing liquid meals with different fat contents. Finally, we measured brain d-serine and l-serine levels after d-serine administration. We found that IP-injected d-serine effectively inhibited the acquisition of a HFD preference, but failed to prevent expression of a previously learned HFD preference. IP-injected d-serine was not sufficient to condition taste aversion. The effect on HFD preference acquisition was associated with increases in d-serine levels in the cerebral cortex, hypothalamus, and cerebellum. IP-injected d-serine most effectively inhibited the intake of liquid meals with high fat content. This effect was dose-dependent, but the responses varied significantly among male C57BL/6J mice. The differential responses to d-serine were consistent among multiple trials in each mouse. In summary, IP-injected d-serine inhibited HFD intake and the acquisition of an HFD preference. Individual mice with the same genetic background showed different sensitivities to d-serine; thus, d-serine sensitivity may be associated with unidentified traits. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  1. The alkaline diet: is there evidence that an alkaline pH diet benefits health?

    Science.gov (United States)

    Schwalfenberg, Gerry K

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.

  2. The Alkaline Diet: Is There Evidence That an Alkaline pH Diet Benefits Health?

    Directory of Open Access Journals (Sweden)

    Gerry K. Schwalfenberg

    2012-01-01

    Full Text Available This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy. Many books written in the lay literature on the alkaline diet were also reviewed and evaluated in light of the published medical literature. There may be some value in considering an alkaline diet in reducing morbidity and mortality from chronic diseases and further studies are warranted in this area of medicine.

  3. Persistently increased intestinal fraction of alkaline phosphatase

    DEFF Research Database (Denmark)

    Nathan, E; Baatrup, G; Berg, H

    1984-01-01

    Persistent elevation of the intestinal fraction of the alkaline phosphatase (API) as an isolated finding has to our knowledge not been reported previously. It was found in a boy followed during a period of 5.5 years. The only symptom was transient periodic fatigue observed at home, but not appare...

  4. Alkaline electrochemical cells and method of making

    Science.gov (United States)

    Hoyt, H. E.; Pfluger, H. L. (Inventor)

    1970-01-01

    Equilibrated cellulose ether membranes of increased electrolytic conductivity for use as separators in concentrated alkaline electrochemical cells are investigated. The method of making such membranes by equilibration to the degree desired in an aqueous alkali solution mantained at a temperature below about 10 C is described.

  5. Use Alkalinity Monitoring to Optimize Bioreactor Performance.

    Science.gov (United States)

    Jones, Christopher S; Kult, Keegan J

    2016-05-01

    In recent years, the agricultural community has reduced flow of nitrogen from farmed landscapes to stream networks through the use of woodchip denitrification bioreactors. Although deployment of this practice is becoming more common to treat high-nitrate water from agricultural drainage pipes, information about bioreactor management strategies is sparse. This study focuses on the use of water monitoring, and especially the use of alkalinity monitoring, in five Iowa woodchip bioreactors to provide insights into and to help manage bioreactor chemistry in ways that will produce desirable outcomes. Results reported here for the five bioreactors show average annual nitrate load reductions between 50 and 80%, which is acceptable according to established practice standards. Alkalinity data, however, imply that nitrous oxide formation may have regularly occurred in at least three of the bioreactors that are considered to be closed systems. Nitrous oxide measurements of influent and effluent water provide evidence that alkalinity may be an important indicator of bioreactor performance. Bioreactor chemistry can be managed by manipulation of water throughput in ways that produce adequate nitrate removal while preventing undesirable side effects. We conclude that (i) water should be retained for longer periods of time in bioreactors where nitrous oxide formation is indicated, (ii) measuring only nitrate and sulfate concentrations is insufficient for proper bioreactor operation, and (iii) alkalinity monitoring should be implemented into protocols for bioreactor management.

  6. Biomass production on saline-alkaline soils

    Energy Technology Data Exchange (ETDEWEB)

    Chaturvedi, A.N.

    1985-01-01

    In a trial of twelve tree species (both nitrogen fixing and non-fixing) for fuel plantations on saline-alkaline soil derived from Gangetic alluvium silty clay, Leucaena leucocephala failed completely after showing rapid growth for six months. Results for other species at age two showed that Prosopis juliflora had the best productivity.

  7. Alkaline Hydrolysis Conversion of Nitrocellulose Fines

    Science.gov (United States)

    1997-10-01

    The conversion of 1,125,000 pounds of bone-dry nitrocellulose fines into a liquid fertilizer was documented. Alkaline hydrolysis was the conversion... fertilizer . Fertilizer nutrient value was 1.3% nitrogen (N), 8.0% potassium (K2O) and 0.9% phosphorus (P2O5). Conversion met all applicable federal and state safety and environmental regulations.

  8. Delignification of biomass using alkaline glycerol

    Energy Technology Data Exchange (ETDEWEB)

    Kucuk, M. [YYU Educational Facility, Van (Turkey)

    2005-10-15

    Ailanthus altissima and Spruce orientalis chips were delignified by using glycerol as a solvent and alkaline-gliycerol with and without catalyst at different temperatures (438, 458, 478, and 498 K) in atmospheric pressure, and results were compared with the other organic solvent systems. (Author)

  9. Gelatinases and serine proteinase inhibitors of seminal plasma and the reproductive tract of turkey (Meleagris gallopavo).

    Science.gov (United States)

    Kotłowska, M; Kowalski, R; Glogowski, J; Jankowski, J; Ciereszko, A

    2005-04-01

    This study examined proteolytic enzymes and serine proteinase inhibitors in turkey seminal plasma with relation to their distribution within the reproductive tract and to yellow semen syndrome (YSS). Proteases of blood plasma, extracts from the reproductive tract, and seminal plasma were analyzed by gelatin zymography. We found a clear regional distribution of proteolytic enzymes in the turkey reproductive tract. Each part was characterized by a unique profile of serine proteolytic enzymes of molecular weights ranging from 29 to 88 kDa. The ductus deferens was found to be a site of very intense proteolytic activity. Two metalloproteases of 58 and 66 kDa were detected in all parts of the reproductive tract and seminal plasma. Using electrophoretic methods for detection of anti-trypsin activity, we found three serine proteinase inhibitors in turkey seminal plasma. Two inhibitors were found in the testis and epididymis and a third in the ductus deferens and seminal plasma. Blood plasma was characterized by the presence of two metalloproteinases and one serine proteinase inhibitor (of low migration rate) that were also detected in the reproductive tract. Amidase and anti-trypsin activities (expressed per gram of protein) differed for yellow and white seminal plasma. We concluded that turkey seminal plasma contains metalloproteases, serine proteinases, and serine proteinase inhibitors. The metalloproteases and one proteinase inhibitor are related to blood proteinases but the other two inhibitors and serine proteinases seem to be unique for the reproductive tract.

  10. Characterization of a novel Kazal-type serine proteinase inhibitor of Arabidopsis thaliana.

    Science.gov (United States)

    Pariani, Sebastián; Contreras, Marisol; Rossi, Franco R; Sander, Valeria; Corigliano, Mariana G; Simón, Francisco; Busi, María V; Gomez-Casati, Diego F; Pieckenstain, Fernando L; Duschak, Vilma G; Clemente, Marina

    2016-04-01

    Many different types of serine proteinase inhibitors have been involved in several kinds of plant physiological processes, including defense mechanisms against phytopathogens. Kazal-type serine proteinase inhibitors, which are included in the serine proteinase inhibitor family, are present in several organisms. These proteins play a regulatory role in processes that involve serine proteinases like trypsin, chymotrypsin, thrombin, elastase and/or subtilisin. In the present work, we characterized two putative Kazal-type serine proteinase inhibitors from Arabidopsis thaliana, which have a single putative Kazal-type domain. The expression of these inhibitors is transiently induced in response to leaf infection by Botrytis cinerea, suggesting that they play some role in defense against pathogens. We also evaluated the inhibitory specificity of one of the Kazal-type serine proteinase inhibitors, which resulted to be induced during the local response to B. cinerea infection. The recombinant Kazal-type serine proteinase inhibitor displayed high specificity for elastase and subtilisin, but low specificity for trypsin, suggesting differences in its selectivity. In addition, this inhibitor exhibited a strong antifungal activity inhibiting the germination rate of B. cinerea conidia in vitro. Due to the important role of proteinase inhibitors in plant protection against pathogens and pests, the information about Kazal-type proteinase inhibitors described in the present work could contribute to improving current methods for plant protection against pathogens.

  11. Reaction pathway of tryptophanase-catalyzed L-tryptophan synthesis from D-serine.

    Science.gov (United States)

    Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Fujii, Noriko

    2011-11-01

    Tryptophanase, L-tryptophan indole-lyase with extremely absolute stereospecificity, can change the stereospecificity in concentrated diammonium hydrogenphosphate solution. While tryptophanase is not inert to D-serine in the absence of diammonium hydrogenphosphate, it can undergo L-tryptophan synthesis from D-serine along with indole in the presence of it. It has been well known that tryptophanase synthesizes L-tryptophan from L-serine through a β-substitution mechanism of the ping-pong type. However, a metabolic pathway of L-tryptophan synthesis from D-serine has remained unclear. The present study aims to elucidate it. Diammonium hydrogenphosphate plays a role in the emergence of catalytic activity on D-serine. The salt gives tryptophanase a small conformational change, which makes it possible to catalyze D-serine. Tryptophanase-bound D-serine produces L-tryptophan synthesis by β-replacement reaction via the enzyme-bound aminoacrylate intermediate. Our result will be valuable in studying the origin of homochirality.

  12. Traditional Food Items in Ogimi, Okinawa: l-Serine Content and the Potential for Neuroprotection.

    Science.gov (United States)

    Cox, Paul Alan; Metcalf, James S

    2017-01-01

    Ogimi village is renowned for its aging population. We sought to determine if the l-serine content of their diet could account for their neurological health. The most frequently consumed food items, including tofu and seaweeds, are rich in the dietary amino acid l-serine. l-serine content of the Ogimi diet >8 grams/day for Ogimi women significantly exceeds the average American dietary intake of 2.5 grams/day for women >70 years old. Our hypothesis that the high l-serine content of the Ogimi diet is related to the paucity of tangle diseases among villagers is buttressed by in vivo results with non-human primates where dietary l-serine slowed development of neurofibrillary tangles and β-amyloid plaques by up to 85% and a human clinical trial finding that l-serine at 15 grams/day twice daily slows functional decline in ALS patients. Analysis of the Ogimi diet suggests that l-serine should be evaluated for therapeutic potential as a neuroprotective agent.

  13. Enzymatic Synthesis of Galactosylated Serine/Threonine Derivatives by β-Galactosidase from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Sooyoun Seo

    2015-06-01

    Full Text Available The transgalactosylations of serine/threonine derivatives were investigated using β-galactosidase from Escherichia coli as biocatalyst. Using ortho-nitrophenyl-β-D-galactoside as donor, the highest bioconversion yield of transgalactosylated N-carboxy benzyl L-serine benzyl ester (23.2% was achieved in heptane:buffer medium (70:30, whereas with the lactose, the highest bioconversion yield (3.94% was obtained in the buffer reaction system. The structures of most abundant galactosylated serine products were characterized by MS/MS. The molecular docking simulation revealed that the binding of serine/threonine derivatives to the enzyme’s active site was stronger (−4.6~−7.9 kcal/mol than that of the natural acceptor, glucose, and mainly occurred through interactions with aromatic residues. For N-tert-butoxycarbonyl serine methyl ester (6.8% and N-carboxybenzyl serine benzyl ester (3.4%, their binding affinities and the distances between their hydroxyl side chain and the 1′-OH group of galactose moiety were in good accordance with the quantified bioconversion yields. Despite its lower predicted bioconversion yield, the high experimental bioconversion yield obtained with N-carboxybenzyl serine methyl ester (23.2% demonstrated the importance of the thermodynamically-driven nature of the transgalactosylation reaction.

  14. Proteome-wide reactivity profiling identifies diverse carbamate chemotypes tuned for serine hydrolase inhibition.

    Science.gov (United States)

    Chang, Jae Won; Cognetta, Armand B; Niphakis, Micah J; Cravatt, Benjamin F

    2013-07-19

    Serine hydrolases are one of the largest and most diverse enzyme classes in Nature. Inhibitors of serine hydrolases are used to treat many diseases, including obesity, diabetes, cognitive dementia, and bacterial and viral infections. Nonetheless, the majority of the 200+ serine hydrolases in mammals still lack selective inhibitors for their functional characterization. We and others have shown that activated carbamates, through covalent reaction with the conserved serine nucleophile of serine hydrolases, can serve as useful inhibitors for members of this enzyme family. The extent to which carbamates, however, cross-react with other protein classes remains mostly unexplored. Here, we address this problem by investigating the proteome-wide reactivity of a diverse set of activated carbamates in vitro and in vivo, using a combination of competitive and click chemistry (CC)-activity-based protein profiling (ABPP). We identify multiple classes of carbamates, including O-aryl, O-hexafluoroisopropyl (HFIP), and O-N-hydroxysuccinimidyl (NHS) carbamates that react selectively with serine hydrolases across entire mouse tissue proteomes in vivo. We exploit the proteome-wide specificity of HFIP carbamates to create in situ imaging probes for the endocannabinoid hydrolases monoacylglycerol lipase (MAGL) and α-β hydrolase-6 (ABHD6). These findings, taken together, designate the carbamate as a privileged reactive group for serine hydrolases that can accommodate diverse structural modifications to produce inhibitors that display exceptional potency and selectivity across the mammalian proteome.

  15. Titanium corrosion in alkaline hydrogen peroxide environments

    Science.gov (United States)

    Been, Jantje

    1998-12-01

    The corrosion of Grade 2 titanium in alkaline hydrogen peroxide environments has been studied by weight loss corrosion tests, electrochemical impedance spectroscopy (EIS), linear polarization resistance (LPR) measurements and potentiodynamic polarography. Calcium ions and wood pulp were investigated as corrosion inhibitors. In alkaline peroxide, the titanium corrosion rate increased with increasing pH, temperature, and hydrogen peroxide concentration. The corrosion controlling mechanism is thought to be the reaction of the oxide with the perhydroxyl ion. No evidence of thermodynamically stable calcium titanate was found in the surface film of test coupons exposed to calcium-inhibited alkaline peroxide solutions. Calcium inhibition is probably the result of low local alkali and peroxide concentrations at the metal surface produced by reaction of adsorbed calcium with hydrogen peroxide. It has been shown that the inhibiting effect of calcium is temporary, possibly through an effect of calcium on the chemical and/or physical stability of the surface oxide. Pulp is an effective and stable corrosion inhibitor. Raising the pulp concentration decreased the corrosion rate. The inhibiting effect of pulp may be related to the adsorption and interaction of the pulp fibers with H 2O2, thereby decreasing the peroxide concentration and rendering the solution less corrosive. The presence of both pulp and calcium led to higher corrosion rates than obtained by either one inhibitor alone. Replacement of hydrofluoric acid with alkaline peroxide for pickling of titanium was investigated. Titanium corrosion rates in alkaline peroxide exceeded those obtained in the conventional hydrofluoric acid bath. General corrosion was observed with extensive roughening of the surface giving a dull gray appearance. Preferred dissolution of certain crystallographic planes was investigated through the corrosion of a titanium single crystal. Whereas the overall effect on the corrosion rate was small

  16. The alkaline diet: is there evidence that an alkaline pH diet benefits health?

    National Research Council Canada - National Science Library

    Schwalfenberg, Gerry K

    2012-01-01

    This review looks at the role of an alkaline diet in health. Pubmed was searched looking for articles on pH, potential renal acid loads, bone health, muscle, growth hormone, back pain, vitamin D and chemotherapy...

  17. Recent Alkaline Lakes: Clues to Understanding the Evolution of Early Planetary Alkaline Oceans and Biogenesis

    Science.gov (United States)

    Kempe, S.; Hartmann, J.; Kazmierczak, J.

    2008-09-01

    Abstract New models suggest that terrestrial weathering consumes 0.26GtC/a (72% silicate-, 28% carbonateweathering), equivalent to a loss of one atmospheric C content every 3700a. Rapid weathering leads in volcanic areas to alkaline conditions, illustrated by the crater lake of Niuafo`ou/Tonga and Lake Van/Turkey, the largest soda lake on Earth. Alkaline conditions cause high CaCO3 supersaturation, permineralization of algal mats and growth of stromatolites. Alkaline conditions can nearly depress free [Ca2+] to levels necessary for proteins to function. Therefore early oceans on Earth (and possibly on Mars) should have been alkaline (i.e. "Soda Oceans"). Recent findings of MgSO4 in top soils on Mars may be misleading about the early history of martian oceans.

  18. D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats.

    Science.gov (United States)

    Seif, Taban; Simms, Jeffrey A; Lei, Kelly; Wegner, Scott; Bonci, Antonello; Messing, Robert O; Hopf, F Woodward

    2015-09-01

    There is considerable interest in NMDAR modulators to enhance memory and treat neuropsychiatric disorders such as addiction, depression, and schizophrenia. D-serine and D-cycloserine, the NMDAR activators at the glycine site, are of particular interest because they have been used in humans without serious adverse effects. Interestingly, D-serine also inhibits some NMDARs active at hyperpolarized potentials (HA-NMDARs), and we previously found that HA-NMDARs within the nucleus accumbens core (NAcore) are critical for promoting compulsion-like alcohol drinking, where rats consume alcohol despite pairing with an aversive stimulus such as quinine, a paradigm considered to model compulsive aspects of human alcohol use disorders (AUDs). Here, we examined the impact of D-serine and D-cycloserine on this aversion-resistant alcohol intake (that persists despite adulteration with quinine) and consumption of quinine-free alcohol. Systemic D-serine reduced aversion-resistant alcohol drinking, without altering consumption of quinine-free alcohol or saccharin with or without quinine. Importantly, D-serine within the NAcore but not the dorsolateral striatum also selectively reduced aversion-resistant alcohol drinking. In addition, D-serine inhibited EPSCs evoked at -70 mV in vitro by optogenetic stimulation of mPFC-NAcore terminals in alcohol-drinking rats, similar to reported effects of the NMDAR blocker AP5. Further, D-serine preexposure occluded AP5 inhibition of mPFC-evoked EPSCs, suggesting that D-serine reduced EPSCs by inhibiting HA-NMDARs. Systemic D-cycloserine also selectively reduced intake of quinine-adulterated alcohol, and D-cycloserine inhibited NAcore HA-NMDARs in vitro. Our results indicate that HA-NMDAR modulators can reduce aversion-resistant alcohol drinking, and support testing of D-serine and D-cycloserine as immediately accessible, FDA-approved drugs to treat AUDs.

  19. D-Serine and D-Cycloserine Reduce Compulsive Alcohol Intake in Rats

    Science.gov (United States)

    Seif, Taban; Simms, Jeffrey A; Lei, Kelly; Wegner, Scott; Bonci, Antonello; Messing, Robert O; Hopf, F Woodward

    2015-01-01

    There is considerable interest in NMDAR modulators to enhance memory and treat neuropsychiatric disorders such as addiction, depression, and schizophrenia. D-serine and D-cycloserine, the NMDAR activators at the glycine site, are of particular interest because they have been used in humans without serious adverse effects. Interestingly, D-serine also inhibits some NMDARs active at hyperpolarized potentials (HA-NMDARs), and we previously found that HA-NMDARs within the nucleus accumbens core (NAcore) are critical for promoting compulsion-like alcohol drinking, where rats consume alcohol despite pairing with an aversive stimulus such as quinine, a paradigm considered to model compulsive aspects of human alcohol use disorders (AUDs). Here, we examined the impact of D-serine and D-cycloserine on this aversion-resistant alcohol intake (that persists despite adulteration with quinine) and consumption of quinine-free alcohol. Systemic D-serine reduced aversion-resistant alcohol drinking, without altering consumption of quinine-free alcohol or saccharin with or without quinine. Importantly, D-serine within the NAcore but not the dorsolateral striatum also selectively reduced aversion-resistant alcohol drinking. In addition, D-serine inhibited EPSCs evoked at −70 mV in vitro by optogenetic stimulation of mPFC–NAcore terminals in alcohol-drinking rats, similar to reported effects of the NMDAR blocker AP5. Further, D-serine preexposure occluded AP5 inhibition of mPFC-evoked EPSCs, suggesting that D-serine reduced EPSCs by inhibiting HA-NMDARs. Systemic D-cycloserine also selectively reduced intake of quinine-adulterated alcohol, and D-cycloserine inhibited NAcore HA-NMDARs in vitro. Our results indicate that HA-NMDAR modulators can reduce aversion-resistant alcohol drinking, and support testing of D-serine and D-cycloserine as immediately accessible, FDA-approved drugs to treat AUDs. PMID:25801502

  20. Involvement of serine proteases in the excystation and metacystic development of Entamoeba invadens.

    Science.gov (United States)

    Makioka, Asao; Kumagai, Masahiro; Kobayashi, Seiki; Takeuchi, Tsutomu

    2009-10-01

    Although the functions of cysteine proteases involved in the pathogenicity and differentiation of Entamoeba histolytica have been demonstrated, little is known about the functions of serine proteases. We examined the involvement of serine proteases in amoebic excystation and metacystic development using inhibitors specific for serine proteases. Entamoeba invadens IP-1 strain was used as the model of excystation and metacystic development of E. histolytica. Four serine protease inhibitors, phenylmethanesulfonyl fluoride (PMSF), 4-(2-aminoethyl) bezensulfonylfluoride hydrochloride, 3, 4-dichloroisocoumarin, and N-tosyl-phe-chloromethylketone, decreased the number of metacystic amoebae in a dose-dependent manner, without showing cytotoxicity to cysts. PMSF inhibited not only the increase but also the development of metacystic amoebae as determined by the change of nucleus number from four- to one-nucleate amoebae. The protease activity in cyst lysates was also inhibited by PMSF and the band of protease on gelatin sodium dodecyl sulfate polyacrylamide gel electrophoresis was weaker than controls when treated with PMSF. Three serine protease families, S28 (three types), S9 (two), and S26 (one) were retrieved from the database of E. invadens. Phylogenetic analysis revealed that amebic enzymes from the serine protease families formed different clades from those from other organisms. The expression levels of these serine proteases in cysts 5 h after the induction of excystation as assessed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) were higher than those observed prior to induction assayed by real-time RT-PCR; the increase in one type of S9 (named S9-3) expression was the highest. The expression of S9 enzymes also increased from cysts to trophozoites higher than the other family serine proteases. Thus, the results show that Entamoeba uses their serine proteases in the excystation and metacystic development, which leads to successful infection.

  1. A Low-temperature Alkaline Protease Producing Marine Psychrophilic Bacterium CHS%产碱性蛋白酶的海洋适冷细菌CHS的初步研究

    Institute of Scientific and Technical Information of China (English)

    陈静; 王淑军; 潘建梅; 许冰; 牛天贵

    2004-01-01

    从连云港海域、港口、远洋捕捞船及鱼市等地采集的海水和各类海鱼、贝类等样品中分离得到一株产碱性蛋白酶的海洋适冷细菌CHS.研究表明,CHS最适生长温度和产酶温度均在15℃左右,0℃下仍可生长;具有一定的耐盐性和嗜盐性,无盐条件下不能生长,在质量分数为5%的NaCl中生长最好;最适生长pH为8.0,最适产酶pH为9.0.

  2. Effect of temperature on sulphate reduction, growth rate and growth yield in five psychrophilic sulphate-reducing bacteria from Arctic sediments

    DEFF Research Database (Denmark)

    Knoblauch, C.; Jørgensen, BB

    1999-01-01

    -41% of those at T(opt). Short-term incubations of exponentially growing cultures showed that the highest sulphate reduction rates occurred 2-9 degrees C above T(opt). In contrast to growth and sulphate reduction rates, growth yields of strains ASv26, LSv54 and PSv29 were almost constant between -1.8 degrees C...... for growth (T(opt)) were 7 degrees C (PSv29), 10 degrees C (ASv26, LSv54) and 18 degrees C (LSv21, LSv514), Although T(opt) was considerably above the in situ temperatures of their habitats (-1.7 degrees C and 2.6 degrees C), relative growth rates were still high at 0 degrees C, accounting for 25...... and T(opt). For strains LSv21 and LSv514, however, growth yields were highest at the lowest temperatures, around 0 degrees C. The results indicate that psychrophilic sulphate-reducing bacteria are specially adapted to permanently low temperatures by high relative growth rates and high growth yields...

  3. Cow, sheep and llama manure at psychrophilic anaerobic co-digestion with low cost tubular digesters in cold climate and high altitude.

    Science.gov (United States)

    Martí-Herrero, J; Alvarez, R; Cespedes, R; Rojas, M R; Conde, V; Aliaga, L; Balboa, M; Danov, S

    2015-04-01

    The aim of this research is to evaluate the co-digestion of cow and llama manure combined with sheep manure, in psychrophilic conditions and real field low cost tubular digesters adapted to cold climate. Four digesters were monitored in cold climate conditions; one fed with cow manure, a second one with llama manure, the third one with co-digestion of cow-sheep manure and the fourth one was fed with llama-sheep manure. The slurry had a mean temperature of 16.6 °C, the organic load rate was 0.44 kgvs m(-3) d(-1) and the hydraulic retention time was 80 days. After one hundred days biogas production was stable, as was the methane content and the pH of the effluent. The co-digestion of cow-sheep manure results in a biogas production increase of 100% compared to the mono-digestion of cow manure, while co-digestion of llama-sheep manure results in a decrease of 50% in biogas production with respect to mono-digestion of llama manure.

  4. In situ activity-based protein profiling of serine hydrolases in E. coli

    Directory of Open Access Journals (Sweden)

    Dmitry Shamshurin

    2014-09-01

    Full Text Available A fluorophosphonate based alkyne activity probe was used for the selective labeling of active serine hydrolases in intact Escherichia coli cells. A biotin-azide tag was subsequently attached to the alkyne functionality of the probe with copper-catalyzed azide-alkyne cycloaddition (CuAAC reaction. Comparison of proteins from in-cell and lysate labeled preparations suggested qualitatively similar patterns of reactivity in both preparations. Approximately 68%, 30 of the total 44 serine hydrolases detectable in E. coli were labeled with the probe indicating significant coverage with a single probe. The methods described here offer a useful tool for profiling and monitoring serine hydrolase activity in situ.

  5. Improved electrodes and gas impurity investigations on alkaline electrolysers

    DEFF Research Database (Denmark)

    Reissner, R.; Schiller, G.; Knoeri, T.;

    Alkaline water electrolysis for hydrogenproduction is a well-established techniquebut some technological issues regarding thecoupling of alkaline water electrolysis andRenewable Energy Sources (RES) remain tobe improved....

  6. Exploring a new serine protease from Cucumis sativus L.

    Science.gov (United States)

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process.

  7. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  8. D-Serine in Neuropsychiatric Disorders: New Advances

    Directory of Open Access Journals (Sweden)

    Andrea R. Durrant

    2014-01-01

    Full Text Available D-Serine (DSR is an endogenous amino acid involved in glia-synapse interactions that has unique neurotransmitter characteristics. DSR acts as obligatory coagonist at the glycine site associated with the N-methyl-D-aspartate subtype of glutamate receptors (NMDAR and has a cardinal modulatory role in major NMDAR-dependent processes including NMDAR-mediated neurotransmission, neurotoxicity, synaptic plasticity, and cell migration. Since either over- or underfunction of NMDARs may be involved in the pathophysiology of neuropsychiatric disorders; the pharmacological manipulation of DSR signaling represents a major drug development target. A first generation of proof-of-concept animal and clinical studies suggest beneficial DSR effects in treatment-refractory schizophrenia, movement, depression, and anxiety disorders and for the improvement of cognitive performance. A related developing pharmacological strategy is the indirect modification of DSR synaptic levels by use of compounds that alter the function of main enzymes responsible for DSR production and degradation. Accumulating data indicate that, during the next decade, we will witness important advances in the understanding of DSR role that will further contribute to elucidating the causes of neuropsychiatric disorders and will be instrumental in the development of innovative treatments.

  9. Homologous inhibitors from potato tubers of serine endopeptidases and metallocarboxypeptidases.

    Science.gov (United States)

    Hass, C M; Venkatakrishnan, R; Ryan, C A

    1976-06-01

    A potent polypeptide inhibitor of chymotrypsin has been purified from Russett Burbank potatoes. The inhibitor has no effect on bovine carboxypeptidases A or B but exhibits homology with a carboxypeptidase inhibitor that is also present in potato tubers. The chymotrypsin inhibitor has a molecular weight of approximately 5400 as estimated by gel filtration, amino acid analysis, and titration with chymotrypsin. The polypeptide chain consists of 49 amino acid residues, of which six are half-cystine, forming three disulfide bonds. Its size is similar to that of the carboxypeptidase inhibitor, which contains 39 amino acid residues and also has three disulfide bridges. In immunological double diffusion assays, the chymotrypsin inhibitor and the carboxypeptidase inhibitor do not crossreact; however, automatic Edman degradation of reduced and alkylated derivatives of the chymotrypsin inhibitor, yielding a partial sequence of 18 amino acid residues at the NH2-terminus, reveals a similarity in sequence to that of the carboxypeptidase inhibitor. Thus, inhibitors directed toward two distinct classes of proteases, the serine endopeptidases and the metallocarboxypeptidases, appear to have evolved from a common ancestor.

  10. [Leucocyte alkaline phosphatase in normal and pathological pregnancy (author's transl)].

    Science.gov (United States)

    Stark, K H; Zaki, I; Sobolewski, K

    1981-01-01

    The activities of leucocyte alkaline phosphatase were determined in 511 patients with normal and pathological pregnancy. Mean values were compared and the enzyme followed up, and the conclusion was drawn that leucocyte alkaline phosphatase was no safe indicator of foetal condition. No direct relationship were found to exist between leucocyte alkaline phosphatase, total oestrogens, HSAP, HLAP, HPL, and oxytocinase.

  11. Improvement in regional CBF by L-serine contributes to its neuroprotective effect in rats after focal cerebral ischemia.

    Directory of Open Access Journals (Sweden)

    Tao-Jie Ren

    Full Text Available To investigate the mechanisms underlying the neuroprotective effect of L-serine, permanent focal cerebral ischemia was induced by occlusion of the middle cerebral artery while monitoring cerebral blood flow (CBF. Rats were divided into control and L-serine-treated groups after middle cerebral artery occlusion. The neurological deficit score and brain infarct volume were assessed. Nissl staining was used to quantify the cortical injury. L-serine and D-serine levels in the ischemic cortex were analyzed with high performance liquid chromatography. We found that L-serine treatment: 1 reduced the neurological deficit score, infarct volume and cortical neuron loss in a dose-dependent manner; 2 improved CBF in the cortex, and this effect was inhibited in the presence of apamin plus charybdotoxin while the alleviation of both neurological deficit score and infarct volume was blocked; and 3 increased the amount of L-serine and D-serine in the cortex, and inhibition of the conversion of L-serine into D-serine by aminooxyacetic acid did not affect the reduction of neurological deficit score and infarct volume by L-serine. In conclusion, improvement in regional CBF by L-serine may contribute to its neuroprotective effect on the ischemic brain, potentially through vasodilation which is mediated by the small- and intermediate-conductance Ca(2+-activated K(+ channels on the cerebral blood vessel endothelium.

  12. Effects of Chronic D-Serine Elevation on Animal Models of Depression and Anxiety-Related Behavior.

    Directory of Open Access Journals (Sweden)

    David-Marian Otte

    Full Text Available NMDA receptors are activated after binding of the agonist glutamate to the NR2 subunit along with a co-agonist, either L-glycine or D-serine, to the NR1 subunit. There is substantial evidence to suggest that D-serine is the most relevant co-agonist in forebrain regions and that alterations in D-serine levels contribute to psychiatric disorders. D-serine is produced through isomerization of L-serine by serine racemase (Srr, either in neurons or in astrocytes. It is released by astrocytes by an activity-dependent mechanism involving secretory vesicles. In the present study we generated transgenic mice (SrrTg expressing serine racemase under a human GFAP promoter. These mice were biochemically and behaviorally analyzed using paradigms of anxiety, depression and cognition. Furthermore, we investigated the behavioral effects of long-term administration of D-serine added to the drinking water. Elevated brain D-serine levels in SrrTg mice resulted in specific behavioral phenotypes in the forced swim, novelty suppression of feeding and olfactory bulbectomy paradigms that are indicative of a reduced proneness towards depression-related behavior. Chronic dietary D-serine supplement mimics the depression-related behavioral phenotype observed in SrrTg mice. Our results suggest that D-serine supplementation may improve mood disorders.

  13. The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons

    Directory of Open Access Journals (Sweden)

    Silverman Ann-Judith

    2002-02-01

    Full Text Available Abstract Background Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. Results Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1, and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. Conclusions These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

  14. Chasing One-Carbon Units to Understand the Role of Serine in Epigenetics.

    Science.gov (United States)

    Parker, Seth J; Metallo, Christian M

    2016-01-21

    In this issue of Molecular Cell, Maddocks et al. (2016) use stable isotope tracing, mass spectrometry, and nutrient modulation in cancer cells to highlight the role of serine in supporting methylation through maintenance of nucleotide levels.

  15. Optical properties of D-serine doped TGS crystals for pyroelectric sensors*

    Directory of Open Access Journals (Sweden)

    Kurlyak V.Yu.

    2015-12-01

    Full Text Available Refractive and birefringence indices in the range of transparency of 300 to 700 nm for triglycine sulphate crystals doped with D-serine molecules have been measured in the temperature range of 290 K to 340 K. The obtained optical properties are discussed together with characteristic electrical features of these materials used as pyroelectric sensors for measurement of temperature. The experimental results obtained in this study will be necessary as the reference data for comparison with the calculated refractive indices of TGS + D-serine on the basis of density functional theory. Determination of the proper position of D-serine, will reveal the features of TGS + D-serine crystal structure necessary to achieve stable unipolarity.

  16. Localization and accessibility of antigenic sites of the extracellular serine proteinase of Lactococcus lactis

    NARCIS (Netherlands)

    Laan, Harm; Kok, Jan; Haandrikman, Alfred J.; Venema, Gerhardus; Konings, Wilhelmus

    1992-01-01

    Lactococcus lactis strains produce an extracellular subtilisin-related serine proteinase in which immunologically different components can be distinguished. Monoclonal antibodies specific for the different proteinase components have been raised and their epitopes were identified. By Western-blot ana

  17. Oxidation catalysts on alkaline earth supports

    Science.gov (United States)

    Mohajeri, Nahid

    2017-03-21

    An oxidation catalyst includes a support including particles of an alkaline earth salt, and first particles including a palladium compound on the support. The oxidation catalyst can also include precious metal group (PMG) metal particles in addition to the first particles intermixed together on the support. A gas permeable polymer that provides a continuous phase can completely encapsulate the particles and the support. The oxidation catalyst may be used as a gas sensor, where the first particles are chemochromic particles.

