The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

Science.gov (United States)

Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

2012-09-01

Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria.

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Ingo L Brand

Full Text Available Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins may underestimate their activity.

Optimization of extraction, characterization and antioxidant activity of polysaccharides from Brassica rapa L.

Science.gov (United States)

Wang, Wei; Wang, Xiaoqing; Ye, Hong; Hu, Bing; Zhou, Li; Jabbar, Saqib; Zeng, Xiaoxiong; Shen, Wenbiao

2016-01-01

The root of Brassica rapa L. has been traditionally used as a Uyghur folk medicine to cure cough and asthma by Uyghur nationality in Xinjiang Uygur Autonomous Region of China. In the present study, therefore, extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from the root of B. rapa L. (BRP) were investigated. The optimal extraction conditions with an extraction yield of 21.48 ± 0.41% for crude BRP were obtained as follows: extraction temperature 93°C, extraction time 4.3h and ratio of extraction solvent (water) to raw material 75 mL/g. The crude BRP was purified by chromatographic columns of DEAE-52 cellulose and Sephadex G-100, affording three purified fractions of BRP-1-1, BRP-2-1 and BRP-2-2 with average molecular weight of 1510, 1110 and 838 kDa, respectively. Monosaccharide composition analysis indicated that BRP-1-1 was composed of mannose, rhamnose, glucose, galactose and arabinose, BRP-2-1 was composed of rhamnose, galacturonic acid, galactose and arabinose, and BRP-2-2 was composed of rhamnose and galacturonic acid in a molar ratio of 1.27: 54.92. Furthermore, the crude BRP exhibited relatively higher antioxidant activity in vitro than purified fractions; hence, it could be used as a natural antioxidant in functional foods or medicines. Copyright © 2015 Elsevier B.V. All rights reserved.

Biocontrol activity of surfactin A purified from Bacillus NH-100 and NH-217 against rice bakanae disease.

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Sarwar, Ambrin; Hassan, Muhammad Nadeem; Imran, Muhammad; Iqbal, Mazhar; Majeed, Saima; Brader, Günter; Sessitsch, Angela; Hafeez, Fauzia Yusuf

2018-04-01

The potential of the Bacillus genus to antagonize phytopathogens is associated with the production of cyclic lipopeptides. Depending upon the type of lipopeptide, they may serve as biocontrol agents that are eco-friendly alternatives to chemical fertilizers. This study evaluates the biocontrol activity of surfactin-producing Bacillus (SPB) strains NH-100 and NH-217 and purified surfactin A from these strains against rice bakanae disease. Biologically active surfactin fractions were purified by HPLC, and surfactin A variants with chain lengths from C12 to C16 were confirmed by LCMS-ESI. In hemolytic assays, a positive correlation between surfactin A production and halo zone formation was observed. The purified surfactin A had strong antifungal activity against Fusarium oxysporum, F. moniliforme, F. solani, Trichoderma atroviride and T. reesei. Maximum fungal growth suppression (84%) was recorded at 2000 ppm against F. moniliforme. Surfactin A retained antifungal activity at different pH levels (5-9) and temperatures (20, 50 and 121 °C). Hydroponic and pot experiments were conducted to determine the biocontrol activity of SPB strains and the purified surfactin A from these strains on Super Basmati rice. Surfactin production in the rice rhizosphere was detected by LCMS-ESI at early growth stages in hydroponics experiments inoculated with SPB strains. However, the maximum yield was observed with a consortium of SPB strains (T4) and purified surfactin A (T5) treatments in the pot experiment. The outcomes of the present study revealed that surfactin A significantly reduced rice bakanae disease by up to 80%. These findings suggest that purified surfactin A could be an effective biocontrol agent against bakanae disease in rice and should be incorporated into strategies for disease management. Copyright © 2018 Elsevier GmbH. All rights reserved.

Purification and Characterization of a Novel and Robust L-Asparaginase Having Low-Glutaminase Activity from Bacillus licheniformis: In Vitro Evaluation of Anti-Cancerous Properties

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Mahajan, Richi V.; Kumar, Vinod; Rajendran, Vinoth; Saran, Saurabh; Ghosh, Prahlad C.; Saxena, Rajendra Kumar

2014-01-01

L-asparaginase having low glutaminase has been a key therapeutic agent in the treatment of acute lymphpoblastic leukemia (A.L.L). In the present study, an extracellular L-asparaginase with low glutaminase activity, produced by Bacillus licheniformis was purified to homogeneity. Protein was found to be a homotetramer of 134.8 KDa with monomeric size of 33.7 KDa and very specific for its natural substrate i.e. L-asparagine. The activity of purified L-asparaginase enhanced in presence of cations including Na+ and K+, whereas it was moderately inhibited in the presence of divalent cations and thiol group blocking reagents. The purified enzyme was maximally active over the range of pH 6.0 to 10.0 and temperature of 40°C and enzyme was stable maximum at pH 9.0 and −20°C. CD spectra of L-asparaginase predicted the enzyme to consist of 63.05% α- helix and 3.29% β-sheets in its native form with T222 of 58°C. Fluorescent spectroscopy showed the protein to be stable even in the presence of more than 3 M GdHCl. Kinetic parameters Km, Vmax and kcat of purified enzyme were found as 1.4×10−5 M, 4.03 IU and 2.68×103 s−1, respectively. The purified L-asparaginase had cytotoxic activity against various cancerous cell lines viz. Jurkat clone E6-1, MCF-7 and K-562 with IC50 of 0.22 IU, 0.78 IU and 0.153 IU respectively. However the enzyme had no toxic effect on human erythrocytes and CHO cell lines hence should be considered potential candidate for further pharmaceutical use as an anticancer drug. PMID:24905227

Oocyte formation by mitotically-active germ cells purified from ovaries of reproductive age women

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White, Yvonne A. R.; Woods, Dori C.; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L.

2012-01-01

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a FACS-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically-active cells that exhibit a gene expression profile consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and spontaneously generate 35–50 µm oocytes, as determined by morphology, gene expression and attainment of haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1–2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, like adult mice, possess rare mitotically-active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo. PMID:22366948

Expression, purification and characterization of hepatitis B virus X protein BH3-like motif-linker-Bcl-xL fusion protein for structural studies

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Hideki Kusunoki

2017-03-01

Full Text Available Hepatitis B virus X protein (HBx is a multifunctional protein that interacts directly with many host proteins. For example, HBx interacts with anti-apoptotic proteins, Bcl-2 and Bcl-xL, through its BH3-like motif, which leads to elevated cytosolic calcium levels, efficient viral DNA replication and the induction of apoptosis. To facilitate sample preparation and perform detailed structural characterization of the complex between HBx and Bcl-xL, we designed and purified a recombinant HBx BH3-like motif-linker-Bcl-xL fusion protein produced in E. coli. The fusion protein was characterized by size exclusion chromatography, circular dichroism and nuclear magnetic resonance experiments. Our results show that the fusion protein is a monomer in aqueous solution, forms a stable intramolecular complex, and likely retains the native conformation of the complex between Bcl-xL and the HBx BH3-like motif. Furthermore, the HBx BH3-like motif of the intramolecular complex forms an α-helix. These observations indicate that the fusion protein should facilitate structural studies aimed at understanding the interaction between HBx and Bcl-xL at the atomic level.

Purification of a Novel Bacteriocin-Like Inhibitory Substance Produced by Enterococcus faecium ICIS 8 and Characterization of Its Mode of Action.

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Vasilchenko, Alexey S; Rogozhin, Eugene A; Valyshev, Alexander V

2017-06-01

The aim of this work was to purify and characterize a bacteriocin-like antimicrobial substance produced by an antagonistic active strain of Enterococcus faecium. A novel bacteriocin-like inhibitory substance (BLIS) produced by the E. faecium ICIS 8 strain was purified and characterized using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and N-terminal amino acid sequencing revealed the following partial sequence: NH 2 -APKEKCFPKYCV. The proteinaceous nature of purified BLIS was assessed by treatment with proteolytic enzyme. Studies of the action of BLIS using bacteriological and bioluminescence assays revealed a dose-dependent inhibition of Listeria monocytogenes 88BK and Escherichia coli K12 TG1 lac::lux viability. The interaction of the BLIS with the bacterial surface led to the compensation of a negative charge value, as shown by zeta-potential measurements. Assessments of membrane integrity using fluorescent probes and atomic force microscopy revealed the permeabilization of the cellular barrier structures in both L. monocytogenes and E. coli. The novel BLIS from E. faecium ICIS 8 was characterized by a unique primary peptide sequence and exerted bactericidal activity against L. monocytogenes and E. coli by disrupting membrane integrity.

Alkaloid profile, antibacterial and allelopathic activities of Lupinus jaimehintoniana B.L. Turner (Fabaceae

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Ruiz-González Nancy

2012-01-01

Full Text Available Herein we describe some aspects of the ethnobotanical use and the first alkaloid profile of Lupinus jaimehintoniana, the 5 to 8 m high arboreous lupine. Five quinolizidine alkaloids identified as sparteine, 5,6-dehydrolupanine, lupanine, nuttalline, and d-thermopsine, were characterized by the respective elution order according to their electronic impact spectra, lupanine being the most abundant in the four different tissues analyzed. Simultaneously, an antibacterial assessment of the four corresponding crude methanolic extracts, as well as the four semi-purified alkaloids was performed on specific Escherichia coli and Agrobacterium tumefaciens strains. These experiments resulted in MIC ranges of 37-61 µg mL-1 and 130-146 µg mL-1, respectively. for both bacterial species. Finally, the allelopathic activity of these extracts on the germination of Lactuca sativa seeds was demonstrated to be in the range of 50-300 µg mL-1 for both semi-purified alkaloid and methanolic extracts.

High specific activity N-Acetyl-3H-α-Aspartyl- L-Glutamic at micro mole scale

International Nuclear Information System (INIS)

Suarez, C.

1984-01-01

High specific activity N-Acetyl-3 H - α -Aspartyl-I-Glutamic acid at micro mole scale in prepared acetylating L- α -Aspartyl-L-glutamic with 3 H -acetic anhydride in re distilled toluene. The product le purified through cationic and anionic columns. The radiochemical purity as determined by thin-layer chromatography is greater then 99% at the time preparation. (Author) 5 refs

Purification and characterization of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Penicillium chrysogenum

DEFF Research Database (Denmark)

Theilgaard, Hanne Birgitte; Kristiansen, K.N.; Henriksen, Claus Maxel

1997-01-01

such as substrates, cofactors and pH on the activity of the purified ACVS was investigated. The K-m values for the three precursor substrates La-aminoadipic acid, L-cysteine and L-valine were determined as 45, 80 and 80 mu M respectively, and the optimal assay concentration of ATP was found to be 5 mM (with 20 mM Mg......delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) from Penicillium chrysogenum was purified to homogeneity by a combination of (NH4)(2)SO4 precipitation, protamine sulphate treatment, ion-exchange chromatography, gel filtration and hydrophobic interaction chromatography......Cl2). The dimer of the reaction product bis-delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (bisACV) gave feedback inhibition of the purified ACVS; the inhibition parameter K-bisACV was determined as 1.4 mM. Furthermore dithiothreitol was shown to inhibit the purified ACVS. From the addition...

Cytotoxic, Anti-Proliferative and Apoptosis Activity of l-Amino Acid Oxidase from Malaysian Cryptelytrops purpureomaculatus (CP-LAAO) Venom on Human Colon Cancer Cells.

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Zainal Abidin, Syafiq Asnawi; Rajadurai, Pathmanathan; Hoque Chowdhury, Md Ezharul; Othman, Iekhsan; Naidu, Rakesh

2018-06-08

The aim of this study is to investigate the potential anti-cancer activity of l-amino acid oxidase (CP-LAAO) purified from the venom of Cryptelytrops purpureomaculatus on SW480 and SW620 human colon cancer cells. Mass spectrometry guided purification was able to identify and purify CP-LAAO. Amino acid variations identified from the partial protein sequence of CP-LAAO may suggest novel variants of these proteins. The activity of the purified CP-LAAO was confirmed with o-phenyldiamine (OPD)-based spectrophotometric assay. CP-LAAO demonstrated time- and dose-dependent cytotoxic activity and the EC 50 value was determined at 13 µg/mL for both SW480 and SW620 cells. Significant increase of caspase-3 activity, reduction of Bcl-2 levels, as well as morphological changes consistent with apoptosis were demonstrated by CP-LAAO. Overall, these data provide evidence on the potential anti-cancer activity of CP-LAAO from the venom of Malaysian C. purpureomaculatus for therapeutic intervention of human colon cancer.