  18. Oxidation catalysts on alkaline earth supports

    Energy Technology Data Exchange (ETDEWEB)

    Mohajeri, Nahid

    2017-03-21

    An oxidation catalyst includes a support including particles of an alkaline earth salt, and first particles including a palladium compound on the support. The oxidation catalyst can also include precious metal group (PMG) metal particles in addition to the first particles intermixed together on the support. A gas permeable polymer that provides a continuous phase can completely encapsulate the particles and the support. The oxidation catalyst may be used as a gas sensor, where the first particles are chemochromic particles.

  19. D-Amino-Acid Oxidase Inhibition Increases D-Serine Plasma Levels in Mouse But not in Monkey or Dog.

    Science.gov (United States)

    Rojas, Camilo; Alt, Jesse; Ator, Nancy A; Thomas, Ajit G; Wu, Ying; Hin, Niyada; Wozniak, Krystyna; Ferraris, Dana; Rais, Rana; Tsukamoto, Takashi; Slusher, Barbara S

    2016-05-01

    D-serine has been shown to improve positive, negative, and cognitive symptoms when used as add-on therapy for the treatment of schizophrenia. However, D-serine has to be administered at high doses to observe clinical effects. This is thought to be due to D-serine undergoing oxidation by D-amino-acid oxidase (DAAO) before it reaches the brain. Consequently, co-administration of D-serine with a DAAO inhibitor could be a way to lower the D-serine dose required to treat schizophrenia. Early studies in rodents to evaluate this hypothesis showed that concomitant administration of structurally distinct DAAO inhibitors with D-serine enhanced plasma and brain D-serine levels in rodents compared with administration of D-serine alone. In the present work we used three potent DAAO inhibitors and confirmed previous results in mice. In a follow-up effort, we evaluated plasma D-serine levels in monkeys after oral administration of D-serine in the presence or absence of these DAAO inhibitors. Even though the compounds reached steady state plasma concentrations exceeding their Ki values by >60-fold, plasma D-serine levels remained the same as those in the absence of DAAO inhibitors. Similar results were obtained with dogs. In summary, in contrast to rodents, DAAO inhibition in monkeys and dogs did not influence the exposure to exogenously administered D-serine. Results could be due to differences in D-serine metabolism and/or clearance mechanisms and suggest that the role of DAAO in the metabolism of D-serine is different across species. These data provide caution regarding the utility of DAAO inhibition for patients with schizophrenia.

  20. Metagenome resource for D-serine utilization in a DsdA-disrupted Escherichia coli.

    Science.gov (United States)

    Lim, Mi Young; Lee, Hyo Jeong; Kim, Pil

    2011-04-01

    To find alternative genetic resources for D-serine dehydratase (E.C. 4.3.1.18, dsdA) mediating the deamination of D-serine into pyruvate, metagenomic libraries were screened. The chromosomal dsdA gene of a wild-type Escherichia coli W3110 strain was disrupted by inserting the tetracycline resistance gene (tet), using double-crossover, for use as a screening host. The W3110 dsdA::tet strain was not able to grow in a medium containing D-serine as a sole carbon source, whereas wild-type W3110 and the complement W3110 dsdA::tet strain containing a dsdA-expression plasmid were able to grow. After introducing metagenome libraries into the screening host, a strain containing a 40-kb DNA fragment obtained from the metagenomic souce derived from a compost was selected based on its capability to grow on the agar plate containing D-serine as a sole carbon source. For identification of the genetic resource responsible for the D-serine degrading capability, transposon- micron was randomly inserted into the 40-kb metagenome. Two strains that had lost their D-serine degrading ability were negatively selected, and the two 6-kb contigs responsible for the D-serine degrading capability were sequenced and deposited (GenBank code: HQ829474.1 and HQ829475.1). Therefore, new alternative genetic resources for D-serine dehydratase was found from the metagenomic resource, and the corresponding ORFs are discussed.

  1. Repurposing Suzuki Coupling Reagents as a Directed Fragment Library Targeting Serine Hydrolases and Related Enzymes

    Science.gov (United States)

    2017-01-01

    Serine hydrolases are susceptible to potent reversible inhibition by boronic acids. Large collections of chemically diverse boronic acid fragments are commercially available because of their utility in coupling chemistry. We repurposed the approximately 650 boronic acid reagents in our collection as a directed fragment library targeting serine hydrolases and related enzymes. Highly efficient hits (LE > 0.6) often result. The utility of the approach is illustrated with the results against autotaxin, a phospholipase implicated in cardiovascular disease. PMID:28564542

  2. Alkaline phosphatase of Physarum polycephalum is insoluble.

    Science.gov (United States)

    Furuhashi, Kiyoshi

    2008-02-01

    The plasmodia of Physarum polycephalum grow as multinucleated cells in the presence of sufficient humidity and nutriment. Under non-illuminating conditions, stresses such as low temperature or high concentrations of salts transform the plasmodia into spherules whereas dehydration induces sclerotization. Some phosphatases including protein phosphatase and acid phosphatase have been purified from the plasmodia, but alkaline phosphatase remains to be elucidated. Phosphatase of the plasmodia, spherules and sclerotia was visualized by electrophoresis gel-staining assay using 5-bromo-4-chloro-3-indolyl phosphate. Insoluble fractions of the sclerotia were abundant in phosphatase activity. The phosphatase which was extracted by nonionic detergent was subjected to column chromatography and preparative electrophoresis. Purified phosphatase showed the highest activity at pH 8.8, indicating that this enzyme belongs to alkaline phosphatase. The apparent molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing condition was estimated to be 100 kDa whereas that under reducing was 105 kDa. An amount of 1% sodium dodecyl sulfate or 0.5 M NaCl had no effects on the activity although the phosphatase showed heat instability, Mg(2+)-dependency and sensitivity to 2-glycerophosphate or NaF. The extracting conditions and enzymatic properties suggest that this alkaline phosphatase which is in a membrane-bound form plays important roles in phosphate metabolism.

  3. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    Energy Technology Data Exchange (ETDEWEB)

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.; (Monash); (Queensland Inst. of Med. Rsrch.)

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  4. Plasmodium serine hydroxymethyltransferase: indispensability and display of distinct localization

    Directory of Open Access Journals (Sweden)

    Pornthanakasem Wichai

    2012-11-01

    Full Text Available Abstract Background Serine hydroxymethyltransferase (SHMT, a pyridoxal phosphate-dependent enzyme, plays a vital role in the de novo pyrimidine biosynthesis pathway in malaria parasites. Two genes have been identified in Plasmodium spp. encoding a cytosolic SHMT (cSHMT and putative mitochondria SHMT (mSHMT, but their roles have not been fully investigated. Methods The presence of Plasmodium SHMT isoforms in the intra-erythrocytic stage was assessed based on their gene expression using reverse transcription PCR (RT-PCR. Localization studies of Plasmodium SHMT isoforms were performed by transfection of fluorescent-tagged gene constructs into P. falciparum and expressions of fluorescent fusion proteins in parasites were observed using a laser scanning confocal microscope. Genetic targeting through homologous recombination was used to study the essentiality of SHMT in Plasmodium spp. Results Semi-quantitative RT-PCR revealed the expression of these two genes throughout intra-erythrocytic development. Localization studies using P. falciparum expressing fluorescent-tagged SHMT showed that PfcSHMT-red fluorescent fusion protein (PfcSHMT-DsRed is localized in the cytoplasm, while PfmSHMT-green fluorescent fusion protein (PfmSHMT-GFP co-localized with Mitotracker™-labelled mitochondria as predicted. The essentiality of plasmodial cSHMT was inferred from transfection experiments where recovery of viable knock-out parasites was not achieved, unless complemented with a functional equivalent copy of shmt. Conclusions Distinct compartment localizations of PfSHMT were observed between cytoplasmic and mitochondrial isoforms, and evidence was provided for the indispensable role of plasmodial cSHMT indicating it as a valid target for development of novel anti-malarials.

  5. A novel serine protease with caspase- and legumain-like activities from edible basidiomycete Flammulina velutipes.

    Science.gov (United States)

    Iketani, Aya; Nakamura, Mayumi; Suzuki, Yuya; Awai, Koichiro; Shioi, Yuzo

    2013-03-01

    A serine protease with caspase- and legumain-like activities from basidiocarps of the edible basidiomycete Flammulina velutipes was characterized. The protease was purified to near homogeneity by three steps of chromatography using acetyl-Tyr-Val-Ala-Asp-4-methylcoumaryl-7-amide (Ac-YVAD-MCA) as a substrate. The enzyme was termed FvSerP (F. velutipes serine protease). This enzyme activity was completely inhibited by the caspase-specific inhibitor, Ac-YVAD-CHO, as well as moderately inhibited by serine protease inhibitors. Based on the N-terminal sequence, the cDNA of FvSerP was identified. The deduced protease sequence was a peptide composed of 325 amino acids with a molecular mass of 34.5 kDa. The amino acid sequence of FvSerP showed similarity to neither caspases nor to the plant subtilisin-like serine protease with caspase-like activity called saspase. FvSerP shared identity to the functionally unknown genes from class of Agaricomycetes, with similarity to the peptidase S41 domain of a serine protease. It was thus concluded that this enzyme is likely a novel serine protease with caspase- and legumain-like activities belonging to the peptidase S41 family and distributed in the class Agaricomycetes. This enzyme possibly functions in autolysis, a type of programmed cell death that occurs in the later stages of development of basidiocarps with reference to their enzymatic functions.

  6. Uropathogenic Escherichia coli use d-serine deaminase to modulate infection of the murine urinary tract.

    Science.gov (United States)

    Roesch, Paula L; Redford, Peter; Batchelet, Stephanie; Moritz, Rebecca L; Pellett, Shahaireen; Haugen, Brian J; Blattner, Frederick R; Welch, Rodney A

    2003-07-01

    Although once thought to be unique to bacteria, d-amino acids are also produced by mammals. For example, d-serine is excreted in human urine at concentrations ranging from 3.0 to 40 micro g ml-1. An epidemiological survey demonstrated that urine isolates of E. coli are more likely to catabolise d-serine via expression of d-serine deaminase, DsdA than enteric disease isolates. The urosepsis strain, CFT073, and an isogenic dsdA mutant have similar growth kinetics in minimal or complex media. However, relative to the wild type, the dsdA mutant has a pleiomorphic cell shape and a prolonged, 4-6 h lag phase when grown in human urine. This suggests that d-serine catabolism provides a growth advantage in the urinary tract. Unexpectedly, in a direct competition model of urinary tract infection, the dsdA mutant was recovered 300-times more frequently than the wild type in the bladders of mice 48 h after infection. A new model of E. coli uropathogenesis is proposed where growth and gene expression are modulated in response to environmental d-serine levels. In support of this, the CFT073 dsdA mutant is hyperflagellated and more motile than the wild type indicating that intracellular levels of d-serine may directly or indirectly influence the expression of regulons associated with E. coli uropathogenesis.

  7. Contribution of the D-Serine-dependent pathway to the cellular mechanisms underlying cognitive aging

    Directory of Open Access Journals (Sweden)

    Emilie Rouaud

    2010-02-01

    Full Text Available An association between age-related memory impairments and changes in functional plasticity in the aging brain has been under intense study within the last decade. In this article, we show that an impaired activation of the strychnine-insensitive glycine site of N-Methyl-D-Aspartate receptors (NMDA-R by its agonist D-serine contributes to deficits of synaptic plasticity in the hippocampus of memory-impaired aged rats. Supplementation with exogenous D-serine prevents the age-related deficits of isolated NMDA-R-dependent synaptic potentials as well as those of theta-burst-induced long-term potentiation and synaptic depotentiation. Endogenous levels of D-serine are reduced in the hippocampus with aging, that correlates with a weaker expression of serine racemase synthesizing the amino acid. On the contrary, the affinity of D-serine binding to NMDA-R is not affected by aging. These results point to a critical role for the D-serine-dependent pathway in the functional alterations of the brain underlying memory impairment and provide key information in the search for new therapeutic strategies for the treatment of memory deficits in the elderly.

  8. Serine, but Not Glycine, Supports One-Carbon Metabolism and Proliferation of Cancer Cells

    Directory of Open Access Journals (Sweden)

    Christiaan F. Labuschagne

    2014-05-01

    Full Text Available Previous work has shown that some cancer cells are highly dependent on serine/glycine uptake for proliferation. Although serine and glycine can be interconverted and either might be used for nucleotide synthesis and one-carbon metabolism, we show that exogenous glycine cannot replace serine to support cancer cell proliferation. Cancer cells selectively consumed exogenous serine, which was converted to intracellular glycine and one-carbon units for building nucleotides. Restriction of exogenous glycine or depletion of the glycine cleavage system did not impede proliferation. In the absence of serine, uptake of exogenous glycine was unable to support nucleotide synthesis. Indeed, higher concentrations of glycine inhibited proliferation. Under these conditions, glycine was converted to serine, a reaction that would deplete the one-carbon pool. Providing one-carbon units by adding formate rescued nucleotide synthesis and growth of glycine-fed cells. We conclude that nucleotide synthesis and cancer cell proliferation are supported by serine—rather than glycine—consumption.

  9. Alkaline and ultrasound assisted alkaline pretreatment for intensification of delignification process from sustainable raw-material.

    Science.gov (United States)

    Subhedar, Preeti B; Gogate, Parag R

    2014-01-01

    Alkaline and ultrasound-assisted alkaline pretreatment under mild operating conditions have been investigated for intensification of delignification. The effect of NaOH concentration, biomass loading, temperature, ultrasonic power and duty cycle on the delignification has been studied. Most favorable conditions for only alkaline pretreatment were alkali concentration of 1.75 N, solid loading of 0.8% (w/v), temperature of 353 K and pretreatment time of 6 h and under these conditions, 40.2% delignification was obtained. In case of ultrasound-assisted alkaline approach, most favorable conditions obtained were alkali concentration of 1N, paper loading of 0.5% (w/v), sonication power of 100 W, duty cycle of 80% and pretreatment time of 70 min and the delignification obtained in ultrasound-assisted alkaline approach under these conditions was 80%. The material samples were characterized by FTIR, SEM, XRD and TGA technique. The lignin was recovered from solution by precipitation method and was characterized by FTIR, GPC and TGA technique.

  10. Processing Methods of Alkaline Hydrolysate from Rice Husk

    Directory of Open Access Journals (Sweden)

    Olga D. Arefieva

    2017-07-01

    Full Text Available This paper devoted to finding processing methods of alkaline hydrolysate produced from rice husk pre-extraction, and discusses alkaline hydrolysate processing schemed and disengagement of some products: amorphous silica of various quality, alkaline lignin, and water and alkaline extraction polysaccharides. Silica samples were characterized: crude (air-dried, burnt (no preliminary water treatment, washed in distilled water, and washed in distilled water and burnt. Waste water parameters upon the extraction of solids from alkaline hydrolysate dropped a few dozens or thousand times depending on the applied processing method. Color decreased a few thousand times, turbidity was virtually eliminated, chemical oxygen demanded about 20–136 times; polyphenols content might decrease 50% or be virtually eliminated. The most prospective scheme obtained the two following solid products from rice husk alkaline hydrolysate: amorphous silica and alkaline extraction polysaccharide. Chemical oxygen demand of the remaining waste water decreased about 140 times compared to the silica-free solution.

  11. Implantation serine proteinase 2 is a monomeric enzyme with mixed serine proteolytic activity and can silence signalling via proteinase activated receptors.

    Science.gov (United States)

    Sharma, Navneet; Fahr, Jochen; Renaux, Bernard; Saifeddine, Mahmoud; Kumar, Rajeev; Nishikawa, Sandra; Mihara, Koichiro; Ramachandran, Rithwik; Hollenberg, Morley D; Rancourt, Derrick E

    2013-12-01

    Implantation serine proteinase 2 (ISP2), a S1 family serine proteinase, is known for its role in the critical processes of embryo hatching and implantation in the mouse uterus. Native implantation serine proteinases (ISPs) are co-expressed and co-exist as heterodimers in uterine and blastocyst tissues. The ISP1-ISP2 enzyme complex shows trypsin-like substrate specificity. In contrast, we found that ISP2, isolated as a 34 kDa monomer from a Pichia pastoris expression system, exhibited a mixed serine proteolytic substrate specificity, as determined by a phage display peptide cleavage approach and verified by the in vitro cleavage of synthetic peptides. Based upon the peptide sequence substrate selectivity, a database search identified many potential ISP2 targets of physiological relevance, including the proteinase activated receptor 2 (PAR2). The in vitro cleavage studies with PAR2-derived peptides confirmed the mixed substrate specificity of ISP2. Treatment of cell lines expressing proteinase-activated receptors (PARs) 1, 2, and 4 with ISP2 prevented receptor activation by either thrombin (PARs 1 and 4) or trypsin (PAR2). The disarming and silencing of PARs by ISP2 may play a role in successful embryo implantation.

  12. Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

    DEFF Research Database (Denmark)

    Jiang, Longguang; Andersen, Lisbeth Moreau; Andreasen, Peter A

    2016-01-01

    BACKGROUND: Serine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a ser...

  13. Mannan-binding lectin (MBL)-associated serine protease-1 (MASP-1), a serine protease associated with humoral pattern-recognition molecules

    DEFF Research Database (Denmark)

    Thiel, Steffen; Degn, Søren Egedal; Nielsen, H J;

    2012-01-01

    The pattern-recognition molecules mannan-binding lectin (MBL) and the three ficolins circulate in blood in complexes with MBL-associated serine proteases (MASPs). When MBL or ficolin recognizes a microorganism, activation of the MASPs occurs leading to activation of the complement system, an impo...

  14. Biodiversity and cold adaptive mechanisms of psychrophiles%低温细菌与古菌的生物多样性及其冷适应机制

    Institute of Scientific and Technical Information of China (English)

    辛玉华; 周宇光; 东秀珠

    2013-01-01

    Cold-adapted bacteria and archaea are widely distributed in cold environments on Earth,such as permafrost,cold soils and deserts,glaciers,lakes,sea ice in the Arctic,Antarctic and high mountains,as well as the deep sea,ice caves and the atmospheric stratosphere etc.Cold-adapted organisms inhabiting these environments exhibit rich diversity.Studies on the biogeography of psychrophiles will enable us to understand their biodiversity,distribution and origins.Due to long-term living in cold regions,cold-adapted bacteria and archeae have developed specific physiological mechanisms of adaptation to cold environments.These mechanisms include:regulating the fluidity of the cytoplasmic membrane through adjusting the composition of membrane lipids; achieving low-temperature protection through compatibility solute,antifreeze proteins,ice-binding proteins,ice-nucleation proteins and anti-nucleating proteins; production of heat-shock and coldshock proteins,cold acclimation protein and DEAD-box RNA helicase at low temperatures; production of cold-active enzymes; increasing energy generation and conservation.With the rapid development of sequencing technology,various omics-based approaches have been used to reveal cold-adaptive mechanisms of psychrophiles at the genomic level.%低温细菌与古菌广泛分布于地球的低温环境,包括南极、北极及高山地带的冻土、低温土壤和荒漠、冰川、湖泊、海冰,以及深海、冰洞和大气平流层等.栖息在这些低温环境中的细菌与古菌具有丰富的多样性,主要为α,p和γ-Proteobacteria分支、CFB类群分支和革兰氏阳性细菌分支等.由于低温环境中的微生物流动性低,因而是研究微生物地理学理想的生态系统,有助于理解地球微生物的多样性、分布规律乃至形成机制.由于长期生活在冰冻环境中,低温细菌与古菌形成了多种适应低温环境的生理机制,如它们通过细胞膜脂类的组成来调节膜的流动性以维

  15. An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of its Binding Model towards its Applications as Detergent Additive

    Directory of Open Access Journals (Sweden)

    Mehak Baweja

    2016-08-01

    Full Text Available A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10˚C -70˚C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50 ºC and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and ̴ 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50ºC and 4ºC with low supplementation (109 U/ml. Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

  16. An Alkaline Protease from Bacillus pumilus MP 27: Functional Analysis of Its Binding Model toward Its Applications As Detergent Additive.

    Science.gov (United States)

    Baweja, Mehak; Tiwari, Rameshwar; Singh, Puneet K; Nain, Lata; Shukla, Pratyoosh

    2016-01-01

    A proteolytic strain of Bacillus pumilus MP 27 was isolated from water samples of Southern ocean produced alkaline protease. Since protease production need expensive ingredients, an economically viable process was developed by using low cost carbon source, wheat straw, supplemented with peptone. This protease was active within temperature ranges 10-70°C at pH 9. This process was optimized by response surface methodology using a Box Bekhman design by Design Expert 7.0 software that increased the protease activity to 776.5 U/ml. Moreover, the enzyme was extremely stable at a broad range of temperature and pH retaining 69% of its activity at 50°C and 70% at pH 11. The enzyme exhibited excellent compatibility with surfactants and commercial detergents, showing 87% stability with triton X-100 and 100% stability with Tide commercial detergent. The results of the wash performance analysis demonstrated considerably good de-staining at 50 and 4°C with low supplementation (109 U/ml). Molecular modeling of the protease revealed the presence of serine proteases, subtilase family and serine active site and further docking supported the association of catalytic site with the various substrates. Certainly, such protease can be considered as a good detergent additive in detergent industry with a possibility to remove the stains effectively even in a cold wash.

  17. Surfactant-enhanced alkaline flooding: Buffering at intermediate alkaline pH

    Energy Technology Data Exchange (ETDEWEB)

    Rudin, J.; Wasan, D.T. (Illinois Inst. of Tech., Chicago, IL (United States))

    1993-11-01

    The alkaline flooding process involves injecting alkaline agents into the reservoir to produce more oil than is produced through conventional waterflooding. The interaction of the alkali in the flood water with the naturally occurring acids in the reservoir oil results in in-situ formation of soaps, which are partially responsible for lowering IFT and improving oil recovery. The extent to which IFT is lowered depends on the specific oil and injection water properties. Numerous investigators have attempted to clarify the relationship between system chemical composition and IFT. An experimental investigation of buffered alkaline flooding system chemistry was undertaken to determine the influence of various species present on interfacial tension (IFT) as a function of pH and ionic strength. IFT was found to go through an ultralow minimum in certain pH ranges. This synergism results from simultaneous adsorption of un-ionized and ionized acid species on the interface.

  18. Analysis of amino acid residues involved in cold activity of monomeric isocitrate dehydrogenase from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea.

    Science.gov (United States)

    Yasuda, Wataru; Kobayashi, Miyuki; Takada, Yasuhiro

    2013-11-01

    Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which was suggested from previous study to be responsible for their distinct catalytic ability, several sequential differences of amino acid residue are present. Among them, ten amino acid residues were exchanged between them by site-directed mutagenesis and several properties of the mutated enzymes were examined in this study. The mutated enzymes of CmIDH-II substituted its Gln671, Leu724 and Phe735 residues with the corresponding residues of CpIDH-M (termed Q671K, L724Q and F735L, respectively) showed lower specific activity and thermostability for activity than the wild-type enzyme. Furthermore, the decreased specific activity was also observed in L693F. In contrast, the corresponding mutants of CpIDH-M, F693L, Q724L and L735F, showed the increased specific activity and thermostability for activity. The catalytic efficiency (k(cat)/K(m)) values of these mutated CmIDH-II and CpIDH-M were lower and higher than those of their wild-type IDHs, respectively. These results suggest that the Gln671, Leu693, Leu724 and Phe735 residues of CmIDH-II are important for exerting its high catalytic ability.

  19. Structural and Chemical Modification of Fe-Rich Smectite Associated with Microbial Fe-Respiration By Psychrophilic Bacteria in King George Island, West Antarctica

    Science.gov (United States)

    Jung, J.; Kim, J.; Lim, H. S.; Yoon, H.; Lee, Y. K.; Park, K.; Lee, J.; Kim, J. W.

    2014-12-01

    Surface soil samples were collected from Antarctic exploration (2010/2011, 2011/2012) at Barton Peninsula, King George Island, West Antarctica to determine the feasible biological alteration of clay minerals in Antarctica where the physical weathering is considered to be a major process. Seven areas (1226-1, 1226-2, 0101-4, 0105-1, 0105-2, 0107-2, 0107-3) from the coast toward the inland were investigated. The duration of exposure of soil samples to the air depending on the retraction of ice to the inland may affect the microbial activity resulting in the biogeochemical mineral alteration. The multiline of techniques for example, X-ray diffraction (XRD), Scanning Electron Microscope (SEM), wet chemistry analysis including the extent of Fe(III) reduction, and batch experiments of microbe-mineral interaction under the low temperature that mimics the Antarctic condition to understand the mechanism of biogeochemical alteration of clay minerals. Clay minerals of smectite, mica, chlorite and kaolinite were detected in the XRD profiles. The variation of relative amount of clay minerals in the regions indicated that the physical/biological alteration might be different depending on the duration of ice retraction. From the batch experiment using Nontronite (NAu-1), moreover, we confirm that Psychrophilic bacteria (Shewanella sp. isolated from King George Island) reduce structural Fe(III) of clay mineral, and occur structural change of smectite at low temperature (4℃ and 15℃). The present study, therefore, would present the feasibility of biological effects on chemical modification through the structural changes in clay mineral in cold environment and suggest a new pathway of Fe-supply into the Antarctic Ocean.

  20. N-methyl-d-aspartate receptor coagonist d-serine suppresses intake of high-preference food.

    Science.gov (United States)

    Sasaki, Tsutomu; Kinoshita, Yoshihiro; Matsui, Sho; Kakuta, Shigeru; Yokota-Hashimoto, Hiromi; Kinoshita, Kuni; Iwasaki, Yusaku; Kinoshita, Toshio; Yada, Toshihiko; Amano, Naoji; Kitamura, Tadahiro

    2015-09-01

    d-Serine is abundant in the forebrain and physiologically important for modulating excitatory glutamatergic neurotransmission as a coagonist of synaptic N-methyl-d-aspartate (NMDA) receptor. NMDA signaling has been implicated in the control of food intake. However, the role of d-serine on appetite regulation is unknown. To clarify the effects of d-serine on appetite, we investigated the effect of oral d-serine ingestion on food intake in three different feeding paradigms (one-food access, two-food choice, and refeeding after 24-h fasting) using three different strains of male mice (C57Bl/6J, BKS, and ICR). The effect of d-serine was also tested in leptin signaling-deficient db/db mice and sensory-deafferented (capsaicin-treated) mice. The expression of orexigenic neuropeptides [neuropeptide Y (Npy) and agouti-related protein (Agrp)] in the hypothalamus was compared in fast/refed experiments. Conditioned taste aversion for high-fat diet (HFD) was tested in the d-serine-treated mice. Under the one-food-access paradigm, some of the d-serine-treated mice showed starvation, but not when fed normal chow. HFD feeding with d-serine ingestion did not cause aversion. Under the two-food-choice paradigm, d-serine suppressed the intake of high-preference food but not normal chow. d-Serine also effectively suppressed HFD intake but not normal chow in db/db mice and sensory-deafferented mice. In addition, d-serine suppressed normal chow intake after 24-h fasting despite higher orexigenic gene expression in the hypothalamus. d-Serine failed to suppress HFD intake in the presence of L-701,324, the selective and full antagonist at the glycine-binding site of the NMDA receptor. Therefore, d-serine suppresses the intake of high-preference food through coagonism toward NMDA receptors. Copyright © 2015 the American Physiological Society.

  1. Cloning of Mouse Enamel Matrix Serine Proteinase Encoding Mature Protein

    Institute of Scientific and Technical Information of China (English)

    MU Ya-bing; SUN Hong-chen; ZHANG Ze-bing; OUYANG Jie

    2003-01-01

    Objective: To clone cDNA of enamel matrix serine proteinase (EMSP1) encoding mature protein from mouse dental germs. Methods: Total RNA was isolated from developing incisors and molars of 7 days mouse pups and reverse-transcribed into cDNA. Two pairs of specific primers was designed to obtain the desired gene by Touchdown PCR and Nested PCR. The segment was inserted into Vector pMD-18T, and recombined vectors was transformed into E.coli JM109.The positive clone was chose and analysed by restriction endonuclease mapping and DNA sequencing. Results:700 bp of cDNA of mouse EMSP1 was sueccessfully cloned from mouse tooth germs tissue. The sequence was consistent with that displayed in PubMed. Conclusion:The mouse EMSP1 cDNA encoding mature protein is obtained for further study.%目的:克隆小鼠牙胚组织中釉基质丝氨酸蛋白酶(EMSP1)成熟肽编码区基因.方法:提取出生后7 d昆明种小白鼠切牙、磨牙牙胚总RNA,逆转录为cDNA,设计两对特异性引物,采用Touchdown PCR 和嵌套PCR方法,扩增出小鼠EMSP1起始密码子至终止密码子基因片段.将目的基因连入载体pMD-18T,转化入大肠杆菌JM109,通过蓝白筛选,挑选阳性克隆培养扩增,纯化重组质粒进行限制性酶切和核苷酸序列分析鉴定.结果:限制性酶切图谱和核苷酸序列分析均表明所克隆cDNA为小鼠700 bp的EMSP1成熟肽基因编码.结论:成功地克隆了小鼠编码EMSP1成熟肽基因片段.

  2. An essential signal peptide peptidase identified in an RNAi screen of serine peptidases of Trypanosoma brucei.

    Directory of Open Access Journals (Sweden)

    Catherine X Moss

    Full Text Available The serine peptidases of Trypanosoma brucei have been viewed as potential drug targets. In particular, the S9 prolyl oligopeptidase subfamily is thought to be a good avenue for drug discovery. This is based on the finding that some S9 peptidases are secreted and active in the mammalian bloodstream, and that they are a class of enzyme against which drugs have successfully been developed. We collated a list of all serine peptidases in T. brucei, identifying 20 serine peptidase genes, of which nine are S9 peptidases. We screened all 20 serine peptidases by RNAi to determine which, if any, are essential for bloodstream form T. brucei survival. All S9 serine peptidases were dispensable for parasite survival in vitro, even when pairs of similar genes, coding for oligopeptidase B or prolyl oligopeptidase, were targeted simultaneously. We also found no effect on parasite survival in an animal host when the S9 peptidases oligopeptidase B, prolyl oligopeptidase or dipeptidyl peptidase 8 were targeted. The only serine peptidase to emerge from the RNAi screen as essential was a putative type-I signal peptide peptidase (SPP1. This gene was essential for parasite survival both in vitro and in vivo. The growth defect conferred by RNAi depletion of SPP1 was rescued by expression of a functional peptidase from an RNAi resistant SPP1 gene. However, expression of catalytically inactive SPP1 was unable to rescue cells from the SPP1 depleted phenotype, demonstrating that SPP1 serine peptidase activity is necessary for T. brucei survival.

  3. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

  4. Allosteric activation and contrasting properties of L-serine dehydratase types 1 and 2.

    Science.gov (United States)

    Chen, Shawei; Xu, Xiao Lan; Grant, Gregory A

    2012-07-01

    Bacterial L-serine dehydratases differ from mammalian L- and D-serine dehydratases and bacterial D-serine dehydratases by the presence of an iron-sulfur center rather than a pyridoxyl phosphate prosthetic group. They exist in two forms, types 1 and 2, distinguished by their sequence and oligomeric configuration. Both types contain an ASB domain, and the type 1 enzymes also contain an ACT domain in a tandem arrangement with the ASB domain like that in type 1 D-3-phosphoglycerate dehydrogenases (PGDHs). This investigation reveals striking kinetic differences between L-serine dehydratases from Bacillus subtilis (bsLSD, type 1) and Legionella pneumophila (lpLSD, type 2). lpLSD is activated by monovalent cations and inhibited by monovalent anions. bsLSD is strongly activated by cations, particularly potassium, and shows a mixed response to anions. Flouride is a competitive inhibitor for lpLSD but an apparent activator for bsLSD at low concentrations and an inhibitor at high concentrations. The reaction products, pyruvate and ammonia, also act as activators but to different extents for each type. Pyruvate activation is competitive with L-serine, but activation of the enzyme is not compatible with it simply competing for binding at the active site and suggests the presence of a second, allosteric site. Because activation can be eliminated by higher levels of L-serine, it may be that this second site is actually a second serine binding site. This is consistent with type 1 PGDH in which the ASB domain functions as a second site for substrate binding and activation.

  5. Alkaline carbonates in blast furnace process

    Directory of Open Access Journals (Sweden)

    P. Besta

    2014-10-01

    Full Text Available The production of iron in blast furnaces is a complex of physical, chemical and mechanical processes. The input raw materials contain not only metallic components, but also a number of negative elements. The most important negative elements include alkaline carbonates. They can significantly affect the course of the blast furnace process and thus the overall performance of the furnace. As a result of that, it is essential to accurately monitor the alkali content in the blast furnace raw materials. The article analyzes the alkali content in input and output raw materials and their impact on the blast furnace process.

  6. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    the operational temperature and pressure to produce pressurized hydrogen at high rate (m3 H2·h-1·m-2 cell area) and high electrical efficiency. This work describes an exploratory technical study of the possibility to produce hydrogen and oxygen with a new type of alkaline electrolysis cell at high temperatures...... for immobilization of aqueous KOH solutions. Electrolysis cells with this electrolyte and metal foam based gas diffusion electrodes were successfully demonstrated at temperatures up to 250 °C at 40 bar. Different electro-catalysts were tested in order to reduce the oxygen and hydrogen overpotentials. Current...

  7. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum

    Directory of Open Access Journals (Sweden)

    Tian Shengli

    2009-08-01

    Full Text Available Abstract Background Serine/arginine (SR protein-specific kinases (SRPKs are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379 from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. Results We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS. Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328 was localized in the alkaline Ω-loop of a helix-loop-helix motif (HLHM of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. Conclusion This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Ω-loop structure could play a crucial role in the NLS function of PSRPK.

  8. Identification of a nuclear localization motif in the serine/arginine protein kinase PSRPK of physarum polycephalum.

    Science.gov (United States)

    Liu, Shide; Zhou, Zhuolong; Lin, Ziyang; Ouyang, Qiuling; Zhang, Jianhua; Tian, Shengli; Xing, Miao

    2009-08-25

    Serine/arginine (SR) protein-specific kinases (SRPKs) are conserved in a wide range of organisms, from humans to yeast. Studies showed that SRPKs can regulate the nuclear import of SR proteins in cytoplasm, and regulate the sub-localization of SR proteins in the nucleus. But no nuclear localization signal (NLS) of SRPKs was found. We isolated an SRPK-like protein PSRPK (GenBank accession No. DQ140379) from Physarum polycephalum previously, and identified a NLS of PSRPK in this study. We carried out a thorough molecular dissection of the different domains of the PSRPK protein involved in its nuclear localization. By truncation of PSRPK protein, deletion of and single amino acid substitution in a putative NLS and transfection of mammalian cells, we observed the distribution of PSRPK fluorescent fusion protein in mammalian cells using confocal microscopy and found that the protein was mainly accumulated in the nucleus; this indicated that the motif contained a nuclear localization signal (NLS). Further investigation with truncated PSPRK peptides showed that the NLS (318PKKGDKYDKTD328) was localized in the alkaline Omega-loop of a helix-loop-helix motif (HLHM) of the C-terminal conserved domain. If the 318PKKGDK322 sequence was deleted from the loop or K320 was mutated to T320, the PSRPK fluorescent fusion protein could not enter and accumulate in the nucleus. This study demonstrated that the 318PKKGDKYDKTD328 peptides localized in the C-terminal conserved domain of PSRPK with the Omega-loop structure could play a crucial role in the NLS function of PSRPK.