[Investigation on the process of sapindus saponin purified with macroporous adsorption resin and screening of its bacteriostasis].

Science.gov (United States)

Fu, Yong; Lei, Peng; Han, Yu-mei; Yan, Dan

2010-02-01

To study the technological parameters of the purification process of saponins with macroporous adsorption resin. The adsorptive characteristics and elutive parameters of the process were studied by taking the elutive and purified ratio of saponins as markers. Bacteriostasis activity of each parts eluted was evaluated by the mean of cup-plate method. 13.6 mL of the extraction of sapindus saponin (crude drugs 0.01 g/mL) was purified with a column of macroporous adsorption resin (phi15 mm x H90 mm, dry weight 2.5 g) and washed with 3BV of distilled water, then eluted with 3BV of 30% ethanol and 3BV of 70% ethanol, most of saponins were collected in the 70% ethanol. With macroporous adsorption resin adsorbing and purifying, the elutive ratio of saponins was 93.8% and the purity reached 250.1%. So this process of applying macroporous adsorption resin to adsorb and purify saponins is feasible, and supplies reference to the purification of other types of saponin.

Purification, characterization, cytotoxicity and anticancer activities of L-asparaginase, anti-colon cancer protein, from the newly isolated alkaliphilic Streptomyces fradiae NEAE-82

Science.gov (United States)

El-Naggar, Noura El-Ahmady; Deraz, Sahar F.; Soliman, Hoda M.; El-Deeb, Nehal M.; El-Ewasy, Sara M.

2016-01-01

L-asparaginase is an important enzyme as therapeutic agents used in combination with other drugs in the treatment of acute lymphoblastic leukemia. A newly isolated actinomycetes strain, Streptomyces sp. NEAE-82, was potentially producing extracellular L-asparaginase, it was identified as Streptomyces fradiae NEAE-82, sequencing product was deposited in the GenBank database under accession number KJ467538. L-asparaginase was purified from the crude enzyme using ammonium sulfate precipitation, dialysis and ion exchange chromatography using DEAE Sepharose CL-6B. Further the kinetic studies of purified enzyme were carried out. The optimum pH, temperature and incubation time for maximum L-asparaginase activity were found to be 8.5, 40 °C and 30 min, respectively. The optimum substrate concentration was found to be 0.06 M. The Km and Vmax of the enzyme were 0.01007 M and 95.08 Uml−1min−1, respectively. The half-life time (T1/2) was 184.91 min at 50 °С, while being 179.53 min at 60 °С. The molecular weight of the subunits of L-asparaginase was found to be approximately 53 kDa by SDS–PAGE analysis. The purified L-asparaginase showed a final specific activity of 30.636 U/mg protein and was purified 3.338-fold. The present work for the first time reported more information in the production, purification and characterization of L-asparaginase produced by newly isolated actinomycetes Streptomyces fradiae NEAE-82. PMID:27605431

Evaluation of the purified fraction of Wilbrandia (c. f. verticillata for antitumour activity

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V. S. N. Rao

1991-01-01

Full Text Available Cucurbatacins are known to produce cytotoxic and anticancer activities. Two novel norcucurbitacin glucosides (Wvl and Wv2 have recently been isolated from a purified fraction obtained from the rhizome of Wilbrandia verticillata. The present study evaluates the cytotoxic and anti-tumour activities of the norcucurbitacins. We have found a regular cytotoxicity in KB cells (Cy50 = 12µg/ml as well as a significant inhibition in the Walker 256 carcinosarcoma growth (approximately 75%.

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

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

2016-01-01

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

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

Science.gov (United States)

Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S

2009-05-04

To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.

Effects of seco-steroids purified from Physalis angulata L., Solanaceae, on the viability of Leishmania sp Efeitos de seco-esteróides purificados de Physalis angulata L., Solanaceae na viabilidade de Leishmania sp

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Elisalva T. Guimarães

2010-12-01

Full Text Available Physalis angulata L., Solanaceae, is an annual herb commonly used in popular medicine in many tropical and subtropical countries. P. angulata extracts contain a variety of substances, but little is known about their pharmacological activities. In this work we investigated the in vitro antileishmanial activity of seco-steroids (physalins purified from P. angulata. Addition of physalins B, F, and G caused a concentration-dependent inhibition in the growth of L. amazonensis promastigotes, being the IC50 values were 6.8, 1.4, and 9.2 μM, respectively. Physalin D was less active and had an IC50 value of 30.5 μM. Physalins were also active in cultures of other Leishmania species (L. major, L. braziliensis, and L. chagasi. Our results demonstrate the potent antileishmanial activity of physalins in cultures of Leishmania species of the New and Old Worlds and suggest the therapeutic potential of these seco-steroids in leishmaniasis.Physalis angulata L., Solanaceae, é uma erva anual utilizada na medicina popular em muitos países tropicais e subtropicais. Apesar dos extratos da P. angulata apresentarem uma grande variedade de substâncias, pouco é conhecido sobre a sua atividade farmacológica. Neste trabalho foi investigado a atividade antileishmania in vitro de seco-esteroides (fisalinas purificados da P. angulata. O tratamento com as fisalinas B, F e G causou uma inibição concentração-dependente do crescimento de promastigotas de Leishmania amazonensis em cultura axênica, com valores de IC50 de 6,8, 1,4, e 9,2 μM respectivamente. A fisalina D foi menos ativa, com valores de IC50 de 30,5 μM. Foi também observada uma atividade leishmanicida em culturas de outras espécies de Leishmania (L. major, L. braziliensis e L. chagasi. Nossos resultados demonstram que as fisalinas inibem o crescimento dos promastigotas com o tratamento de espécies de Leishmania do Velho e do Novo Mundos e sugerem o potencial terapêutico destas moléculas na

Comparative study of two purified inulinases from thermophile Thielavia Terrestris NRRL 8126 and mesophile Aspergillus Foetidus NRRL 337 grown on Cichorium Intybus l

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Eman Mohamed Fawzi

2011-06-01

Full Text Available Thirty fungal species grown on Cichorium intybus L. root extract as a sole carbon source, were screened for the production of exo-inulinase activities. The thermophile Thielavia terrestris NRRL 8126 and mesophile Aspergillus foetidus NRRL 337 gave the highest production levels of inulinases I & II at 50 and 24 ºC respectively. Yeast extract and peptone were the best nitrogen sources for highest production of inulinases I & II at five and seven days of incubation respectively. The two inulinases I & II were purified to homogeneity by gel-filtration and ion-exchange chromatography with 66.0 and 42.0 fold of purification respectively. The optimum temperatures of purified inulinases I & II were 75 and 50 ºC respectively. Inulinase I was more thermostable than the other one. The optimum pH for activity was found to be 4.5 and 5.5 for inulinases I & II respectively. A comparatively lower Michaelis-Menten constant (2.15 mg/ml and higher maximum initial velocity (115 µmol/min/mg of protein for inulinase I on inulin demonstrated the exoinulinase's greater affinity for inulin substrate. These findings are significant for its potential industrial application. The molecular mass of the inulinases I & II were estimated to be 72 & 78 kDa respectively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Structural characterization of the thermally-tolerant pectin methylesterase purified from Citrus sinensis fruit and its gene sequence

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Despite the longstanding importance for the thermally-tolerant pectin methylesterase (TT-PME) activity in citrus juice processing and product quality, unequivocal identification of the protein and its corresponding gene has remained elusive. We purified TT-PME from sweet orange [Citrus sinensis (L.)...

CHARACTERIZATION OF THE PARTIALLY PURIFIED PLANTARCIN SR18 PRODUCED BY LACTOBACILLUS PLANTARUM SR18

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Wagih El-Shouny

2013-04-01

Full Text Available The bacteriocin bound to the cells and that secreted into the culture filtrate of Lactobacillus plantarum SR18 were precipitated by 75% ammomium sulphate, dialysed and further purified by Gel filtration on Sephadex G-100. Bacteriocins were purified from proteins bound to the cell of L. plantarum SR18 (plantarcin SR18 a and culture filtrate proteins (plantarcin SR18 b, respectively. The SDS-PAGE of partially purified Plantarcin SR18a showed a molecular weight of 3.5 KDa. While, plantarcin SR18 b had a molecular weight of 10.3 KDa. The antibacterial activity of the tested plantarcin SR18 preparations suffered no measurable loss after 45 min at 80ºC. Whereas, At 100ºC, significant decrease in the activity of bacteriocin preparations (60- 80 % took place by the end of 45 min. At pH ranged from 5-8, the activity of the plantarcin SR18 preparations suffered no measurable loss. Dissociating agents significantly affected the bacteriocin activity. Thus, tween 80 and mercaptoethanol increased the activity of bacteriocin preparations to 1.2-1.4 fold. Sodium dodecyl sulphate (SDS increased the activity of the tested bacteriocin preparations by about 20%.The lowest residual activity (60% was recorded after treatment with Triton X100 for 45 min. Protease completely inhibited the activities of all forms of plantarcin SR18 after 45 min at 37ºC.

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

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Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2017-07-05

Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

Structural and functional characterization of recombinant napin-like protein of Momordica charantia expressed in methylotrophic yeast Pichia pastoris.

Science.gov (United States)

Yadav, Shailesh Kumar R; Sahu, Tejram; Dixit, Aparna

2016-08-01

Napin and napin-like proteins belong to the 2S albumin seed storage family of proteins and have been shown to display a variety of biological activities. However, due to a high degree of polymorphism, purification of a single napin or napin-like protein exhibiting biological activity is extremely difficult. In the present study, we have produced the napin-like protein of Momordica charantia using the methylotrophic Pichia pastoris expression system. The recombinant napin-like protein (rMcnapin) secreted in the extracellular culture supernatant was enriched by ammonium sulfate precipitation, and purified using size exclusion chromatography at a yield of ∼290 mg/L of culture. Secondary structure analysis of the purified rMcnapin revealed it to be predominantly α-helical with minimal β strand content. CD spectroscopic and fluorescence spectroscopic analyses revealed the rMcnapin to be stable at a wide range of temperatures and pH. The rMcnapin exhibited antifungal activity against Trichoderma viride with an IC50 of ∼3.7 μg/ml and trypsin inhibitor activity with an IC50 of 4.2 μM. Thus, large amounts of homogenous preparations of the biologically active rMcnapin could be obtained at shake flask level, which is otherwise difficult from its natural source.

Investigating the characteristic strength of flocs formed from crude and purified Hibiscus extracts in water treatment.

Science.gov (United States)

Jones, Alfred Ndahi; Bridgeman, John

2016-10-15

The growth, breakage and re-growth of flocs formed using crude and purified seed extracts of Okra (OK), Sabdariffa (SB) and Kenaf (KE) as coagulants and coagulant aids was assessed. The results showed floc size increased from 300 μm when aluminium sulphate (AS) was used as a coagulant to between 696 μm and 722 μm with the addition of 50 mg/l of OK, KE and SB crude samples as coagulant aids. Similarly, an increase in floc size was observed when each of the purified proteins was used as coagulant aid at doses of between 0.123 and 0.74 mg/l. The largest floc sizes of 741 μm, 460 μm and 571 μm were obtained with a 0.123 mg/l dose of purified Okra protein (POP), purified Sabdariffa (PSP) and purified Kenaf (PKP) respectively. Further coagulant aid addition from 0.123 to 0.74 mg/l resulted in a decrease in floc size and strength in POP and PSP. However, an increase in floc strength and reduced d50 size was observed in PKP at a dose of 0.74 mg/l. Flocs produced when using purified and crude extract samples as coagulant aids exhibited high recovery factors and strength. However, flocs exhibited greater recovery post-breakage when the extracts were used as a primary coagulant. It was observed that the combination of purified proteins and AS improved floc size, strength and recovery factors. Therefore, the applications of Hibiscus seeds in either crude or purified form increases floc growth, strength, recoverability and can also reduce the cost associated with the import of AS in developing countries. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Characterization of chickpea (Cicer arietinum L.) lectin for biological activity.