  9. Affinity and Western blotting reveal homologies between ovine intervertebral disc serine proteinase inhibitory proteins and bovine pancreatic trypsin inhibitor.

    Science.gov (United States)

    Melrose, J; Shen, B; Ghosh, P

    2001-12-01

    The objective of this study was to assess any similarities between ovine intervertebral disc (IVD) serine proteinase inhibitory proteins (SPIs) and known mammalian IVD SPIs. Ovine IVDs were dissected into the annulus fibrosus and nucleus pulposus and the tissue finely diced then extracted with 4 M guanidine hydrochloride. The tissue extracts were subjected to caesium chloride density gradient ultracentrifugation to separate the large high buoyant density (rho > 1.5 g/mL) proteoglycans from the SPI proteins of low buoyant density (rho top two ultracentrifuge fractions containing the SPIs of interest were subjected to enzyme linked immunosorbent analysis (ELISA) and also examined by Western and Affinity blotting using an antibody to bovine pancreatic trypsin inhibitor and biotinylated trypsin respectively for detection and an alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium system for visualisation. The major SPI proteins present in the Western and Affinity blots were 34-36 kDa species, minor 12 and 16, and 85 and 120 kDa species were also present. Qualitatively similar results were obtained for each respective tissue zone of the lumbar and lumbosacral disc specimens examined. Densitometric analysis of the major 34-36 kDa SPI bands visualised on Western and Affinity blots using NIH 1.61.1 image analysis software indicated that lumbar IVD samples contained higher levels of this SPI species than lumbosacral IVD samples. ELISA confirmed that lumbar IVD extracts contained quantitatively higher levels of BPTI equivalents per g of tissue extracted than lumbosacral IVDs. This study therefore has demonstrated that the ovine disc contains a range of SPI species which share some homology with bovine pancreatic trypsin inhibitor and in this respect are similar to SPIs previously demonstrated in canine IVDs.

  10. Enzymatic production of L-serine with a feedback control system for formaldehyde addition.

    Science.gov (United States)

    Hsiao, H Y; Wei, T

    1986-10-01

    Serine hydroxymethyltransferase (SHMT) in the form of crude extract from a recombinant strain of Klebsiella aerogenes was used for the production of L-serine from glycine and formaldehyde (HCHO). A stirred tank bio-reactor with a continuous feed of HCHO (37%) was employed. Since the performance of the serine bioreactor was heavily dependent on how HCHO was fed, an automatic feedback control system was developed for HCHO delivery utilizing the phenomenon of formol titration. This control procedure was based on the following circumstance: as a bioconversion proceeded, if the rate of HCHO feed was balanced by the rate of serine synthesis so that HCHO concentration was maintained near zero, then there was no pH change in the bioreactor. Once the rate of HCHO addition exceeded that of serine synthesis, the HCHO concentration built up and the excess HCHO reacted with the amino group of an amino acid (e.g. glycine or serine) to produce a Schiff base and a proton which lowered the pH. A pH controller detected and relayed this pH change to the on-off switch of the HCHO feed pump. Thus, HCHO infusion stopped when the pH was lower than the set point, which was the initial pH of the reaction. With this control system, the maximum concentration of HCHO that was reached in the bioreactor was only 1mM-3.3mM depending on the pH and amino acid composition in the bioreactor. Moreover, a decrease in pH also signaled the use of a slower feed rate at which HCHO was to be, delivered once the pH resumed its initial value after excess HCHO was consumed by the reaction. Employing this control system, we have optimized the performance of the serine bioreactor to give a serine titer of 450 g/L with an 88% molar conversion of glycine at a volumetric serine productivity of 8.9 g/L/h.

  11. Alkaline chemistry of transuranium elements and technetium and the treatment of alkaline radioactive wastes

    Energy Technology Data Exchange (ETDEWEB)

    Delegard, C.H. [Westinghouse Hanford Co., Richland, WA (United States); Peretrukhin, V.F.; Shilov, V.P.; Pikaev, A.K. [Russian Academy of Sciences (Russian Federation). Inst. of Physical Chemistry

    1995-05-01

    Goal of this survey is to generalize the known data on fundamental physical-chemical properties of TRUs and Tc, methods for their isolation, and to provide recommendations that will be useful for partitioning them from alkaline high-level wastes.

  12. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

    Science.gov (United States)

    Qin, Zhen; Yan, Qiaojuan; Ma, Qingjun; Jiang, Zhengqiang

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering.

  13. D-Serine and Glycine Differentially Control Neurotransmission during Visual Cortex Critical Period.

    Directory of Open Access Journals (Sweden)

    Claire N J Meunier

    Full Text Available N-methyl-D-aspartate receptors (NMDARs play a central role in synaptic plasticity. Their activation requires the binding of both glutamate and d-serine or glycine as co-agonist. The prevalence of either co-agonist on NMDA-receptor function differs between brain regions and remains undetermined in the visual cortex (VC at the critical period of postnatal development. Here, we therefore investigated the regulatory role that d-serine and/or glycine may exert on NMDARs function and on synaptic plasticity in the rat VC layer 5 pyramidal neurons of young rats. Using selective enzymatic depletion of d-serine or glycine, we demonstrate that d-serine and not glycine is the endogenous co-agonist of synaptic NMDARs required for the induction and expression of Long Term Potentiation (LTP at both excitatory and inhibitory synapses. Glycine on the other hand is not involved in synaptic efficacy per se but regulates excitatory and inhibitory neurotransmission by activating strychnine-sensitive glycine receptors, then producing a shunting inhibition that controls neuronal gain and results in a depression of synaptic inputs at the somatic level after dendritic integration. In conclusion, we describe for the first time that in the VC both D-serine and glycine differentially regulate somatic depolarization through the activation of distinct synaptic and extrasynaptic receptors.

  14. Quantitative serine protease assays based on formation of copper(II)-oligopeptide complexes.

    Science.gov (United States)

    Ding, Xiaokang; Yang, Kun-Lin

    2015-01-07

    A quantitative protease assay based on the formation of a copper-oligopeptide complex is developed. In this assay, when a tripeptide GGH fragment is cleaved from an oligopeptide chain by serine proteases, the tripeptide quickly forms a pink GGH/Cu(2+) complex whose concentration can be determined quantitatively by using UV-Vis spectroscopy. Therefore, activities of serine proteases can be determined from the formation rate of the GGH/Cu(2+) complex. This principle can be used to detect the presence of serine protease in a real-time manner, or measure proteolytic activities of serine protease cleaving different oligopeptide substrates. For example, by using this assay, we demonstrate that trypsin, a model serine protease, is able to cleave two oligopeptides GGGGKGGH () and GGGGRGGH (). However, the specificity constant (kcat/Km) for is higher than that of (6.4 × 10(3) mM(-1) min(-1)vs. 1.3 × 10(3) mM(-1) min(-1)). This result shows that trypsin is more specific toward arginine (R) than lysine (K) in the oligopeptide sequence.

  15. Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma.

    Science.gov (United States)

    Guo, Min; Chong, Yeeting E; Shapiro, Ryan; Beebe, Kirk; Yang, Xiang-Lei; Schimmel, Paul

    2009-12-10

    Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

  16. Cysteine biosynthesis in Lactobacillus casei: identification and characterization of a serine acetyltransferase.

    Science.gov (United States)

    Bogicevic, Biljana; Berthoud, Hélène; Portmann, Reto; Bavan, Tharmatha; Meile, Leo; Irmler, Stefan

    2016-02-01

    In bacteria, cysteine can be synthesized from serine by two steps involving an L-serine O-acetyltransferase (SAT) and a cysteine synthase (CysK). While CysK is found in the publicly available annotated genome from Lactobacillus casei ATCC 334, a gene encoding SAT (cysE) is missing. In this study, we found that various strains of L. casei grew in a chemically defined medium containing sulfide as the sole sulfur source, indicating the presence of a serine O-acetyltransferase. The gene lying upstream of cysK is predicted to encode a homoserine trans-succinylase (metA). To study the function of this gene, it was cloned from L. casei FAM18110. The purified, recombinant protein did not acylate L-homoserine in vitro. Instead, it catalyzed the formation of O-acetyl serine from L-serine and acetyl-CoA. Furthermore, the plasmid expressing the L. casei gene complemented an Escherichia coli cysE mutant strain but not an E. coli metA mutant. This clearly demonstrated that the gene annotated as metA in fact encodes the SAT function and should be annotated as cysE.

  17. Cell-type specific mechanisms of D-serine uptake and release in the brain

    Directory of Open Access Journals (Sweden)

    Magalie eMartineau

    2014-05-01

    Full Text Available Accumulating evidence during the last decade established that D-serine is a key signaling molecule utilized by neurons and astroglia in the mammalian central nervous system. D-serine is increasingly appreciated as the main physiological endogenous coagonist for synaptic NMDA receptors at central excitatory synapses; it is mandatory for long-term changes in synaptic strength, memory, learning, and social interactions. Alterations in the extracellular levels of D-serine leading to disrupted cell-cell signaling are a trademark of many chronic or acute neurological (i.e. Alzheimer disease, epilepsy, stroke and psychiatric (i.e. schizophrenia disorders, and are associated with addictive behavior (i.e. cocaine addiction. Indeed, fine tuning of the extracellular levels of D-serine, achieved by various molecular machineries and signaling pathways, is necessary for maintenance of accurate NMDA receptor functions. Here, we review the experimental data supporting the notion that astroglia and neurons use different pathways to regulate levels of extracellular D-serine.

  18. The uropathogenic species Staphylococcus saprophyticus tolerates a high concentration of D-serine.

    Science.gov (United States)

    Sakinç, Türkân; Michalski, Nadine; Kleine, Britta; Gatermann, Sören G

    2009-10-01

    Human urine contains a relatively high concentration of d-serine, which is toxic to several nonuropathogenic bacteria, but can be utilized or detoxified by uropathogenic Escherichia coli (UPEC). The sequenced genome of uropathogenic Staphylococcus saprophyticus contains a gene with homology to the d-serine deaminase gene (dsdA) of UPEC. We found the gene in several clinical isolates of S. saprophyticus; however, the gene was absent in Staphylococcus xylosus and Staphylococcus cohnii, phylogenetically close relatives of S. saprophyticus, and could also not be detected in isolates of Staphylococcus aureus, Staphylococcus epidermidis and 13 other staphylococcal species. In addition, the genomes of other sequenced staphylococci do not harbor homologues of this operon. Interestingly, S. saprophyticus could grow in media supplemented with relatively high concentrations of d-serine, whereas S. aureus, S. epidermidis and other staphylococcal species could not. The association of the dsdA gene with growth in media including d-serine was proved by introducing the gene into S. aureus Newman. Given the fact that UPEC and S. saprophyticus tolerate this compound, d-serine utilization and detoxification may be a general property of uropathogenic bacteria.

  19. Covalent binding of the organophosphorus agent FP-biotin to tyrosine in eight proteins that have no active site serine

    OpenAIRE

    Grigoryan, Hasmik; Li, Bin; Anderson, Erica K.; Xue, Weihua; Nachon, Florian; Lockridge, Oksana; Schopfer, Lawrence M.

    2009-01-01

    Organophosphorus esters (OP) are known to bind covalently to the active site serine of enzymes in the serine hydrolase family. It was a surprise to find that proteins with no active site serine are also covalently modified by OP. The binding site in albumin, transferrin, and tubulin was identified as tyrosine. The goal of the present work was to determine whether binding to tyrosine is a general phenomenon. Fourteen proteins were treated with a biotin-tagged organophosphorus agent called FP-b...

  20. Allosteric inactivation of a trypsin-like serine protease by an antibody binding to the 37- and 70-loops

    DEFF Research Database (Denmark)

    Kromann-Hansen, Tobias; Lund, Ida K; Liu, Zhuo

    2013-01-01

    Serine protease catalytic activity is in many cases regulated by conformational changes initiated by binding of physiological modulators to exosites located distantly from the active site. Inhibitory monoclonal antibodies binding to such exosites are potential therapeutics and offer opportunities...... criteria similar to the E* conformation described for other serine proteases. Hence, agents targeting serine protease conformation through binding to exosites in the 37- and 70-loops represent a new class of potential therapeutics....

  1. Low serum alkaline phosphatase activity in Wilson's disease.

    Science.gov (United States)

    Shaver, W A; Bhatt, H; Combes, B

    1986-01-01

    Low values for serum alkaline phosphatase activity were observed early in the course of two patients with Wilson's disease presenting with the combination of severe liver disease and Coombs' negative acute hemolytic anemia. A review of other cases of Wilson's disease revealed that 11 of 12 patients presenting with hemolytic anemia had values for serum alkaline phosphatase less than their respective sex- and age-adjusted mean values; in eight, serum alkaline phosphatase activity was less than the lower value for the normal range of the test. Low values for serum alkaline phosphatase were much less common in Wilson's disease patients with more chronic forms of presentation. Copper added in high concentration to serum in vitro did not have an important effect on serum alkaline phosphatase activity. The mechanism responsible for the decrease in serum alkaline phosphatase activity in patients is uncertain.

  2. DNA DAMAGE QUANTITATION BY ALKALINE GEL ELECTROPHORESIS.

    Energy Technology Data Exchange (ETDEWEB)

    SUTHERLAND,B.M.; BENNETT,P.V.; SUTHERLAND, J.C.

    2004-03-24

    Physical and chemical agents in the environment, those used in clinical applications, or encountered during recreational exposures to sunlight, induce damages in DNA. Understanding the biological impact of these agents requires quantitation of the levels of such damages in laboratory test systems as well as in field or clinical samples. Alkaline gel electrophoresis provides a sensitive (down to {approx} a few lesions/5Mb), rapid method of direct quantitation of a wide variety of DNA damages in nanogram quantities of non-radioactive DNAs from laboratory, field, or clinical specimens, including higher plants and animals. This method stems from velocity sedimentation studies of DNA populations, and from the simple methods of agarose gel electrophoresis. Our laboratories have developed quantitative agarose gel methods, analytical descriptions of DNA migration during electrophoresis on agarose gels (1-6), and electronic imaging for accurate determinations of DNA mass (7-9). Although all these components improve sensitivity and throughput of large numbers of samples (7,8,10), a simple version using only standard molecular biology equipment allows routine analysis of DNA damages at moderate frequencies. We present here a description of the methods, as well as a brief description of the underlying principles, required for a simplified approach to quantitation of DNA damages by alkaline gel electrophoresis.

  3. The effect of alkaline agents on retention of EOR chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Lorenz, P.B.

    1991-07-01

    This report summarizes a literature survey on how alkaline agents reduce losses of surfactants and polymers in oil recovery by chemical injection. Data are reviewed for crude sulfonates, clean anionic surfactants, nonionic surfactants, and anionic and nonionic polymers. The role of mineral chemistry is briefly described. Specific effects of various alkaline anions are discussed. Investigations needed to improve the design of alkaline-surfactant-polymer floods are suggested. 62 refs., 28 figs., 6 tabs.

  4. Enumeration of Psychrophilic Bacteria in Raw Milk by Rapid Real-time Optoelectronic Microbiological Method%实时光电微生物法快速检测生鲜乳中的嗜冷菌

    Institute of Scientific and Technical Information of China (English)

    许振伟; 韩奕奕; 郑小平; 孟瑾; 邹明辉; 周众; 朱红; 高磊

    2012-01-01

    Psychrophilic bacteria from 50 raw milk samples were enumerated by rapid real-time optoelectronic microbiological method and aerobic plate counting method in the same time. Testing data of psychrophilic bacteria was assayed and compared deriving from the two detection methods. The results showed that counts of psychrophilic bacteria using standard curve: Lg CFU/mL= -0.417*DT+7.91 of rapid real-time optoelectronic microbiological method were 2.3-3.7x 106 CFU/mL and the detection time was of 3.2 to 18.1 h. The result of aerobic plate counting was 2.3~3.7x106 CFU/mL. When the detection time of rapid real-time optoelectronic microbiological method was more than 18 h, the psychrophilic bacteria was not detected. Meanwhile the results of aerobic plate counting were <1 CFU/mL. The difference of logarithm of testing results between the two detection method was -0.9~0.8 with the absolute values of <1. The testing results from the two methods were quite consistent These results indicated that using rapid real-time optoelectronic microbiological method to detect psychrophilic bacteria in raw milk had high applicability, greatly shorten the testing time, and the method was also accurate, simple and easy.%对50份生鲜牛乳样品中的嗜冷菌同时采用实时光电微生物快速检测方法和平板计数法进行检测,分析比较两种检测方法所得的嗜冷菌检测数据.结果表明:采用嗜冷菌检测标准曲线:Lg CFU/mL=-0.417*DT+7.91的实时光电微生物法检测的嗜冷茵含量在2.3~3.7×106 CFU/mL,检出时间在3.2~18.1 h.采用平板计数法的检出结果范围在4.0~3.0×106 CFU/mL.当实时光电微生物检测方法的检出时间大于18h时,样品检测结果均为未检出,此时平板计数法的检测结果均为<1CFU/mL.实时光电微生物法检测结果对数值与平板计数法检测结果对数值的差值范围在-0.9~0.8,两者差值的绝对值均<1.采用实时光电微生物法和平板计数法两种检测

  5. Enzymatic dehairing of goat skins using alkaline protease from Bacillus sp. SB12.

    Science.gov (United States)

    Briki, Selmen; Hamdi, Olfa; Landoulsi, Ahmed

    2016-05-01

    The present paper reports the production, purification and biochemical characterization of an extracellular alkaline protease from Bacillus sp. SB12. The enzyme has been used as an alternative to conventional chemicals treatment for dehairing of goat skins. The protease was optimally active at 37 °C and pH 9. Starch at 2% (w/v) was used as the best carbon source and the addition of yeast extract and peptone at 1% each supported the maximum level of protease production in the presence of 5 mM Ca(2+). Protease purification was performed with ammonium sulphate precipitation at 70% saturated fraction followed by dialysis and gel filtration chromatography using Sephadex G-100. The purified enzyme was homogeneous on non-denaturing PAGE and appeared as a single band with an apparent molecular weight of 41 kDa. This enzyme was moderately thermostable and has a wide pH stability range extending from pH 7 to 11. It showed high tolerance toward surfactants agents and organic solvents while it was completely inhibited by PMSF indicating the serine protease type. Purified protease was used to remove hair from goat skin proving its potential application in leather processing industry. The results revealed that the protease has enhanced the quality and physico-chemical properties of the skins while reducing the pollution.

  6. Role of a conserved arginine residue during catalysis in serine palmitoyltransferase.

    Science.gov (United States)

    Lowther, Jonathan; Charmier, Guillaume; Raman, Marine C; Ikushiro, Hiroko; Hayashi, Hideyuki; Campopiano, Dominic J

    2011-06-23

    All sphingolipid-producing organisms require the pyridoxal 5'-phosphate (PLP)-dependent serine palmitoyltransferase (SPT) to catalyse the first reaction on the de novo sphingolipid biosynthetic pathway. SPT is a member of the alpha oxoamine synthase (AOS) family that catalyses a Claisen-like condensation of palmitoyl-CoA and L-serine to form 3-ketodihydrosphingosine (KDS). Protein sequence alignment across various species reveals an arginine residue, not involved in PLP binding, to be strictly conserved in all prokaryotic SPTs, the lcb2 subunits of eukaryotic SPTs and all members of the AOS family. Here we use UV-vis spectroscopy and site-directed mutagenesis, in combination with a substrate analogue, to show that the equivalent residue (R370) in the SPT from Sphingomonas wittichii is required to form the key PLP:L-serine quinonoid intermediate that condenses with palmitoyl-CoA and thus plays an essential role enzyme catalysis.

  7. Modulatory effect of a serine protease inhibitor on surgical stress: its clinical implications.

    Directory of Open Access Journals (Sweden)

    Iwagaki H

    1999-10-01

    Full Text Available The relationship between endogenous cytokine antagonists and surgical stress is poorly understood. Surgical stress induces immunosuppression, and the reversed therapy of postoperative immunosuppression has been expected. The aim of the present study was to assess the effect of a serine protease inhibitor on postoperative immune reactivity. Twenty patients with colorectal cancer were randomly separated into experimental and control groups of 10 patients each. The experimental group received perioperative administration of a serine protease inhibitor while the control group did not. Plasma levels of cytokine antagonists, which suppress cell-mediated immunity, such as cortisol, interleukin-1 receptor antagonist, soluble interleukin-2 receptor (sIL-2R and soluble tumor necrosis factors p55, p75 (sTNF-R55, -R75 were simultaneously measured. Significant reductions of plasma concentration of sIL-2R and sTNF-R55 were observed. Perioperative administration of a serine protease inhibitor may contribute to ameliorating immunosuppression after major surgery.

  8. Conservation of sequence and function in fertilization of the cortical granule serine protease in echinoderms.

    Science.gov (United States)

    Oulhen, Nathalie; Xu, Dongdong; Wessel, Gary M

    2014-08-01

    Conservation of the cortical granule serine protease during fertilization in echinoderms was tested both functionally in sea stars, and computationally throughout the echinoderm phylum. We find that the inhibitor of serine protease (soybean trypsin inhibitor) effectively blocks proper transition of the sea star fertilization envelope into a protective sperm repellent, whereas inhibitors of the other main types of proteases had no effect. Scanning the transcriptomes of 15 different echinoderm ovaries revealed sequences of high conservation to the originally identified sea urchin cortical serine protease, CGSP1. These conserved sequences contained the catalytic triad necessary for enzymatic activity, and the tandemly repeated LDLr-like repeats. We conclude that the protease involved in the slow block to polyspermy is an essential and conserved element of fertilization in echinoderms, and may provide an important reagent for identification and testing of the cell surface proteins in eggs necessary for sperm binding.

  9. Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.

    Science.gov (United States)

    McFall, E

    1975-03-01

    A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis.

  10. Serine Proteases of Malaria Parasite Plasmodium falciparum: Potential as Antimalarial Drug Targets

    Directory of Open Access Journals (Sweden)

    Asrar Alam

    2014-01-01

    Full Text Available Malaria is a major global parasitic disease and a cause of enormous mortality and morbidity. Widespread drug resistance against currently available antimalarials warrants the identification of novel drug targets and development of new drugs. Malarial proteases are a group of molecules that serve as potential drug targets because of their essentiality for parasite life cycle stages and feasibility of designing specific inhibitors against them. Proteases belonging to various mechanistic classes are found in P. falciparum, of which serine proteases are of particular interest due to their involvement in parasite-specific processes of egress and invasion. In P. falciparum, a number of serine proteases belonging to chymotrypsin, subtilisin, and rhomboid clans are found. This review focuses on the potential of P. falciparum serine proteases as antimalarial drug targets.

  11. The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

    DEFF Research Database (Denmark)

    Jürgensen, Vibeke Würtz; Jalkanen, Karl J.

    2006-01-01

    investigated for one conformer, with the goal to determine which level of theory is appropriate to use in the systematic search of the conformational space. In addition, the effects of the counterion, here Cl- anion, are also investigated. Here we present the current state of the art in nanobiology, where......The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory...... with the Becke 3LYP hybrid exchange correlation (XC) functional and the PW91 GGA XC functional and the 6-31G* and aug-cc-pVDZ basis sets. Subsequently, the vibrational absorption, vibrational circular dichroism, Raman and Raman optical activity spectra were simulated in order to compare them to experimentally...

  12. Possible Role of Mg2+ ion in the Reaction of Organophosphate (dichlorvos with Serine

    Directory of Open Access Journals (Sweden)

    Seyed Vahid Shetab-Boushehri

    2012-01-01

    Full Text Available Organophosphate pesticides (OPs inhibit both true and pseudo-cholinesterases by reaction with the hydroxyl group of serine in their active sites. Poisoning with OPs is commonly seen in clinics. A common antidote for OP poisoning is atropine but, after ageing and OP dealkylation, even oximes could not be effective. It has been shown that oximes are not always useful in management of OP poisoning. On the other hand, magnesium has been found effective in both clinical and experimental studies. Studies to find more effective antidotes for OP poisoning are in progress. Presently, the possible role of magnesium ion in catalysis of reaction of dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP, a water-soluble OP, with serine is proposed. The hydroxyl group of serine could be a target to which DDVP can react. Nucleophilic attack of pralidoxime to DDVP was previously investigated. To confirm the idea, data were derived from recent and previous research on the role of magnesium in phosphoryl group transfer reactions. Possible reactions of serine and pralidoxime with DDVP in the absence and the presence of magnesium ion were separately investigated theoretically.We propose that the chemical reaction of serine with DDVP exclusively occurs in the presence of a magnesium divalent cation, whereas the reaction of pralidoxime with DDVP occurs independent of the presence of the magnesium ion. The role of the dissociation constant (pKa of functional groups in these reactions seems important.It is suggested that application of serine in combination with the magnesium cation can become a more efficient antidote for treatment of OP poisoning.

  13. Ligand Exchange Between Penta-Coordinated Phosphoryl Serine and Histidine Compounds

    Institute of Scientific and Technical Information of China (English)

    曹书霞; 牛明玉; 苏玉倩; 廖新成; 卢奎; 赵玉芬

    2003-01-01

    With the assistance of HPLC-ESI-MS/MS, the self-assembly products of serine and histidine penta-coordinated phosphorus compound were separated and identified. The expectative product was seryl-histidine dipeptide, but it was found that there was almost equimolar amount of histidyl-histidine dipeptide as well as seryl-histidine dipeptide. The mechanism was speculated that there was iigand exchange between penta-coordinated phosphoryl serine and histidine in the reaction process. As a result,two types of dipeptide were produced.

  14. Expression and biochemical properties of a protein serine/threonine phosphatase encoded by bacteriophage lambda.

    OpenAIRE

    Barik, S

    1993-01-01

    The predicted amino acid sequence encoded by the open reading frame 221 (orf221) of bacteriophage lambda exhibited a high degree of similarity to the catalytic subunits of a variety of protein serine/threonine phosphatases belonging to PP1, PP2A, and PP2B groups. Cloning and expression of the orf221 gene in Escherichia coli provided direct evidence that the gene codes for a protein serine/threonine phosphatase. The single-subunit recombinant enzyme was purified in soluble form and shown to po...

  15. Propargyloxycarbonyl as a protecting group for the side chains of serine, threonine and tyrosine

    Indian Academy of Sciences (India)

    Ramapanicker Ramesh; Kavita De; Shipra Gupta; Srinivasan Chandrasekaran

    2008-01-01

    Propargyloxycarbonyl group is used as a protecting group for the hydroxyl groups of serine, threonine and tyrosine. The propargyloxycarbonyl derivatives of these hydroxy amino acids are stable to acidic and basic reagents commonly employed in peptide synthesis. The deprotection of the -Poc derivatives using tetrathiomolybdate does not affect commonly used protecting groups such as -Boc, -Cbz, -Fmoc, methyl and benzyl esters. The di- and tripeptides synthesized using -Poc derivatives of serine, threonine and tyrosine are stable, isolable compounds and give the hydroxy peptides in good yields when treated with tetrathiomolybdate.

  16. Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases In Vivo

    DEFF Research Database (Denmark)

    Glenthøj, Andreas; Nickles, Katrin; Cowland, Jack;

    2015-01-01

    in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which are able to process recombinant proHNP into HNP in vitro. We investigated whether serine proteases are in fact responsible for processing of proHNP in human bone marrow cells and in human and murine myeloid cell...... lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of 35S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease pro...

  17. Molecular cloning of complementary DNA for human medullasin: an inflammatory serine protease in bone marrow cells.

    Science.gov (United States)

    Okano, K; Aoki, Y; Sakurai, T; Kajitani, M; Kanai, S; Shimazu, T; Shimizu, H; Naruto, M

    1987-07-01

    Medullasin, an inflammatory serine protease in bone marrow cells, modifies the functions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural meduallasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41% homology with pig elastase 1.

  18. DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073.

    Science.gov (United States)

    Anfora, Andrew T; Welch, Rodney A

    2006-09-01

    d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.

  19. The Cryptic dsdA Gene Encodes a Functional D-Serine Dehydratase in Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Li, Guoqing; Lu, Chung-Dar

    2016-06-01

    D-Serine, an important neurotransmitter, also contributes to bacterial adaptation and virulence in humans. It was reported that Pseudomonas aeruginosa PAO1 can grow on D-serine as the sole nitrogen source, and growth was severely reduced in the dadA mutant devoid of the D-alanine dehydrogenase with broad substrate specificity. In this study, the dsdA gene (PA3357) encoding a putative D-serine dehydratase was subjected to further characterization. Growth on D-serine as the sole source of nitrogen was retained in the ∆dsdA mutant and was abolished completely in the ∆dadA and ∆dadA-∆dsdA mutants. However, when complemented by dsdA on a plasmid, the double mutant was able to grow on D-serine as the sole source of carbon and nitrogen, supporting the proposed biochemical function of DsdA in the conversion of D-serine into pyruvate and ammonia. Among D- and L-amino acids tested, only D-serine and D-threonine could serve as the substrates of DsdA, and the Km of DsdA with D-serine was calculated to be 330 μM. Comparative genomics revealed that this cryptic dsdA gene was highly conserved in strains of P. aeruginosa, and that most strains of Pseudomonas putida possess putative dsdCAX genes encoding a transcriptional regulator DsdC and a D-serine transporter DsdX as in enteric bacteria. In conclusion, this study supports the presence of a cryptic dsdA gene encoding a functional D-serine dehydratase in P. aeruginosa, and the absence of dsdA expression in response to exogenous D-serine might be due to the loss of regulatory elements for gene activation during evolution.

  20. Advanced-capability alkaline fuel cell powerplant

    Science.gov (United States)

    Deronck, Henry J.

    The alkaline fuel cell powerplant utilized in the Space Shuttle Orbiter has established an excellent performance and reliability record over the past decade. Recent AFC technology programs have demonstrated significant advances in cell durability and power density. These capabilities provide the basis for substantial improvement of the Orbiter powerplant, enabling new mission applications as well as enhancing performance in the Orbiter. Improved durability would extend the powerplant's time between overhaul fivefold, and permit longer-duration missions. The powerplant would also be a strong candidate for lunar/planetary surface power systems. Higher power capability would enable replacement of the Orbiter's auxiliary power units with electric motors, and benefits mass-critical applications such as the National AeroSpace Plane.

  1. Corrosion of copper in alkaline chloride environments

    Energy Technology Data Exchange (ETDEWEB)

    King, F. [Integrity Corrosion Consulting Ltd., Calgary (Canada)

    2002-08-01

    The available literature information on the corrosion and electrochemical behaviour of copper in alkaline environments has been reviewed. The purpose of the review was to assess the impact of an alkaline plume from cementitious material on the corrosion behaviour of a copper canister in an SKB-3 type repository. The effect of the evolution of the environmental conditions within the repository have been considered, including the effects of temperature, redox conditions, pore-water salinity and pH. If the pore-water pH increases prior to the establishment of anoxic conditions, the canister surface will passivate as the pore-water pH exceeds a value of {approx} pH 9. Passivation will result from the formation of a duplex Cu{sub 2}O/Cu(OH){sub 2} film. The corrosion potential will be determined by the equilibrium potential for the Cu{sub 2}O/Cu(OH){sub 2} couple under oxic conditions, or by the Cu/Cu{sub 2}O redox couple under anoxic conditions (in the absence of sulphide). Pitting corrosion is only likely to occur early in the evolution of the repository environment, whilst the canister is still relatively cool (<40 deg C), whilst there is still O{sub 2} available to support localised corrosion, and prior to the increase in pore-water pH and salinity. The subsequent increase in canister surface temperature, pore-water pH and salinity, and decrease in O{sub 2} will make pit initiation less likely, although the canister will remain passive provided the pore-water pH is maintained above pH 9. The higher the pore-water pH, the more strongly the canister is passivated and the less likely the surface is to undergo localised attack. If the pore-water salinity increases prior to the increase in pH, there could be a period of active canister corrosion before passivation occurs.Under these circumstances, the corrosion potential will be a true mixed potential, determine by the relative kinetics of Cu dissolution as CuCl{sub 2} - and of the reduction of O{sub 2}. The development

  2. High Temperature and Pressure Alkaline Electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank

    and oxygen with a new type of alkaline electrolysis cell at high temperatures and pressures. To perform measurements under high pressure and at elevated temperatures it was necessary to build a measurement system around an autoclave which could stand high temperatures up to 250 °C and pressures up to 200 bar...... as well as extremely caustic environments. Based on a literature study to identify resistant materials for these conditions, Inconel 600 was selected among the metals which are available for autoclave construction. An initial single atmosphere high temperature and pressure measurement setup was build...... comprising this autoclave. A second high temperature and pressure measurement setup was build based on experiences from the first setup in order to perform automatized measurements. The conductivity of aqueous KOH at elevated temperatures and high concentrations was investigated using the van der Pauw method...

  3. Polyvinyl alcohol membranes as alkaline battery separators

    Science.gov (United States)

    Sheibley, D. W.; Gonzalez-Sanabria, O.; Manzo, M. A.

    1982-01-01

    Polyvinly alcohol (PVA) cross-linked with aldehyde reagents yields membranes that demonstrate properties that make them suitable for use as alkaline battery separators. Film properties can be controlled by the choice of cross-linker, cross-link density and the method of cross-linking. Three methods of cross-linking and their effects on film properties are discussed. Film properties can also be modified by using a copolymer of vinyl alcohol and acrylic acid as the base for the separator and cross-linking it similarly to the PVA. Fillers can be incorporated into the films to further modify film properties. Results of separator screening tests and cell tests for several variations of PBA films are discussed.

  4. Acylglucuronide in alkaline conditions: migration vs. hydrolysis.

    Science.gov (United States)

    Di Meo, Florent; Steel, Michele; Nicolas, Picard; Marquet, Pierre; Duroux, Jean-Luc; Trouillas, Patrick

    2013-06-01

    This work rationalizes the glucuronidation process (one of the reactions of the phase II metabolism) for drugs having a carboxylic acid moiety. At this stage, acylglucuronides (AG) metabolites are produced, that have largely been reported in the literature for various drugs (e.g., mycophenolic acid (MPA), diclofenac, ibuprofen, phenylacetic acids). The competition between migration and hydrolysis is rationalized by adequate quantum calculations, combing MP2 and density functional theory (DFT) methods. At the molecular scale, the former process is a real rotation of the drug around the glucuconic acid. This chemical-engine provides four different metabolites with various toxicities. Migration definitely appears feasible under alkaline conditions, making proton release from the OH groups. The latter reaction (hydrolysis) releases the free drug, so the competition is of crucial importance to tackle drug action and elimination. From the theoretical data, both migration and hydrolysis appear kinetically and thermodynamically favored, respectively.

  5. Hydrogen production by alkaline water electrolysis

    Directory of Open Access Journals (Sweden)

    Diogo M. F. Santos

    2013-01-01

    Full Text Available Water electrolysis is one of the simplest methods used for hydrogen production. It has the advantage of being able to produce hydrogen using only renewable energy. To expand the use of water electrolysis, it is mandatory to reduce energy consumption, cost, and maintenance of current electrolyzers, and, on the other hand, to increase their efficiency, durability, and safety. In this study, modern technologies for hydrogen production by water electrolysis have been investigated. In this article, the electrochemical fundamentals of alkaline water electrolysis are explained and the main process constraints (e.g., electrical, reaction, and transport are analyzed. The historical background of water electrolysis is described, different technologies are compared, and main research needs for the development of water electrolysis technologies are discussed.

  6. RES Hydrogen: efficient pressurised alkaline electrolysers

    DEFF Research Database (Denmark)

    Bowen, Jacob R.; Bentzen, Janet Jonna; Jørgensen, Peter Stanley;

    including BoP. Investigation of cathodes revealed highly heterogeneous microstructures and 3D microstructure quantification methods were developed. Nanometre scale -Ni(OH)2 formation was identified on tested cathode surfaces and is considered a potential degradation mechanism that is not presently well......The RESelyser project addresses issues associated with coupling alkaline electrolysis to renewable energy sources such as electrode stability and gas purity by implementing improved electrodes and a new separator membrane concept. The project aims to improve performance, operation pressure...... and reduce system cost. The project supports DTU Energy's activities on electrodes within the larger FCH-JU project. The overall project demonstrated: improved electrode efficiency also during cyclic operation, safe gas purity at a system pressure of 30 bar, 10 kW stack operation and estimated system costs...

  7. Enzymatic Hydrolysis of Alkaline Pretreated Coconut Coir

    Directory of Open Access Journals (Sweden)

    Akbarningrum Fatmawati

    2013-06-01

    Full Text Available The purpose of this research is to study the effect of concentration and temperature on the cellulose and lignin content, and the reducing sugars produced in the enzymatic hydrolysis of coconut coir. In this research, the coconut coir is pretreated using 3%, 7%, and 11% NaOH solution at 60oC, 80oC, and 100oC. The pretreated coir were assayed by measuring the amount of cellulose and lignin and then hydrolysed using Celluclast and Novozyme 188 under various temperature (30oC, 40oC, 50oC and pH (3, 4, 5. The hydrolysis results were assayed for the reducing sugar content. The results showed that the alkaline delignification was effective to reduce lignin and to increase the cellulose content of the coir. The best delignification condition was observed at 11% NaOH solution and 100oC which removed 14,53% of lignin and increased the cellulose content up to 50,23%. The best condition of the enzymatic hydrolysis was obtained at 50oC and pH 4 which produced 7,57 gr/L reducing sugar. © 2013 BCREC UNDIP. All rights reservedReceived: 2nd October 2012; Revised: 31st January 2013; Accepted: 6th February 2013[How to Cite: Fatmawati, A., Agustriyanto, R., Liasari, Y. (2013. Enzymatic Hydrolysis of Alkaline Pre-treated Coconut Coir. Bulletin of Chemical Reaction Engineering & Catalysis, 8 (1: 34-39 (doi:10.9767/bcrec.8.1.4048.34-39[Permalink/DOI: http://dx.doi.org/10.9767/bcrec.8.1.4048.34-39] | View in  |

  8. On electrochemical devices using alkaline polymer electrolytes

    Energy Technology Data Exchange (ETDEWEB)

    Zhuang, L. [Wuhan Univ., Wuhan (China). Dept. of Chemistry

    2010-07-01

    Solid polymer electrolytes (SPEs) enable a compact assembly of fuel cells and electrolyzers, thereby increasing the space-specific conversion efficiency and avoiding electrolyte leakage. The most widely used SPE in proton exchange membrane fuel cells (PEMFC) and chloro-alkali electrolyzers is Nafion. However, this strongly acidic polyelectrolyte allows only noble metals to be used as the catalysts in the electrochemical devices, which poses a problem in terms of price and resource limits. In principle, alkaline polymer electrolytes (APEs) should be used to eliminate the dependence on noble metal catalysts. The general structure of alkaline polymer electrolytes is a positively charged polymer, notably, a polymer chain attached with fixed cations such as quaternary ammonia group, and dissociated anion, OH-, to act as the charge carrier. This presentation described the challenges of developing APEs in terms of the chemical stability of quaternary ammonia group, the mobility of OH-, and high ionic concentration. The authors have been working on developing high-performance APEs since 2001. The most recent APEs were quaternary ammonia polysulfone (QAPS), which were found to be suitable for fuel cell and electrolyzer applications. The ionic conductivity was high and the crosslinked membrane had excellent mechanical strength, enabling operation at 90 degrees C. Non-precious metal catalysts were used in the APEs. For APE-based fuel cells (APEFC), chromium decorated nickel was used as the anode catalyst for hydrogen oxidation, and silver was used as the cathode catalyst for oxygen reduction. The preliminary performance of such an APEFC with non-Pt catalysts was found to be much better than that of traditional water electrolyzers using KOH solutions. 2 refs.

  9. Advanced membranes for alkaline primary and rechargeable alkaline cells with zinc anodes

    Science.gov (United States)

    Lewis, Harlan; Jackson, Patricia; Salkind, Alvin; Danko, Thomas; Bell, Roger

    Several advanced cellulosic and radiation grafted polypropylene membrane materials are currently under evaluation in the laboratories at Navsea Crane and Rutgers University, for application to alkaline primary and rechargeable cell chemistries which employ zinc as the anode material. A portion of these tests involve model cell evaluations of cellulosic membranes for silver migration rates through the membranes as a function of separation layers and changes in the degree of polymerisation (DP), wet tensile strength (WTS) and voltage changes at both electrodes as a function of model rechargeable cell life cycle. Other testing on the actual membranes is generating data for both cellulosic and polypropylene materials on impedance, swelling properties, and silver and zinc penetration rates. The overall goal of these investigations is to obtain candidate separation membranes which will reduce zinc anode shape change and shedding and resist alkaline oxidative degradation to extend the active wet life in primary cells and both wet and life cycle in rechargeable cells.

  10. The thesis of the alkaline milieu in oncology: a review.

    Science.gov (United States)

    Malhotra, S L

    1993-02-01

    An alkaline milieu is a common factor in some carcinomas of the oropharynx and oesophagus, the stomach, the bronchus, the cervix and the large bowel. The hypothesis is advanced that a change to an alkaline pH enhances the mitotic activity of mucous cells and that this change can be often avoided by alterations in diet and habit.

  11. Space-time variability of alkalinity in the Mediterranean Sea

    Directory of Open Access Journals (Sweden)

    G. Cossarini

    2014-09-01

    Full Text Available The paper provides a basin assessment of the spatial distribution of ocean alkalinity in the Mediterranean Sea. The assessment is made using a 3-D transport-biogeochemical-carbonate model to integrate the available experimental findings, which also constrains model output. The results indicate that the Mediterranean Sea shows alkalinity values that are much higher than those observed in the Atlantic Ocean on a basin-wide scale. A marked west-to-east surface gradient of alkalinity is reproduced as a response to the terrestrial discharges, the mixing effect with the Atlantic water entering from the Gibraltar Strait and the Black Sea water from Dardanelles, and the surface flux of evaporation minus precipitation. Dense water production in marginal seas (Adriatic and Aegean Seas, where alkaline inputs are relevant, and the Mediterranean thermohaline circulation sustains the west-to-east gradient along the entire water column. In the surface layers, alkalinity has a relevant seasonal cycle (up to 40 μmol kg−1 that is driven both by physical and biological processes. A comparison of alkalinity vs. salinity indicates that different regions present different relationships. In regions of freshwater influence, the two measures are negatively correlated due to riverine alkalinity input, whereas they are positively correlated in open seas. Alkalinity always is much higher than in the Atlantic waters, which might indicate a higher than usual buffering capacity towards ocean acidification, even at high concentrations of dissolved inorganic carbon.

  12. Increased river alkalinization in the Eastern U.S

    Science.gov (United States)

    Kaushal, S.; Likens, G. E.; Utz, R.; Pace, M.; Grese, M.; Yepsen, M.

    2013-12-01

    The interaction between human activities and watershed geology is accelerating long-term changes in the carbon cycle of rivers. We evaluated changes in bicarbonate alkalinity, a product of chemical weathering, and tested for long-term trends at 97 sites in the eastern United States draining over 260,000 km2. We observed statistically significant increasing trends in alkalinity at 62 of the 97 sites, while remaining sites exhibited no significant decreasing trends. Over 50% of study sites also had statistically significant increasing trends in concentrations of calcium (another product of chemical weathering) where data were available. River alkalinization rates were significantly related to watershed carbonate lithology, acid deposition, and topography. These 3 variables explained ~40% of variation in river alkalinization rates. The strongest predictor of river alkalinization rates was carbonate lithology. The most rapid rates of river alkalinization occurred at sites with highest inputs of acid deposition and highest elevation. The rise of alkalinity in many rivers throughout the eastern U.S. suggests human-accelerated chemical weathering, in addition to previously documented impacts of mining and land use. Increased river alkalinization has major environmental implications including impacts on water hardness and salinization of drinking water, alterations of air-water exchange of CO2, coastal ocean acidification, and the influence of bicarbonate availability on primary production.

  13. Increased river alkalinization in the Eastern U.S.

    Science.gov (United States)

    Kaushal, Sujay S; Likens, Gene E; Utz, Ryan M; Pace, Michael L; Grese, Melissa; Yepsen, Metthea

    2013-09-17

    The interaction between human activities and watershed geology is accelerating long-term changes in the carbon cycle of rivers. We evaluated changes in bicarbonate alkalinity, a product of chemical weathering, and tested for long-term trends at 97 sites in the eastern United States draining over 260,000 km(2). We observed statistically significant increasing trends in alkalinity at 62 of the 97 sites, while remaining sites exhibited no significant decreasing trends. Over 50% of study sites also had statistically significant increasing trends in concentrations of calcium (another product of chemical weathering) where data were available. River alkalinization rates were significantly related to watershed carbonate lithology, acid deposition, and topography. These three variables explained ~40% of variation in river alkalinization rates. The strongest predictor of river alkalinization rates was carbonate lithology. The most rapid rates of river alkalinization occurred at sites with highest inputs of acid deposition and highest elevation. The rise of alkalinity in many rivers throughout the Eastern U.S. suggests human-accelerated chemical weathering, in addition to previously documented impacts of mining and land use. Increased river alkalinization has major environmental implications including impacts on water hardness and salinization of drinking water, alterations of air-water exchange of CO2, coastal ocean acidification, and the influence of bicarbonate availability on primary production.

  14. The Pochonia chlamydosporia serine protease gene vcp1 is subject to regulation by carbon, nitrogen and pH: implications for nematode biocontrol.

    Directory of Open Access Journals (Sweden)

    Elaine Ward

    Full Text Available The alkaline serine protease VCP1 of the fungus Pochonia chlamydosporia belongs to a family of subtilisin-like enzymes that are involved in infection of nematode and insect hosts. It is involved early in the infection process, removing the outer proteinaceous vitelline membrane of nematode eggs. Little is known about the regulation of this gene, even though an understanding of how nutrients and other factors affect its expression is critical for ensuring its efficacy as a biocontrol agent. This paper provides new information on the regulation of vcp1 expression. Sequence analysis of the upstream regulatory region of this gene in 30 isolates revealed that it was highly conserved and contained sequence motifs characteristic of genes that are subject to carbon, nitrogen and pH-regulation. Expression studies, monitoring enzyme activity and mRNA, confirmed that these factors affect VCP1 production. As expected, glucose reduced VCP1 expression and for a few hours so did ammonium chloride. Surprisingly, however, by 24 h VCP1 levels were increased in the presence of ammonium chloride for most isolates. Ambient pH also regulated VCP1 expression, with most isolates producing more VCP1 under alkaline conditions. There were some differences in the response of one isolate with a distinctive upstream sequence including a variant regulatory-motif profile. Cryo-scanning electron microscopy studies indicated that the presence of nematode eggs stimulates VCP1 production by P. chlamydosporia, but only where the two are in close contact. Overall, the results indicate that readily-metabolisable carbon sources and unfavourable pH in the rhizosphere/egg-mass environment may compromise nematode parasitism by P. chlamydosporia. However, contrary to previous indications using other nematophagous and entomopathogenic fungi, ammonium nitrate (e.g. from fertilizers may enhance biocontrol potential in some circumstances.

  15. Alkaline stability of quaternary ammonium cations for alkaline fuel cell membranes and ionic liquids.

    Science.gov (United States)

    Marino, M G; Kreuer, K D

    2015-02-01

    The alkaline stability of 26 different quaternary ammonium groups (QA) is investigated for temperatures up to 160 °C and NaOH concentrations up to 10 mol L(-1) with the aim to provide a basis for the selection of functional groups for hydroxide exchange membranes in alkaline fuel cells and of ionic-liquid cations stable in basic conditions. Most QAs exhibit unexpectedly high alkaline stability with the exception of aromatic cations. β-Protons are found to be far less susceptible to nucleophilic attack than previously suggested, whereas the presence of benzyl groups, nearby hetero-atoms, or other electron-withdrawing species promote degradation reactions significantly. Cyclic QAs proved to be exceptionally stable, with the piperidine-based 6-azonia-spiro[5.5]undecane featuring the highest half-life at the chosen conditions. Absolute and relative stabilities presented herein stand in contrast to literature data, the differences being ascribed to solvent effects on degradation.

  16. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.

    Science.gov (United States)

    Matthews, S A; Rozengurt, E; Cantrell, D

    1999-09-10

    Activation of the serine kinase protein kinase D (PKD)/PKCmicro is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKCmu. An antibody that recognized PKD/PKCmu proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, we show that serine 916 is not trans-phosphorylated by an upstream kinase but is rather an autophosphorylation event that occurs following activation of PKD/PKCmu.

  17. Feasibility study on the reaction of 1,4-diazabicyclo[2.2.2]octane (DABCO with (L-Serine-L-Serine and (L-Phenylalanine-L-Serine diketopiperazines

    Directory of Open Access Journals (Sweden)

    Sitaram Bhavaraju

    2016-09-01

    Full Text Available This paper summarizes the reaction of DABCO with the enol tosylate derivatives made from (L-Ser-L-Ser and (L-Phe-L-Ser diketopiperazines (DKP’s. The reaction between DABCO and EE-di-tosylate (L-Ser-L-Ser DKP (2, results in the isomerization of the serine di-tosylate from EE-2 to ZZ-2. This is the first direct example of the utility of DABCO as a reagent demonstrating the successful isomerization in a DKP derivative. The E-enol tosylate of (L-Phe-L-Ser DKP (4 upon reaction with DABCO provided a unique bis-ylidiene product (5.

  18. Phosphatidylinositol anchor of HeLa cell alkaline phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Jemmerson, R.; Low, M.G.

    1987-09-08

    Alkaline phosphatase from cancer cells, HeLa TCRC-1, was biosynthetically labeled with either /sup 3/H-fatty acids or (/sup 3/H)ethanolamine as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of immunoprecipitated material. Phosphatidylinositol-specific phospholipase C (PI-PLC) released a substantial proportion of the /sup 3/H-fatty acid label from immunoaffinity-purified alkaline phosphatase but had no effect on the radioactivity of (/sup 3/H)ethanolamine-labeled material. PI-PLC also liberated catalytically active alkaline phosphatase from viable cells, and this could be selectively blocked by monoclonal antibodies to alkaline phosphatase. However, the alkaline phosphatase released from /sup 3/H-fatty acid labeled cells by PI-PLC was not radioactive. By contrast, treatment with bromelain removed both the /sup 3/H-fatty acid and the (/sup 3/H)ethanolamine label from purified alkaline phosphatase. Subtilisin was also able to remove the (/sup 3/H)ethanolamine label from the purified alkaline phosphatase. The /sup 3/H radioactivity in alkaline phosphatase purified from (/sup 3/H)ethanolamine-labeled cells comigrated with authentic (/sup 3/H)ethanolamine by anion-exchange chromatography after acid hydrolysis. The data suggest that the /sup 3/H-fatty acid and (/sup 3/H)ethanolamine are covalently attached to the carboxyl-terminal segment since bromelain and subtilisin both release alkaline phosphatase from the membrane by cleavage at that end of the polypeptide chain. The data are consistent with findings for other proteins recently shown to be anchored in the membrane through a glycosylphosphatidylinositol structure and indicate that a similar structure contributes to the membrane anchoring of alkaline phosphatase.

  19. Serine Protease Catalysis: A Computational Study of Tetrahedral Intermediates and Inhibitory Adducts.

    Science.gov (United States)

    Ngo, Phong D; Mansoorabadi, Steven O; Frey, Perry A

    2016-08-04

    Peptide boronic acids and peptidyl trifluoromethyl ketones (TFKs) inhibit serine proteases by forming monoanionic, tetrahedral adducts to serine in the active sites. Investigators regard these adducts as analogs of monoanionic, tetrahedral intermediates. Density functional theory (DFT) calculations and fractional charge analysis show that tetrahedral adducts of model peptidyl TFKs are structurally and electrostatically very similar to corresponding tetrahedral intermediates. In contrast, the DFT calculations show the structures and electrostatic properties of analogous peptide boronate adducts to be significantly different. The peptide boronates display highly electrostatically positive boron, with correspondingly negative ligands in the tetrahedra. In addition, the computed boron-oxygen and boron-carbon bond lengths in peptide boronates (which are identical or very similar to the corresponding bonds in a peptide boronate adduct of α-lytic protease determined by X-ray crystallography at subangstrom resolution) are significantly longer than the corresponding bond lengths in model tetrahedral intermediates. Since protease-peptidyl TFKs incorporate low-barrier hydrogen bonds (LBHBs) between an active site histidine and aspartate, while the protease-peptide boronates do not, these data complement the spectroscopic and chemical evidence for the participation of LBHBs in catalysis by serine proteases. Moreover, while the potency of these classes of inhibitors can be correlated to the structures of the peptide moieties, the present results indicate that the strength of their bonds to serine contribute significantly to their inhibitory properties.

  20. Cloning, Expression and Activity Analysis of a Novel Fibrinolytic Serine Protease fromArenicola cristata

    Institute of Scientific and Technical Information of China (English)

    ZHAO Chunling; JU Jiyu

    2015-01-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplifi-cation of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encod-ing a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (<40%) to other serine proteases. The gene encoding the active form ofA. cristata serine protease was cloned and expressed inE. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result sug-gested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clotin vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene inA. cristata.

  1. Serine protease immunohistochemistry and lectin histochemistry in the small intestine of weaned and unweaned pigs

    DEFF Research Database (Denmark)

    Brown, P J; Poulsen, Steen Seier; Wells, M

    1991-01-01

    The distribution of goblet cells containing serine protease and of those binding the lectin Ulex europaeus agglutinin-1 (UEA-1) in the pig small intestine is altered during the period after weaning. Goblet cells exhibiting binding of other lectins were not altered. These alterations and other...

  2. HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

    NARCIS (Netherlands)

    SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase a

  3. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

    NARCIS (Netherlands)

    Siezen, Roland J.; Vos, Willem M. de; Leunissen, Jack A.M.; Dijkstra, Bauke W.

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and

  4. Identification and characterization of a surface-associated, subtilisin-like serine protease in Trichomonas vaginalis.

    Science.gov (United States)

    Hernández-Romano, Pablo; Hernández, Roberto; Arroyo, Rossana; Alderete, John F; López-Villaseñor, Imelda

    2010-09-01

    Trichomonas vaginalis is a protozoan parasite causing trichomonosis, a sexually transmitted infection in humans. This parasite has numerous proteases, most of which are cysteine proteases that appear to be involved in adherence and cytotoxicity of host cells. In this report we identify and characterize a putative subtilisin-like serine protease (SUB1). The sub1 gene encodes a 101-kDa protein. In silico analyses predict signal and pro-peptides at the N-terminus, and a transmembrane helix at the carboxy-terminal region. The sub1 gene was found as single copy by Southern analysis, albeit additional serine protease related genes are annotated in the T. vaginalis genome. The expression of sub1 could only be detected by RT-PCR and Ribonuclease Protection Assays, suggesting a low abundant mRNA. The sub1 gene transcription start site was correctly assigned by RPA. The transcript abundance was found to be modulated by the availability of iron in the growth medium. Antibodies raised to a specific SUB1 peptide recognized a single protein band (approximately 82 kDa) in Western blots, possibly representing the mature form of the protein. Immunofluorescence showed SUB1 on the trichomonad surface, and in dispersed vesicles throughout the cytoplasm. A bioinformatic analysis of genes annotated as serine proteases in the T. vaginalis genome is also presented. To our knowledge this is the first putative serine protease experimentally described for T. vaginalis.

  5. Serine aspartate repeat protein D increases Staphylococcus aureus virulence and survival in blood

    NARCIS (Netherlands)

    Askarian, Fatemeh; Uchiyama, Satoshi; Valderrama, J. Andrés; Ajayi, Clement; Sollid, Johanna U E; van Sorge, Nina M.; Nizet, Victor; van Strijp, Jos A G; Johannessen, Mona

    2017-01-01

    Staphylococcus aureus expresses a panel of cell wall-anchored adhesins, including proteins belonging to the microbial surface components recognizing adhesive matrix molecule (MSCRAMM) family, exemplified by the serine-aspartate repeat protein D (SdrD), which serve key roles in colonization and

  6. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  7. CSF d-serine concentrations are similar in Alzheimer's disease, other dementias, and elderly controls

    NARCIS (Netherlands)

    Biemans, Elisanne A L M; Verhoeven-Duif, Nanda M; Gerrits, Johan; Claassen, Jurgen A H R; Kuiperij, H Bea; Verbeek, Marcel M

    2016-01-01

    Cerebrospinal fluid (CSF) levels of d-serine were recently reported as a potential new biomarker for Alzheimer's disease (AD), showing a perfect distinction between AD patients and healthy controls. In this study, we aimed to confirm these results and extend these previous findings to dementia with

  8. Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone

    Science.gov (United States)

    Serine acetyltransferase (SAT) catalyzes the limiting reaction in plant and microbial biosynthesis of cysteine. In addition to its enzymatic function, SAT forms a macromolecular complex with O-acetylserine sulfhydrylase (OASS). Formation of the cysteine regulatory complex (CRC) is a critical biochem...

  9. An Epithelial-Derived, Integral Membrane, Kunitz-Type Serine Protease Inhibitor in Breast Cancer

    Science.gov (United States)

    2005-08-01

    an epithelial membrane serine protease. J Biol Chein 275: 36720-36725, 2000. prevents15. Lin CY, Anders J, Johnson M, and Dickson RB. Purification and...functions properly, thus trypsin-like activity. J Biol Chein 274: 18231--18236, 1999. 0 avoiding harmful effects. 17. Lin CY, Wang JK, Torri J, Dou L, Sang

  10. HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

    NARCIS (Netherlands)

    SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase a

  11. Identification and purification of O-acetyl-L-serine sulphhydrylase in Penicillium chrysogenum

    DEFF Research Database (Denmark)

    østergaard, Simon; Theilgaard, Hanne Birgitte; Nielsen, Jens Bredal

    1998-01-01

    in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/ polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K-m value for O-acetyl-L-serine and V-max of O...

  12. Entamoeba dispar: genetic diversity of Iranian isolates based on serine-rich Entamoeba dispar protein gene.

    Science.gov (United States)

    Haghighi, A; Rasti, S; Nazemalhosseini Mojarad, E; Kazemi, B; Bandehpour, M; Nochi, Z; Hooshyar, H; Rezaian, M

    2008-12-01

    The nucleotide sequences of Serine-Rich Entamoeba histolytica Protein (SREHP) gene have already exhibited stable and significant polymorphism in the gene studies. Serine-rich protein is also present and polymorphic in Entamoeba dispar which called SREDP. The polymorphism of the Serine-Rich Entamoeba dispar Protein (SREDP) gene among 8 isolates obtained from Iranian cyst carriers were analyzed by a nested PCR-RFLP followed by sequencing of the PCR products. From those isolates, six distinct DNA patterns were observed after PCR-RFLP of the nested PCR, whereas sequencing showed 8 different patterns among the isolates. The results demonstrate an extensive genetic variability among Iranian E. dispar isolates. The repeat-containing region of the SREDP was found extensively polymorphic in size, number and order of repeat units. Genetic diversity of Iranian E. dispar isolates based on the SREDP was more polymorphic in comparison of Serine-Rich Entamoeba histolytica Protein (SREHP) of the E. histolytica isolates as well as were different from a few known SREDP genes.

  13. Ketamine induces tau hyperphosphorylation at serine 404 in the hippocampus of neonatal rats

    Institute of Scientific and Technical Information of China (English)

    Haiyan Jin; Zhiyong Hu; Mengjie Dong; Yidong Wu; Zhirui Zhu; Lili Xu

    2013-01-01

    Male Wistar 7-day-old rats were injected with 40 mg/kg ketamine intraperitoneally, followed by three additional injections of 20 mg/kg ketamine each upon restoration of the righting reflex. Neonatal rats injected with equivalent volumes of saline served as controls. Hippocampal samples were collected at 1, 7 or 14 days following administration. Electron microscopy showed that neuronal structure changed noticeably following ketamine treatment. Specifically, microtubular structure became irregular and disorganized. Quantitative real time-PCR revealed that phosphorylated tau mRNA was upregulated after ketamine. Western blot analysis demonstrated that phosphorylated tau levels at serine 396 initially decreased at 1 day after ketamine injection, and then gradually returned to control values. At 14 days after injection, levels of phosphorylated tau were higher in the ketamine group than in the control group. Tau protein phosphorylated at serine 404 significantly increased after ketamine injection, and then gradually decreased with time. However, the levels of tau protein at serine 404 were significantly greater in the ketamine group than in the control group until 14 days. The present results indicate that ketamine induces an increase of phosphorylated tau mRNA and excessive phosphorylation of tau protein at serine 404, causing disruption of microtubules in the neonatal rat hippocampus and potentially resulting in damage to hippocampal neurons.

  14. Kazal-type serine proteinase inhibitors in the midgut of Phlebotomus papatasi

    Directory of Open Access Journals (Sweden)

    Leah Theresa Sigle

    2013-09-01

    Full Text Available Sandflies (Diptera: Psychodidae are important disease vectors of parasites of the genus Leishmania, as well as bacteria and viruses. Following studies of the midgut transcriptome of Phlebotomus papatasi, the principal vector of Leishmania major, two non-classical Kazal-type serine proteinase inhibitors were identified (PpKzl1 and PpKzl2. Analyses of expression profiles indicated that PpKzl1 and PpKzl2 transcripts are both regulated by blood-feeding in the midgut of P. papatasi and are also expressed in males, larva and pupa. We expressed a recombinant PpKzl2 in a mammalian expression system (CHO-S free style cells that was applied to in vitro studies to assess serine proteinase inhibition. Recombinant PpKzl2 inhibited α-chymotrypsin to 9.4% residual activity and also inhibited α-thrombin and trypsin to 33.5% and 63.9% residual activity, suggesting that native PpKzl2 is an active serine proteinase inhibitor and likely involved in regulating digestive enzymes in the midgut. Early stages of Leishmania are susceptible to killing by digestive proteinases in the sandfly midgut. Thus, characterising serine proteinase inhibitors may provide new targets and strategies to prevent transmission of Leishmania.

  15. Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis.

    Science.gov (United States)

    Manuse, Sylvie; Fleurie, Aurore; Zucchini, Laure; Lesterlin, Christian; Grangeasse, Christophe

    2016-01-01

    Bacteria possess a repertoire of versatile protein kinases modulating diverse aspects of their physiology by phosphorylating proteins on various amino acids including histidine, cysteine, aspartic acid, arginine, serine, threonine and tyrosine. One class of membrane serine/threonine protein kinases possesses a catalytic domain sharing a common fold with eukaryotic protein kinases and an extracellular mosaic domain found in bacteria only, named PASTA for 'Penicillin binding proteins And Serine/Threonine kinase Associated'. Over the last decade, evidence has been accumulating that these protein kinases are involved in cell division, morphogenesis and developmental processes in Firmicutes and Actinobacteria. However, observations differ from one species to another suggesting that a general mechanism of activation of their kinase activity is unlikely and that species-specific regulation of cell division is at play. In this review, we survey the latest research on the structural aspects and the cellular functions of bacterial serine/threonine kinases with PASTA motifs to illustrate the diversity of the regulatory mechanisms controlling bacterial cell division and morphogenesis.

  16. Taraxalisin -- a serine proteinase from dandelion Taraxacum officinale Webb s.l.

    Science.gov (United States)

    Rudenskaya, G N; Bogacheva, A M; Preusser, A; Kuznetsova, A V; Dunaevsky YaE; Golovkin, B N; Stepanov, V M

    1998-10-23

    Latex of dandelion roots contains a serine proteinase that hydrolyzes a chromogenic peptide substrate Glp-Ala-Ala-Leu-pNA optimally at pH 8.0. Maximal activity of the proteinase in the roots is attained in April, at the beginning of plant development after the winter period. The protease was isolated by ammonium sulfate precipitation of the root extract followed by affinity chromatography on a Sepharose-Ala-Ala-Leu-mrp and gel filtration on Superose 6R performed in FPLC regime. Pure serine proteinase named taraxalisin was inactivated by specific inhibitors of serine proteinases, diisopropylfluorophosphate (DFP) and phenylmethylsulfonylfluoride (PMSF). Its molecular mass is 67 kDa and pI 4.5. pH stability range is 6-9 in the presence of 2 mM Ca2+, temperature optimum is at 40 degrees C; Km=0.37+/-0.06 mM. The substrate specificity of taraxalisin towards synthetic peptides and insulin B-chain is comparable with that of two other subtilisin-like serine proteinases, cucumisin and macluralisin. The taraxalisin N-terminal sequence traced for 15 residues revealed 40% coinciding residues when aligned with that of subtilisin Carlsberg.

  17. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    Science.gov (United States)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  18. Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

    NARCIS (Netherlands)

    Siezen, Roland J.; Vos, Willem M. de; Leunissen, Jack A.M.; Dijkstra, Bauke W.

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, >50 subtilases are known, >40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase and

  19. HOMOLOGY MODELING AND PROTEIN ENGINEERING STRATEGY OF SUBTILASES, THE FAMILY OF SUBTILISIN-LIKE SERINE PROTEINASES

    NARCIS (Netherlands)

    SIEZEN, RJ; DEVOS, WM; LEUNISSEN, JAM

    1991-01-01

    Subtilases are members of the family of subtilisin-like serine proteases. Presently, > 50 subtilases are known, > 40 of which with their complete amino acid sequences. We have compared these sequences and the available three-dimensional structures (subtilisin BPN', subtilisin Carlsberg, thermitase

  20. Identification and characterization of a chymotrypsin-like serine protease from periodontal pathogen, Tannerella forsythia.

    Science.gov (United States)

    Hockensmith, K; Dillard, K; Sanders, B; Harville, B A

    2016-11-01

    Tannerella forsythia is a bacteria associated with severe periodontal disease. This study reports identification and characterization of a membrane-associated serine protease from T. forsythia. The protease was isolated from T. forsythia membrane fractions and shown to cleave both gelatin and type I collagen. The protease was able to cleave both substrates over a wide range of pH values, however optimal cleavage occurred at pH 7.5 for gelatin and 8.0 for type I collagen. The protease was also shown to cleave both gelatin and type I collagen at the average reported temperature for the gingival sulcus however it showed a lack of thermal stability with a complete loss of activity by 60 °C. When treated with protease inhibitors the enzyme's activity could only be completely inhibited by serine protease inhibitors antipain and phenylmethanesulfonyl fluoride (PMSF). Further characterization of the protease utilized serine protease synthetic peptides. The protease cleaved N-succinyl-Ala-Ala-Pro-Phe p-nitroanilide but not Nα-benzoyl-dl-arginine p-nitroanilide (BAPNA) or N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide indicating that the protease is a chymotrypsin-like serine protease. Since type I collagen is a major component in the gingival tissues and periodontal ligament, identification and characterization of this enzyme provides important information regarding the role of T. forsythia in periodontal disease.

  1. The serine-threonine kinase LKB1 is essential for survival under energetic stress in zebrafish

    NARCIS (Netherlands)

    Velden, Y.U.; Wang, L.; Zevenhoven, J.; van Rooijen, E.; van Lohuizen, M.; Giles, R.H.; Clevers, H.; Haramis, A.P.

    2011-01-01

    Mutations in the serine-threonine kinase (LKB1) lead to a gastrointestinal hamartomatous polyposis disorder with increased predisposition to cancer (Peutz-Jeghers syndrome). LKB1 has many targets, including the AMP-activated protein kinase (AMPK) that is phosphorylated under low-energy conditions.

  2. Tryptophanase-catalyzed L-tryptophan synthesis from D-serine in the presence of diammonium hydrogen phosphate.

    Science.gov (United States)

    Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Noriko, Fujii

    2009-06-01

    Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a beta-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH(4))(2)HPO(4) solution. The present study provided the same stereospecific change seen in the d-tryptophan degradation reaction also occurs in tryptophan synthesis from d-serine. Tryptophanase became active to d-serine to synthesize l-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. d-serine seems to undergo beta-replacement via an enzyme-bonded alpha-aminoacylate intermediate to yield l-tryptophan.

  3. The effect of irrigated rice cropping on the alkalinity of two alkaline rice soils in the Sahel

    NARCIS (Netherlands)

    Asten, van P.J.A.; Zelfde, van 't J.A.; Zee, van der S.E.A.T.M.; Hammecker, C.

    2004-01-01

    Irrigated rice cropping is practiced to reclaim alkaline-sodic soils in many parts of the world. This practice is in apparent contrast with earlier studies in the Sahel, which suggests that irrigated rice cropping may lead to the formation of alkaline-sodic soils. Soil column experiments were done w

  4. Distinct biochemical properties of human serine hydroxymethyltransferase compared with the Plasmodium enzyme: implications for selective inhibition.