Science.gov (United States)

Gautam, Ajay Kumar; Gupta, Neha; Narvekar, Dakshita T; Bhadkariya, Rajni; Bhagyawant, Sameer S

2018-05-01

Lectins are proteins that are subject of intense investigations. Information on lectin from chickpea ( Cicer arietinum L.) with respect to its biological activities are very limited. In this study, we purified lectin from the seeds of chickpea employing DEAE-cellulose and SP-Sephadex ion exchange chromatography and identified its molecular subunit mass as 35 kDa. The free radical scavenging activity of lectin measured by the DPPH assay has IC 50 of 0.88 µg/mL. Lectin exerted antifungal activity against Candida krusei , Fusarium oxysporium oxysporium , Saccharomyces cerevisiae and Candida albicans , while antibacterial activity against E. coli , B. subtilis , S. marcescens and P. aeruginosa. The minimum inhibitory concentrations were 200, 240, 160 and 140 µg for C. krusei, F. oxysporium , S. cerevisiae and C. albicans respectively. Lectin was further examined for its antiproliferative potential against cancerous cell line. The cell viability assay indicated a high inhibition activity on Ishikawa, HepG2, MCF-7 and MDA-MB-231 with IC 50 value of 46.67, 44.20, 53.58 and 37.46 µg/mL respectively. These results can provide a background for future research into the benefits of chickpea lectin to pharmacological perspective.

Characterization of a protein kinase activity associated with purified capsids of the granulosis virus infecting Plodia interpunctella.

Science.gov (United States)

Wilson, M E; Consigli, R A

1985-06-01

A cyclic-nucleotide independent protein kinase activity has been demonstrated in highly purified preparations of the granulosis virus infecting the Indian meal moth, Plodia interpunctella. A divalent cation was required for activity. Manganese was the preferred cation and a pH of 8.0 resulted in optimal incorporation of 32P radiolabel into acid-precipitable protein. Although both ATP and GTP could serve as phosphate donors, ATP was utilized more efficiently by the enzyme. The kinase activity was localized to purified capsids; and the basic, internal core protein, VP12, was found to be the predominant viral acceptor. Histones and protamine sulfate could also serve as acceptors for the capsid-associated kinase activity. Using acid hydrolysis and phosphoamino acid analysis of phosphorylated nucleocapsid protein and nuclear magnetic resonance of phosphorylated VP12, it was determined that the enzyme catalyzes the transfer of phosphate to both serine and arginine residues of acceptor proteins. We believe this kinase activity may play a significant role in the viral replication cycle.

Identification of cancer stem-like side population cells in purified primary cultured human laryngeal squamous cell carcinoma epithelia.

Directory of Open Access Journals (Sweden)

Chun-Ping Wu

Full Text Available Cancer stem-like side population (SP cells have been identified in many solid tumors; however, most of these investigations are performed using established cancer cell lines. Cancer cells in tumor tissue containing fibroblasts and many other types of cells are much more complex than any cancer cell line. Although SP cells were identified in the laryngeal squamous cell carcinoma (LSCC cell line Hep-2 in our pilot study, it is unknown whether the LSCC tissue contains SP cells. In this study, LSCC cells (LSCCs were primary cultured and purified from a surgically resected LSCC specimen derived from a well-differentiated epiglottic neoplasm of a Chinese male. This was followed by the verification of epithelium-specific characteristics, such as ultrastructure and biomarkers. A distinct SP subpopulation (4.45±1.07% was isolated by Hoechst 33342 efflux analysis from cultured LSCCs by using a flow cytometer. Cancer stem cell (CSC-associated assays, including expression of self-renewal and CSC marker genes, proliferation, differentiation, spheroid formation, chemotherapy resistance, and tumorigenicity were then conducted between SP and non-SP (NSP LSCCs. In vitro and in vivo assays revealed that SP cells manifested preferential expression of self-renewal and CSC marker genes, higher capacity for proliferation, differentiation, and spheroid formation; enhanced resistance to chemotherapy; and greater xenograft tumorigenicity in immunodeficient mice compared with NSP cells. These findings suggest that the primary cultured and purified LSCCs contain cancer stem-like SP cells, which may serve as a valuable model for CSC research in LSCC.

Mixed protocols: Multiple ratios of FSH and LH bioactivity using highly purified, human-derived FSH (BRAVELLE and highly purified hMG (MENOPUR are unaltered by mixing together in the same syringe

Directory of Open Access Journals (Sweden)

Raike Elizabeth

2005-11-01

Full Text Available Abstract Background The use of mixed or blended protocols, that utilize both FSH and hMG, for controlled ovarian hyperstimulation is increasing in use. To reduce the number of injections a patient must administer, many physicians instruct their patients to mix their FSH and hMG together to be given as a single injection. Therefore, the goal of this study was to definitively determine if the FSH and LH bioactivities of highly purified, human-derived FSH (Bravelle(R and highly purified hMG (Menopur(R were altered by reconstituting in 0.9% saline and mixing in the same syringe. Methods Bravelle(R and Menopur(R were reconstituted in 0.9% saline and mixed in a Becton Dickinson plastic syringe. The FSH and LH bioactivities of the products were determined after injecting female and male rats, respectively, with Bravelle(R, Menopur(R, or a mixture of Bravelle(R and Menopur(R. Ratios of FSH:LH activity tested were 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1 vial Bravelle(R: 3 vials of Menopur(R. Results There were no statistically significant changes in either FSH or LH bioactivity that occurred after mixing Bravelle(R with Menopur(R in the same syringe. The theoretical vs. actual FSH bioactivity for Bravelle(R and Menopur(R were 75 vs. 76.58 IU/mL and 75 vs. 76.0 IU/mL, respectively. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1 vial Bravelle(R: 3 vials of Menopur(R tested, the theoretical vs. actual FSH bioactivities were 150 vs. 156.86 IU/mL, 300 vs. 308.69 IU/mL and 300 vs. 306.58 IU/mL, respectively. The theoretical vs. actual LH bioactivity for Menopur(R in the above mentioned ratios tested were 75 vs. 77.50 IU/mL. For the 3 ratios of FSH:LH activity tested, 150:75 IU (1 vial Bravelle(R: 1 vial Menopur(R, 300:75 IU (3 vials Bravelle(R: 1 vial Menopur(R or 300:225 IU (1

Purification of a lectin from Eugenia uniflora L. seeds and its potential antibacterial activity.

Science.gov (United States)

Oliveira, M D L; Andrade, C A S; Santos-Magalhães, N S; Coelho, L C B B; Teixeira, J A; Carneiro-da-Cunha, M G; Correia, M T S

2008-03-01

The aim of this work was to analyse the antimicrobial properties of a purified lectin from Eugenia uniflora L. seeds. The E. uniflora lectin (EuniSL) was isolated from the seed extract and purified by ion-exchange chromatography in DEAE-Sephadex with a purification factor of 11.68. The purified lectin showed a single band on denaturing electrophoresis, with a molecular mass of 67 kDa. EuniSL agglutinated rabbit and human erythrocytes with a higher specificity for rabbit erythrocytes. The haemagglutination was not inhibited by the tested carbohydrates but glycoproteins exerted a strong inhibitory action. The lectin proved to be thermo resistant with the highest stability at pH 6.5 and divalent ions did not affect its activity. EuniSL demonstrated a remarkable nonselective antibacterial activity. EuniSL strongly inhibited the growth of Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella sp. with a minimum inhibitory concentration (MIC) of 1.5 microg ml(-1), and moderately inhibited the growth of Bacillus subtilis, Streptococcus sp. and Escherichia coli with a MIC of 16.5 microg ml(-1). EuniSL was found to be effective against bacteria. The strong antibacterial activity of the studied lectin indicates a high potential for clinical microbiology and therapeutic applications.

Kinetic characterization of recombinant Bacillus coagulans FDP-activated l-lactate dehydrogenase expressed in Escherichia coli and its substrate specificity.

Science.gov (United States)

Jiang, Ting; Xu, Yanbing; Sun, Xiucheng; Zheng, Zhaojuan; Ouyang, Jia

2014-03-01

Bacillus coagulans is a homofermentative, acid-tolerant and thermophilic sporogenic lactic acid bacterium, which is capable of producing high yields of optically pure lactic acid. The l-(+)-lactate dehydrogenase (l-LDH) from B. coagulans is considered as an ideal biocatalyst for industrial production. In this study, the gene ldhL encoding a thermostable l-LDH was amplified from B. coagulans NL01 genomic DNA and successfully expressed in Escherichia coli BL21 (DE3). The recombinant enzyme was partially purified and its enzymatic properties were characterized. Sequence analysis demonstrated that the l-LDH was a fructose 1,6-diphosphate-activated NAD-dependent lactate dehydrogenase (l-nLDH). Its molecular weight was approximately 34-36kDa. The Km and Vmax values of the purified l-nLDH for pyruvate were 1.91±0.28mM and 2613.57±6.43μmol(minmg)(-1), respectively. The biochemical properties of l-nLDH showed that the specific activity were up to 2323.29U/mg with optimum temperature of 55°C and pH of 6.5 in the pyruvate reduction and 351.01U/mg with temperature of 55°C and pH of 11.5 in the lactate oxidation. The enzyme also showed some activity in the absence of FDP, with a pH optimum of 4.0. Compared to other lactic acid bacterial l-nLDHs, the enzyme was found to be relatively stable at 50°C. Ca(2+), Ba(2+), Mg(2+) and Mn(2+) ions had activated effects on the enzyme activity, and the enzyme was greatly inhibited by Ni(2+) ion. Besides these, l-nLDH showed the higher specificity towards pyruvate esters, such as methyl pyruvate and ethyl pyruvate. Copyright © 2014 Elsevier Inc. All rights reserved.

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

Science.gov (United States)

Santiago, Margarita; Gardner, Richard C

2015-07-01

Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize l-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized l-cystine and some other cysteine conjugates, but not l-cystathionine or l-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on l-cysteine as a nitrogen source, and that overexpression of the gene results in increased H2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-l-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis. Copyright © 2015 John Wiley & Sons, Ltd.

Argon activation analysis, application to dating by the potassium-argon method; Analyse par activation de l'argon. Application a la datation par la methode potassium-argon

Energy Technology Data Exchange (ETDEWEB)

Dumesnil, P [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1967-07-01

Activation analysis using radiogenic argon-40 has been applied to rock-dating by the K-Ar method. The argon is extracted from the sample, purified, activated to saturation in a flux of 2 X 10{sup 13} neutrons/cm{sup 2} sec{sup -1} and measured by gamma spectroscopy. The sensitivity obtained is such that it is possible to measure amounts of argon corresponding to age of a few thousand years only. However since it has not been possible to measure the amount of pollution of radiogenic argon by atmospheric argon with any accuracy, the measurable age limit is in fact of the order of MY. The method has been applied to basalts from the Mont-Dore region. The results obtained are in fairly good agreement with geological, stratigraphic and paleomagnetic data. (author) [French] L'analyse par activation de l'argon 40 radiogenique a ete appliquee a la datation des roches par la methode K-Ar. L'argon est extrait de l'echantillon, purifie, active a saturation dans un flux de 2.10{sup 13} neutrons.cm{sup -2}.s{sup -1} et mesure en spectrometrie gamma. La sensibilite obtenue est telle qu'il est possible de mesurer des quantites d'argon correspondant a des ages de quelques milliers d'annees seulement. Cependant la correction de pollution de l'argon radiogenique par l'argon atmospherique n'ayant pu etre etablie avec precision, la limite d'age mesurable pratique est de l'ordre de 1 Ma. La methode a ete appliquee aux basaltes de la region du Mont-Dore. Les ages obtenus sont en assez bon accord avec les donnees geologiques, stratigraphiques et paleomagnetiques. (auteur)

Simultaneous purifying of Hg0, SO2, and NOx from flue gas by Fe3+/H2O2: the performance and purifying mechanism.