    Science.gov (United States)

    Pinthong, Chatchadaporn; Maenpuen, Somchart; Amornwatcharapong, Watcharee; Yuthavong, Yongyuth; Leartsakulpanich, Ubolsree; Chaiyen, Pimchai

    2014-06-01

    Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a hydroxymethyl group from l-serine to tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Our previous investigations have shown that SHMTs from Plasmodium spp. (P. falciparum, Pf; P. vivax, Pv) are different from the enzyme from rabbit liver in that Plasmodium SHMT can use d-serine as a substrate. In this report, the biochemical and biophysical properties of the Plasmodium and the human cytosolic form (hcSHMT) enzymes including ligand binding and kinetics were investigated. The data indicate that, similar to Plasmodium enzymes, hcSHMT can use d-serine as a substrate. However, hcSHMT displays many properties that are different from those of the Plasmodium enzymes. The molar absorption coefficient of hcSHMT-bound pyridoxal-5'-phosphate (PLP) is much greater than PvSHMT-bound or PfSHMT-bound PLP. The binding interactions of hcSHMT and Plasmodium SHMT with d-serine are different, as only the Plasmodium enzyme undergoes formation of a quinonoid-like species upon binding to d-serine. Furthermore, it has been noted that hcSHMT displays strong substrate inhibition by tetrahydrofolate (THF) (at THF > 40 μm), compared with SHMTs from Plasmodium and other species. The pH-activity profile of hcSHMT shows higher activities at lower pH values corresponding to a pKa value of 7.8 ± 0.1. Thiosemicarbazide reacts with hcSHMT following a one-step model [k1 of 12 ± 0.6 m(-1) ·s(-1) and k-1 of (1.0 ± 0.6) × 10(-3) s(-1) ], while the same reaction with PfSHMT involves at least three steps. All data indicated that the ligand binding environment of SHMT from human and Plasmodium are different, indicating that it should be possible to develop species-selective inhibitors in future studies. serine hydroxymethyltransferase, EC 2.1.2.1; 5,10-methylenetetrahydrofolate dehydrogenase, EC 1.5.1.5. © 2014 FEBS.

  5. Batteries: from alkaline to zinc-air.

    Science.gov (United States)

    Dondelinger, Robert M

    2004-01-01

    There is no perfect disposable battery--one that will sit on the shelf for 20 years, then continually provide unlimited current, at a completely constant voltage until exhausted, without producing heat. There is no perfect rechargeable battery--one with all of the above characteristics and will also withstand an infinite overcharge while providing an equally infinite cycle life. There are only compromises. Every battery selection is a compromise between the ideally required characteristics, the advantages, and the limitations of each battery type. General selection of a battery type to power a medical device is largely outside the purview of the biomed. Initially, these are engineering decisions made at the time of medical equipment design and are intended to be followed in perpetuity. However, since newer cell types evolve and the manufacturer's literature is fixed at the time of printing, some intelligent substitutions may be made as long as the biomed understands the characteristics of both the recommended cell and the replacement cell. For example, when the manufacturer recommends alkaline, it is usually because of the almost constant voltage it produces under the devices' design load. Over time, other battery types may be developed that will meet the intent of the manufacturer, at a lower cost, providing longer operational life, at a lower environmental cost, or with a combination of these advantages. In the Obstetrical Doppler cited at the beginning of this article, the user had put in carbon-zinc cells, and the biomed had unknowingly replaced them with carbonzinc cells. If the alkaline cells recommended by the manufacturer had been used, there would have been the proper output voltage at the battery terminals when the [table: see text] cells were at their half-life. Instead, the device refused to operate since the battery voltage was below presumed design voltage. While battery-type substitutions may be easily and relatively successfully made in disposable

  6. Roles of serine accumulation and catabolism in the colonization of the murine urinary tract by Escherichia coli CFT073.

    Science.gov (United States)

    Anfora, Andrew T; Haugen, Brian J; Roesch, Paula; Redford, Peter; Welch, Rodney A

    2007-11-01

    A D-serine deaminase (DsdA) mutant of uropathogenic Escherichia coli strain CFT073 has a hypercolonization phenotype in a murine model of urinary tract infection (UTI) due to increased virulence gene expression by an unknown mechanism (B. J. Haugen et al., Infect. Immun. 75:278-289, 2007). DsdC is a D-serine-dependent activator of dsdXA transcription. DsdC may regulate the virulence genes responsible for hypercolonization. The loss of DsdA leads to increased intracellular accumulation of D-serine. In this study we show that deletion of the genes encoding L-serine deaminases SdaA and SdaB resulted in a mutant that accumulates higher intracellular levels of L-serine than CFT073. CFT073 sdaA sdaB has a mild competitive colonization defect whereas a CFT073 dsdA sdaA sdaB triple mutant shows a greater loss in competitive colonization ability. Thus, the inability to generate serine-specific catabolic products does not result in hypercolonization and the ability to catabolize serine represents a positive physiological trait during murine UTI. CFT073 dsdC and CFT073 dsdC dsdA mutants continue to outcompete the wild type in the UTI model. These results confirm that loss of DsdA activity results in the hypercolonization phenotype and that DsdC does not play a direct role in the elevated-colonization phenotype. Interestingly, a CFT073 dsdA mutant with deletions of D-serine transporter genes dsdX and cycA shows wild-type colonization levels of the bladder but is attenuated for kidney colonization. Thus, D-serine acts as a signal for hypercolonization and virulence gene expression by CFT073 dsdA, whereas overall catabolism of serine represents a positive Escherichia coli fitness trait during UTI.

  7. Roles of Serine Accumulation and Catabolism in the Colonization of the Murine Urinary Tract by Escherichia coli CFT073▿

    Science.gov (United States)

    Anfora, Andrew T.; Haugen, Brian J.; Roesch, Paula; Redford, Peter; Welch, Rodney A.

    2007-01-01

    A d-serine deaminase (DsdA) mutant of uropathogenic Escherichia coli strain CFT073 has a hypercolonization phenotype in a murine model of urinary tract infection (UTI) due to increased virulence gene expression by an unknown mechanism (B. J. Haugen et al., Infect. Immun. 75:278-289, 2007). DsdC is a d-serine-dependent activator of dsdXA transcription. DsdC may regulate the virulence genes responsible for hypercolonization. The loss of DsdA leads to increased intracellular accumulation of d-serine. In this study we show that deletion of the genes encoding l-serine deaminases SdaA and SdaB resulted in a mutant that accumulates higher intracellular levels of l-serine than CFT073. CFT073 sdaA sdaB has a mild competitive colonization defect whereas a CFT073 dsdA sdaA sdaB triple mutant shows a greater loss in competitive colonization ability. Thus, the inability to generate serine-specific catabolic products does not result in hypercolonization and the ability to catabolize serine represents a positive physiological trait during murine UTI. CFT073 dsdC and CFT073 dsdC dsdA mutants continue to outcompete the wild type in the UTI model. These results confirm that loss of DsdA activity results in the hypercolonization phenotype and that DsdC does not play a direct role in the elevated-colonization phenotype. Interestingly, a CFT073 dsdA mutant with deletions of d-serine transporter genes dsdX and cycA shows wild-type colonization levels of the bladder but is attenuated for kidney colonization. Thus, d-serine acts as a signal for hypercolonization and virulence gene expression by CFT073 dsdA, whereas overall catabolism of serine represents a positive Escherichia coli fitness trait during UTI. PMID:17785472

  8. Roscovitine inhibits EBNA1 serine 393 phosphorylation, nuclear localization, transcription, and episome maintenance.

    Science.gov (United States)

    Kang, Myung-Soo; Lee, Eun Kyung; Soni, Vishal; Lewis, Timothy A; Koehler, Angela N; Srinivasan, Viswanathan; Kieff, Elliott

    2011-03-01

    Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.

  9. Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance▿ †

    Science.gov (United States)

    Kang, Myung-Soo; Lee, Eun Kyung; Soni, Vishal; Lewis, Timothy A.; Koehler, Angela N.; Srinivasan, Viswanathan; Kieff, Elliott

    2011-01-01

    Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and 486RALL489 and 580KDLVM584 as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence. PMID:21209116

  10. Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function.

    Science.gov (United States)

    Lahey, Kelcie A; Ronaghan, Natalie J; Shang, Judie; Dion, Sébastien P; Désilets, Antoine; Leduc, Richard; MacNaughton, Wallace K

    2017-01-01

    Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial

  11. Microbial thiocyanate utilization under highly alkaline conditions.

    Science.gov (United States)

    Sorokin, D Y; Tourova, T P; Lysenko, A M; Kuenen, J G

    2001-02-01

    Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS-) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (the Halomonas group for the heterotrophs and the genus Thioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genus Thioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase

  12. Titratable Acidity and Alkalinity of Red Soil Surfaces

    Institute of Scientific and Technical Information of China (English)

    SHAOZONG-CHEN; HEQUN; 等

    1993-01-01

    The surfaces of red soils have an apparent amphoteric character,carrying titratable acidity and titratable alkalinity simultaneously.The titratable acidity arises from deprotonation of hydroxyl groups of hydrous oxide-type surfaces and dissociation of weak-acid functional groups of soil organic matter,while the titratable alkalinity is derived from release of hydroxyl groups of hydrous oxide-type surfaces.The titratable acidity and titratable alkalinity mainly depended on the composition and content of iron and aluminum oxides in the soils.The results showed that the titratable acidity and titratable alkalinity were in significantly positive correlation not only with the content of amorphous aluminum oxide(Alo) and iron oxide(Feo) extracted with acid ammonium oxalate solution,free iron oxide(Fed) extracted with sodium dithionite-citrate-bicarbonate(DCB) and clays,but also with the zero point of charge (ZPC) of the samples.Organic matter made an important contribution to the titratable acidity.the titratable alkalinity was closely correlated with the amount of fluoride ions adsorbed.The titratable acidity and titratable alkalinity of red soils were influenced by parent materials,being in the order of red soil derived from basalt> that from tuff> that from granite.The titratable acidity and titratable alkalinity ware closely related with origination of the variable charges of red soils,and to a certain extent were responsible for variable negative and positive charges of the soils.

  13. Alkaline Phosphatase, an Unconventional Immune Protein.

    Science.gov (United States)

    Rader, Bethany A

    2017-01-01

    Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs), revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP) proteins, most is known about tissue non-specific AP (TNAP) and intestinal AP (IAP). This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer's disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

  14. Alkaline Phosphatase, an Unconventional Immune Protein

    Science.gov (United States)

    Rader, Bethany A.

    2017-01-01

    Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs), revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP) proteins, most is known about tissue non-specific AP (TNAP) and intestinal AP (IAP). This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer’s disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont. PMID:28824625

  15. Response of Desulfovibrio vulgaris to Alkaline Stress

    Energy Technology Data Exchange (ETDEWEB)

    Stolyar, S.; He, Q.; He, Z.; Yang, Z.; Borglin, S.E.; Joyner, D.; Huang, K.; Alm, E.; Hazen, T.C.; Zhou, J.; Wall, J.D.; Arkin, A.P.; Stahl, D.A.

    2007-11-30

    The response of exponentially growing Desulfovibrio vulgarisHildenborough to pH 10 stress was studied using oligonucleotidemicroarrays and a study set of mutants with genes suggested by microarraydata to be involved in the alkaline stress response deleted. The datashowed that the response of D. vulgaris to increased pH is generallysimilar to that of Escherichia coli but is apparently controlled byunique regulatory circuits since the alternative sigma factors (sigma Sand sigma E) contributing to this stress response in E. coli appear to beabsent in D. vulgaris. Genes previously reported to be up-regulated in E.coli were up-regulated in D. vulgaris; these genes included three ATPasegenes and a tryptophan synthase gene. Transcription of chaperone andprotease genes (encoding ATP-dependent Clp and La proteases and DnaK) wasalso elevated in D. vulgaris. As in E. coli, genes involved in flagellumsynthesis were down-regulated. The transcriptional data also identifiedregulators, distinct from sigma S and sigma E, that are likely part of aD. vulgaris Hildenborough-specific stress response system.Characterization of a study set of mutants with genes implicated inalkaline stress response deleted confirmed that there was protectiveinvolvement of the sodium/proton antiporter NhaC-2, tryptophanase A, andtwo putative regulators/histidine kinases (DVU0331 andDVU2580).

  16. Alkaline phosphatase as a periodontal disease marker

    Directory of Open Access Journals (Sweden)

    Malhotra Ranjan

    2010-01-01

    Full Text Available Background: The potential of alkaline phosphatase (ALP as an important diagnostic marker of gingival crevicular fluid (GCF has been the subject to investigation since 1970. ALP is stored in specific granules and secretory vesicles of the neutrophils and is mainly released during their migration to the site of infection. It is also present in bacteria within dental plaque, osteoblasts and fibroblasts. It has, thus, become important to elucidate whether GCF levels of ALP are potential measures of the inflammatory activity occurring in the adjacent periodontal tissues. Objective: The aim of this study was to assess the total activity of ALP in the GCF collected from healthy sites, sites with gingivitis and with chronic adult periodontitis. An attempt was also made to establish the correlation of ALP activity with plaque index, gingival index, bleeding index and probing depth. Materials and Methods: A total of 18 patients were divided into three groups: viz., healthy sites, Group I; gingivitis, Group II; chronic periodontitis, Group III. Clinical parameters like plaque index, bleeding index, gingival index and probing depth were recorded. The ALP level in GCF of all three groups was determined by spectrophotometric analysis. Results: Total enzyme activity of ALP was significantly higher in periodontitis as compared with that in healthy and gingivitis sites, and was significantly and positively correlated with probing depth. Conclusion: ALP can be considered as a periodontal disease marker as it can distinguish between healthy and inflamed sites. However, to better define its capacity for periodontal diagnosis, additional longitudinal studies are required.

  17. Alkaline Phosphatase, an Unconventional Immune Protein

    Directory of Open Access Journals (Sweden)

    Bethany A. Rader

    2017-08-01

    Full Text Available Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs, revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP proteins, most is known about tissue non-specific AP (TNAP and intestinal AP (IAP. This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer’s disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

  18. 2nd Generation alkaline electrolysis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Yde, L. [Aarhus Univ. Business and Social Science - Centre for Energy Technologies (CET), Aarhus (Denmark); Kjartansdottir, C.K. [Technical Univ. of Denmark. DTU Mechanical Engineering, Kgs. Lyngby (Denmark); Allebrod, F. [Technical Univ. of Denmark. DTU Energy Conversion, DTU Risoe Campus, Roskilde (Denmark)] [and others

    2013-03-15

    The overall purpose of this project has been to contribute to this load management by developing a 2{sup nd} generation of alkaline electrolysis system characterized by being compact, reliable, inexpensive and energy efficient. The specific targets for the project have been to: 1) Increase cell efficiency to more than 88% (according to the higher heating value (HHV)) at a current density of 200 mA /cm{sup 2}; 2) Increase operation temperature to more than 100 degree Celsius to make the cooling energy more valuable; 3) Obtain an operation pressure more than 30 bar hereby minimizing the need for further compression of hydrogen for storage; 4) Improve stack architecture decreasing the price of the stack with at least 50%; 5) Develop a modular design making it easy to customize plants in the size from 20 to 200 kW; 6) Demonstrating a 20 kW 2{sup nd} generation stack in H2College at the campus of Arhus University in Herning. The project has included research and development on three different technology tracks of electrodes; an electrochemical plating, an atmospheric plasma spray (APS) and finally a high temperature and pressure (HTP) track with operating temperature around 250 deg. C and pressure around 40 bar. The results show that all three electrode tracks have reached high energy efficiencies. In the electrochemical plating track a stack efficiency of 86.5% at a current density of 177mA/cm{sup 2} and a temperature of 74.4 deg. C has been shown. The APS track showed cell efficiencies of 97%, however, coatings for the anode side still need to be developed. The HTP cell has reached 100 % electric efficiency operating at 1.5 V (the thermoneutral voltage) with a current density of 1. 1 A/cm{sup 2}. This track only tested small cells in an externally heated laboratory set-up, and thus the thermal loss to surroundings cannot be given. The goal set for the 2{sup nd} generation electrolyser system, has been to generate 30 bar pressure in the cell stack. An obstacle to be

  19. Engineering challenges of ocean alkalinity enhancement

    Science.gov (United States)

    Kruger, T.; Renforth, P.

    2012-04-01

    The addition of calcium oxide (CaO) to the ocean as a means of enhancing the capacity of the ocean as a carbon sink was first proposed by Haroon Kheshgi in 1995. Calcium oxide is created by heating high purity limestone in a kiln to temperatures of approximately 1000°C. Addition of this material to the ocean draws carbon dioxide out of the atmosphere (approximately 1 tonne of CaO could sequester 1.3 tonnes of CO2). Abiotic carbonate precipitation is inhibited in the surface ocean. This is a carbon and energy expensive process, where approximately 0.8 tonnes of CO2 are produced at a point source for every tonne sequestered. The feasibility of ocean alkalinity enhancement requires capture and storage of the point source of CO2. We present details of a feasibility study of the engineering challenges of Kheshgi's method focusing on the potential scalability and costs of the proposed process. To draw down a PgC per year would require the extraction and processing of ~6Pg of limestone per year, which is similar in scale to the current coal industry. Costs are estimated at ~USD30-40 per tonne of CO2 sequestered through the process, which is favourable to comparative processes. Kheshgi, H. (1995) Energy 20 (9) 915-922

  20. Alkaline fuel cell with nitride membrane

    Science.gov (United States)

    Sun, Shen-Huei; Pilaski, Moritz; Wartmann, Jens; Letzkus, Florian; Funke, Benedikt; Dura, Georg; Heinzel, Angelika

    2017-06-01

    The aim of this work is to fabricate patterned nitride membranes with Si-MEMS-technology as a platform to build up new membrane-electrode-assemblies (MEA) for alkaline fuel cell applications. Two 6-inch wafer processes based on chemical vapor deposition (CVD) were developed for the fabrication of separated nitride membranes with a nitride thickness up to 1 μm. The mechanical stability of the perforated nitride membrane has been adjusted in both processes either by embedding of subsequent ion implantation step or by optimizing the deposition process parameters. A nearly 100% yield of separated membranes of each deposition process was achieved with layer thickness from 150 nm to 1 μm and micro-channel pattern width of 1μm at a pitch of 3 μm. The process for membrane coating with electrolyte materials could be verified to build up MEA. Uniform membrane coating with channel filling was achieved after the optimization of speed controlled dip-coating method and the selection of dimethylsulfoxide (DMSO) as electrolyte solvent. Finally, silver as conductive material was defined for printing a conductive layer onto the MEA by Ink-Technology. With the established IR-thermography setup, characterizations of MEAs in terms of catalytic conversion were performed successfully. The results of this work show promise for build up a platform on wafer-level for high throughput experiments.

  1. Process for treating alkaline wastes for vitrification

    Science.gov (United States)

    Hsu, Chia-lin W.

    1994-01-01

    According to its major aspects and broadly stated, the present invention is a process for treating alkaline waste materials, including high level radioactive wastes, for vitrification. The process involves adjusting the pH of the wastes with nitric acid, adding formic acid (or a process stream containing formic acid) to reduce mercury compounds to elemental mercury and MnO{sub 2} to the Mn(II) ion, and mixing with class formers to produce a melter feed. The process minimizes production of hydrogen due to noble metal-catalyzed formic acid decomposition during, treatment, while producing a redox-balanced feed for effective melter operation and a quality glass product. An important feature of the present invention is the use of different acidifying and reducing, agents to treat the wastes. The nitric acid acidifies the wastes to improve yield stress and supplies acid for various reactions; then the formic acid reduces mercury compounds to elemental mercury and MnO{sub 2}) to the Mn(II) ion. When the pH of the waste is lower, reduction of mercury compounds and MnO{sub 2}) is faster and less formic acid is needed, and the production of hydrogen caused by catalytically-active noble metals is decreased.

  2. High temperature and pressure alkaline electrolysis

    DEFF Research Database (Denmark)

    Allebrod, Frank; Chatzichristodoulou, Christodoulos; Mogensen, Mogens Bjerg

    2013-01-01

    and pressures. Two measurement systems were built to perform measurements under high pressures and at elevated temperatures of up to 95 bar and 250 °C, respectively. The conductivity of aqueous KOH and aqueous KOH immobilized in a porous SrTiO3 structure were investigated at elevated temperatures and high...... the operational temperature and pressure to produce pressurized hydrogen at high rate (m3 H2·h-1·m-2 cell area) and high electrical efficiency. This work describes an exploratory technical study of the possibility to produce hydrogen and oxygen with a new type of alkaline electrolysis cell at high temperatures...... concentrations of the electrolyte using the van der Pauw method in combination with electrochemical impedance spectroscopy (EIS). Conductivity values as high as 2.9 S cm-1 for 45 wt% KOH aqueous KOH and 0.84 S cm-1 for the immobilized KOH of the same concentration were measured at 200 °C. Porous SrTiO3 was used...

  3. 乳源嗜冷菌产胞外耐热酶检测方法的对比分析%Comparing Detection Methods for Extracellular Thermostable Enzymes Secreted by Psychrophile in Milk

    Institute of Scientific and Technical Information of China (English)

    王伟军; 李延华

    2015-01-01

    Extracellular protease and lipase could be secreted by psychrotrophic bacteria in dairy products. These enzymes directly influenced the quality of dairy products. The study described the species of psychrophile isolated from milk, pointing out that pseudomonas fluorescens were the main strains which could metabolize protease and lipase. Thermal stability of these enzymes was elaborated and the methods of detection them were compared. It was expected to provide theoretical thoughts to predict the amount of contaminated psychrophile in dairy and dairy products, online detect and control the activity of thermostable enzyme and improve the quality of dairy products.%乳及乳制品中污染的嗜冷菌可分泌耐热的胞外蛋白酶和脂肪酶,直接影响产品品质。介绍从乳体系中分离鉴定的嗜冷菌种类,指出荧光假单胞菌是产胞外蛋白酶和脂肪酶的主要嗜冷菌菌株;分别阐述乳中污染的嗜冷菌所分泌蛋白酶和脂肪酶的热稳定性,并比较乳体系中耐热酶的测定方法,期望为有效预测乳中嗜冷菌污染程度、在线检测和控制耐热酶活性、提升乳制品的质量提供理论参考。

  4. Optimization of Soilless Media for Alkaline Irrigation Water

    OpenAIRE

    Tramp, Cody Alexander; Chard, Julie K.; Bugbee, Bruce

    2009-01-01

    High root zone pH reduces nutrient availability and high alkalinity water is strongly buffered around an alkaline pH. Soilless media can be altered to improve nutrient availability. This study was conducted to optimize the composition of soilless media for use with high alkalinity water. Mixes of peat and/or perlite or vermiculite in 50/50 and 33/33/33 volumetric ratios were tested. In some studies, mixes were also amended with up to 2.4 g/L of dolomite limestone to neutralize the initial aci...

  5. Mixed alkaline earth effect in sodium aluminosilicate glasses

    DEFF Research Database (Denmark)

    Kjeldsen, Jonas; Smedskjær, Morten Mattrup; Mauro, John C.

    2013-01-01

    While the mixed alkali effect has received significant attention in the glass literature, the mixed alkaline earth effect has not been thoroughly studied. Here, we investigate the latter effect by partial substitution of magnesium for calcium in sodium aluminosilicate glasses. We use Raman and NMR...... spectroscopies to obtain insights into the structural and topological features of these glasses, and hence into the mixed alkaline earth effect. We demonstrate that the mixed alkaline earth effect manifests itself as a maximum in the amount of bonded tetrahedral units and as a minimum in liquid fragility index...

  6. Inhibition of homocysteine-induced endoplasmic reticulum stress and endothelial cell damage by l-serine and glycine.

    Science.gov (United States)

    Sim, Woo-Cheol; Han, Inhoi; Lee, Wonseok; Choi, You-Jin; Lee, Kang-Yo; Kim, Dong Gwang; Jung, Seung-Hwan; Oh, Seon-Hee; Lee, Byung-Hoon

    2016-08-01

    Hyperhomocysteinemia is an independent risk factor for several cardiovascular diseases. The use of vitamins to modulate homocysteine metabolism substantially lowers the risk by reducing plasma homocysteine levels. In this study, we evaluated the effects of l-serine and related amino acids on homocysteine-induced endoplasmic reticulum (ER) stress and endothelial cell damage using EA.hy926 human endothelial cells. Homocysteine treatment decreased cell viability and increased apoptosis, which were reversed by cotreatment with l-serine. l-Serine inhibited homocysteine-induced ER stress as verified by decreased glucose-regulated protein 78kDa (GRP78) and C/EBP homologous protein (CHOP) expression as well as X-box binding protein 1 (xbp1) mRNA splicing. The effects of l-serine on homocysteine-induced ER stress are not attributed to intracellular homocysteine metabolism, but instead to decreased homocysteine uptake. Glycine exerted effects on homocysteine-induced ER stress, apoptosis, and cell viability that were comparable to those of l-serine. Although glycine did not affect homocysteine uptake or export, coincubation of homocysteine with glycine for 24h reduced the intracellular concentration of homocysteine. Taken together, l-serine and glycine cause homocysteine-induced endothelial cell damage by reducing the level of intracellular homocysteine. l-Serine acts by competitively inhibiting homocysteine uptake in the cells. However, the mechanism(s) by which glycine lowers homocysteine levels are unclear.

  7. Optimisation of freeze drying conditions for purified serine protease from mango (Mangifera indicaCv. Chokanan) peel.

    Science.gov (United States)

    Mehrnoush, Amid; Tan, Chin Ping; Hamed, Mirhosseini; Aziz, Norashikin Ab; Ling, Tau Chuan

    2011-09-01

    This study investigated the possible relationship between the encapsulation variables, namely serine protease content (9-50mg/ml, X1), Arabic gum (0.2-10%(w/w), X2), maltodextrin (2-5%(w/w), X3) and calcium chloride (1.3-5.5%(w/w), X4) on the enzymatic properties of encapsulated serine protease. The study demonstrated that Arabic gum, maltodextrin and calcium chloride, as coating agents, protected serine protease from activity loss during freeze-drying. The overall optimum region resulted in a suitable freeze drying condition with a yield of 92% for the encapsulated serine protease, were obtained using 29.5mg/ml serine protease content, 5.1%(w/w) Arabic gum, 3.5%(w/w) maltodextrin and 3.4%(w/w) calcium chloride. It was found that the interaction effect of Arabic gum and calcium chloride improved the serine protease activity, and Arabic gum was the most effective amongst the examined coating agents. Thus, Arabic gum should be considered as potential protection in freeze drying of serine protease.

  8. Serine dipeptide lipids of Porphyromonas gingivalis inhibit osteoblast differentiation: Relationship to Toll-like receptor 2.

    Science.gov (United States)

    Wang, Yu-Hsiung; Nemati, Reza; Anstadt, Emily; Liu, Yaling; Son, Young; Zhu, Qiang; Yao, Xudong; Clark, Robert B; Rowe, David W; Nichols, Frank C

    2015-12-01

    Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential

  9. Endogenous D-serine contributes to NMDA-receptor-mediated light-evoked responses in the vertebrate retina.

    Science.gov (United States)

    Gustafson, Eric C; Stevens, Eric R; Wolosker, Herman; Miller, Robert F

    2007-07-01

    We have combined electrophysiology and chemical separation and measurement techniques with capillary electrophoresis (CE) to evaluate the role of endogenous d-serine as an NMDA receptor (NMDAR) coagonist in the salamander retina. Electrophysiological experiments were carried out using whole cell recordings from retinal ganglion cells and extracellular recordings of the proximal negative response (PNR), while bath applying two D-serine degrading enzymes, including d-amino acid oxidase (DAAO) and D-serine deaminase (DsdA). The addition of either enzyme resulted in a significant and rapid decline in the light-evoked responses observed in ganglion cell and PNR recordings. The addition of exogenous D-serine in the presence of the enzymes restored the light-evoked responses to the control or supracontrol amplitudes. Heat-inactivated enzymes had no effect on the light responses and blocking NMDARs with AP7 eliminated the suppressive influence of the enzymes as well as the response enhancement normally associated with exogenous d-serine application. CE was used to separate amino acid racemates and to study the selectivity of DAAO and DsdA against D-serine and glycine. Both enzymes showed high selectivity for D-serine without significant effects on glycine. Our results strongly support the concept that endogenous D-serine plays an essential role as a coagonist for NMDARs, allowing them to contribute to the light-evoked responses of retinal ganglion cells. Furthermore under our experimental conditions, these coagonist sites are not saturated so that modulation of NMDAR sensitivity can be achieved with further modulaton of d-serine.

  10. Sulfate—Exchange Alkalinity of Ferralsol Colloid

    Institute of Scientific and Technical Information of China (English)

    ZHANGGANGYA; ZHANGXIAONIAN

    1999-01-01

    The amount of OH- replaced by sulfate,i.e.,sulfate-exchange alkalinity,from the electric double layer of ferralsol colloid was measured quantitatively in different conditions with an automatic titration equipment.The amount of OH- release increased with the amount of Na2SO4 added and decreased with raising pH in the suspension of ferralsol colloid.The exchange acidity was displayed as pH was higher than 5.6,If the negative effect of sodium ions was offset,the amount of OH- replaced by sulfate was larger than the original amount of OH- released in the pH range of lower than 5.8.The amount of OH- released decreased rapidly as pH was higher than 6.0 and dropped to zero when pH reached 6.5.In the solution of 2.0molL-1 NaClO4,the amount of OH- repleaced by sulfate from the surface of ferralsol colloid could be considered as the amount of OH- adsorbed by ligand exchange reaction.The amount of OH- released in the solution of NaClO4 concentration below 2.0mol L-1 from which the amount of OH- adsorbed by ligand exchange reaction was subtracted could be conidered as the OH- adsorbed by electrostatic force,The OH- adsorbed by electrostatic force decreased with increases in the concentration of NaClO4 and pH and increased almost linearly with the increasing amount of Na2SON4 added.The percentages of OH- adsorbed by electrostatic force in water and in the electrolyte solutions of 0.05 and 0.5mol L-1 NaClO4 in the total OH- released were calculated,respectively.

  11. Photovoltaic hydrogen production with commercial alkaline electrolysers

    Energy Technology Data Exchange (ETDEWEB)

    Ursua, A.; Lopez, J.; Gubia, E.; Marroyo, L.; Sanchis, P. [Public Univ. of Navarra, Pamplona (Spain). Dept. of Electric and Electronic Engineering

    2010-07-01

    Renewable energy sources and Electrolysis generate the so-called green Hydrogen, a zero-emission and potentially fossil fuel independent energy source. However, the inherent variability of the renewable energy sources implies a mode of operation for which most current electrolysers have not been designed. This paper analyses the operation of a water electrolyser fed with photovoltaic (PV) generator electric profile. The system, Integrated by a 1 Nm{sup 3}/h Hydrogenics alkaline electrolyser and a 5100 W PV generator with 60 BP585 modules, is installed at the Public University of Navarra (Spain). The PV generator profile fed to the electrolyser is emulated by a custom-made apparatus designed and built by the authors of this paper. The profile is designed according to real irradiance data measured by a calibration cell. The irradiance data are converted to the electric power profile that the PV generator would have delivered in case of having been connected to the electrolyser by means of a DC/DC converter with maximum power point tracking (MPPT). Finally, from previously measured power-current electrolyser characteristic curves, the current profile to be delivered to the electrolyser is obtained and programmed to the electronic device. The electrolyser was tested for two types of days. During the first day, the irradiance was very stable, whereas during the second day, the irradiance was very variable. The experimental results show an average power consumption rate and an efficiency of 4908 Wh/Nm{sup 3} and 72.1%, on the first day, and 4842 Wh/Nm{sup 3} and 73.3% on the second day. The electrolyser performance was particularly good in spite of the high variability of the electric supply of the second day. (orig.)

  12. Chlorine solubility in evolved alkaline magmas

    Directory of Open Access Journals (Sweden)

    M. R. Carroll

    2005-06-01

    Full Text Available Experimental studies of Cl solubility in trachytic to phonolitic melts provide insights into the capacity of alkaline magmas to transport Cl from depth to the earth?s surface and atmosphere, and information on Cl solubility variations with pressure, temperature and melt or fluid composition is crucial for understanding the reasons for variations in Cl emissions at active volcanoes. This paper provides a brief review of Cl solubility experiments conducted on a range of trachytic to phonolitic melt compositions. Depending on the experimental conditions the melts studied were in equilibrium with either a Cl-bearing aqueous fluid or a subcritical assemblage of low- Cl aqueous fluid + Cl-rich brine. The nature of the fluid phase(s was identified by examination of fluid inclusions present in run product glasses and the fluid bulk composition was calculated by mass balance. Chlorine concentrations in the glass increase with increasing Cl molality in the fluid phase until a plateau in Cl concentration is reached when melt coexists with aqueous fluid + brine. With fluids of similar Cl molality, higher Cl concentrations are observed in peralkaline phonolitic melts compared with peraluminous phonolitic melts; overall the Cl concentrations observed in phonolitic and trachytic melts are approximately twice those found in calcalkaline rhyolitic melts under similar conditions. The observed negative pressure dependence of Cl solubility implies that Cl contents of melts may actually increase during magma decompression if the magma coexists with aqueous fluid and Cl-rich brine (assuming melt-vapor equilibrium is maintained. The high Cl contents (approaching 1 wt% Cl observed in some melts/glasses from the Vesuvius and Campi Flegrei areas suggest saturation with a Cl-rich brine prior to eruption.

  13. Oxygen electrodes for rechargeable alkaline fuel cells

    Science.gov (United States)

    Swette, L.; Kackley, N.

    1989-12-01

    Electrocatalysts and supports for the positive electrode of moderate temperature single-unit rechargeable alkaline fuel cells are being investigated and developed. Candidate support materials were drawn from transition metal carbides, borides, nitrides and oxides which have high conductivity (greater than 1 ohm/cm). Candidate catalyst materials were selected largely from metal oxides of the form ABO sub x (where A = Pb, Cd, Mn, Ti, Zr, La, Sr, Na, and B = Pt, Pd, Ir, Ru, Ni (Co) which were investigated and/or developed for one function only, O2 reduction or O2 evolution. The electrical conductivity requirement for catalysts may be lower, especially if integrated with a higher conductivity support. All candidate materials of acceptable conductivity are subjected to corrosion testing. Materials that survive chemical testing are examined for electrochemical corrosion activity. For more stringent corrosion testing, and for further evaluation of electrocatalysts (which generally show significant O2 evolution at at 1.4 V), samples are held at 1.6 V or 0.6 V for about 100 hours. The surviving materials are then physically and chemically analyzed for signs of degradation. To evaluate the bifunctional oxygen activity of candidate catalysts, Teflon-bonded electrodes are fabricated and tested in a floating electrode configuration. Many of the experimental materials being studied have required development of a customized electrode fabrication procedure. In advanced development, the goal is to reduce the polarization to about 300 to 350 mV. Approximately six support materials and five catalyst materials were identified to date for further development. The test results will be described.

  14. Alkaline Peroxide Delignification of Corn Stover

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Ashutosh [Biosciences; Katahira, Rui [National; Donohoe, Bryon S. [Biosciences; Black, Brenna A. [National; Pattathil, Sivakumar [Complex; Stringer, Jack M. [National; Beckham, Gregg T. [National

    2017-05-30

    Selective biomass fractionation into carbohydrates and lignin is a key challenge in the conversion of lignocellulosic biomass to fuels and chemicals. In the present study, alkaline hydrogen peroxide (AHP) pretreatment was investigated to fractionate lignin from polysaccharides in corn stover (CS), with a particular emphasis on the fate of the lignin for subsequent valorization. The influence of peroxide loading on delignification during AHP pretreatment was examined over the range of 30-500 mg H2O2/g dry CS at 50 degrees C for 3 h. Mass balances were conducted on the solid and liquid fractions generated after pretreatment for each of the three primary components, lignin, hemicellulose, and cellulose. AHP pretreatment at 250 mg H2O2/g dry CS resulted in the pretreated solids with more than 80% delignification consequently enriching the carbohydrate fraction to >90%. Two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy of the AHP pretreated residue shows that, under high peroxide loadings (>250 mg H2O2/g dry CS), most of the side chain structures were oxidized and the aryl-ether bonds in lignin were partially cleaved, resulting in significant delignification of the pretreated residues. Gel permeation chromatography (GPC) analysis shows that AHP pretreatment effectively depolymerizes CS lignin into low molecular weight (LMW) lignin fragments in the aqueous fraction. Imaging of AHP pretreated residues shows a more granular texture and a clear lamellar pattern in secondary walls, indicative of layers of varying lignin removal or relocalization. Enzymatic hydrolysis of this pretreated residue at 20 mg/g of glucan resulted in 90% and 80% yields of glucose and xylose, respectively, after 120 h. Overall, AHP pretreatment is able to selectively remove more than 80% of the lignin from biomass in a form that has potential for downstream valorization processes and enriches the solid pulp into a highly digestible material.

  15. Marked Transient Alkaline Phosphatemia Following Pediatric Liver Transplantation

    Science.gov (United States)

    Koneru, Baburao; Carone, Eduardo; Malatack, J. Jeffrey; Esquivel, Carlos O.; Starzl, Thomas E.

    2010-01-01

    An isolated marked transient rise in serum alkaline phosphatase levels in otherwise healthy children is a well-documented occurrence. However, in children undergoing liver transplantation, elevated alkaline phosphatase values raise the possibility of biliary obstruction, rejection, or both. During a 6-year period, 6 of 278 children undergoing liver transplantation exhibited a similar phenomenon as an isolated abnormality. None had rejection, biliary obstruction, or other allograft dysfunction during a long follow-up. Eventually and without intervention, the alkaline phosphatase levels returned to normal. These instructive cases suggest that caution be used in advocating Invasive procedures if elevated alkaline phosphatase levels are an isolated abnormality, and close observation with noninvasive testing is recommended. PMID:2658549

  16. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... sodium chloride concentration), production by submerged fermentation and analytical ... subtilis AKRS3 for alkaline protease production indicated that 3% of .... using eight different carbon sources namely, glucose, lactose,.

  17. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline...... proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically...... active molecules and carriers, and in synthesis of carbohydrate derivatives with designed functional properties.  When it comes to regio-selectivity of alkaline proateses on carbohydrates both the properties of the particular enzyme and the influence of the solvent is determining for the position...

  18. Microbial alkaline pectinases and their industrial applications: a review.

    Science.gov (United States)

    Hoondal, G S; Tiwari, R P; Tewari, R; Dahiya, N; Beg, Q K

    2002-08-01

    The biotechnological potential of pectinolytic enzymes from microorganisms has drawn a great deal of attention from various researchers worldwide as likely biological catalysts in a variety of industrial processes. Alkaline pectinases are among the most important industrial enzymes and are of great significance in the current biotechnological arena with wide-ranging applications in textile processing, degumming of plant bast fibers, treatment of pectic wastewaters, paper making, and coffee and tea fermentations. The present review features the potential applications and uses of microbial alkaline pectinases, the nature of pectin, and the vast range of pectinolytic enzymes that function to mineralize pectic substances present in the environment. It also emphasizes the environmentally friendly applications of microbial alkaline pectinases thereby revealing their underestimated potential. The review intends to explore the potential of these enzymes and to encourage new alkaline pectinase-based industrial technology.

  19. Combined wet oxidation and alkaline hydrolysis of polyvinylchloride

    DEFF Research Database (Denmark)

    Sørensen, E.; Bjerre, A.B.

    1992-01-01

    In view of the widespread aversion to burning polyvinylchloride (PVC) together with municipal waste, we have attempted an alternative approach to its decomposition. This paper describes a combined wet oxidation/alkaline hydrolysis yielding water soluble, biodegradable products. Experiments were...

  20. Effects of alkaline treatment for fibroblastic adhesion on titanium

    Directory of Open Access Journals (Sweden)

    Miryam Cuellar-Flores

    2016-01-01

    Conclusion: The treatment of Ti plates with NaOH enhances cell adhesion and the proliferation of HPLF cells. Clinically, the alkaline treatment of Ti-based implants could be an option to improve and accelerate osseointegration.

  1. Alkaline protease production on date waste by an alkalophilic ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... activity and stability towards anionic surfactants like SDS and oxidants ... Alkaline protease activity was determined by the method of. Higahara et al. ... rpm, the media was analyzed for cell mass, total protein concentration ...

  2. Optimization of alkaline protease production by Streptomyces sp ...

    African Journals Online (AJOL)

    Hacene

    2016-06-29

    Jun 29, 2016 ... The results showed the presence of an alkaline protease with optimal pH and ... significant effect on the production of the enzyme (fructose and malt extract), then defining theirs ..... Urease test. + ..... terminating inhibitors. Proc.

  3. Palladium-based nanocatalysts for alcohol electrooxidation in alkaline media

    CSIR Research Space (South Africa)

    Modibedi, RM

    2013-01-01

    Full Text Available Direct alcohol alkaline fuel cells (DAAFCs) are potential power sources for a variety of portable applications as they provide unique advantages over hydrogen-based fuel cell devices. Alcohols (such as methanol, ethanol, ethylene glycol...

  4. AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at COOH-terminal serine residues

    DEFF Research Database (Denmark)

    Assentoft, Mette; Larsen, Brian R; Olesen, Emma T B;

    2014-01-01

    . Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser...... heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4...

  5. Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1) at Serine 284 and Serine 292 during Mitosis.

    Science.gov (United States)

    Xu, Chang; Wang, Yan; Wang, Lu; Wang, Qin; Du, Li-Qing; Fan, Saijun; Liu, Qiang; Li, Lei

    2015-11-04

    Chromosome instability usually leads to tumorigenesis. Bloom syndrome (BS) is a genetic disease associated with chromosome instability. The BS gene product, BLM, has been reported to function in the spindle assembly checkpoint (SAC) to prevent chromosome instability. BTR complex, composed of BLM, topoisomerase IIIα (Topo IIIα), RMI1 (RecQ-mediated genome instability protein 1, BLAP75) and RMI2 (RecQ-mediated genome instability protein 2, BLAP18), is crucial for maintaining genome stability. Recent work has demonstrated that RMI2 also plays critical role in SAC. However, little is know about RMI1 regulation during the cell cycle. Here we present that RMI1 protein level does not change through G1, S and G2 phases, but significantly increases in M phase. Moreover, phosphorylation of RMI1 occurs in mitosis. Upon microtubule-disturbing agent, RMI1 is phosphorylated primarily at the sites of Serine 284 and Serine 292, which does not interfere with the formation of BTR complex. Additionally, this phosphorylation is partially reversed by roscovitine treatment, implying cycling-dependent kinase 1 (CDK1) might be one of the upstream kinases.

  6. Accumulation and Phosphorylation of RecQ-Mediated Genome Instability Protein 1 (RMI1 at Serine 284 and Serine 292 during Mitosis

    Directory of Open Access Journals (Sweden)

    Chang Xu

    2015-11-01

    Full Text Available Chromosome instability usually leads to tumorigenesis. Bloom syndrome (BS is a genetic disease associated with chromosome instability. The BS gene product, BLM, has been reported to function in the spindle assembly checkpoint (SAC to prevent chromosome instability. BTR complex, composed of BLM, topoisomerase IIIα (Topo IIIα, RMI1 (RecQ-mediated genome instability protein 1, BLAP75 and RMI2 (RecQ-mediated genome instability protein 2, BLAP18, is crucial for maintaining genome stability. Recent work has demonstrated that RMI2 also plays critical role in SAC. However, little is know about RMI1 regulation during the cell cycle. Here we present that RMI1 protein level does not change through G1, S and G2 phases, but significantly increases in M phase. Moreover, phosphorylation of RMI1 occurs in mitosis. Upon microtubule-disturbing agent, RMI1 is phosphorylated primarily at the sites of Serine 284 and Serine 292, which does not interfere with the formation of BTR complex. Additionally, this phosphorylation is partially reversed by roscovitine treatment, implying cycling-dependent kinase 1 (CDK1 might be one of the upstream kinases.

  7. 丝氨酸羧肽酶及其类蛋白的研究进展%Research Progress of Serine Carboxypeptidases and Serine Carboxypeptidase-Like Proteins

    Institute of Scientific and Technical Information of China (English)

    叶玲飞; 罗光宇; 向建华; 刘爱玲; 陈信波

    2013-01-01

    Serine carboxypeptidases (SCP) widely exist in animals, plants and fungi and form a large protease family. This review briefly introduces the structural characteristics and classification of SCP and serine carboxy-peptidase-like proteins (SCPLs), and summarizes the current research advances of SCP/SCPLs in their subcel-lular localization, gene expression, the regulatory role in stress tolerance, cellular regulation and synthesis of plant secondary metabolites.%丝氨酸羧肽酶(SCP)是一个庞大的蛋白酶家族,普遍存在于动物、植物和真菌中.本文简要介绍了SCP及丝氨酸羧肽酶类蛋白(SCPLs)的结构特点和分类,综述了它们的亚细胞定位、基因表达水平、提高植物耐逆性、调控植物生长发育和影响次生代谢产物合成等方面的最新研究进展.

  8. Alkaline mineral water lowers bone resorption even in calcium sufficiency: alkaline mineral water and bone metabolism.

    Science.gov (United States)

    Wynn, Emma; Krieg, Marc-Antoine; Aeschlimann, Jean-Marc; Burckhardt, Peter

    2009-01-01

    Dietary acid charge enhances bone loss. Bicarbonate or alkali diet decreases bone resorption in humans. We compared the effect of an alkaline mineral water, rich in bicarbonate, with that of an acid one, rich in calcium only, on bone markers, in young women with a normal calcium intake. This study compared water A (per litre: 520 mg Ca, 291 mg HCO(3)(-), 1160 mg SO(4)(-), Potential Renal Acid load (PRAL) +9.2 mEq) with water B (per litre: 547 mg Ca, 2172 mg HCO(3)(-), 9 mg SO(4)(-), PRAL -11.2 mEq). 30 female dieticians aged 26.3 yrs (SD 7.3) were randomized into two groups, followed an identical weighed, balanced diet (965 mg Ca) and drank 1.5 l/d of the assigned water. Changes in blood and urine electrolytes, C-telopeptides (CTX), urinary pH and bicarbonate, and serum PTH were measured after 2 and 4 weeks. The two groups were not different at baseline, and showed a similar increase in urinary calcium excretion. Urinary pH and bicarbonate excretion increased with water B, but not with water A. PTH (p=0.022) and S-CTX (p=0.023) decreased with water B but not with water A. In calcium sufficiency, the acid calcium-rich water had no effect on bone resorption, while the alkaline water rich in bicarbonate led to a significant decrease of PTH and of S-CTX.

  9. Extracellular Alkalinization as a Defense Response in Potato Cells

    OpenAIRE

    Moroz, Natalia; Fritch, Karen R.; Marcec, Matthew J.; Tripathi, Diwaker; Smertenko, Andrei; Tanaka, Kiwamu

    2017-01-01

    A quantitative and robust bioassay to assess plant defense response is important for studies of disease resistance and also for the early identification of disease during pre- or non-symptomatic phases. An increase in extracellular pH is known to be an early defense response in plants. In this study, we demonstrate extracellular alkalinization as a defense response in potatoes. Using potato suspension cell cultures, we observed an alkalinization response against various pathogen- and plant-de...

  10. Physical processing efficiency of saline vs. alkaline spent batteries

    OpenAIRE

    Cabral,Marta; Nogueira, C. A.; Margarido, F.

    2008-01-01

    Physical processing of spent batteries which includes shredding and sieving operation is the first step for chemical treatment by hydrometallurgy. A laboratory study was carried out to evaluate physical processing efficiency, by analysing the resulting particle size, of alkaline and saline mignon-type Zn-MnO2 batteries. After shredding with a tip shredder, results obtained showed that alkaline batteries were more efficiently size reduced than saline batteries. Difference in particle size dist...

  11. Characterization and molecular cloning of a serine hydroxymethyltransferase 1 (OsSHM1) in rice.

    Science.gov (United States)

    Wang, Dekai; Liu, Heqin; Li, Sujuan; Zhai, Guowei; Shao, Jianfeng; Tao, Yuezhi

    2015-09-01

    Serine hydroxymethyltransferase (SHMT) is important for one carbon metabolism and photorespiration in higher plants for its participation in plant growth and development, and resistance to biotic and abiotic stresses. A rice serine hydroxymethyltransferase gene, OsSHM1, an ortholog of Arabidopsis SHM1, was isolated using map-based cloning. The osshm1 mutant had chlorotic lesions and a considerably smaller, lethal phenotype under natural ambient CO2 concentrations, but could be restored to wild type with normal growth under elevated CO2 levels (0.5% CO2 ), showing a typical photorespiratory phenotype. The data from antioxidant enzymes activity measurement suggested that osshm1 was subjected to significant oxidative stress. Also, OsSHM1 was expressed in all organs tested (root, culm, leaf, and young panicle) but predominantly in leaves. OsSHM1 protein is localized to the mitochondria. Our study suggested that molecular function of the OsSHM1 gene is conserved in rice and Arabidopsis.

  12. The importance of SERINE DECARBOXYLASE1 (SDC1) and ethanolamine biosynthesis during embryogenesis of Arabidopsis thaliana.

    Science.gov (United States)

    Yunus, Ian Sofian; Liu, Yu-Chi; Nakamura, Yuki

    2016-11-01

    In plants, ethanolamine is considered a precursor for the synthesis of choline, which is an essential dietary nutrient for animals. An enzyme serine decarboxylase (SDC) has been identified and characterized in Arabidopsis, which directly converts serine to ethanolamine, a precursor to phosphorylethanolamine and its subsequent metabolites in plants. However, the importance of SDC and ethanolamine production in plant growth and development remains unclear. Here, we show that SDC is required for ethanolamine biosynthesis in vivo and essential in plant embryogenesis in Arabidopsis. The knockout of SDC1 caused an embryonic lethal defect due to the developmental arrest of the embryos at the heart stage. During embryo development, the expression was observed at the later stages, at which developmental defect occurred in the knockout mutant. Overexpression of SDC1 in planta increased levels of ethanolamine, phosphatidylethanolamine, and phosphatidylcholine both in leaves and siliques. These results suggest that SDC1 plays an essential role in ethanolamine biosynthesis during the embryogenesis in Arabidopsis.

  13. Structural basis of serine/threonine phosphatase inhibition by the archetypal small molecules cantharidin and norcantharidin.

    Science.gov (United States)

    Bertini, I; Calderone, V; Fragai, M; Luchinat, C; Talluri, E

    2009-08-13

    The inhibition of a subgroup of human serine/threonine protein phosphatases is responsible for the cytotoxicity of cantharidin and norcantharidin against tumor cells. It is shown that the anhydride rings of cantharidin and norcantharidin are hydrolyzed when bound to the catalytic domain of the human serine/threonine protein phosphatases 5 (PP5c), and the high-resolution crystal structures of PP5c complexed with the corresponding dicarboxylic acid derivatives of the two molecules are reported. Norcantharidin shows a unique binding conformation with the catalytically active Mn2PP5c, while cantharidin is characterized by a double conformation in its binding mode to the protein. Different binding modes of norcantharidin are observed depending of whether the starting ligand is in the anhydride or in the dicarboxylic acid form. All these structures will provide the basis for the rational design of new cantharidin-based drugs.

  14. Characterization of the Usage of the Serine Metabolic Network in Human Cancer

    Directory of Open Access Journals (Sweden)

    Mahya Mehrmohamadi

    2014-11-01

    Full Text Available The serine, glycine, one-carbon (SGOC metabolic network is implicated in cancer pathogenesis, but its general functions are unknown. We carried out a computational reconstruction of the SGOC network and then characterized its expression across thousands of cancer tissues. Pathways including methylation and redox metabolism exhibited heterogeneous expression indicating a strong context dependency of their usage in tumors. From an analysis of coexpression, simultaneous up- or downregulation of nucleotide synthesis, NADPH, and glutathione synthesis was found to be a common occurrence in all cancers. Finally, we developed a method to trace the metabolic fate of serine using stable isotopes, high-resolution mass spectrometry, and a mathematical model. Although the expression of single genes didn’t appear indicative of flux, the collective expression of several genes in a given pathway allowed for successful flux prediction. Altogether, these findings identify expansive and heterogeneous functions for the SGOC metabolic network in human cancer.

  15. Nucleotide sequences of three tRNA(Ser) from Drosophila melanogaster reading the six serine codons.

    Science.gov (United States)

    Cribbs, D L; Gillam, I C; Tener, G M

    1987-10-05

    The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.

  16. Serine protease inhibitor 6-deficient mice have increased neutrophil immunity to Pseudomonas aeruginosa.

    Science.gov (United States)

    Zhang, Manling; Liu, Ni; Park, Sun-Mi; Wang, Yue; Byrne, Susan; Murmann, Andrea E; Bahr, Scott; Peter, Marcus E; Olson, Steven T; Belaaouaj, Abderrazzaq; Ashton-Rickardt, Philip G

    2007-10-01

    Inflammation is a localized, protective response to trauma or microbial invasion that destroys the injurious agent and the injured tissue. Neutrophil elastase (NE), a serine protease stored in the azurophil granules of polymorphonuclear neutrophils, digests microbes after phagocytosis. NE can also digest microbes extracellularly but is associated with tissue damage and inflammatory disease. In this study, we show that polymorphonuclear neutrophils from mice deficient in serine protease inhibitor 6, a weak intracellular NE inhibitor, had increased susceptibility to self-inflicted lysis because of increased NE activity. The resulting transient increase in local extracellular NE activity was within a narrow range that resulted in the clearance of Pseudomonas aeruginosa but did not damage the lung. Therefore, deficiency in a weak intracellular inhibitor of NE results in an acute inflammatory response that protects from P. aeruginosa but does not cause lung disease.

  17. The Occurrence of Type S1A Serine Proteases in Sponge and Jellyfish

    Science.gov (United States)

    Rojas, Ana; Doolittle, Russell F.

    2003-01-01

    Although serine proteases are found in all kinds of cellular organisms and many viruses, the classic "chymotrypsin family" (Group S1A by th e 1998 Barrett nomenclature) has an unusual phylogenetic distribution , being especially common in animals, entirely absent from plants and protists, and rare among fungi. The distribution in Bacteria is larg ely restricted to the genus Streptomyces, although a few isolated occ urrences in other bacteria have been reported. The family may be enti rely absent from Archaea. Although more than a thousand sequences have been reported for enzymes of this type from animals, none of them ha ve been from early diverging phyla like Porifera or Cnidaria, We now report the existence of Group SlA serine proteases in a sponge (phylu m Porifera) and a jellyfish (phylum Cnidaria), making it safe to conc lude that all animal groups possess these enzymes.

  18. Acid transformation of bauxite residue: Conversion of its alkaline characteristics.

    Science.gov (United States)

    Kong, Xiangfeng; Li, Meng; Xue, Shengguo; Hartley, William; Chen, Chengrong; Wu, Chuan; Li, Xiaofei; Li, Yiwei

    2017-02-15

    Bauxite residue (BR) is a highly alkaline solid hazardous waste produced from bauxite processing for alumina production. Alkaline transformation appears to reduce the environmental risk of bauxite residue disposal areas (BRDAs) whilst potentially providing opportunities for the sustainable reuse and on-going management of BR. Mineral acids, a novel citric acid and a hybrid combination of acid-gypsum treatments were investigated for their potential to reduce residue pH and total alkalinity and transform the alkaline mineral phase. XRD results revealed that with the exception of andradite, the primary alkaline solid phases of cancrinite, grossular and calcite were transformed into discriminative products based on the transformation used. Supernatants separated from BR and transformed bauxite residue (TBR) displayed distinct changes in soluble Na, Ca and Al, and a reduction in pH and total alkalinity. SEM images suggest that mineral acid transformations promote macro-aggregate formation, and the positive promotion of citric acid, confirming the removal or reduction in soluble and exchangeable Na. NEXAFS analysis of Na K-edge revealed that the chemical speciation of Na in TBRs was consistent with BR. Three acid treatments and gypsum combination had no effect on Na speciation, which affects the distribution of Na revealed by sodium STXM imaging. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Acid/alkaline ash diets: time for assessment and change.

    Science.gov (United States)

    Dwyer, J; Foulkes, E; Evans, M; Ausman, L

    1985-07-01

    The purpose of this article is to review critically the assumptions made to predict the effects of different diets on the pH of urine by calculations from food tables and lists of acid or alkaline ash in foods. Acid/alkaline ash calculations were completed for 7 days' worth of omnivore, lacto-ovo, and vegan diets. The vegetarian diets were significantly more alkaline than the omnivore diets, and the vegan diets were more alkaline than lacto-ovo vegetarian diets. The article discusses the history of the acid/alkaline ash concept, assumptions underlying it, mechanisms by which urine is acidified, how the acid/alkaline ash content of diets is calculated from food tables, difficulties arising in acid/ash calculations, and their validity in predicting urine pH. The authors conclude that while diet does influence the pH of urine, present calculation methods are time consuming, imprecise, and do not permit quantitative prediction of urine pH. Better methods for calculating the effects of diet on acid-base balance are needed.

  20. Cation exchange properties of zeolites in hyper alkaline aqueous media.

    Science.gov (United States)

    Van Tendeloo, Leen; de Blochouse, Benny; Dom, Dirk; Vancluysen, Jacqueline; Snellings, Ruben; Martens, Johan A; Kirschhock, Christine E A; Maes, André; Breynaert, Eric

    2015-02-03

    Construction of multibarrier concrete based waste disposal sites and management of alkaline mine drainage water requires cation exchangers combining excellent sorption properties with a high stability and predictable performance in hyper alkaline media. Though highly selective organic cation exchange resins have been developed for most pollutants, they can serve as a growth medium for bacterial proliferation, impairing their long-term stability and introducing unpredictable parameters into the evolution of the system. Zeolites represent a family of inorganic cation exchangers, which naturally occur in hyper alkaline conditions and cannot serve as an electron donor or carbon source for microbial proliferation. Despite their successful application as industrial cation exchangers under near neutral conditions, their performance in hyper alkaline, saline water remains highly undocumented. Using Cs(+) as a benchmark element, this study aims to assess the long-term cation exchange performance of zeolites in concrete derived aqueous solutions. Comparison of their exchange properties in alkaline media with data obtained in near neutral solutions demonstrated that the cation exchange selectivity remains unaffected by the increased hydroxyl concentration; the cation exchange capacity did however show an unexpected increase in hyper alkaline media.

  1. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  2. Purification and properties of detergent-compatible extracellular alkaline protease from Scopulariopsis spp.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2014-10-03

    A fungal alkaline protease of Scopulariopsis spp. was purified to homogeneity with a recovery of 32.2% and 138.1 U/mg specific activity on lectin-agarose column. The apparent molecular mass was 15 ± 1 kD by sodium dodecyl sulfate polyacryalamide gel electrophoresis (SDS-PAGE). It was a homogenous monomeric glycoprotein as shown by a single band and confirmed by native PAGE and gelatin zymography. The enzyme was active and stable over pH range 8.0-12.0 with optimum activity at pH 9.0. The maximum activity was recorded at 50°C and remained unaltered at 50°C for 24 hr. The enzyme was stimulated by Co(2+) and Mn(2+) at 10 mM but was unaffected by Ba(2+), Mg(2+), Cu(2+), Na(+), K(+), and Fe(2+). Ca(2+) and Fe(3+) moderately reduced the activity (∼18%); however, a reduction of about 40% was seen for Zn(2+) and Hg(2+). The enzyme activity was completely inhibited by 5 mM phenylmethylsulfonyl fluoride (PMSF) and partially by N-bromosuccinimide (NBS) and tocylchloride methylketone (TLCK). The serine, tryptophan, and histidine may therefore be at or near the active site of the enzyme. The protease was more active against gelatin compared to casein, fibrinogen, egg albumin, and bovine serum albumin (BSA). With casein as substrate, Km and Vmax were 4.3 mg/mL and 15.9 U/mL, respectively. An activation was observed with sodium dodecyl sulfate (SDS), Tween-80, and Triton X-100 at 2% (v/v); however, H2O2 and NaClO did not affect the protease activity. Storage stability was better for all the temperatures tested (-20, 4, and 28 ± 2°C) with a retention of more than 85% of initial activity after 40 days. The protease retained more than 50% activity after 24 hr of incubation at 28, 60, and 90°C in the presence (0.7%, w/v) of commercial enzymatic and nonenzymatic detergents. The Super Wheel-enzyme solution was able to completely remove blood staining, differing from the detergent solution alone. The stability at alkaline pH and high temperatures, broad substrate specificity

  3. Selective Inhibition of Plant Serine Hydrolases by Agrochemicals Revealed by Competitive ABPP

    OpenAIRE

    Kaschani, Farnusch; Nickel, Sabrina; Pandey, Bikram; Benjamin F Cravatt; Kaiser, Markus; van der Hoorn, Renier A L

    2011-01-01

    Organophosphate and –phosphonates and their thiol derivatives are often used in agroindustry as herbicides and insecticides, but their potential off-targets in the plant and their consumers are poorly investigated. Here, we use competitive Activity-based Protein Profiling (ABPP) of serine hydrolases (SHs) to detect targets of these agrochemicals and other compounds in Arabidopsis thaliana. Using broad-range and specific probes, and by overexpression of various SHs in planta, we are able to co...

  4. The Self-catalytic Esterification Reaction of O-Phosphoryl Serine Derivative

    Institute of Scientific and Technical Information of China (English)

    Jin Tang DU; Yan Mei LI; Zhong Zhou CHEN; Shi Zhong LUO; Yu Fen ZHAO

    2005-01-01

    O-Phosphoryl serine derivative can perform self-catalytic esterification reaction in the mixture of CH3OH and CHCl3 at the room temperature. The phosphoryl group participation was the key step of the esterification. This type of reactions were proposed through an intermediate of mixed phosphoric-carboxylic anhydride that might provide a clue to the function of the phosphoryl group in the phosphorylated enzymes and in the prebiotic synthesis of protein.

  5. Molecular Recognition of Cobalt(III)-ligated Peptides by Serine Proteases: The Role of Electrostatic Effects

    DEFF Research Database (Denmark)

    Bagger, Sven; Wagner, Kim

    1998-01-01

    A series of peptides with a positively charged cobalt(III)-complex group attached to the carboxylate terminal was synthesized. The behaviour of these metallopeptides as acceptor nucleophiles in acyl transfer reactions catalyzed by the three serine proteases bovine pancreatic à-chymotrypsin, porci...... and charged residues on the enzyme surfaces. The idea of using the metallopeptides in practical enzymatic peptide synthesis is put forward....

  6. Potential role of serine proteases in modulating renal sodium transport in vivo.

    Science.gov (United States)

    Jacquillet, G; Rubera, I; Unwin, R J

    2011-01-01

    The maintenance of sodium (Na+) homeostasis is an essential function of the kidney. It is achieved by a variety of transport processes localized all along the highly specialised segments of the nephron. Impairment of these transport mechanisms, and thereby Na+ handling, is associated with disturbed Na+ and water balance, leading to hypertension and oedema. This review focuses on the novel regulation of sodium reabsorption by serine proteases acting along the entire nephron.

  7. Distribution and evolution of the serine/aspartate racemase family in invertebrates.

    Science.gov (United States)

    Uda, Kouji; Abe, Keita; Dehara, Yoko; Mizobata, Kiriko; Sogawa, Natsumi; Akagi, Yuki; Saigan, Mai; Radkov, Atanas D; Moe, Luke A

    2016-02-01

    Free D-amino acids have been found in various invertebrate phyla, while amino acid racemase genes have been identified in few species. The purpose of this study is to elucidate the distribution, function, and evolution of amino acid racemases in invertebrate animals. We searched the GenBank databases, and found 11 homologous serine racemase genes from eight species in eight different invertebrate phyla. The cloned genes were identified based on their maximum activity as Acropora millepora (Cnidaria) serine racemase (SerR) and aspartate racemase (AspR), Caenorhabditis elegans (Nematoda) SerR, Capitella teleta (Annelida) SerR, Crassostrea gigas (Mollusca) SerR and AspR, Dugesia japonica (Platyhelminthes) SerR, Milnesium tardigradum (Tardigrada) SerR, Penaeus monodon (Arthropoda) SerR and AspR and Strongylocentrotus purpuratus (Echinodermata) AspR. We found that Acropora, Aplysia, Capitella, Crassostrea and Penaeus had two amino acid racemase paralogous genes and these paralogous genes have evolved independently by gene duplication at their recent ancestral species. The transcriptome analyses using available SRA data and enzyme kinetic data suggested that these paralogous genes are expressed in different tissues and have different functions in vivo. Phylogenetic analyses clearly indicated that animal SerR and AspR are not separated by their particular racemase functions and form a serine/aspartate racemase family cluster. Our results revealed that SerR and AspR are more widely distributed among invertebrates than previously known. Moreover, we propose that the triple serine loop motif at amino acid positions 150-152 may be responsible for the large aspartate racemase activity and the AspR evolution from SerR.

  8. Distinct and site-specific phosphorylation of the retinoblastoma protein at serine 612 in differentiated cells.

    Directory of Open Access Journals (Sweden)

    Takayuki Hattori

    Full Text Available The retinoblastoma susceptibility protein (pRB is a phosphoprotein that regulates cell cycle progression at the G1/S transition. In quiescent and early G1 cells, pRB predominantly exists in the active hypophosphorylated form. The cyclin/cyclin-dependent protein kinase complexes phosphorylate pRB at the late G1 phase to inactivate pRB. This event leads to the dissociation and activation of E2F family transcriptional factors. At least 12 serine/threonine residues in pRB are phosphorylated in vivo. Although there have been many reports describing bulk phosphorylation of pRB, detail research describing the function of each phosphorylation site remains unknown. Besides its G1/S inhibitory function, pRB is involved in differentiation, prevention of cell death and control of tissue fate. To uncover the function of phosphorylation of pRB in various cellular conditions, we have been investigating phosphorylation of each serine/threonine residue in pRB with site-specific phospho-serine/threonine antibodies. Here we demonstrate that pRB is specifically phosphorylated at Ser612 in differentiated cells in a known kinase-independent manner. We also found that pRB phosphorylated at Ser612 still associates with E2F-1 and tightly binds to nuclear structures including chromatin. Moreover, expression of the Ser612Ala mutant pRB failed to induce differentiation. The findings suggest that phosphorylation of Ser612 provides a distinct function that differs from the function of phosphorylation of other serine/threonine residues in pRB.

  9. Acanthamoeba polyphaga mimivirus prevents amoebal encystment-mediating serine proteinase expression and circumvents cell encystment.

    Science.gov (United States)

    Boratto, Paulo; Albarnaz, Jonas Dutra; Almeida, Gabriel Magno de Freitas; Botelho, Lucas; Fontes, Alide Caroline Lima; Costa, Adriana Oliveira; Santos, Daniel de Assis; Bonjardim, Cláudio Antônio; La Scola, Bernard; Kroon, Erna Geessien; Abrahão, Jônatas Santos

    2015-03-01

    Acanthamoeba is a genus of free-living amoebas distributed worldwide. Few studies have explored the interactions between these protozoa and their infecting giant virus, Acanthamoeba polyphaga mimivirus (APMV). Here we show that, once the amoebal encystment is triggered, trophozoites become significantly resistant to APMV. Otherwise, upon infection, APMV is able to interfere with the expression of a serine proteinase related to amoebal encystment and the encystment can no longer be triggered.

  10. Localization of a new serine protease, ingobsin, in goblet cells in rat, pig and man

    DEFF Research Database (Denmark)

    Poulsen, Steen Seier; Nexø, Ebba

    1985-01-01

    A serine protease, ingobsin, that cleaves Lys-x and Arg-x, has been purified from rat duodenal tissue. By immunohistochemical methods, the enzyme was localized in goblet cells in the small intestine of rat, pig, and man. The immunoreactive cells were most numerous in the proximal part...... of the intestine. In the electron microscope, the immunoreaction was localized mainly to the rough endoplasmic reticulum of the goblet cells and to the secretion being extruded from the cells....

  11. Alkali metal-cationized serine clusters studied by sonic spray ionization tandem mass spectrometry.

    Science.gov (United States)

    Nanita, Sergio C; Sokol, Ewa; Cooks, R Graham

    2007-05-01

    Serine solutions containing salts of alkali metals yield magic number clusters of the type (Ser(4)+C)(+), (Ser(8)+C)(+), (Ser(12)+C)(+), and (Ser(17)+2C)(+2) (where C = Li(+), Na(+), K(+), Rb(+), or Cs(+)), in relative abundances which are strongly dependent on the cation size. Strong selectivity for homochirality is involved in the formation of serine tetramers cationized by K(+), Rb(+), and Cs(+). This is also the case for the octamers cationized by the smaller alkalis but there is a strong preference for heterochirality in the octamers cationized by the larger alkali cations. Tandem mass spectrometry shows that the octamers and dodecamers cationized by K(+), Rb(+), and Cs(+) dissociate mainly by the loss of Ser(4) units, suggesting that the neutral tetramers are the stable building blocks of the observed larger aggregates, (Ser(8)+C)(+) and (Ser(12)+C)(+). Remarkably, although the Ser(4) units are formed with a strong preference for homochirality, they aggregate further regardless of their handedness and, therefore, with a preference for the nominally racemic 4D:4L structure and an overall strong heterochiral preference. The octamers cationized by K(+), Rb(+), or Cs(+) therefore represent a new type of cluster ion that is homochiral in its internal subunits, which then assemble in a random fashion to form octamers. We tentatively interpret the homochirality of these tetramers as a consequence of assembly of the serine molecules around a central metal ion. The data provide additional evidence that the neutral serine octamer is homochiral and is readily cationized by smaller ions.

  12. A serine protease inhibitor from hemolymph of green mussel, Perna viridis

    Digital Repository Service at National Institute of Oceanography (India)

    Khan, M.S.; Goswami, U.; Rojatkar, S.R.; Khan, M.I.

    of different bacteria were mixed and dissolved in 1500ll saline buffer. Animals were injected with 100ll of mixed cocktail of bacteria into the posterior adductor muscles. Hemolymph collection was done by slightly opening the animals with the help of forceps so...- sin and are novel anticoagulants isolated from the marine animals. 5 In vertebrates, serine protease inhibitors have been studied extensively and they are known to be involved in phagocytosis, coagulation, complement activation, fibrinolysis, blood...