Science.gov (United States)

Xing, Yi; Li, Liuliu; Lu, Pei; Cui, Jiansheng; Li, Qianli; Yan, Bojun; Jiang, Bo; Wang, Mengsi

2018-03-01

Hg 0 , SO 2 , and NOx result in heavily global environmental pollution and serious health hazards. Up to now, how to efficiently remove mercury with SO 2 and NOx from flue gas is still a tough task. In this study, series of high oxidizing Fenton systems were employed to purify the pollutants. The experimental results showed that Fe 3+ /H 2 O 2 was more suitable to purify Hg 0 than Fe 2+ /H 2 O 2 and Cu 2+ /H 2 O 2. The optimal condition includes Fe 3+ concentration of 0.008 mol/L, Hg 0 inlet concentration of 40 μg/m 3 , solution temperature of 50 °C, pH of 3, H 2 O 2 concentration of 0.7 mol/L, and O 2 percentage of 6%. When SO 2 and NOx were taken into account under the optimal condition, Hg 0 removal efficiency could be enhanced to 91.11% while the removal efficiency of both NOx and SO 2 was slightly declined, which was consistent to the analysis of purifying mechanism. The removal efficiency of Hg 0 was stimulated by accelerating the conversion of Fe 2+ to Fe 3+ , which resulted from the existence of SO 2 and NOx. The results of this study suggested that simultaneously purifying Hg 0 , SO 2 , and NOx from flue gas is feasible.

Assay of partially purified glutamate dehydrogenase isolated from ...

African Journals Online (AJOL)

Glutamate dehydrogenase (E C 1.4.1.1) isolated from the seeds of asparagus beans was partially purified to a factor of 22 by dialysis after fractional precipitation with solid ammonium sulphate at 40 and 60% saturation. A specific activity of 11.78μmol min-1 mg-1 protein was calculated for the partially purified enzyme when ...

Partially purified polygalacturonase from Aspergillus niger (SA6 ...

African Journals Online (AJOL)

Polygalacturonase (PG) was isolated from Aspergillus niger (A. niger) (SA6), partially purified and characterized. The PG showed two bands on SDS-PAGE suggesting an “endo and exo PG with apparent molecular weights of 35 and 40 KDa, respectively. It was purified 9-fold with a yield of 0.18% and specific activity of 246 ...

Purifying Nanomaterials

Science.gov (United States)

Hung, Ching-Cheh (Inventor); Hurst, Janet (Inventor)

2014-01-01

A method of purifying a nanomaterial and the resultant purified nanomaterial in which a salt, such as ferric chloride, at or near its liquid phase temperature, is used to penetrate and wet the internal surfaces of a nanomaterial to dissolve impurities that may be present, for example, from processes used in the manufacture of the nanomaterial.

In vitro and in vivo antioxidant activities of polysaccharide purified from aloe vera (Aloe barbadensis) gel.

Science.gov (United States)

Kang, Min-Cheol; Kim, Seo Young; Kim, Yoon Taek; Kim, Eun-A; Lee, Seung-Hong; Ko, Seok-Chun; Wijesinghe, W A J P; Samarakoon, Kalpa W; Kim, Young-Sun; Cho, Jin Hun; Jang, Hyeang-Su; Jeon, You-Jin

2014-01-01

The in vitro and in vivo antioxidant potentials of a polysaccharide isolated from aloe vera gel were investigated. Enzymatic extracts were prepared from aloe vera gel by using ten digestive enzymes including five carbohydrases and five proteases. Among them, the highest yield was obtained with the Viscozyme extract and the same extract showed the best radical scavenging activity. An active polysaccharide was purified from the Viscozyme extract using ethanol-added separation and anion exchange chromatography. Purified aloe vera polysaccharide (APS) strongly scavenged radicals including DPPH, hydroxyl and alkyl radicals. In addition, APS showed a protective effect against AAPH-induced oxidative stress and cell death in Vero cells as well as in the in vivo zebrafish model. In this study, it is proved that both the in vitro and in vivo antioxidant potentials of APS could be further utilized in relevant industrial applications. Copyright © 2013 Elsevier Ltd. All rights reserved.

76 FR 29191 - Purified Carboxymethylcellulose From Finland and the Netherlands: Continuation of Antidumping...

Science.gov (United States)

2011-05-20

... Carboxymethylcellulose From Finland and the Netherlands: Continuation of Antidumping Duty Orders AGENCY: Import... antidumping duty orders on purified carboxymethylcellulose from Finland and the Netherlands would likely lead...) from Finland and the Netherlands. See Notice of Antidumping Duty Orders: Purified...

Cultured rat and purified human Pneumocystis carinii stimulate intra- but not extracellular free radical production in human neutrophils

DEFF Research Database (Denmark)

Jensen, T; Aliouat, E M; Lundgren, B

1998-01-01

The production of free radicals in human neutrophils was studied in both Pneumocystis carinii derived from cultures of L2 rat lung epithelial-like cells and Pneumocystis carinii purified from human lung. Using the cytochrome C technique, which selectively measured extracellular superoxide...... generation, hardly any free radical production was observed after stimulation with cultured rat-derived P. carinii. A chemiluminescence technique, which separately measured intra- and extracellular free radical production, was subsequently employed to differentiate the free radical generation....... It was established that 1) P. carinii stimulated intra- but not extracellular free radical production in human neutrophils, 2) opsonized cultured rat-derived P. carinii stimulated human neutrophils to a strong intracellular response of superoxide production, and 3) opsonized P. carinii, purified from human lung also...

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

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Tapati Bhanja Dey

2014-01-01

Full Text Available Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103 using wheat bran by solid state fermentation (SSF process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL and 0.4% α-amylase (899 U/mL, maximum clarity (%T660nm = 97.0% of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification.

Science.gov (United States)

Dey, Tapati Bhanja; Banerjee, Rintu

2014-01-01

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T(660 nm) = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Catalytic Properties and Immobilization Studies of Catalase from Malva sylvestris L.

OpenAIRE

Arabaci, G.; Usluoglu, A.

2013-01-01

Catalase was partially purified from Malva sylvestris L. and immobilized onto chitosan. Then, its catalytic properties were investigated. (NH4)2SO4 precipitation and dialysis were performed in the extracted enzyme. Further purification was performed with sephadex G-200 column. Kinetic studies of the purified enzyme activity were measured and characterized. The inhibitory effects of KCN, NaN3, CuSO4, and EDTA on M. sylvestris L. catalase activity were observed except NaCl. Furthermore, M. sylv...

Study of NSILA-s (nonsuppressible insulin-like activity soluble in acid ethanol) by a new radio-receptor assay

International Nuclear Information System (INIS)

Megyeri, K.

1977-01-01

The insulin-like activity nonsuppressible with insulin-antibodies (NSILA) accounts for 90% of the insulin activity of the blood plasma. A peptid, soluble in acid ethanol, was purified (NSILA-s) and specific NSILA-s receptors were found on the plasma membrane of liver cells. The specificity, kinetics, affinity and pH-optimum of NSILA-s receptors significantly differed from those of insulin-receptors. A new, highly specific radio-receptor assay was developed, applying 125 I NSILA-s and liver cell membranes or lymphocytes. By this means the NSILA-s concentration of blood plasma was determined under normal and pathological (hypoglycaemizing tumours, hypopituritarism, acromegaly, anorexia nervosa, etc.) conditions. It is concluded that, 90% of the NSILA-s concentration of blood plasma is bound. In cases of hypoglycaemizing tumours increased NSILA-s activity was demonstrated both in blood serum and in the extracts of the tumour-tissue. Pharmacological doses of growth hormon (GH) increased plasma NSILA-s concentration, however, in the case of stimulation- and inhibition-tests carried out in normal patients, no unambiguous relationship could be demonstrated between plasma GH- and NSILA-s-levels. (L.E.)

Effect of purified gambir leaves extract to prevent atherosclerosis in rats

Directory of Open Access Journals (Sweden)

Nanang Yunarto

2016-03-01

, antiaterosklerosis AbstractBackground: Atherosclerosis is a risk factor for coronary heart disease (CHD. Catechin have highantioxidant activity that can prevent atherosclerosis. Gambir (Uncaria gambir, Roxb. leaves extract havehigh catechin content thereby potentially inhibiting atherosclerosis. This research was aimed to examineeffect of purified gambir leaves extract to prevent atherosclerosis in rats.Methods: The experimental laboratory study was conducted in Pharmacy Laboratory and Animal Laboratory,National Institute of Health Research and Development, Ministry of Health, Republic of Indonesia in 2014.Gambir leaves extract were purified to gain optimum catechin. Afterwards, antioxidant activity was testedusing 2.2-diphenyl-1-picrylhydrazyl (DPPH method, with ascorbic acid as positive control. Thirty six whitemale Sprague Dawley rats aged 2.5 months were randomly divided into six groups, i.e. normal control group,negative control group (aquadest, positive control group (atorvastatin 2 mg/200 g bw,extract dose I (20mg/200 g bw, dose II (40 mg/200 g bw and dose III (80 mg/200 g bw. The rats were given high fat diet andtreatment according to their group for 60 days, except for normal control group.Results: Catechin content in the purified gambir leaves extract was 92,69%. From antioxidant activity test, IC50 wasfound to be 11,76 μg/mL. Anti-atherosclerotic activity study shown that compared to negative control, all three dosesof purified gambir leaves extract were able to prevent atherosclerosis through inhibition of aortic wall thickening andfoam cell formation due to high fat diet (p<0.05. Anti-atherosclerotic activity increased with increasing dose.Conclusion: Gambir leaves purified extract had the effect of preventing the thickening of the walls andfoam cell formation rat aorta. (Health Science Journal of Indonesia 2015;6:105-10Keywords: gambir, catechin, antiatherosclerosis

Argon activation analysis, application to dating by the potassium-argon method; Analyse par activation de l'argon. Application a la datation par la methode potassium-argon

Energy Technology Data Exchange (ETDEWEB)

Dumesnil, P. [Commissariat a l' Energie Atomique, Saclay (France). Centre d' Etudes Nucleaires

1967-07-01

Activation analysis using radiogenic argon-40 has been applied to rock-dating by the K-Ar method. The argon is extracted from the sample, purified, activated to saturation in a flux of 2 X 10{sup 13} neutrons/cm{sup 2} sec{sup -1} and measured by gamma spectroscopy. The sensitivity obtained is such that it is possible to measure amounts of argon corresponding to age of a few thousand years only. However since it has not been possible to measure the amount of pollution of radiogenic argon by atmospheric argon with any accuracy, the measurable age limit is in fact of the order of MY. The method has been applied to basalts from the Mont-Dore region. The results obtained are in fairly good agreement with geological, stratigraphic and paleomagnetic data. (author) [French] L'analyse par activation de l'argon 40 radiogenique a ete appliquee a la datation des roches par la methode K-Ar. L'argon est extrait de l'echantillon, purifie, active a saturation dans un flux de 2.10{sup 13} neutrons.cm{sup -2}.s{sup -1} et mesure en spectrometrie gamma. La sensibilite obtenue est telle qu'il est possible de mesurer des quantites d'argon correspondant a des ages de quelques milliers d'annees seulement. Cependant la correction de pollution de l'argon radiogenique par l'argon atmospherique n'ayant pu etre etablie avec precision, la limite d'age mesurable pratique est de l'ordre de 1 Ma. La methode a ete appliquee aux basaltes de la region du Mont-Dore. Les ages obtenus sont en assez bon accord avec les donnees geologiques, stratigraphiques et paleomagnetiques. (auteur)

Purifying hydrocarbons in the gaseous stage

Energy Technology Data Exchange (ETDEWEB)

1937-02-01

Gaseous tar oils are subjected, at temperatures of 320 to 380/sup 0/C, to the action of a mixture of activated carbon mixed with powdered metal which removes the sulfur contamination from the substance to be purified.