  13. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

    Directory of Open Access Journals (Sweden)

    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  14. Effect of Plant Growth Regulators on Calcium-stimulated Serine Transport into Tobacco Cells

    Science.gov (United States)

    Smith, Ivan K.

    1978-01-01

    The transport of serine into tobacco cells (Nicotiana tabacum L.) cultured in liquid medium was examined. Transport was inhibited approximately 50% by 2,4-dichlorophenoxyacetic acid, indoleacetic acid, α-naphthalene acetic acid, and kinetin at a concentration of 10 micrograms per milliliter. Transport was not inhibited by 2,6-dichlorophenoxyacetic acid and inhibited less than 25% by p-chlorophenoxyacetic acid at this concentration. Removal of 2,4-dichlorophenoxyacetic acid from the transport medium resulted in an alleviation of inhibition. Gibberellic acid at concentrations from 2 to 20 micrograms per milliliter stimulated transport. It was previously shown that inhibition of transport by La3+ was due to removal of Ca2+ from surface sites and inhibition of Ca2+ uptake by cells. None of the growth regulators tested had any significant effect on Ca2+ binding and/or transport. A contributing factor to the low transport rates in the absence of Ca2+ is the increased rate of serine efflux. None of the growth regulators tested had any significant effect on the rate of serine efflux. PMID:16660646

  15. Cardioprotection by a novel recombinant serine protease inhibitor in myocardial ischemia and reperfusion injury.

    Science.gov (United States)

    Murohara, T; Guo, J P; Lefer, A M

    1995-09-01

    Polymorphonuclear neutrophils (PMN) play an important role in myocardial ischemia/reperfusion (MI/R) injury; however, the role of neutrophilic proteases is less understood. The effects of a novel serine protease inhibitor (serpin), LEX032, were investigated in a murine model of MI (20 min) and R (24 hr) injury in vivo. LEX032 is a recombinant human alpha 1-antichymotrypsin in which six amino acid residues were replaced around the active center with those of alpha-1 protease inhibitor. LEX032 has the ability to inhibit both neutrophil elastase and cathepsin G, two major neutral serine proteases in neutrophils, as well as superoxide generation. LEX032 (25 or 50 mg/kg) administered i.v. 1 min before reperfusion significantly attenuated myocardial necrotic injury evaluated by cardiac creatine kinase loss compared to MI/R rats receiving only vehicle (P LEX032 as compared with rats receiving vehicle (P LEX032 also moderately attenuated leukotriene B4-stimulated PMN adherence to rat superior mesenteric artery endothelium and markedly diminished superoxide radical release from LTB4-stimulated PMN in vitro. In a glycogen-induced rat peritonitis model, LEX032 (50 mg/kg) significantly attenuated PMN transmigration into the peritoneal cavity in vivo. In conclusion, the recombinant serine protease inhibitor, LEX032, appears to be an effective agent for attenuating MI/R injury by inhibiting neutrophil-accumulation into the ischemic-reperfused myocardium and by inactivating cytotoxic metabolites (proteases and superoxide radical) released from neutrophils.

  16. Artemether Exhibits Amoebicidal Activity against Acanthamoeba castellanii through Inhibition of the Serine Biosynthesis Pathway.

    Science.gov (United States)

    Deng, Yihong; Ran, Wei; Man, Suqin; Li, Xueping; Gao, Hongjian; Tang, Wei; Tachibana, Hiroshi; Cheng, Xunjia

    2015-08-01

    Acanthamoeba sp. parasites are the causative agents of Acanthamoeba keratitis, fatal granulomatous amoebic encephalitis, and cutaneous infections. However, there are currently no effective drugs for these organisms. Here, we evaluated the activity of the antimalarial agent artemether against Acanthamoeba castellanii trophozoites and identified potential targets of this agent through a proteomic approach. Artemether exhibited in vitro amoebicidal activity in a time- and dose-dependent manner and induced ultrastructural modification and cell apoptosis. The iTRAQ quantitative proteomic analysis identified 707 proteins that were differentially expressed after artemether treatment. We focused on phosphoglycerate dehydrogenase and phosphoserine aminotransferase in the serine biosynthesis pathway because of their importance to the growth and proliferation of protozoan and cancer cells. The expression of these proteins in Acanthamoeba was validated using quantitative real-time PCR and Western blotting after artemether treatment. The changes in the expression levels of phosphoserine aminotransferase were consistent with those of phosphoglycerate dehydrogenase. Therefore, the downregulation of phosphoserine aminotransferase may be due to the downregulation of phosphoglycerate dehydrogenase. Furthermore, exogenous serine might antagonize the activity of artemether against Acanthamoeba trophozoites. These results indicate that the serine biosynthesis pathway is important to amoeba survival and that targeting these enzymes would improve the treatment of Acanthamoeba infections. Artemether may be used as a phosphoglycerate dehydrogenase inhibitor to control or block Acanthamoeba infections.

  17. Modeling and structural analysis of evolutionarily diverse S8 family serine proteases.

    Science.gov (United States)

    Laskar, Aparna; Rodger, Euan James; Chatterjee, Aniruddha; Mandal, Chhabinath

    2011-01-01

    Serine proteases are an abundant class of enzymes that are involved in a wide range of physiological processes and are classified into clans sharing structural homology. The active site of the subtilisin-like clan contains a catalytic triad in the order Asp, His, Ser (S8 family) or a catalytic tetrad in the order Glu, Asp and Ser (S53 family). The core structure and active site geometry of these proteases is of interest for many applications. The aim of this study was to investigate the structural properties of different S8 family serine proteases from a diverse range of taxa using molecular modeling techniques. In conjunction with 12 experimentally determined three-dimensional structures of S8 family members, our predicted structures from an archaeon, protozoan and a plant were used for analysis of the catalytic core. Amino acid sequences were obtained from the MEROPS database and submitted to the LOOPP server for threading based structure prediction. The predicted structures were refined and validated using PROCHECK, SCRWL and MODELYN. Investigation of secondary structures and electrostatic surface potential was performed using MOLMOL. Encompassing a wide range of taxa, our structural analysis provides an evolutionary perspective on S8 family serine proteases. Focusing on the common core containing the catalytic site of the enzyme, the analysis presented here is beneficial for future molecular modeling strategies and structure-based rational drug design.

  18. Serine proteinase inhibitors in seeds of Cycas siamensis and other gymnosperms.

    Science.gov (United States)

    Konarev, Alexander V; Lovegrove, Alison; Shewry, Peter R

    2008-10-01

    Seeds of 32 species selected from two of the four major groups of gymnosperms, the ancient Cycadales and the economically important Coniferales, were analysed for inhibitors (I) of the serine proteinases trypsin (T), chymotrypsin (C), subtilisin (S) and elastase (E) using isoelectric focusing (IEF) combined with gelatin replicas. Subtilisin inhibitors were detected in 17 species, being particularly active in the Cycadales. Several species of the genera Cephalotaxus, Pseudotsuga and Cycas contained inhibitors active against elastase while strong CSTIs and CSIs were also present in Cycas pectinata and C. siamensis. No inhibitors were detected in seeds of Chamaecyparis, Thuja, Abies, Larix, Picea and Pinus spp. Serine proteinase inhibitors were purified from seeds of C. siamensis by affinity chromatography using trypsin and chymotrypsin, IEF and SDS-PAGE. Several CSTI components with M(r) ranging from 4000 to 18,000 were partially sequenced using Edman degradation and mass spectrometry. Most of the sequences were similar to a hypothetical protein encoded by an mRNA from sporophylls of C. rumphii which in turn was similar to Kunitz-type proteinase inhibitors from flowering plants. Analysis of expressed sequence tag (EST) databases confirmed the presence of mRNAs encoding Kunitz-type inhibitors in the Cycadales and Coniferales and also demonstrated their presence in a third major group of gymnosperms, the Ginkgoales. This is the first report of Kunitz-type serine proteinase inhibitors from plants other than Angiosperms.

  19. Structure-Based Mechanism for Early PLP-Mediated Steps of Rabbit Cytosolic Serine Hydroxymethyltransferase Reaction

    Directory of Open Access Journals (Sweden)

    Martino L. Di Salvo

    2013-01-01

    Full Text Available Serine hydroxymethyltransferase catalyzes the reversible interconversion of L-serine and glycine with transfer of one-carbon groups to and from tetrahydrofolate. Active site residue Thr254 is known to be involved in the transaldimination reaction, a crucial step in the catalytic mechanism of all pyridoxal 5′-phosphate- (PLP- dependent enzymes, which determines binding of substrates and release of products. In order to better understand the role of Thr254, we have expressed, characterized, and determined the crystal structures of rabbit cytosolic serine hydroxymethyltransferase T254A and T254C mutant forms, in the absence and presence of substrates. These mutants accumulate a kinetically stable gem-diamine intermediate, and their crystal structures show differences in the active site with respect to wild type. The kinetic and crystallographic data acquired with mutant enzymes permit us to infer that conversion of gem-diamine to external aldimine is significantly slowed because intermediates are trapped into an anomalous position by a misorientation of the PLP ring, and a new energy barrier hampers the transaldimination reaction. This barrier likely arises from the loss of the stabilizing hydrogen bond between the hydroxymethyl group of Thr254 and the ε-amino group of active site Lys257, which stabilizes the external aldimine intermediate in wild type SHMTs.

  20. Histone H3 Serine 28 Is Essential for Efficient Polycomb-Mediated Gene Repression in Drosophila

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    Philip Yuk Kwong Yung

    2015-06-01

    Full Text Available Trimethylation at histone H3K27 is central to the polycomb repression system. Juxtaposed to H3K27 is a widely conserved phosphorylatable serine residue (H3S28 whose function is unclear. To assess the importance of H3S28, we generated a Drosophila H3 histone mutant with a serine-to-alanine mutation at position 28. H3S28A mutant cells lack H3S28ph on mitotic chromosomes but support normal mitosis. Strikingly, all methylation states of H3K27 drop in H3S28A cells, leading to Hox gene derepression and to homeotic transformations in adult tissues. These defects are not caused by active H3K27 demethylation nor by the loss of H3S28ph. Biochemical assays show that H3S28A nucleosomes are a suboptimal substrate for PRC2, suggesting that the unphosphorylated state of serine 28 is important for assisting in the function of polycomb complexes. Collectively, our data indicate that the conserved H3S28 residue in metazoans has a role in supporting PRC2 catalysis.

  1. Osmotic stress-dependent serine phosphorylation of the histidine kinase homologue DokA

    Directory of Open Access Journals (Sweden)

    Oehme Felix

    2001-03-01

    Full Text Available Abstract Background Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. Results We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. Conclusions A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.

  2. Molecular characterization of a serine/threonine kinase in the DiGeorge minimal critical region.

    Science.gov (United States)

    Goldmuntz, E; Fedon, J; Roe, B; Budarf, M L

    1997-10-01

    The majority of patients with DiGeorge, velocardiofacial or conotruncal anomaly facial syndromes share a common genetic etiology, deletion of chromosomal region 22q11.2. This report describes a computational approach toward the identification and molecular characterization of a newly identified serine/threonine kinase from the minimal critical deleted region (MDGCR). A cosmid contig of the minimal critical region has been assembled and sequenced in its entirety. Database searches and computer analysis of one cosmid (111f11) for coding sequences identified two regions with high similarity to the mouse serine/threonine kinase, Tsk1. Our investigations demonstrate that one of these regions contains a testis-specific gene that undergoes differential splicing, while the other region is most likely a pseudogene. Northern blot analysis and cDNA cloning demonstrate that there is alternate processing of the 3'UTR without altering the conserved kinase domains within the open reading frame. Serine/threonine kinases can play a regulatory role and have been found to be expressed during early embryogenesis. Based on its position in the MDGCR and possible function, the gene reported here is a candidate for the features seen in the 22q11 deletion syndrome.

  3. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Science.gov (United States)

    Connolly, James P R; Gabrielsen, Mads; Goldstone, Robert J; Grinter, Rhys; Wang, Dai; Cogdell, Richard J; Walker, Daniel; Smith, David G E; Roe, Andrew J

    2016-01-01

    The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC) in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  4. A Highly Conserved Bacterial D-Serine Uptake System Links Host Metabolism and Virulence.

    Directory of Open Access Journals (Sweden)

    James P R Connolly

    2016-01-01

    Full Text Available The ability of any organism to sense and respond to challenges presented in the environment is critically important for promoting or restricting colonization of specific sites. Recent work has demonstrated that the host metabolite D-serine has the ability to markedly influence the outcome of infection by repressing the type III secretion system of enterohaemorrhagic Escherichia coli (EHEC in a concentration-dependent manner. However, exactly how EHEC monitors environmental D-serine is not understood. In this work, we have identified two highly conserved members of the E. coli core genome, encoding an inner membrane transporter and a transcriptional regulator, which collectively help to "sense" levels of D-serine by regulating its uptake from the environment and in turn influencing global gene expression. Both proteins are required for full expression of the type III secretion system and diversely regulated prophage-encoded effector proteins demonstrating an important infection-relevant adaptation of the core genome. We propose that this system acts as a key safety net, sampling the environment for this metabolite, thereby promoting colonization of EHEC to favorable sites within the host.

  5. Mirolase, a novel subtilisin-like serine protease from the periodontopathogen Tannerella forsythia.

    Science.gov (United States)

    Ksiazek, Miroslaw; Karim, Abdulkarim Y; Bryzek, Danuta; Enghild, Jan J; Thøgersen, Ida B; Koziel, Joanna; Potempa, Jan

    2015-03-01

    The genome of Tannerella forsythia, an etiological factor of chronic periodontitis, contains several genes encoding putative proteases. Here, we characterized a subtilisin-like serine protease of T. forsythia referred to as mirolase. Recombinant full-length latent promirolase [85 kDa, without its signal peptide (SP)] processed itself through sequential autoproteolytic cleavages into a mature enzyme of 40 kDa. Mirolase latency was driven by the N-terminal prodomain (NTP). In stark contrast to almost all known subtilases, the cleaved NTP remained non-covalently associated with mirolase, inhibiting its proteolytic, but not amidolytic, activity. Full activity was observed only after the NTP was gradually, and fully, degraded. Both activity and processing was absolutely dependent on calcium ions, which were also essential for enzyme stability. As a consequence, both serine protease inhibitors and calcium ions chelators inhibited mirolase activity. Activity assays using an array of chromogenic substrates revealed that mirolase specificity is driven not only by the substrate-binding subsite S1, but also by other subsites. Taken together, mirolase is a calcium-dependent serine protease of the S8 family with the unique mechanism of activation that may contribute to T. forsythia pathogenicity by degradation of fibrinogen, hemoglobin, and the antimicrobial peptide LL-37.

  6. Serine Protease Variants Encoded by Echis ocellatus Venom Gland cDNA: Cloning and Sequencing Analysis

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    S. S. Hasson

    2010-01-01

    Full Text Available Envenoming by Echis saw-scaled viper is the leading cause of death and morbidity in Africa due to snake bite. Despite its medical importance, there have been few investigations into the toxin composition of the venom of this viper. Here, we report the cloning of cDNA sequences encoding four groups or isoforms of the haemostasis-disruptive Serine protease proteins (SPs from the venom glands of Echis ocellatus. All these SP sequences encoded the cysteine residues scaffold that form the 6-disulphide bonds responsible for the characteristic tertiary structure of venom serine proteases. All the Echis ocellatus EoSP groups showed varying degrees of sequence similarity to published viper venom SPs. However, these groups also showed marked intercluster sequence conservation across them which were significantly different from that of previously published viper SPs. Because viper venom SPs exhibit a high degree of sequence similarity and yet exert profoundly different effects on the mammalian haemostatic system, no attempt was made to assign functionality to the new Echis ocellatus EoSPs on the basis of sequence alone. The extraordinary level of interspecific and intergeneric sequence conservation exhibited by the Echis ocellatus EoSPs and analogous serine proteases from other viper species leads us to speculate that antibodies to representative molecules should neutralise (that we will exploit, by epidermal DNA immunization the biological function of this important group of venom toxins in vipers that are distributed throughout Africa, the Middle East, and the Indian subcontinent.

  7. Purification and characterization of an alkaline protease from Bacillus licheniformis UV-9 for detergent formulations

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    Muhammad Nadeem

    2013-04-01

    Full Text Available Alkaline protease produced by mutant strain B. licheniformis UV-9 was purified and characterized for its exploitationin detergent formulation. The enzyme was purified to homogeneity by employing ammonium sulphate precipitation andsephadex G-100 gel filtration chromatography with a 36.83 fold increase in specific activity and 11% recovery. The molecularweight of the protease was found to be 36.12 kDa by SDS-PAGE. The Km and Vmax values exhibited by purified proteasewere 5 mg/ml and 61.58ìM/ml/min, respectively, using casein as substrate. The enzyme exhibited highest activity at pH 11 andtemperature 60°C. Stability studies showed that the enzyme retained higher than 80% residual activity in the pH and temperature ranges of 8 to 11 and 30 to 50°C, respectively. However, in the presence of 10 mM Ca2+ ions the enzyme tained morethan 90% of its residual activity at pH 11 and temperature 60°C. Phenyl methyl sulphonyl fluoride (PMSF completelyinhibited the enzyme activity suggesting that it was serine protease. Among metal ions, the Mg2+ and Ca2+ ions enhancedactivity up to 128% and 145%, respectively. The purified enzyme showed extreme stability towards various surfactantssuch as Tween-20, Tween- 45, Tween-65 and Triton X-45. In addition, the enzyme also exhibited more than 100% residualactivity in the presence of oxidizing agents, H2O2 and sodium perborate. These biochemical properties indicate the potentialuse of B. licheniformis UV-9 enzyme in laundry detergents.

  8. Purification and characterization of a unique alkaline elastase from Micrococcus luteus.

    Science.gov (United States)

    Clark, D J; Hawrylik, S J; Kavanagh, E; Opheim, D J

    2000-02-01

    Micrococcus luteus isolated from human skin secretes an alkaline protease which degrades elastin. M. luteus protease (MLP) was produced in the late logarithmic and stationary phases of growth. MLP, purified to homogeneity by a three-step process, had a molecular mass of 32,812 Da and an isoelectric point of 9.3. MLP was active and highly stable in solution for 24 h from pH 6.0 to 10.5; it had maximal activity at temperatures between 57 and 59 degrees C. The presence of calcium in the solution was essential for enzyme activity and to prevent autolysis. Optimal activity occurred between pH 9.0 and 9.5, with 60% maximal activity from pH 6.5 to 11.0. The enzyme was inhibited by the serine enzyme inhibitors phenylmethylsulfonyl fluoride and chymostatin but not by the metalloenzyme inhibitor 1,10-phenanthroline or sulfhydryl enzyme inhibitors. Casein, bovine serum albumin, ovalbumin, beta-lactoglobulin, and elastin were digested by the protease while collagen and keratin were resistant to digestion. MLP demonstrated both esterase and amidase activity on synthetic peptide substrates. MLP preferentially cleaved the Leu(15)-Tyr(16) and Phe(24)-Phe(25) bonds of the oxidized beta-chain of insulin. Longer digests of insulin and the pattern of activity against synthetic substrates suggest that MLP has a cleavage specificity for bulky, hydrophobic, or aromatic amino acids in the P(1) or P(1)' positions. Amino acid sequences from the N-terminus and internal peptides of MLP were unique.

  9. Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

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    Arlem Nascimento de Oliveira

    2010-10-01

    Full Text Available Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100 and stable in oxidizing (H2O2 and reducing agents (β-mercaptoethanol, and organic solvents (acetone, hexane, methanol, 1-propanol and toluene.Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100, e estáveis na presença de agentes oxidantes (H2O2 e redutores (β-mercaptoetanol e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno.

  10. Immunogold detection of L-glutamate and D-serine in small synaptic-like microvesicles in adult hippocampal astrocytes.

    Science.gov (United States)

    Bergersen, L H; Morland, C; Ormel, L; Rinholm, J E; Larsson, M; Wold, J F H; Røe, A T; Stranna, A; Santello, M; Bouvier, D; Ottersen, O P; Volterra, A; Gundersen, V

    2012-07-01

    Glutamate and the N-methyl-D-aspartate receptor ligand D-serine are putative gliotransmitters. Here, we show by immunogold cytochemistry of the adult hippocampus that glutamate and D-serine accumulate in synaptic-like microvesicles (SLMVs) in the perisynaptic processes of astrocytes. The estimated concentration of fixed glutamate in the astrocytic SLMVs is comparable to that in synaptic vesicles of excitatory nerve terminals (≈ 45 and ≈ 55 mM, respectively), whereas the D-serine level is about 6 mM. The vesicles are organized in small spaced clusters located near the astrocytic plasma membrane. Endoplasmic reticulum is regularly found in close vicinity to SLMVs, suggesting that astrocytes contain functional nanodomains, where a local Ca(2+) increase can trigger release of glutamate and/or D-serine.

  11. Structural basis of metallo-β-lactamase, serine-β-lactamase and penicillin-binding protein inhibition by cyclic boronates

    Science.gov (United States)

    Brem, Jürgen; Cain, Ricky; Cahill, Samuel; McDonough, Michael A.; Clifton, Ian J.; Jiménez-Castellanos, Juan-Carlos; Avison, Matthew B.; Spencer, James; Fishwick, Colin W. G.; Schofield, Christopher J.

    2016-08-01

    β-Lactamases enable resistance to almost all β-lactam antibiotics. Pioneering work revealed that acyclic boronic acids can act as `transition state analogue' inhibitors of nucleophilic serine enzymes, including serine-β-lactamases. Here we report biochemical and biophysical analyses revealing that cyclic boronates potently inhibit both nucleophilic serine and zinc-dependent β-lactamases by a mechanism involving mimicking of the common tetrahedral intermediate. Cyclic boronates also potently inhibit the non-essential penicillin-binding protein PBP 5 by the same mechanism of action. The results open the way for development of dual action inhibitors effective against both serine- and metallo-β-lactamases, and which could also have antimicrobial activity through inhibition of PBPs.

  12. Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

    DEFF Research Database (Denmark)

    Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

    2005-01-01

    processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

  13. Effects of structural changes in the dsdA-dsdC intergenic region on D-serine deaminase synthesis.

    OpenAIRE

    McFall, E; Nikam, S S; Palchaudhuri, S

    1991-01-01

    Single-base-pair changes well upstream of its transcription initiation site resulted in partially to fully constitutive expression of the D-serine deaminase structural gene, dsdA, independently of the cyclic AMP-cyclic AMP-binding protein complex and of the specific D-serine deaminase activator protein. These promoter mutations appear to define a consensus sequence that is repeated several times. Basal expression of dsdA+ was also strongly enhanced by subcloning on multicopy plasmids, by the ...

  14. Tryptophanase-Catalyzed l-Tryptophan Synthesis from d-Serine in the Presence of Diammonium Hydrogen Phosphate

    OpenAIRE

    Fujii Noriko; Haruka Ozaki; Takeshi Saito; Akihiko Shimada

    2009-01-01

    Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of L-tryptophan from L-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on D-serine. We previously reported that tryptophanase changed its stereospecificity to degrade D-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH4)2HPO4 solution. The present study provided the same stereospecific change seen in the...

  15. Tryptophanase-Catalyzed l-Tryptophan Synthesis from d-Serine in the Presence of Diammonium Hydrogen Phosphate

    OpenAIRE

    Shimada, Akihiko; Ozaki, Haruka; Saito, Takeshi; Fujii, Noriko

    2009-01-01

    Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of l-tryptophan from l-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on d-serine. We previously reported that tryptophanase changed its stereospecificity to degrade d-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH4)2HPO4 solution. The present study provided the same stereospecific change seen in the...

  16. L-serine treatment may improve neurorestoration of rats after permanent focal cerebral ischemia potentially through improvement of neurorepair.

    Directory of Open Access Journals (Sweden)

    Li Sun

    Full Text Available The present study was conducted to clarify whether treatment with L-serine can improve the brain repair and neurorestoration of rats after permanent middle cerebral artery occlusion (pMCAO. After pMCAO, the neurological functions, brain lesion volume, and cortical injury were determined. GDNF, NGF, NCAM L1, tenascin-C, and Nogo-A levels were measured. Proliferation and differentiation of the neural stem cells (NSCs and proliferation of the microvessels in the ischemic boundary zone of the cortex were evaluated. Treatment with L-serine (168 mg/kg body weight, i.p. began 3 h after pMCAO and was repeated every 12 h for 7 days or until the end of the experiment. L-Serine treatment: 1 reduced the lesion volume and neuronal loss; 2 improved the recovery of neurological functions; 3 elevated the expression of nerve growth-related factors; and 4 facilitated the proliferation of endogenous NSCs and microvessels activated after pMCAO and increased the number of new-born neurons. 5 D-cycloserine, an inhibitor of serine hydroxymethyltransferase, blunted the effects of L-serine on NSC proliferation, differentiation, microvascular proliferation. In conclusions, L-serine treatment in pMCAO rats can reduce brain injury and facilitate neurorestoration which is partly associated with the improvement of proliferation of NSCs and microvessels, reconstruction of neurovascular units and resultant neurorepair. The effects of L-serine on endogenous NSC proliferation and microvascular proliferation are partly mediated by the action of L-serine as a substrate for the production of one-carbon groups used for purine and pyrimidine synthesis and modulation of the expression of some nerve growth-related factors.

  17. An idiosyncratic serine ordering loop in methanogen seryl-tRNA synthetases guides substrates through seryl-tRNASer formation.

    Science.gov (United States)

    Dulic, Morana; Pozar, Josip; Bilokapic, Silvija; Weygand-Durasevic, Ivana; Gruic-Sovulj, Ita

    2011-10-01

    Seryl-tRNA synthetases (SerRS) covalently attach serine to cognate tRNA(Ser). Atypical SerRSs, considerably different from canonical enzymes, have been found in methanogenic archaea. A crystal structure of methanogenic-type SerRS revealed a motif within the active site (serine ordering loop; SOL), which undergoes a notable induced-fit rearrangement during serine binding. The loop rearranges from a disordered conformation in the unliganded enzyme, to an ordered structure comprising an α-helix followed by a loop. We performed kinetic and thermodynamic analyses of SerRS variants to establish the role of the SOL in serylation. Thermodynamic data confirmed a linkage between binding of serine and α-helix formation, previously described by the crystallographic analysis. The ability of the SOL to adopt the observed secondary structure was recognized as essential for serine activation. Mutation of Gln400, which according to the structural data establishes the main connection between the serine and the SOL, produced only modest kinetic effects. Kinetic data offer new insights into the coupling of the conformational change with active site assembly. Productive positioning of the SOL may be driven by the interaction between Trp396 and the serine α-amino group. Rapid kinetics reveals that His250, a non-SOL residue, is essential for transfer of serine to tRNA. Modeling data established that accommodation of the tRNA within the active site may require movement of the SOL. This would enable His250 to assist in productive positioning of the 3'-end of the tRNA for the aminoacyl transfer. Thus, the rearrangements of the SOL conformationally adjust the active site for both reaction steps.

  18. Purification and Functional Characterisation of Rhinocerase, a Novel Serine Protease from the Venom of Bitis gabonica rhinoceros

    Science.gov (United States)

    Vaiyapuri, Sakthivel; Harrison, Robert A.; Bicknell, Andrew B.; Gibbins, Jonathan M.; Hutchinson, Gail

    2010-01-01

    Background Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes. PMID:20300193

  19. Molekulare und funktionelle Charakterisierung der Serin/Threonin-Proteinkinase PknB und -Phosphatase Stp in Staphylococcus aureus

    OpenAIRE

    Donat, Stefanie

    2009-01-01

    Um Änderungen in seiner Umwelt wahrnehmen zu können, benötigt S. aureus unterschiedliche Signaltransduktionssysteme. In dieser Arbeit wurde erstmals die Eukaryoten-ähnliche Serin/Threonin-Proteinkinase (STPK) PknB umfassend charakterisiert. Die posttranslationale Proteinmodifikation mittels Phosphorylierung spielt sowohl in Eukaryoten als auch in Prokaryoten eine wichtige Rolle. Man glaubte lange, dass die Phosphorylierung von Serin-, Threonin- und Tyrosinresten ein nur auf Eukaryoten beschrä...

  20. Purification and functional characterisation of rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros.

    Directory of Open Access Journals (Sweden)

    Sakthivel Vaiyapuri

    Full Text Available BACKGROUND: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. METHODOLOGY/PRINCIPAL FINDINGS: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36 kDa, which reduces to 31 kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading alpha and beta chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. CONCLUSIONS/SIGNIFICANCE: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

  1. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

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    Hu Peng

    Full Text Available Endothelial nitric oxide synthase (eNOS is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-. in the absence of the cofactor tetrahydrobiopterin (BH4. Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM. S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS. The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (P<0.01. VEGF-mediated Serine 1179 phosphorylation further decreased the cellular viability in eNOS-overexpressing 293 cells (P<0.01. Our data demonstrate that eNOS serine 1179 phosphorylation, in addition to enhancing NO production, also profoundly affects superoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  2. Alkalinity Enrichment Enhances Net Calcification of a Coral Reef Flat

    Science.gov (United States)

    Albright, R.; Caldeira, K.

    2015-12-01

    Ocean acidification is projected to shift reefs from a state of net accretion to one of net dissolution sometime this century. While retrospective studies show large-scale changes in coral calcification over the last several decades, it is not possible to unequivocally link these results to ocean acidification due to confounding factors of temperature and other environmental parameters. Here, we quantified the calcification response of a coral reef flat to alkalinity enrichment to test whether reef calcification increases when ocean chemistry is restored to near pre-industrial conditions. We used sodium hydroxide (NaOH) to increase the total alkalinity of seawater flowing over a reef flat, with the aim of increasing carbonate ion concentrations [CO32-] and the aragonite saturation state (Ωarag) to values that would have been attained under pre-industrial atmospheric pCO2 levels. We developed a dual tracer regression method to estimate alkalinity uptake (i.e., calcification) in response to alkalinity enrichment. This approach uses the change in ratios between a non-conservative tracer (alkalinity) and a conservative tracer (a non-reactive dye, Rhodamine WT) to assess the fraction of added alkalinity that is taken up by the reef as a result of an induced increase in calcification rate. Using this method, we estimate that an average of 17.3% ± 2.3% of the added alkalinity was taken up by the reef community. In providing results from the first seawater chemistry manipulation experiment performed on a natural coral reef community (without artificial confinement), we demonstrate that, upon increase of [CO32-] and Ωarag to near pre-industrial values, reef calcification increases. Thus, we conclude that, the impacts of ocean acidification are already being felt by coral reefs. This work is the culmination of years of work in the Caldeira lab at the Carnegie Institution for Science, involving many people including Jack Silverman, Kenny Schneider, and Jana Maclaren.

  3. Evaluation of some bean lines tolerance to alkaline soil

    Directory of Open Access Journals (Sweden)

    Abeer A. Radi

    2012-01-01

    Full Text Available Introduction: In less arid climates, salts are less concentrated and sodium dominates in carbonate and bicarbonate forms, which enhance the formation of alkaline soils. The development and identification of salt-tolerant crop cultivars or lines would complement salt management programs to improve the productivity and yields of salt stressed plants.Materials and methods: This work was to study the evaluation of alkalinity tolerance of some bean lines grown under different levels of sodium carbonate (Na2CO3 to select the most alkalinity tolerant lines versus the most-sensitive ones out of 6 lines of the test plants.Results: The symptoms induced by alkalinity included reduction in root, shoot growth, and leaf area which were more severe in some bean lines. Potassium leakage was severely affected by alkalinity in some lines at all tested levels, while in some others a moderate damage was manifested only at the higher levels. The increase in Na2CO3 level was associated with a gradual fall in chlorophyll a and b biosynthesis of all the test bean lines. However, alkalinity at low and moderate levels had a favorable effect on the biosynthesis of carotenoids in all the test bean lines. The increase in Na2CO3 supply had a considerable stimulatory effect on sodium accumulation, while potassium accumulation fluctuated in organs of bean lines.Conclusion: Assiut 1104 out of all the different lines investigated was found to display the lowest sensitivity to alkalinity stress, while Assiut 12/104 was the most sensitive one.

  4. CHARACTERIZATION OF BULK SOIL HUMIN AND ITS ALKALINE-SOLUBLE AND ALKALINE-INSOLUBLE FRACTIONS

    Directory of Open Access Journals (Sweden)

    Cuilan Li

    2015-02-01

    Full Text Available Humic substances are the major components of soil organic matter. Among the three humic substance components (humic acid, fulvic acid, and humin, humin is the most insoluble in aqueous solution at any pH value and, in turn, the least understood. Humin has poor solubility mainly because it is tightly bonded to inorganic soil colloids. By breaking the linkage between humin and inorganic soil colloids using inorganic or organic solvents, bulk humin can be partially soluble in alkali, enabling a better understanding of the structure and properties of humin. However, the structural relationship between bulk humin and its alkaline-soluble (AS and alkaline-insoluble (AIS fractions is still unknown. In this study, we isolated bulk humin from two soils of Northeast China by exhaustive extraction (25 to 28 times with 0.1 mol L-1 NaOH + 0.1 mol L-1 Na4P2O7, followed by the traditional treatment with 10 % HF-HCl. The isolated bulk humin was then fractionated into AS-humin and AIS-humin by exhaustive extraction (12 to 15 times with 0.1 mol L-1 NaOH. Elemental analysis and solid-state 13C cross-polarization magic angle spinning nuclear magnetic resonance (13C CPMAS NMR spectroscopy were used to characterize and compare the chemical structures of bulk humin and its corresponding fractions. The results showed that, regardless of soil types, bulk humin was the most aliphatic and most hydrophobic, AS-humin was the least aliphatic, and AIS-humin was the least alkylated among the three humic components. The results showed that bulk humin and its corresponding AS-humin and AIS-humin fractions are structurally differed from one another, implying that the functions of these humic components in the soil environment differed.

  5. Active site detection by spatial conformity and electrostatic analysis--unravelling a proteolytic function in shrimp alkaline phosphatase.

    Directory of Open Access Journals (Sweden)

    Sandeep Chakraborty

    Full Text Available Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP. In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP, one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP, where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in

  6. Active site detection by spatial conformity and electrostatic analysis--unravelling a proteolytic function in shrimp alkaline phosphatase.

    Science.gov (United States)

    Chakraborty, Sandeep; Minda, Renu; Salaye, Lipika; Bhattacharjee, Swapan K; Rao, Basuthkar J

    2011-01-01

    Computational methods are increasingly gaining importance as an aid in identifying active sites. Mostly these methods tend to have structural information that supplement sequence conservation based analyses. Development of tools that compute electrostatic potentials has further improved our ability to better characterize the active site residues in proteins. We have described a computational methodology for detecting active sites based on structural and electrostatic conformity - CataLytic Active Site Prediction (CLASP). In our pipelined model, physical 3D signature of any particular enzymatic function as defined by its active sites is used to obtain spatially congruent matches. While previous work has revealed that catalytic residues have large pKa deviations from standard values, we show that for a given enzymatic activity, electrostatic potential difference (PD) between analogous residue pairs in an active site taken from different proteins of the same family are similar. False positives in spatially congruent matches are further pruned by PD analysis where cognate pairs with large deviations are rejected. We first present the results of active site prediction by CLASP for two enzymatic activities - β-lactamases and serine proteases, two of the most extensively investigated enzymes. The results of CLASP analysis on motifs extracted from Catalytic Site Atlas (CSA) are also presented in order to demonstrate its ability to accurately classify any protein, putative or otherwise, with known structure. The source code and database is made available at www.sanchak.com/clasp/. Subsequently, we probed alkaline phosphatases (AP), one of the well known promiscuous enzymes, for additional activities. Such a search has led us to predict a hitherto unknown function of shrimp alkaline phosphatase (SAP), where the protein acts as a protease. Finally, we present experimental evidence of the prediction by CLASP by showing that SAP indeed has protease activity in vitro.