Studying the fate of non-volatile organic compounds in a commercial plasma air purifier

Energy Technology Data Exchange (ETDEWEB)

Schmid, Stefan [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Seiler, Cornelia; Gerecke, Andreas C. [Swiss Federal Laboratories for Material Science and Technology (EMPA), CH-8600 Dübendorf (Switzerland); Hächler, Herbert [University of Zürich, Institute for Food Safety and Hygiene, National Centre for Enteropathogenic Bacteria and Listeria (NENT), CH-8057 Zürich (Switzerland); Hilbi, Hubert [Ludwig-Maximilians-Universität München Max von Pettenkofer-Institut, D-80336 München (Germany); Frey, Joachim [University of Bern, Institute for Veterinary Bacteriology, CH-3001 Bern (Switzerland); Weidmann, Simon; Meier, Lukas; Berchtold, Christian [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland); Zenobi, Renato, E-mail: zenobi@org.chem.ethz.ch [ETH Zürich, Department of Chemistry and Applied Biosciences, CH-8093 Zürich (Switzerland)

2013-07-15

Highlights: • Degradation of environmental toxins, a protein, and bioparticles were studied. • A commercial air purifier based on a cold plasma was used. • Passage through the device reduced the concentration of the compounds/particles. • Deposition inside the plasma air purifier was the main removal process. -- Abstract: Degradation of non-volatile organic compounds–environmental toxins (methyltriclosane and phenanthrene), bovine serum albumin, as well as bioparticles (Legionella pneumophila, Bacillus subtilis, and Bacillus anthracis)–in a commercially available plasma air purifier based on a cold plasma was studied in detail, focusing on its efficiency and on the resulting degradation products. This system is capable of handling air flow velocities of up to 3.0 m s{sup −1} (3200 L min{sup −1}), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10 L min{sup −1}. Mass balance studies consistently indicated a reduction in concentration of the compounds/particles after passage through the plasma air purifier, 31% for phenanthrene, 17% for methyltriclosane, and 80% for bovine serum albumin. L. pneumophila did not survive passage through the plasma air purifier, and cell counts of aerosolized spores of B. subtilis and B. anthracis were reduced by 26- and 15-fold, depending on whether it was run at 10 Hz or 50 Hz, respectively. However rather than chemical degradation, deposition on the inner surfaces of the plasma air purifier occured. Our interpretation is that putative “degradation” efficiencies were largely due to electrostatic precipitation rather than to decomposition into smaller molecules.

The industrial applicability of purified cellulase complex indigenously produced by Trichoderma viride through solid-state bio-processing of agro-industrial and municipal paper wastes

Directory of Open Access Journals (Sweden)

Muhammad Irshad

2013-02-01

Full Text Available An indigenous strain of Trichoderma viride produced high titers of cellulase complex in solid-state bio-processing of agro-industrial orange peel waste, which was used as the growth-supporting substrate. When the conditions of the SSF medium containing 15 g orange peel (50% w/w moisture inoculated with 5 mL of inoculum were optimal, the maximum productions of endoglucanase (655 ± 5.5 U/mL, exoglucanase (412 ± 4.3 U/mL, and β-glucosidase (515 ± 3.7 U/mL were recorded after 4 days of incubation at pH 5 and 35 °C. The enzyme with maximum activity (endoglucanase was purified by ammonium sulfate fractionation and Sephadex G-100 column gel filtration chromatographic technique. Endoglucanase was 5.5-fold purified with specific activity of 498 U/mg in comparison to the crude enzyme. The enzyme was shown to have a molecular weight of 58 kDa by sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDS-PAGE. The shelf life profile revealed that the enzyme could be stored at room temperature (30 °C for up to 45 days without losing much of its activity.

Staphylococcal superantigen-like 5 activates platelets and supports platelet adhesion under flow conditions, which involves glycoprotein Ib alpha and alpha(IIb)beta(3)

NARCIS (Netherlands)

De Haas, C. J. C.; Weeterings, C.; Vughs, M. M.; De Groot, P. G.; Van Strijp, J. A.; Lisman, T.

2009-01-01

Objectives: Staphylococcal superantigen-like 5 (SSL5) is an exoprotein secreted by Staphylococcus aureus that has been shown to inhibit neutrophil rolling over activated endothelial cells via a direct interaction with P-selectin glycoprotein ligand 1 (PSGL-1). Methods and Results: When purified

Purification, characterization and antiproliferative activity of l-asparaginase from Aspergillus oryzae CCT 3940 with no glutaminase activity

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Fernanda Furlan Gonçalves Dias

2016-09-01

Conclusions: The sensitivity of the cells lines to purified l-asparaginase from A. oryzae CCT 3940 appeared to be concentration dependent affording a more significant decrease in cell growth than that observed for the commercial l-asparaginase from Escherichia coli. The l-asparaginase from A. oryzae CCT 3940 has a high potential for pharmaceutical exploitation in the treatment of leukemia.

Antitumor activity of Portulaca oleracea L. polysaccharides against cervical carcinoma in vitro and in vivo.

Science.gov (United States)

Zhao, Rui; Gao, Xu; Cai, Yaping; Shao, Xingyue; Jia, Guiyan; Huang, Yulan; Qin, Xuegong; Wang, Jingwei; Zheng, Xiaoliang

2013-07-25

Portulaca oleracea L. has been used as folk medicine in different countries to treat different ailments in humans. P. oleracea L. polysaccharide (POL-P), extracted from P. oleracea L., is found to have bioactivities such as hypoglycemic and hypolipidemic activities, antioxidant and antitumor activities. In our study, a water-soluble polysaccharide (POL-P3b) was successfully purified from Galium verum L. by DEAE cellulose and Sephadex G-200 column chromatography. To evaluate the anticancer efficacy and associated mechanisms of POL-P3b on cervical cancer in vitro and in vivo, we showed that treatment of HeLa cell with POL-P3b inhibited cell proliferation. In addition, POL-P3b significantly inhibited tumor growth in U14-bearing mice. Further analysis indicated that POL-P3b possesses the activity of inhibiting cervical cancer cell growth in vitro and in vivo at a concentration- and time-dependent manner, and the mechanisms were associated with Sub-G1 phase cell cycle arrest, triggering DNA damage and inducing apoptosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

International Nuclear Information System (INIS)

Guo, Leilei; Huang, Kaixun; Liu, Hongmei

2016-01-01

Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na 2 SeO 3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K m ) values and maximal reaction velocity (V max ) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

Science.gov (United States)

Guo, Leilei; Huang, Kaixun; Liu, Hongmei

2016-03-01

Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na2SeO3 as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3',5,5'-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant ( K m) values and maximal reaction velocity ( V max) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

Small surfactant-like peptides can drive soluble proteins into active aggregates

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Zhou Bihong

2012-01-01

Full Text Available Abstract Background Inactive protein inclusion bodies occur commonly in Escherichia coli (E. coli cells expressing heterologous proteins. Previously several independent groups have found that active protein aggregates or pseudo inclusion bodies can be induced by a fusion partner such as a cellulose binding domain from Clostridium cellulovorans (CBDclos when expressed in E. coli. More recently we further showed that a short amphipathic helical octadecapeptide 18A (EWLKAFYEKVLEKLKELF and a short beta structure peptide ELK16 (LELELKLKLELELKLK have a similar property. Results In this work, we explored a third type of peptides, surfactant-like peptides, for performing such a "pulling-down" function. One or more of three such peptides (L6KD, L6K2, DKL6 were fused to the carboxyl termini of model proteins including Aspergillus fumigatus amadoriase II (AMA, all three peptides were used, Bacillus subtilis lipase A (LipA, only L6KD was used, hereinafter the same, Bacillus pumilus xylosidase (XynB, and green fluorescent protein (GFP, and expressed in E. coli. All fusions were found to predominantly accumulate in the insoluble fractions, with specific activities ranging from 25% to 92% of the native counterparts. Transmission electron microscopic (TEM and confocal fluorescence microscopic analyses confirmed the formation of protein aggregates in the cell. Furthermore, binding assays with amyloid-specific dyes (thioflavin T and Cong red to the AMA-L6KD aggregate and the TEM analysis of the aggregate following digestion with protease K suggested that the AMA-L6KD aggregate may contain structures reminiscent of amyloids, including a fibril-like structure core. Conclusions This study shows that the surfactant-like peptides L6KD and it derivatives can act as a pull-down handler for converting soluble proteins into active aggregates, much like 18A and ELK16. These peptide-mediated protein aggregations might have important implications for protein aggregation in

Purification and immunolocalization of an annexin-like protein in pea seedlings

Science.gov (United States)

Clark, G. B.; Dauwalder, M.; Roux, S. J.

1992-01-01

As part of a study to identify potential targets of calcium action in plant cells, a 35-kDa, annexin-like protein was purified from pea (Pisum sativum L.) plumules by a method used to purify animal annexins. This protein, called p35, binds to a phosphatidylserine affinity column in a calcium-dependent manner and binds 45Ca2+ in a dot-blot assay. Preliminary sequence data confirm a relationship for p35 with the annexin family of proteins. Polyclonal antibodies have been raised which recognize p35 in Western and dot blots. Immunofluorescence and immunogold techniques were used to study the distribution and subcellular localization of p35 in pea plumules and roots. The highest levels of immunostain were found in young developing vascular cells producing wall thickenings and in peripheral root-cap cells releasing slime. This localization in cells which are actively involved in secretion is of interest because one function suggested for the animal annexins is involvement in the mediation of exocytosis.

Endurance Pump Test with MIL-PRF-83282 Hydraulic Fluid, Purified with Malabar Purifier

National Research Council Canada - National Science Library

Sharma, Shashi

2004-01-01

.... Endurance aircraft hydraulic pump tests under carefully controlled conditions were previously conducted using hydraulic fluid purified with a rotating-disk and vacuum type purifier, the portable...

The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

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Schoeman Leann

2008-05-01

Full Text Available Abstract Background The female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay. Methods Pregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells. Results The pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity. Conclusion Although it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions, is insufficient to cause viral inhibition or aggregation.

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

We have demonstrated that Penicillium chrysogenum possesses the L-cysteine biosynthetic enzyme O-acetyI-L-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates...... the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-L-serine sulphhydrylase and O-acetyl-L-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-L-serine as substrate for the formation of L-cysteine....... The purified enzyme did not catalyse the formation of L-homocysteine from O-acetyl-L-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyI-L-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold...

Phospholipid analysis and fractional reconstitution of the ice nucleation protein activity purified from Escherichia coli overexpressing the inaZ gene of Pseudomonas syringae.

Science.gov (United States)

Palaiomylitou, M A; Kalimanis, A; Koukkou, A I; Drainas, C; Anastassopoulos, E; Panopoulos, N J; Ekateriniadou, L V; Kyriakidis, D A

1998-08-01

Ice nucleation protein was partially purified from the membrane fraction of E. coli carrying inaZ from Pseudomonas syringae. The ice nucleation protein was totally localized in the bacterial envelope and was extracted by either salt (0.25 M NH4Cl) or the nonionic detergent Tween 20. The extracted protein was partially purified by sequential passage through DEAE-52 cellulose and Sephacryl-S400 columns. The activity of the purified protein was lost after treatment with phospholipase C, and its activity was subsequently restored by addition of the naturally occurring lipid phosphatidylethanolamine. These results suggest that ice nucleation proteins have a requirement for lipids that reconstitute a physiological hydrophobic environment similar to the one existing in vivo, to attain and maintain a structure that enables ice catalysis. Copyright 1998 Academic Press.

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity.

Science.gov (United States)

Ogino, Minako; Ito, Naoto; Sugiyama, Makoto; Ogino, Tomoaki

2016-05-21

The large (L) protein of rabies virus (RABV) plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5'-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase) activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5'-triphosphorylated but not 5'-diphosphorylated RABV mRNA-start sequences, 5'-AACA(C/U), with GDP to generate the 5'-terminal cap structure G(5')ppp(5')A. The 5'-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286) in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

Energy Technology Data Exchange (ETDEWEB)

Guo, Leilei; Huang, Kaixun; Liu, Hongmei, E-mail: hmliu2004@126.com [Huazhong University of Science and Technology, School of Chemistry and Chemical Engineering (China)

2016-03-15

Selenium nanoparticles (SeNPs) are considered to be the new selenium supplement forms with high biological activity and low toxicity; however, the molecular mechanism by which SeNPs exert the biological function is unclear. Here, we reported that biocompatibility SeNPs possessed intrinsic oxidase-like activity. Using Na{sub 2}SeO{sub 3} as a precursor and glutathione as a reductant, biocompatibility SeNPs were synthesized by the wet chemical reduction method in the presence of bovine serum albumin (BSA). The results of structure characterization revealed that synthesized SeNPs were amorphous red elementary selenium with spherical morphology, and ranged in size from 25 to 70 nm size with a narrow distribution (41.4 ± 6.7 nm). The oxidase-like activity of the as-synthesized SeNPs was tested with 3,3′,5,5′-tetramethylbenzidine (TMB) as a substrate. The results indicated that SeNPs could catalyze the oxidization of TMB by dissolved oxygen. These SeNPs showed an optimum catalytic activity at pH 4 and 30 °C, and the oxidase-like activity was higher as the concentration of SeNPs increased and the size of SeNPs decreased. The Michaelis constant (K{sub m}) values and maximal reaction velocity (V{sub max}) of the SeNPs for TMB oxidation were 0.0083 mol/L and 3.042 μmol/L min, respectively.