  7. Inhibition of lung serine proteases in mice: a potentially new approach to control influenza infection

    Directory of Open Access Journals (Sweden)

    Błazejewska Paulina

    2011-01-01

    Full Text Available Abstract Background Host serine proteases are essential for the influenza virus life cycle because the viral haemagglutinin is synthesized as a precursor which requires proteolytic maturation. Therefore, we studied the activity and expression of serine proteases in lungs from mice infected with influenza and evaluated the effect of serine protease inhibitors on virus replication both in cell culture and in infected mice. Results Two different inbred mouse strains were investigated: DBA/2J as a highly susceptible and C57Bl/6J as a more resistant strain to influenza virus infection. The serine proteases from lung homogenates of mice exhibited pH optima of 10.00. Using the substrate Bz-Val-Gly-Arg-p-nitroanilide or in zymograms, the intensities of proteolysis increased in homogenates from both mouse strains with time post infection (p.i. with the mouse-adapted influenza virus A/Puerto Rico/8/34 (H1N1; PR8. In zymograms at day 7 p.i., proteolytic bands were stronger and numerous in lung homogenates from DBA/2J than C57Bl/6J mice. Real-time PCR results confirmed differential expression of several lung proteases before and after infecting mice with the H1N1 virus. The most strongly up-regulated proteases were Gzma, Tmprss4, Elane, Ctrl, Gzmc and Gzmb. Pretreatment of mouse and human lung cell lines with the serine protease inhibitors AEBSF or pAB or a cocktail of both prior to infection with the H1N1 or the A/Seal/Massachusetts/1/80 (H7N7; SC35M virus resulted in a decrease in virus replication. Pretreatment of C57Bl/6J mice with either AEBSF or a cocktail of AEBSF and pAB prior to infection with the H1N1 virus significantly reduced weight loss and led to a faster recovery of treated versus untreated mice while pAB alone exerted a very poor effect. After infection with the H7N7 virus, the most significant reduction of weight loss was obtained upon pretreatment with either the protease inhibitor cocktail or pAB. Furthermore, pretreatment of C57BL/6J

  8. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis.

  9. Bacillus thuringiensis Cry3Aa protoxin intoxication of Tenebrio molitor induces widespread changes in the expression of serine peptidase transcripts.

    Science.gov (United States)

    Oppert, Brenda; Martynov, Alexander G; Elpidina, Elena N

    2012-09-01

    The yellow mealworm, Tenebrio molitor, is a pest of stored grain products and is sensitive to the Bacillus thuringiensis (Bt) Cry3Aa toxin. As digestive peptidases are a determining factor in Cry toxicity and resistance, we evaluated the expression of peptidase transcripts in the midgut of T. molitor larvae fed either a control or Cry3Aa protoxin diet for 24 h (RNA-Seq), or in larvae exposed to the protoxin for 6, 12, or 24 h (microarrays). Cysteine peptidase transcripts (9) were similar to cathepsins B, L, and K, and their expression did not vary more than 2.5-fold in control and Cry3Aa-treated larvae. Serine peptidase transcripts (48) included trypsin, chymotrypsin and chymotrypsin-like, elastase 1-like, and unclassified serine peptidases, as well as homologs lacking functional amino acids. Highly expressed trypsin and chymotrypsin transcripts were severely repressed, and most serine peptidase transcripts were expressed 2- to 15-fold lower in Cry3Aa-treated larvae. Many serine peptidase and homolog transcripts were found only in control larvae. However, expression of a few serine peptidase transcripts was increased or found only in Cry3Aa-treated larvae. Therefore, Bt intoxication significantly impacted the expression of serine peptidases, potentially important in protoxin processing, while the insect maintained the production of critical digestive cysteine peptidases.

  10. Martian alkaline basites chemically resemble basic rocks of the Lovozero alkaline massif, Kola peninsula

    Science.gov (United States)

    Kochemasov, G.

    The comparative wave planetology [1, 5] successfully overcomes the most principal martian test having now analyses of alkaline rocks from Columbia Hills [2, 3, 4]. This kind of rocks was predicted earlier on basis of the wave paradigm having stated that "the higher planetary relief range - the higher density difference between lithologies composing hypsometrically (tectonically) contrasting blocks [5]. This paradigm declares that "celestial bodies are dichotomic"(Theorem 1), "celestial bodies are sectoral" (Theorem 2), "celestial bodies are granular"(Theorem 3), "angular momenta of different level blocks tend to be equal" (Theorem 4)[1, 5]. Mars is a typical terrestrial planet but the farthest from Sun and thus with the smallest tide effects. Nevertheless it has the highest relief range and seems to be most distorted (ellipsoid in shape) and broken by deep fissures. The wave approach explains this by a warping action of standing waves of 4 ortho- and diagonal directions - they are the longest and highest in the martian case. These interfering warping waves caused by the elliptic keplerian orbits implying periodically changing accelerations and inertia-gravity forces produce inevitable tectonic dichotomy (the fundamental wave 1 long 2πR), sectoring (wave 2, πR, and other overtones), granulation. A granule size depends on an orbital frequency: the higher frequency the smaller granule. The Earth's granule, as a scale, is πR/4 (see it in NASA's PIA04159), Venus ` πR/6, Mercury's πR/16, Mars' πR/2 (the sizes are strictly tied to orb. fr.). Along with the granule sizes increase relief ranges ( Mercury ˜5 km, Venus 14, Earth 20, Mars ˜30) and compositional (density) difference between lowland and highland lithologies [5]. The lowland compositions become Fericher and denser: enstatite (Mercury), Mg-basalt (Venus), tholeiite (Earth), Fe-basalt (Mars). The highland compositions get less dense, lighter: anorthosite, alkaline basalt, andesite and conditional "albitite

  11. Studies on Fast Remediation of Soda Meadow Alkaline Soil

    Institute of Scientific and Technical Information of China (English)

    ZHOU Lianren; SUN Yankun; LI Dawei

    2010-01-01

    Researches on models of remediation quickly in soda meadow alkaline soil, and dynamic variation of water-salt in saline soil of Zhaozhou County were studied systematically from 2001 to 2006. Realize the vegetation cover of those years through the artificial planting, mixed seeding lymc grass (Elymus dahuricus Turcz) and melilot in the mode of rotary tillage and deep loosening in lower and medium saline soils. The results showed that there was remarkable relationship between net evaporation (difference of precipitation and evaporation) and total salt content in the soil. The net evaporation could be used as a new method to forecast the dynamics variation of salt to ensure the pasture optimum sowing time. Realize the autumnal vegetation cover of those years through direct planting on the bourgeon layer of soda meadow alkaline soil, on the other hand, the covered pasture made the function of restraining salt and alkaline content to realize the biology reverse succession quickly. Forage seeds were seeded directly on the seeding bed of soda alkaline meadow at the end of July. In fall of the same year, a certain amount of biomass was obtained. The model, which has remarkable economical efficiency and use widely, represented the innovative model for the fast vegetation restoration on the soda alkaline meadow soil.

  12. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  13. 南极石油烃降解嗜冷菌的筛选及其降解特性的研究%Antarctic Psychrophile Bacteria Screening for Oil Degradation and Their Degrading Characteristics

    Institute of Scientific and Technical Information of China (English)

    郑洲; 金青; 缪锦来; 刘芳明

    2008-01-01

    从 385 株南极海洋细菌中筛选出 2 株石油烃降解菌,并对其降解特性进行了初步研究.以柴油为唯一碳源进行降解实验的结果表明,南极嗜冷菌 NJ276 和 NJ341 在 5 °C、20 d 内对柴油的降解率分别达到 23.47 % 和32.15 %,在 15 °C、20 d 内降解率分别达到 43.95 % 和 62.47 %,其降解能力随着培养温度的升高而显著增强;石油烃降解残油组分的GC~MS分析表明,柴油经过 NJ276 降解后的残油组分中能检测到 C15~C21 七种烷烃,柴油经过 NJ341 降解后的残油组分只能检测到少量 C16,C17 和 C18 三种烷烃.对它们进行 16S rDNA 基因序列的同源性和系统发育分析表明,菌株 NJ276 属于假交替单胞菌属( Pseudoalteromonas),NJ341 属于科尔韦尔氏属(Colwellia).%Two hydrocarbon degrading bacteria NJ276 and NJ341 were screened from 385 Antarctic marine bacteria and their degrading characteristics were studied.Diesel oil as sole carbon source was used in this study.The results showed that the oil degradation rates of Antarctic psychrophile bacteria NJ276 and NJ341 were 23.47 % and 32.15 %,respectively,after 20 days culturation at 5 °C,and the rates were 43.95 % and 62.47 % respectively after 20 days culturation at 15 °C.The oil degradation abilities were enhanced remarkably with the increasing culture temperature.GC-MSs indicated the residual oil contained C15-C21 7 alkyls after degradation by NJ276,and C16,C17 and C18 3 alkyls after degradation by NJ341.The 16S rDNA gene sequences homology and phylogenetic analysis of the two Antarctic psychrophile bacteria showed that NJ276 belonged to the described genus Pseudoalteromonas and NJ341 belonged to the genus Colwellia.

  14. Alkaline Comet Assay for Assessing DNA Damage in Individual Cells.

    Science.gov (United States)

    Pu, Xinzhu; Wang, Zemin; Klaunig, James E

    2015-08-06

    Single-cell gel electrophoresis, commonly called a comet assay, is a simple and sensitive method for assessing DNA damage at the single-cell level. It is an important technique in genetic toxicological studies. The comet assay performed under alkaline conditions (pH >13) is considered the optimal version for identifying agents with genotoxic activity. The alkaline comet assay is capable of detecting DNA double-strand breaks, single-strand breaks, alkali-labile sites, DNA-DNA/DNA-protein cross-linking, and incomplete excision repair sites. The inclusion of digestion of lesion-specific DNA repair enzymes in the procedure allows the detection of various DNA base alterations, such as oxidative base damage. This unit describes alkaline comet assay procedures for assessing DNA strand breaks and oxidative base alterations. These methods can be applied in a variety of cells from in vitro and in vivo experiments, as well as human studies.

  15. Alkaline direct alcohol fuel cells using an anion exchange membrane

    Energy Technology Data Exchange (ETDEWEB)

    Matsuoka, Koji; Iriyama, Yasutoshi; Abe, Takeshi; Ogumi, Zempachi [Department of Energy and Hydrocarbon Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510 (Japan); Matsuoka, Masao [Faculty of Science and Engineering, Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2005-10-04

    Alkaline direct alcohol fuel cells using an OH-form anion exchange membrane and polyhydric alcohols were studied. A high open circuit voltage of ca. 800mV was obtained for a cell using Pt-Ru/C (anode) and Pt/C (cathode) at 323K, which was about 100-200mV higher than that for a DMFC using Nafion{sup R}. The maximum power densities were in the order of ethylene glycol>glycerol>methanol>erythritol>xylitol. Silver catalysts were used as a cathode catalyst to fabricate alkaline fuel cells, since silver catalyst is almost inactive in the oxidation of polyhydric alcohols. Alkaline direct ethylene glycol fuel cells using silver as a cathode catalyst gave excellent performance because higher concentrations of fuel could be supplied to the anode. (author)

  16. Metasomatized lithosphere and the origin of alkaline lavas.

    Science.gov (United States)

    Pilet, Sébastien; Baker, Michael B; Stolper, Edward M

    2008-05-16

    Recycled oceanic crust, with or without sediment, is often invoked as a source component of continental and oceanic alkaline magmas to account for their trace-element and isotopic characteristics. Alternatively, these features have been attributed to sources containing veined, metasomatized lithosphere. In melting experiments on natural amphibole-rich veins at 1.5 gigapascals, we found that partial melts of metasomatic veins can reproduce key major- and trace-element features of oceanic and continental alkaline magmas. Moreover, experiments with hornblendite plus lherzolite showed that reaction of melts of amphibole-rich veins with surrounding lherzolite can explain observed compositional trends from nephelinites to alkali olivine basalts. We conclude that melting of metasomatized lithosphere is a viable alternative to models of alkaline basalt formation by melting of recycled oceanic crust with or without sediment.

  17. ADVANCES IN THE MODEL OF CYLINDRICAL ALKALINE CELLS

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The advancement of a systematic investigation on the modeling of cylindrical alkaline cells is presented.Initial analysis utilizes thermodynamic and kinetic information to predict alkaline cell performance under low discharge rates.Subsequent modling has taken into consideration detailed information on the chemistry of electrode reactions,mass tranport of dissolved species,physical and chemical properties of the electrolyte and solid phases,and internal geonetry of cell systems.The model is capable of predicting alkaline cell performance under a variety of dicharge conditions.The model also provides information regarding internal cell changes during discharge.The model is the basis of a rational approach for the optimal design of cells.

  18. POLYETHER POLYURETHANE FROM MODIFIED WHEAT STRAW OXYGEN-ALKALINE LIGNIN

    Institute of Scientific and Technical Information of China (English)

    QuanxiaoLiu; HuaiyuZhan; BeihaiHe; ShuhuiYang; JianhuaLiu; JianluLiu(1); ZhenxingPang

    2004-01-01

    Polyether polyurethane was synthesized from modified wheat straw oxygen-alkaline lignin, polyethylene glycol and two different diisocyanates (diphenylemethane-4, 4'-diisocyanate, tolulene diisocyanate) by solution casting method, its properties were investigated. The results show that modified wheat straw oxygen-alkaline lignin can substitute part of polyethylene glycol to react with diisocyanate to synthesize polyurethane. The molar ratio of NCO to OH and modified wheat straw oxygen-alkaline lignin content affect the properties of lignin-based polyether polyurethane respectively. The addition of plasticizer in the polyurethane synthesis process improves the properties of synthesized polyurethane, especially the elasticity of polyurethane. The synthesized polyurethane from modified wheat straw oxygen-alkali lignin can be used as both engineering plastic and hard foam plastic in future.

  19. POLYETHER POLYURETHANE FROM MODIFIED WHEAT STRAW OXYGEN-ALKALINE LIGNIN

    Institute of Scientific and Technical Information of China (English)

    Quanxiao Liu; Huaiyu Zhan; Beihai He; Shuhui Yang; Jianhua Liu; Jianlu Liu; Zhenxing Pang

    2004-01-01

    Polyether polyurethane was synthesized from modified wheat straw oxygen-alkaline lignin,polyethylene glycol and two different diisocyanates (diphenylemethane-4, 4′-diisocyanate, tolulene diisocyanate) by solution casting method, its properties were investigated. The results show that modified wheat straw oxygen-alkaline lignin can substitute part of polyethylene glycol to react with diisocyanate to synthesize polyurethane. The molar ratio of NCO to OH and modified wheat straw oxygen-alkaline lignin content affect the properties of lignin-based polyether polyurethane respectively. The addition of plasticizer in the polyurethane synthesis process improves the properties of synthesized polyurethane, especially the elasticity of polyurethane. The synthesized polyurethane from modified wheat straw oxygen-alkali lignin can be used as both engineering plastic and hard foam plastic in future.

  20. A statistical approach for optimization of alkaline lipase production by ascidian associated—Halobacillus trueperi RSK CAS9

    Directory of Open Access Journals (Sweden)

    Ramamoorthy Sathishkumar

    2015-12-01

    Full Text Available A marine ascidian-associated bacterium, Halobacillus trueperi RSK CAS9, was optimized for lipase production by response surface methodology using marine waste as substrate. The central composite design was employed, and the optimal medium constituents for maximum lipase production (1355.81 U/ml were determined to be tuna powder (14.58 g/l, olive oil (5.05 ml/l; NaCl (72.42 g/l, temperature (45 °C and pH 9.0. An alkaline lipase was purified to 8.46 fold with 1193.59 U mg−1 specific activities with the molecular weight of 44 kDa. The activity was substantially inhibited by EDTA and PMSF, indicating that it was a metalloenzyme serine residue which was essential for catalytic activity. Thus, lipase production by microbial conversion of marine fish wastes in this study suggested its potential utilization for the production of high value products.

  1. 2nd Generation alkaline electrolysis. Final report

    Energy Technology Data Exchange (ETDEWEB)

    Yde, L. [Aarhus Univ. Business and Social Science - Centre for Energy Technologies (CET), Aarhus (Denmark); Kjartansdottir, C.K. [Technical Univ. of Denmark. DTU Mechanical Engineering, Kgs. Lyngby (Denmark); Allebrod, F. [Technical Univ. of Denmark. DTU Energy Conversion, DTU Risoe Campus, Roskilde (Denmark)] [and others

    2013-03-15

    The overall purpose of this project has been to contribute to this load management by developing a 2{sup nd} generation of alkaline electrolysis system characterized by being compact, reliable, inexpensive and energy efficient. The specific targets for the project have been to: 1) Increase cell efficiency to more than 88% (according to the higher heating value (HHV)) at a current density of 200 mA /cm{sup 2}; 2) Increase operation temperature to more than 100 degree Celsius to make the cooling energy more valuable; 3) Obtain an operation pressure more than 30 bar hereby minimizing the need for further compression of hydrogen for storage; 4) Improve stack architecture decreasing the price of the stack with at least 50%; 5) Develop a modular design making it easy to customize plants in the size from 20 to 200 kW; 6) Demonstrating a 20 kW 2{sup nd} generation stack in H2College at the campus of Arhus University in Herning. The project has included research and development on three different technology tracks of electrodes; an electrochemical plating, an atmospheric plasma spray (APS) and finally a high temperature and pressure (HTP) track with operating temperature around 250 deg. C and pressure around 40 bar. The results show that all three electrode tracks have reached high energy efficiencies. In the electrochemical plating track a stack efficiency of 86.5% at a current density of 177mA/cm{sup 2} and a temperature of 74.4 deg. C has been shown. The APS track showed cell efficiencies of 97%, however, coatings for the anode side still need to be developed. The HTP cell has reached 100 % electric efficiency operating at 1.5 V (the thermoneutral voltage) with a current density of 1. 1 A/cm{sup 2}. This track only tested small cells in an externally heated laboratory set-up, and thus the thermal loss to surroundings cannot be given. The goal set for the 2{sup nd} generation electrolyser system, has been to generate 30 bar pressure in the cell stack. An obstacle to be

  2. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    Science.gov (United States)

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (Psuperoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  3. The action of neutrophil serine proteases on elastin and its precursor.

    Science.gov (United States)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther; Rusciani, Anthony; Duca, Laurent; Debelle, Laurent; Weiss, Anthony S; Neubert, Reinhard H H; Schmelzer, Christian E H

    2012-01-01

    This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr. CG shows a strong preference for the charged amino acid Lys at P(1) in tropoelastin, whereas Lys was not identified at P(1) in CG digests of elastin due to extensive cross-linking at Lys residues in mature elastin. All three serine proteases showed a clear preference for Pro at P(2) and P(4)'. With respect to the liberation of potentially bioactive peptides from elastin, the study revealed that all three serine proteases have a similar ability to release bioactive sequences, with CG producing the highest number of these peptides. In bioactivity studies, potentially bioactive peptides that have not been investigated on their bioactivity to date, were tested. Three new bioactive GxxPG motifs were identified; GVYPG, GFGPG and GVLPG. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  4. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    Science.gov (United States)

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  5. A Mycobacterium avium subsp. paratuberculosis predicted serine protease is associated with acid stress and intraphagosomal survival

    Directory of Open Access Journals (Sweden)

    Abirami Kugadas

    2016-08-01

    Full Text Available AbstractThe ability to maintain intra-cellular pH is crucial for bacteria and other microbes to survive in diverse environments, particularly those that undergo fluctuations in pH. Mechanisms of acid resistance remain poorly understood in mycobacteria. Although studies investigating acid stress in M. tuberculosis are gaining traction, few center on Mycobacterium avium subsp. paratuberculosis (MAP, the etiological agent of chronic enteritis in ruminants. We identified a MAP acid stress response network involved in macrophage infection. The central node of this network was MAP0403, a predicted serine protease that shared an 86% amino acid identity with MarP in M. tuberculosis. Previous studies confirmed MarP as a serine protease integral to maintaining intra-bacterial pH and survival in acid in vitro and in vivo. We show that MAP0403 is upregulated in infected macrophage and MAC-T cells and coincided with phagosome acidification. Treatment of mammalian cells with bafilomcyin A1, a potent inhibitor of phagosomal vATPases, diminished MAP0403 transcription. MAP0403 expression was also noted in acidic medium. A surrogate host, M. smegmatis mc2 155, was designed to express MAP0403 and when exposed to either macrophages or in vitro acid stress had increase bacterial cell viability, which corresponds to maintenance of intra-bacterial pH in acidic (pH = 5 conditions. These data suggest that MAP0403 may be the equivalent of MarP in MAP. Future studies confirming MAP0403 as a serine protease and exploring its structure and possible substrates are warranted.

  6. Functional suppression of HAMP domain signaling defects in the E. coli serine chemoreceptor.

    Science.gov (United States)

    Lai, Run-Zhi; Parkinson, John S

    2014-10-23

    HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations.

  7. Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

    Science.gov (United States)

    Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

    2016-02-19

    Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

  8. Human Parturition Involves Phosphorylation of Progesterone Receptor-A at Serine-345 in Myometrial Cells.

    Science.gov (United States)

    Amini, Peyvand; Michniuk, Daniel; Kuo, Kelly; Yi, Lijuan; Skomorovska-Prokvolit, Yelenna; Peters, Gregory A; Tan, Huiqing; Wang, Junye; Malemud, Charles J; Mesiano, Sam

    2016-11-01

    The hypothesis that phosphorylation of progesterone receptor (PR) isoforms, PR-A and PR-B, in myometrial cells affects progesterone action in the context of human parturition was tested. Immunodetection of phosphoserine (pSer) PR forms in term myometrium revealed that the onset of labor is associated with increased phosphorylation of PR-A at serine-345 (pSer345-PRA) and that pSer345-PRA localized to the nucleus of myometrial cells. In explant cultures of term myometrium generation of pSer345-PRA was induced by interleukin-1β and dependent on progesterone, suggesting that pSer345-PRA generation is induced by a proinflammatory stimulus. In the hTERT-HM(A/B) human myometrial cell line, abundance of pSer345-PRA was induced by progesterone in a dose- (EC50 ∼1 nM) and time-dependent manner. Prevention of pSer345 (by site-directed mutagenesis) abolished the capacity for PR-A to inhibit anti-inflammatory actions of progesterone mediated by PR-B but had no effect on the transrepressive activity of PR-A at a canonical progesterone response element. Taken together, the data show that human parturition involves the phosphorylation of PR-A at serine-345 in myometrial cells and that this process is ligand dependent and induced by a proinflammatory stimulus. We also found that in myometrial cells, pSer345 activates the capacity for PR-A to inhibit antiinflammatory actions of progesterone mediated by PR-B. Phosphorylation of PR-A at serine-345 may be an important functional link between tissue-level inflammation and PR-A-mediated functional progesterone withdrawal to trigger parturition.

  9. Roles of quaternary structure and cysteine residues in the activity of human serine racemase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2011-12-01

    Full Text Available Abstract Background D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. It is chiefly synthesized in the CNS by serine racemase (SR. Regulation of SR activity is still poorly understood. As step toward developing reagents and methods for investigating SR in vitro, we analyzed structure-function relationships of a recombinant enzyme of human sequence. Results Michaelis-Menten kinetic analysis indicated a KM value of 14 mM and Vmax value of 3.66 μmol·mg-1·hr-1 when L-serine was used as a substrate for purified SR. Gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major oligomeric form of recombinant SR in aqueous solution, though the proportions of monomer, tetramer, and larger aggregates differed somewhat with the specific buffer used. These buffers also altered activity in a manner correlating with the relative abundance of dimer. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Chemical reduction with DTT increased the activity of SR by elevating Vmax; cystamine, a reagent that blocks sulfhydryl groups, abolished SR activity. Gel-filtration chromatography and western blot analysis indicated that DTT enhanced the recovery of noncovalent SR dimer. Conclusions These data suggest that SR is most active as a noncovalent dimer containing one or more free sulfhydryls in the enzyme's active center or a modulatory site. Buffer composition and reduction/oxidation status during preparation can dramatically impact interpretations of SR activity. These findings also highlight the possibility that SR is sensitive to oxidative stress in vivo.

  10. IDENTIFICATION OF SERINE CARBAPENEMASE AND METALLOCARBAPENEMASE ENZYMES IN PSEUDOMONAS AERUGINOSA IN GEMS MEDICAL COLLEGE, RAGOLU, SRIKAKULAM

    Directory of Open Access Journals (Sweden)

    Radhika

    2016-06-01

    Full Text Available Various carbapenems have been reported in Pseudomonas aeruginosa such as VIM, NDM & OXA-48, etc. In addition, carbapenemase producers are usually associated with many other non–β-lactam resistance determinants which give rise to multidrug and pan drug resistant isolates. Detection of these enzymes in infected patients and in carriers are the two main approaches for prevention of their spread. Potential carbapenemase producers are currently screened first by susceptibility testing, using breakpoint values for carbapenems. However, many carbapenemase producers do not confer obvious resistance levels to carbapenems. So there is need for Laboratories to search for carbapenemase producers. In such instance, phenotypic based test such as Modified Hodge Test (MHT is very much useful in confirming in vitro production of carbapenemase enzymes. But this test does not differentiate serine carbapenemase enzyme (i.e. Ambler class A & C from metallocarbapenemase (i.e. Ambler class B. To differentiate these two enzymes, MHT positive isolates can be subjected to Disc Synergy test. These two tests are highly sensitive and specific and adaptable to any laboratory in the world. Out of 100 ceftazidime resistant Pseudomonas aeruginosa, 75(75% were sensitive, 7(7% were intermediate sensitive and 18(18% were resistant to imipenem. When the 18 imipenem resistant strains were subjected to Modified Hodge test, 15 gave positive results. When the 15 MHT positive strains subjected to disc synergy test, 8 were positive and 7 were negative showing that 8 were producing metallocarbapenemases and 7 were producing serine carbapenemases. Out of 7 intermediately imipenem sensitive isolates, 2 were producing metallocarbapenemase and 3 were producing serine carbapenemase. Hence, total number of imipenem resistant Pseudomonas aeruginosa isolates were 23.

  11. Research on Alkaline Filler Flame-Retarded Asphalt Pavement

    Institute of Scientific and Technical Information of China (English)

    HU Shuguang; ZHANG Houji; WANG Jiaolan

    2006-01-01

    Used as flame retardant of tunnel asphalt pavement, organic bromides produce a large amount of poisons and smoke in construction and flame retardation stage. The alkaline filler was found to replace mineral filler, and the flame-retarded asphalt mixtures were produced. Experimental results show that these asphalt mixtures are smoke restrained; the performances and construction technology of asphalt pavement are not influenced; also the alkaline filler is of low-price. So this kind of flame-retarded asphalt mixtures is suitable for tunnel pavement.

  12. Direct Promotion of Collagen Calcification by Alkaline Phosphatase

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Alkaline phosphatase promotes hydrolysis of phosphate containing substrates, causes a rise in inorganic phosphate and, therefore, enhances calcification of biological tissues. In this work, the calcification of collagen in a model serum was used as a model of collagenous tissue biomaterials to study the possible calcification promotion mechanism of alkaline phosphatase. In the enzyme concentration range of 0.10.5mg/mL, the enzyme shows a direct calcification promoting effect which is independent of the hydrolysis of its phosphate containing substrates but proportional to the enzyme concentration. Potassium pyrophosphate somewhat inhibits the calcification promotion.

  13. Surface-treatment of Alkaline Earth Sulfides Based Phosphor

    Institute of Scientific and Technical Information of China (English)

    GUO Chong-feng; CHU Ben-li; XU Jian; SU Qiang

    2004-01-01

    A series of alkaline earth sulfides based phosphors Ca0.8Sr0.2S∶Eu2+, Tm3+ were covered with a layer of protective coating with alkaline earth fluorides by heating the mixture of phosphor and NH4HF2 at elevated temperatures. The coatings were characterized by means of XRD and SEM. The optical properties of the coated phosphors and the influences of the coating on their properties have been discussed extensively. The stabilities of the coated and uncoated phosphors have been compared.

  14. Application conditions for ester cured alkaline phenolic resin sand

    Institute of Scientific and Technical Information of China (English)

    Ren-he Huang; Bao-ping Zhang; Yao-ji Tang

    2016-01-01

    Five organic esters with different curing speeds: propylene carbonate (i.e. high-speed ester A); 1, 4-butyrolactone; glycerol triacetate (i.e. medium-speed ester B); glycerol diacetate; dibasic ester (DBE) (i.e. low-speed ester C), were chosen to react with alkaline phenolic resin to analyze the application conditions of ester cured alkaline phenolic resin. The relationships between the curing performances of the resin (including pH value, gel pH value, gel time of resin solution, heat release rate of the curing reaction and tensile strength of the resin sand) and the amount of added organic ester and curing temperature were investigated. The results indicated the folowing: (1) The optimal added amount of organic ester should be 25wt.%-30wt.% of alkaline phenolic resin and it must be above 20wt.%-50 wt.% of the organic ester hydrolysis amount. (2) High-speed ester A (propylene carbonate) has a higher curing speed than 1, 4-butyrolactone, and they were both used as high-speed esters. Glycerol diacetate is not a high-speed ester in alkaline phenolic resin although it was used as a high-speed ester in ester cured sodium silicate sand; glycerol diacetate and glycerol triacetate can be used as medium-speed esters in alkaline phenolic resin. (3) High-speed ester A, medium-speed ester B (glycerol triacetate) and low-speed ester C (dibasic ester, i.e., DBE) should be used below 15 ºC, 35 ºC and 50 ºC, respectively. High-speed ester A or low-speed ester C should not be used alone but mixed with medium-speed ester B to improve the strength of the resin sand. (4) There should be a suitable solid content (generaly 45wt.%-65wt.% of resin), alkali content (generaly 10wt.%-15wt.% of resin) and viscosity of alkaline phenolic resin (generaly 50-300 mPa·s) in the preparation of alkaline phenolic resin. Finaly, the technique conditions of alkaline phenolic resin preparation and the application principles of organic ester were discussed.

  15. Advances in alkaline cooling water treatment technology: An update

    Energy Technology Data Exchange (ETDEWEB)

    Shaffer, A.E. Jr.; Klatskin, S.D.

    1985-01-01

    A series of chromate and non-chromate treatment programs, specifically designed for alkaline pH cooling waters, have been developed. The treatments provide excellent corrosion and scale control over a broad range of water chemistries and are applicable to high conductivity and iron contaminated waters. Low levels of zinc are used to reduce the dependency on alkalinity, chromate and calcium carbonate supersaturation for corrosion control. The precipitation and fouling problems previously encountered with zinc containing treatments have been eliminated by the use of polymeric dispersants.

  16. Immobilization of cesium in alkaline activated fly ash matrix

    Science.gov (United States)

    Fernandez-Jimenez, A.; Macphee, D. E.; Lachowski, E. E.; Palomo, A.

    2005-11-01

    The immobilization potential of alkaline activated fly ash (AAFA) matrices for cesium has been investigated. The presence of Cs in the AAFA pastes, prepared using 8M NaOH solution as activator, showed no significant adverse effects on mechanical strength or microstructure, nor were significant quantities of Cs leached following application of the Toxic Characteristic Leaching Procedure (TCLP) and American Nuclear Society (ANS) 16.1 leaching protocols. Microstructural analysis shows Cs associated with the main reaction product in the AAFA suggesting that cesium is chemically bound rather than physically encapsulated. It is proposed that cesium is incorporated into the alkaline aluminosilicate gel, a precursor for zeolite formation.

  17. Impact of Alkaline Dust Pollution on Soil Microbial Biomass Carbon

    OpenAIRE

    Kara, Ömer; Bolat, İlyas

    2007-01-01

    The effect of alkaline dust pollution emitted from Bartın cement plant on the soil microbial biomass carbon was investigated using the chloroform fumigation-extraction (CFE) method. Microbial biomass C (Cmic) values ranged from 157.82 to 1201.51 µg g-1 soils in the polluted area and from 726.70 to 1529.14 µg g-1 soils in the control area. Soils polluted with alkaline cement dust resulted in significant reductions in Cmic levels compared to control soils. Microbial biomass C correlated negativ...

  18. Impact of Alkaline Dust Pollution on Soil Microbial Biomass Carbon

    OpenAIRE

    KARA, Ömer; Bolat, İlyas

    2014-01-01

    The effect of alkaline dust pollution emitted from Bartın cement plant on the soil microbial biomass carbon was investigated using the chloroform fumigation-extraction (CFE) method. Microbial biomass C (Cmic) values ranged from 157.82 to 1201.51 µg g-1 soils in the polluted area and from 726.70 to 1529.14 µg g-1 soils in the control area. Soils polluted with alkaline cement dust resulted in significant reductions in Cmic levels compared to control soils. Microbial biomass C correlated negativ...

  19. Human placental alkaline phosphatase electrophoretic alleles: Quantitative studies

    Science.gov (United States)

    Lucarelli, Paola; Scacchi, Renato; Corbo, Rosa Maria; Benincasa, Alberto; Palmarino, Ricciotti

    1982-01-01

    Human placental alkaline phosphatase (ALP) activity has been determined in specimens obtained from 562 Italian subjects. The mean activities of the three common homozygotes (Pl 2 = 4.70 ± 0.24, Pl 1 = 4.09 ± 0.08, and Pl 3 = 2.15 ± 0.71 μmol of p-nitrophenol produced) were significantly different. The differences among the various allelic forms account for 10% of the total quantitative variation of the human placental alkaline phosphatase. PMID:7072721

  20. Alkaline protease production by solid state fermentation on polyurethane foam

    OpenAIRE

    Hongzhang, Chen; Hui, Wang; Aijun, Zhang; Zuohu, Li

    2006-01-01

    This paper investigated the process of solid state fermentation (SSF) using PUF (polyurethane foam) as inert solid support to produce alkaline protease. Maximal enzyme activity was 2185U/ml at pH 9.0, incubation temperature 32 0C inoculum amount of 1.0 % (v/v) , nutrient solution3.75 ml/g PUF, incubation time for 2 h and 15.0 mM of added CaCl2. Under the same conditions, the yield of alkaline protease produced by SSF using PUF as support is higher than that by submerged fermentation (SMF).