An Experiment with Air Purifiers in Delhi during Winter 2015-2016.

Science.gov (United States)

Vyas, Sangita; Srivastav, Nikhil; Spears, Dean

2016-01-01

Particulate pollution has important consequences for human health, and is an issue of global concern. Outdoor air pollution has become a cause for alarm in India in particular because recent data suggest that ambient pollution levels in Indian cities are some of the highest in the world. We study the number of particles between 0.5μm and 2.5μm indoors while using affordable air purifiers in the highly polluted city of Delhi. Though substantial reductions in indoor number concentrations are observed during air purifier use, indoor air quality while using an air purifier is frequently worse than in cities with moderate pollution, and often worse than levels observed even in polluted cities. When outdoor pollution levels are higher, on average, indoor pollution levels while using an air purifier are also higher. Moreover, the ratio of indoor air quality during air purifier use to two comparison measures of air quality without an air purifier are also positively correlated with outdoor pollution levels, suggesting that as ambient air quality worsens there are diminishing returns to improvements in indoor air quality during air purifier use. The findings of this study indicate that although the most affordable air purifiers currently available are associated with significant improvements in the indoor environment, they are not a replacement for public action in regions like Delhi. Although private solutions may serve as a stopgap, reducing ambient air pollution must be a public health and policy priority in any region where air pollution is as high as Delhi's during the winter.

Hydrodynamic and Membrane Binding Properties of Purified Rous Sarcoma Virus Gag Protein

Energy Technology Data Exchange (ETDEWEB)

Dick, Robert A.; Datta, Siddhartha A.K.; Nanda, Hirsh; Fang, Xianyang; Wen, Yi; Barros, Marilia; Wang, Yun-Xing; Rein, Alan; Vogt, Volker M. (NCI); (Cornell); (CM); (NIST)

2016-05-06

Previously, no retroviral Gag protein has been highly purified in milligram quantities and in a biologically relevant and active form. We have purified Rous sarcoma virus (RSV) Gag protein and in parallel several truncation mutants of Gag and have studied their biophysical properties and membrane interactionsin vitro. RSV Gag is unusual in that it is not naturally myristoylated. From its ability to assemble into virus-like particlesin vitro, we infer that RSV Gag is biologically active. By size exclusion chromatography and small-angle X-ray scattering, Gag in solution appears extended and flexible, in contrast to previous reports on unmyristoylated HIV-1 Gag, which is compact. However, by neutron reflectometry measurements of RSV Gag bound to a supported bilayer, the protein appears to adopt a more compact, folded-over conformation. At physiological ionic strength, purified Gag binds strongly to liposomes containing acidic lipids. This interaction is stimulated by physiological levels of phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] and by cholesterol. However, unlike HIV-1 Gag, RSV Gag shows no sensitivity to acyl chain saturation. In contrast with full-length RSV Gag, the purified MA domain of Gag binds to liposomes only weakly. Similarly, both an N-terminally truncated version of Gag that is missing the MA domain and a C-terminally truncated version that is missing the NC domain bind only weakly. These results imply that NC contributes to membrane interactionin vitro, either by directly contacting acidic lipids or by promoting Gag multimerization.

Retroviruses like HIV assemble at and bud from the plasma membrane of cells. Assembly requires the interaction between thousands of Gag molecules to form a lattice. Previous work indicated that lattice formation at the plasma membrane is influenced by the conformation of monomeric HIV. We have extended this work to the more tractable RSV Gag. Our

Phytochemical analysis and antioxidant activity of Hyssopus officinalis L. from Iran

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Fatemeh Fathiazad

2011-12-01

Full Text Available Introduction: Hyssopus officinalis (L (Hyssop, Family: Lamiaceae, one of the endemic Iranian perennial herb with a long history of medicinal use, was studied to detect some biologically active chemical constituents of the plant. Methods: The flavonoids of the hydromethanolic extract of the aerial parts of Hyssopus officinalis (L. were studied by VLC and crystalisation of the major compound in subsequent fractions. Furthermore, the composition of its essential oil, total phenolic content and antioxidant activities were studied by GC-MS, Folin–Ciocalteau and DPPH reagents respectively. Results: Apigenin 7-O-β-D-glucuronide was isolated as the major flavonoid. All structural elucidation was performed by spectral means. A total of 20 compounds representing 99.97% of the oil have been identified. Myrtenylacetate , Camphor, Germacrene, Spathulenol were the main compounds The total phenol content of the n-butanol and ethylacetate extracts were determined spectrophotometrically according to the Folin–Ciocalteau procedure to be 246 mgGAE g-1 and 51 mgGAE g-1 in the aerial parts of Hyssopus officinalis. The antioxidant activities of apigenin 7-O-β-D-glucuronide, ethylacetate and n-butanol extracts were also determined by DPPH radical scavenging assay with IC50 values of 116×10_3, 103×10_3, 25×10_3 mg mL-1 respectively. The purified flavonoid showed weak radical scavenging activity (IC50 = 116×10_3mg mL_1. N-butanol extract, because of the highest content of total phenolic compounds (246 mgGAE100_1g had the best antioxidant activity (IC50 = 25mg mL_1. Conclusion: On the whole, the findings of the study revealed that Hyssop possesses valuable antioxidant properties for culinary and possible medicinal use.

Handbook of purified gases

CERN Document Server

Schoen, Helmut

2015-01-01

Technical gases are used in almost every field of industry, science and medicine and also as a means of control by government authorities and institutions and are regarded as indispensable means of assistance. In this complete handbook of purified gases the physical foundations of purified gases and mixtures as well as their manufacturing, purification, analysis, storage, handling and transport are presented in a comprehensive way. This important reference work is accompanied with a large number of Data Sheets dedicated to the most important purified gases.  

Optimization of freeze drying conditions for purified pectinase from mango (Mangifera indica cv. Chokanan) peel.

Science.gov (United States)

Mehrnoush, Amid; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

2012-01-01

Response surface methodology (RSM) along with central composite design (CCD) was applied to optimize the freeze drying conditions for purified pectinase from mango (Mangifera indica cv. Chokanan) peel. The effect of pectinase content (-2.66, 62.66 mg/mL), Arabic gum (-1.21, 10.21%, w/v), and maltodextrin (0.73, 7.26%, w/v) as independent variables on activity, yield, and storage stability of freeze-dried enzyme was evaluated. Storage stability of pectinase was investigated after one week at 4 °C and yield percentage of the enzyme after encapsulation was also determined. The independent variables had the most significant (p mango peel were obtained using 30 mg/mL of pectinase content, 4.5 (%, w/v) of Arabic gum, and 4 (%, w/v) of maltodextrin. Under these conditions, the maximum activity (11.12 U/mL), yield (86.4%) and storage stability (84.2%) of encapsulated pectinase were achieved.

Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.

Science.gov (United States)

Cheng, Lifang; Mu, Wanmeng; Jiang, Bo

2010-06-01

D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose. The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%. The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production. Copyright (c) 2010 Society of Chemical Industry.

Purification and characterization of l,(l/d)-aminopeptidase from Guinea pig serum.

Science.gov (United States)

Krstanović, Marina; Brgles, Marija; Halassy, Beata; Frkanec, Ruza; Vrdoljak, Anto; Branović, Karmen; Tomasić, Jelka; Benedetti, Fabio

2006-01-01

Mammalian sera contain enzymes that catalyze the hydrolytic degradation of peptidoglycans and molecules of related structure and are relevant for the metabolism of peptidoglycans. We now report on a novel L,(L/D)-aminopeptidase found in human and mammalian sera. The enzyme hydrolyses the pentapeptide L-Ala-D-iso-Gln-meso-DAP(omegaNH(2))-D-Ala-D-Ala yielding the free L-alanine and the respective tetrapeptide (K(M) 18 mM). L,(L/D)-aminopeptidase from guinea pig serum was highly purified in four chromatographic steps, up to 700-fold. Molecular weight of the enzyme was estimated by HPLC to be approximately 175,000. The configuration of alanine obtained by hydrolysis of the pentapeptide was determined by oxidation with L-amino acid oxidase. The amino acids sequence in the respective tetrapeptide was deduced from the results of mass spectrometry. The novel L,(L/D)-aminopeptidase also hydrolyzed alanine-4-nitroanilide (K(M)=0.6 mM) and several peptides comprising L-amino acids. Peptides containing D-amino acid at the amino end and L-Asp-L-Asp were not the substrates for this enzyme. The purified enzyme also exhibited enkephalin degrading activity, hydrolyzing enkephalins comprising L,L- and L,D-peptide bonds. The enzyme was inhibited strongly by metal chelating agents, bestatin and amastatin.

A Facile synthesis of superparamagnetic Fe3O4 nanofibers with superior peroxidase-like catalytic activity for sensitive colorimetric detection of L-cysteine

Science.gov (United States)

Chen, Sihui; Chi, Maoqiang; Zhu, Yun; Gao, Mu; Wang, Ce; Lu, Xiaofeng

2018-05-01

Superaramagnetic Fe3O4 nanomaterials are good candidates as enzyme mimics due to their excellent catalytic activity, high stability and facile synthesis. However, the morphology of Fe3O4 nanomaterials has much influence on their enzyme-like catalytic activity. In this work, we have developed a simple polymer-assisted thermochemical reduction approach to prepare Fe3O4 nanofibers for peroxidase-like catalytic applications. The as-prepared Fe3O4 nanofibers show a higher catalytic activity than commercial Fe3O4 nanoparticles. The steady-state kinetic assay result shows that the Michaelis-Menten constant value of the as-obtained Fe3O4 nanofibers is similar to that of horseradish peroxidase (HRP), indicating their superior affinity to the 3,3‧,5,5‧-tetramethylbenzidine (TMB) and H2O2 substrate. Based on the outstanding catalytic activity, a sensing platform for the detection of L-cysteine has been performed and the limit of detection is as low as 0.028 μM. In addition, an excellent selectivity toward L-cysteine over other types of amino acids, glucose and metal ions has been achieved as well. This work offers an original means for the fabrication of superparamagnetic Fe3O4 nanofibers and demonstrates their delightful potential applications in the fields of biosensing, environmental monitoring, and medical diagnostics.

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

Directory of Open Access Journals (Sweden)

Jenkins Jeremy L

2001-10-01

Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

Science.gov (United States)

Jenkins, Jeremy L; Dean, Donald H

2001-01-01

Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

Synthetic protease substrate n-benzoyl-L-argininyl-p-nitroanilide activates specific binding of [3H]estradiol to a protein in rat pancreas: relationship of structure to activity

International Nuclear Information System (INIS)

Grossman, A.

1984-01-01

N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of [ 3 H]estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of [ 3 H]estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the [ 3 H]estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of [ 3 H]estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables

Production of phenylpyruvic acid from L-phenylalanine using an L-amino acid deaminase from Proteus mirabilis: comparison of enzymatic and whole-cell biotransformation approaches.

Science.gov (United States)

Hou, Ying; Hossain, Gazi Sakir; Li, Jianghua; Shin, Hyun-Dong; Liu, Long; Du, Guocheng

2015-10-01

Phenylpyruvic acid (PPA) is an important organic acid that has a wide range of applications. In this study, the membrane-bound L-amino acid deaminase (L-AAD) gene from Proteus mirabilis KCTC 2566 was expressed in Escherichia coli BL21(DE3) and then the L-AAD was purified. After that, we used the purified enzyme and the recombinant E. coli whole-cell biocatalyst to produce PPA via a one-step biotransformation from L-phenylalanine. L-AAD was solubilized from the membrane and purified 52-fold with an overall yield of 13 %, which corresponded to a specific activity of 0.94 ± 0.01 μmol PPA min(-1)·mg(-1). Then, the biotransformation conditions for the pure enzyme and the whole-cell biocatalyst were optimized. The maximal production was 2.6 ± 0.1 g·L(-1) (specific activity of 1.02 ± 0.02 μmol PPA min(-1)·mg(-1) protein, 86.7 ± 5 % mass conversion rate, and 1.04 g·L(-1)·h(-1) productivity) and 3.3 ± 0.2 g L(-1) (specific activity of 0.013 ± 0.003 μmol PPA min(-1)·mg(-1) protein, 82.5 ± 4 % mass conversion rate, and 0.55 g·L(-1)·h(-1) productivity) for the pure enzyme and whole-cell biocatalyst, respectively. Comparative studies of the enzymatic and whole-cell biotransformation were performed in terms of specific activity, production, conversion, productivity, stability, need of external cofactors, and recycling. We have developed two eco-friendly and efficient approaches for PPA production. The strategy described herein may aid the biotransformational synthesis of other α-keto acids from their corresponding amino acids.

The Rabies Virus L Protein Catalyzes mRNA Capping with GDP Polyribonucleotidyltransferase Activity

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Minako Ogino

2016-05-01

Full Text Available The large (L protein of rabies virus (RABV plays multiple enzymatic roles in viral RNA synthesis and processing. However, none of its putative enzymatic activities have been directly demonstrated in vitro. In this study, we expressed and purified a recombinant form of the RABV L protein and verified its guanosine 5′-triphosphatase and GDP polyribonucleotidyltransferase (PRNTase activities, which are essential for viral mRNA cap formation by the unconventional mechanism. The RABV L protein capped 5′-triphosphorylated but not 5′-diphosphorylated RABV mRNA-start sequences, 5′-AACA(C/U, with GDP to generate the 5′-terminal cap structure G(5′ppp(5′A. The 5′-AAC sequence in the substrate RNAs was found to be strictly essential for RNA capping with the RABV L protein. Furthermore, site-directed mutagenesis showed that some conserved amino acid residues (G1112, T1170, W1201, H1241, R1242, F1285, and Q1286 in the PRNTase motifs A to E of the RABV L protein are required for cap formation. These findings suggest that the putative PRNTase domain in the RABV L protein catalyzes the rhabdovirus-specific capping reaction involving covalent catalysis of the pRNA transfer to GDP, thus offering this domain as a target for developing anti-viral agents.

Antidepressant-like activity of Sonchus oleraceus in mouse models of immobility tests.

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Vilela, Fabiana Cardoso; Padilha, Marina de Mesquita; Alves-da-Silva, Geraldo; Soncini, Roseli; Giusti-Paiva, Alexandre

2010-02-01

The aim of the present work is to evaluate the putative antidepressant-like effects of hydroethanolic and dichloromethanic extracts from the aerial parts of Sonchus oleraceus (Family Asteraceae) on the performance of male mice in the forced swimming test (FST) and tail suspension test (TST) models predictive of depression. The hydroethanolic and dichloromethanic extracts, both in doses of 30, 100, and 300 mg/kg, were orally administered 1 hour before carrying out the FST or the TST. The immobility time in both the FST and the TST was significantly reduced by acute oral treatment with the extracts (dose range, 100-300 mg/kg), without accompanying changes in ambulation, as assessed in an open-field test. This excluded the possibility that the effect of the extracts is due to an activation of locomotion. The efficacy of the extracts was found to be comparable to that of amitriptyline (10 mg/kg, p.o.). The present study provides evidence for an antidepressant-like effect of the active principle(s) present in the extracts of S. oleraceus in mice. Therefore, a standardized S. oleraceus extract or its purified constituents could be of potential interest for the treatment of depressive disorders.

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum).

Science.gov (United States)

Shamsi, Tooba Naz; Parveen, Romana; Sen, Priyankar; Fatima, Sadaf

2017-05-28

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20 mM tris-CaCl 2 buffer (pH 8.2) with a flow rate of 0.5 mL min -1 . The molecular weight and purity of ∼23 kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697 U mg -1 , fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.

Antibacterial activity of Mangifera indica L. seeds against some ...

African Journals Online (AJOL)

Antibacterial activity of methanol extract of Mangifera indica L. seeds was done against 41 clinically isolated and 20 standard bacterial strains. Clinical bacterial strains were isolated from different specimens like blood, urine, catheter, stool and pus. Antibacterial activity was done by agar disc diffusion method at two different ...

Optimization of Freeze Drying Conditions for Purified Pectinase from Mango (Mangifera indica cv. Chokanan Peel

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Abdul Manap Mohd Yazid

2012-03-01

Full Text Available Response surface methodology (RSM along with central composite design (CCD was applied to optimize the freeze drying conditions for purified pectinase from mango (Mangifera indica cv. Chokanan peel. The effect of pectinase content (−2.66, 62.66 mg/mL, Arabic gum (−1.21, 10.21%, w/v, and maltodextrin (0.73, 7.26%, w/v as independent variables on activity, yield, and storage stability of freeze-dried enzyme was evaluated. Storage stability of pectinase was investigated after one week at 4 °C and yield percentage of the enzyme after encapsulation was also determined. The independent variables had the most significant (p < 0.05 effect on pectinase activity and yield of the enzyme. It was observed that the interaction effect of Arabic gum and maltodextrin improved the enzymatic properties of freeze-dried pectinase. The optimal conditions for freeze-dried pectinase from mango peel were obtained using 30 mg/mL of pectinase content, 4.5 (%, w/v of Arabic gum, and 4 (%, w/v of maltodextrin. Under these conditions, the maximum activity (11.12 U/mL, yield (86.4% and storage stability (84.2% of encapsulated pectinase were achieved.

Optimization of Freeze Drying Conditions for Purified Pectinase from Mango (Mangifera indica cv. Chokanan) Peel

Science.gov (United States)

Mehrnoush, Amid; Mustafa, Shuhaimi; Yazid, Abdul Manap Mohd

2012-01-01

Response surface methodology (RSM) along with central composite design (CCD) was applied to optimize the freeze drying conditions for purified pectinase from mango (Mangifera indica cv. Chokanan) peel. The effect of pectinase content (−2.66, 62.66 mg/mL), Arabic gum (−1.21, 10.21%, w/v), and maltodextrin (0.73, 7.26%, w/v) as independent variables on activity, yield, and storage stability of freeze-dried enzyme was evaluated. Storage stability of pectinase was investigated after one week at 4 °C and yield percentage of the enzyme after encapsulation was also determined. The independent variables had the most significant (p < 0.05) effect on pectinase activity and yield of the enzyme. It was observed that the interaction effect of Arabic gum and maltodextrin improved the enzymatic properties of freeze-dried pectinase. The optimal conditions for freeze-dried pectinase from mango peel were obtained using 30 mg/mL of pectinase content, 4.5 (%, w/v) of Arabic gum, and 4 (%, w/v) of maltodextrin. Under these conditions, the maximum activity (11.12 U/mL), yield (86.4%) and storage stability (84.2%) of encapsulated pectinase were achieved. PMID:22489134

TLR9 and NF-κB are partially involved in activation of human neutrophils by Helicobacter pylori and its purified DNA.

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Lourdes Alvarez-Arellano

Full Text Available Helicobacter pylori infection represents one of the most common bacterial infections worldwide. The inflammatory response to this bacterium involves a large influx of neutrophils to the lamina propria of the gastric mucosa. However, little is known about the receptors and molecular mechanisms involved in activation of these neutrophils. In this study, we aimed to determine the role of toll-like receptor 9 (TLR9 in the response of human neutrophils to H. pylori and purified H. pylori DNA (Hp-DNA. Neutrophils were isolated from the blood of adult volunteers and challenged with either H. pylori or Hp-DNA. We found that both, H. pylori and Hp-DNA induced increased expression and release of IL-8. Furthermore, we showed that TLR9 is involved in the induction of IL-8 production by H. pylori and Hp-DNA. IL-8 production induced by H. pylori but not by Hp-DNA was partially mediated by NF-κB. In conclusion, this study showed for first time that both, H. pylori and Hp-DNA activate TLR9 and induce a different inflammatory response that leads to activation of neutrophils.

Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.

Science.gov (United States)

Visessanguan, Wonnop; Benjakul, Soottawat; An, Haejung

2003-03-01

A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.

Insulin-like growth factors: assay methods and their implications

International Nuclear Information System (INIS)

Guyda, H.J.; Posner, B.I.; Schiffrin, A.; Rappaport, R.; Postel-Vinay, M.C.; Corvol, M.T.

1981-01-01

The insulin-like growth factors (IGF's) are small molecular weight peptides (6-10 x 10 3 daltons) that circulate in blood plasma almost entirely bound to macromolecular carrier proteins. The growth-promoting and insulin-like activities of IGF's can be explained by the observed ability of these peptides to interact with the IGF receptor on the one hand and with the insulin receptor on the other. These observations have led to the establishment of radioreceptor assays (RRA's), competitive protein binding assays (CPBA's), and more recently radioimmunoassays (RIA's) for the IGF's that have different specificities. Because of their ease of performance and sensitivity, the radioligand assays have largely supplanted the biological assays originally utilized to identify and characterize these anabolic peptides. In this report the authors' studies are summarised which utilize a slightly acidic IGF which has been purified on the basis of its insulin-like activity in an insulin RRA and which was termed ILAs. They refer to purified insulin-like peptides that have the properties of a somatomedin by the generic term insulin-like growth factor (IGF). Somatomedin (SM) activity will be utilized to connote that activity in plasma or serum determined by bioassay. The competitive dose-response curves for IGF peptides in the insulin RRA as well as those in the ILAs RRA are presented. A combination of bioassays, RRA and RIA were employed to assess somatomedin activity and IGF peptide levels in a number of clinical circumstances. The correlations are discussed. (Auth.)

Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women.

Science.gov (United States)

White, Yvonne A R; Woods, Dori C; Takai, Yasushi; Ishihara, Osamu; Seki, Hiroyuki; Tilly, Jonathan L

2012-02-26

Germline stem cells that produce oocytes in vitro and fertilization-competent eggs in vivo have been identified in and isolated from adult mouse ovaries. Here we describe and validate a fluorescence-activated cell sorting-based protocol that can be used with adult mouse ovaries and human ovarian cortical tissue to purify rare mitotically active cells that have a gene expression profile that is consistent with primitive germ cells. Once established in vitro, these cells can be expanded for months and can spontaneously generate 35- to 50-μm oocytes, as determined by morphology, gene expression and haploid (1n) status. Injection of the human germline cells, engineered to stably express GFP, into human ovarian cortical biopsies leads to formation of follicles containing GFP-positive oocytes 1-2 weeks after xenotransplantation into immunodeficient female mice. Thus, ovaries of reproductive-age women, similar to adult mice, possess rare mitotically active germ cells that can be propagated in vitro as well as generate oocytes in vitro and in vivo.

Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

International Nuclear Information System (INIS)

Senogles, S.E.; Caron, M.G.

1986-01-01

The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

Strong antimicrobial activity of xanthohumol and other derivatives from hops (Humulus lupulus L.) on gut anaerobic bacteria.

Science.gov (United States)

Cermak, Pavel; Olsovska, Jana; Mikyska, Alexandr; Dusek, Martin; Kadleckova, Zuzana; Vanicek, Jiri; Nyc, Otakar; Sigler, Karel; Bostikova, Vanda; Bostik, Pavel

2017-11-01

Anaerobic bacteria, such as Bacteroides fragilis or Clostridium perfringens, are part of indigenous human flora. However, Clostridium difficile represents also an important causative agent of nosocomial infectious antibiotic-associated diarrhoea. Treatment of C. difficile infection is problematic, making it imperative to search for new compounds with antimicrobial properties. Hops (Humulus lupulus L.) contain substances with antibacterial properties. We tested antimicrobial activity of purified hop constituents humulone, lupulone and xanthohumol against anaerobic bacteria. The antimicrobial activity was established against B. fragilis, C. perfringens and C. difficile strains according to standard testing protocols (CLSI, EUCAST), and the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBC) were calculated. All C. difficile strains were toxigenic and clinically relevant, as they were isolated from patients with diarrhoea. Strongest antimicrobial effects were observed with xanthohumol showing MIC and MBC values of 15-107 μg/mL, which are close to those of conventional antibiotics in the strains of bacteria with increased resistance. Slightly higher MIC and MBC values were obtained with lupulone followed by higher values of humulone. Our study, thus, shows a potential of purified hop compounds, especially xanthohumol, as alternatives for treatment of infections caused by select anaerobic bacteria, namely nosocomial diarrhoea caused by resistant strains. © 2017 APMIS. Published by John Wiley & Sons Ltd.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets.

Science.gov (United States)

Marcinkiewicz, Cezary; Gerstenhaber, Jonathan A; Sternberg, Mark; Lelkes, Peter I; Feuerstein, Giora

2017-01-01

Thromboembolic events (TEE) underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs) functionalized with bitistatin (Bit), a disintegrin that specifically binds to the α IIb β 3 integrin, platelet fibrinogen receptor (PFR) on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP-Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, α IIb β 3 receptor antagonist, specifically blocked F-NDP-Bit-PFR complex formation. Moreover, F-NDP-Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP). Our results suggest that engineered F-NDP-Bit particles could serve as noninvasive, "real-time" optical diagnostics for clots present in blood vessels.

Effect of preparation conditions on the characteristics and photocatalytic activity of TiO2/purified diatomite composite photocatalysts

International Nuclear Information System (INIS)

Sun, Zhiming; Hu, Zhibo; Yan, Yang; Zheng, Shuilin

2014-01-01

Highlights: • TiO 2 /purified diatomite composites were synthesized under different conditions. • The optimum preparation conditions of composites were obtained. • The obtained photocatalyst showed good photocatalytic activity. • The dispersity and grain size of loaded TiO 2 NPs are the critical factors. - Abstract: TiO 2 /purified diatomite composite materials were prepared through a modified hydrolysis-deposition method under low temperature using titanium tetrachloride as precursor combined with a calcination crystallization process. The microstructure and crystalline phases of the obtained composites prepared under different preparation conditions were characterized by high resolution scanning electron microscope (SEM) and X-ray diffraction (XRD), respectively. The photocatalytic performance of TiO 2 /purified diatomite composites was evaluated by Rhodamine B as the target pollutant under UV irradiation, and the optimum preparation conditions of composites were obtained. The TiO 2 crystal form in composites prepared under optimum conditions was anatase, the grain size of which was 34.12 nm. The relationships between structure and property of composite materials were analyzed and discussed. It is indicated that the TiO 2 nanoparticles uniformly dispersed on the surface of diatoms, and the photocatalytic performance of the composite materials was mainly determined by the dispersity and grain size of loaded TiO 2 nanoparticles

Excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in experimental approaches of Parkinsonism.

Science.gov (United States)

Fernández-Espejo, Emilio; Bis-Humbert, Cristian

2018-06-06

3-iodo-l-tyrosine might play a role in Parkinson's disease since this molecule is able, at high concentration, to inhibit tyrosine-hydroxylase activity, the rate-limiting enzyme in dopamine biosynthesis. The possible Parkinson-like effects of 3-iodo-l-tyrosine were tested on three experimental approaches in mice: cultured substantia nigra neurons, the enteric nervous system of the jejunum after intra-peritoneal infusions, and the nigrostriatal system following unilateral intrabrain injections. 3-iodo-l-tyrosine, a physiological molecule, was used at concentrations higher than its serum levels in humans. Parkinson-like signs were evaluated through abnormal aggregation of α-synuclein and tyrosine-hydroxylase, loss of tyrosine-hydroxylase-expressing and striatum-projecting neurons and fibers, reduced tyrosine-hydroxylase density, and Parkinson-like motor and non-motor deficits. The retrograde tracer FluoroGold was used in the brain model. The findings revealed that excess amounts of 3-iodo-l-tyrosine induce Parkinson-like effects in the three experimental approaches. Thus, culture neurons of substantia nigra show, after 3-iodo-l-tyrosine exposure, intracytoplasmic inclusions that express α-synuclein and tyrosine-hydroxylase. Intra-peritoneal infusions of 3-iodo-l-tyrosine cause, in the long-term, α-synuclein aggregation, thicker α-synuclein-positive fibers, and loss of tyrosine-hydroxylase-positive cells and fibers in intramural plexuses and ganglia of the jejunum. Infusion of 3-iodo-l-tyrosine into the left dorsal striata of mice damages the nigrostriatal system, as revealed through lower striatal tyrosine-hydroxylase density, reduced number of tyrosine-hydroxylase-expressing and striatum-projecting neurons in the left substantia nigra, as well as the emergence of Parkinson-like behavioral deficits such as akinesia, bradykinesia, motor disbalance, and locomotion directional bias. In conclusion, excess amounts of 3-iodo-l-tyrosine induce Parkinson-like features in

Antioxidant and antibacterial activities of polysaccharides isolated and purified from Diaphragma juglandis fructus.

Science.gov (United States)

Meng, Qingran; Li, Yinghao; Xiao, Tiancun; Zhang, Lianfu; Xu, Dan

2017-12-01

A water-soluble polysaccharide fraction (DJP-2) isolated from Diaphragma juglandis was successfully purified by ion-exchange chromatography (DEAE-cellulose) and gel-permeation chromatography (Sephadex G-100). The weight-average molecular weight (Mw) and number-average molecular weight (Mn) of DJP-2 were 4.95 and 3.99kDa, respectively. Monosaccharide component analysis indicated that DJP-2 comprised arabinose, galactose, glucose, xylose, and mannose in a molar ratio of 0.27:0.55:1:0.14:0.08. The evaluation of the antioxidant and antibacterial activities of polysaccharides from Diaphragma juglandis fructus indicated that they could be explored as promising natural antioxidant and bacteriostatic agents in the food and pharmaceutical industries. Copyright © 2017 Elsevier B.V. All rights reserved.

Pharmacological and biological activities of Mirabilis jalapa L.

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Rozina Rozina

2016-05-01

Full Text Available AbstractPlants have been used for health care and medical purposes for several thousands of years. The number of higher plant species on earth is about 250000. It is estimated that 35000 to 70000 species have, at one time or another, been used in some cultures for medicinal purposes. One of these is Mirabilis jalapa L. which is traditionally used as Purgative and emetic, for treatment of many gastrointestinal disorders, including dysentery, diarrhea, muscle pain and abdominal colic. Besides this it also exhibits certain useful activities like Antiviral activity, Antimicrobial activity, Antimalarial activity, Anthelmintic activity, Antioxidant activity, Aytotoxic activity Anti-tubercular drugs induced hepatotoxicity, Antinociceptive activity, Antifungal activity, Anti-corrosion activity, Antispasmodic activity, Anti-inflammatory activity and many others. Pharmacologically active compounds include active alcoholic extract, ether compound-3,3’-Methylenebis (4- hydroxycoumarin N-D-alpha-Phenylyglycinelaminaribiitol-3-(4-(dimethylamino cinnamoyl 4hydroxycoumarin. The purpose of my review is to find out the areas of scope and to give the detail of the work done on Mirabilis Jalapa L. for future research work.

Glutamine Triggers and Potentiates Glucagon-Like Peptide-1 Secretion by Raising Cytosolic Ca2+ and cAMP

OpenAIRE

Tolhurst, Gwen; Zheng, Yue; Parker, Helen E.; Habib, Abdella M.; Reimann, Frank; Gribble, Fiona M.

2011-01-01

L-glutamine stimulates glucagon-like peptide 1 (GLP-1) secretion in human subjects and cell lines. As recent advances have enabled the study of primary GLP-1–releasing L cells, this study aimed to characterize glutamine-sensing pathways in native murine L cells. L cells were identified using transgenic mice with cell-specific expression of fluorescent markers. Cells were studied in primary colonic cultures from adult mice, or purified by flow cytometry for expression analysis. Intracellular C...

Phospholipase B activity of a purified phospholipase A from Vipera palestinae venom.

Science.gov (United States)

Shiloah, J; Klibansky, C; de Vries, A; Berger, A

1973-05-01

Phospholipase was isolated (in two fractions) from Vipera palestinae venom and it was shown to possess phospholipase A activity (hydrolyzing diacyl-sn-glycerophosphorylcholines, e.g., lecithin, in the 2-position) as well as lysophospholipase (phospholipase B) activity (hydrolyzing 1-monoacyl-sn-glycerophosphorylcholines, e.g., lysolecithin, yielding free fatty acid and glycerophosphorylcholine). Each of the two purified enzyme fractions was homogeneous as judged by electrophoresis on acrylamide gel and by immunodiffusion and immunoelectrophoresis, and both had essentially equal activities. The ratio of the specific activity, at various purification stages, to the specific activity of the whole venom was the same for A activity (substrate lecithin) as for B activity (substrate lysolecithin). The enzyme has a molecular weight of 16,000, six S-S bridges, and no free thiol groups. At pH 7, dimerization was observed in the ultracentrifuge. A dissociation constant of about 10(-5) m was estimated. The amino acid composition for both fractions (140 amino acid residues) was found to be essentially the same. The A activity had a pH optimum at 9; B activity was low at this pH but increased steadily beyond pH 10.5. For the hydrolysis of lysolecithin the Lineweaver-Burk plot was found to be linear, giving K(m) = 1.1 mm and k(cat) = 0.55 sec(-1) at 37 degrees C and pH 10. 2-Deoxylysolecithin was also hydrolyzed by the enzyme at pH 10, with k(cat) = 0.01 sec(-1) (zero-order kinetics in the range 0.5-2.5 mm). For lecithin these constants could not be determined, but at 0.25 mm substrate the hydrolysis rate (at pH 9) of lecithin was about 1000 times the hydrolysis rate of lysolecithin (at pH 10).

High-level water purifying technology. Kodo josui shori gijutsu

Energy Technology Data Exchange (ETDEWEB)

Tsugura, H; Tsukiashi, K [Meidensha Corp., Tokyo (Japan)

1993-07-01

Research and development have been carried out on a high-level water purifying system using ozone and activated charcoals to supply drinking water free of carcinogenic matters and odors. This system comprises a system to utilize ozone by using silent discharge and oxygen enriching device, and a living organism/activated charcoal treatment system. The latter system utilizes living organisms deposited on activated charcoal surfaces to remove polluting substances including ammonia. The treatment experimenting equipment comprises an ozone generating system, an ozone treating column, an activated charcoal treating column, an ozone/activated charcoal control device, and a water amount and quality measuring system. An experiment was carried out using an experimental plant with a capacity of 20 m[sup 3]/day on water taken from the sedimentation process at an actual water purifying plant. As a result, trihalomethane formation potential was removed at about 40% in the ozone treatment, and at 70% in the whole treatment combining the ozone and living organism/activated charcoal treatments. For parameterization of palatability of water, a method is being studied that utilizes nuclear magnetic resonance to investigate degrees of water cluster. The method is regarded promising. 1 ref., 4 figs.

Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

Science.gov (United States)

Patel, Arti T; Akhani, Rekha C; Patel, Manisha J; Dedania, Samir R; Patel, Darshan H

2017-06-01

Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 μM and 2 M, respectively. The catalytic efficiency (k cat /K m ) for L-aspartic acid (14.18 s -1  mM -1 ) was higher than that for L-phenylalanine (4.65 s -1  mM -1 ). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 μM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

Extraction, purification and anti-fatigue activity of γ-aminobutyric acid from mulberry (Morus alba L.) leaves

Science.gov (United States)

Chen, Hengwen; He, Xuanhui; Liu, Yan; Li, Jun; He, Qingyong; Zhang, Cuiying; Wei, Benjun; Zhang, Ye; Wang, Jie

2016-01-01

Mulberry (Morus alba L.) is a tree species of Moraceae widely distributed in Southern China. In the present study, the white crystal of γ-aminobutyric acid (GABA) was purified from mulberry leaves, and its bioactivity was also investigated. The main results were as follows: first, the crude GABA was extracted from mulberry leaves by using biochemical methods. Then, the crude was purified by chromatography over an S-8 macroporous resin, Sephadex G-10, and 732 cation exchange resin to yield a white crystal. Lavage administration and exposure of GABA to male NIH mice showed no adverse effects on their growth and development. In an endurance capacity test, the average loaded-swimming time of medium dose was 111.60% longer than the control (P levels of blood urea nitrogen (BUN) were 36.83% and 40.54% lower (P levels were 12.81% and 17.22% lower (P GABA has an advantage over taurine of anti-fatigue effect. These findings were indicative of the anti-fatigue activity of GABA.

Purifying hydrocarbons

Energy Technology Data Exchange (ETDEWEB)

Dunstan, A E

1918-06-03

Ligroin, kerosene, and other distillates from petroleum and shale oil, are purified by treatment with a solution of a hypochlorite containing an excess of alkali. The hydrocarbon may be poured into brine, the mixture stirred, and an electric current passed through. Heat may be applied.

Effect of partially purified angiotensin converting enzyme inhibitory ...

African Journals Online (AJOL)

This study evaluated the effect of partially-purified angiotensin converting enzyme (ACE) inhibitory proteins obtained from the leaves of Moringa oleifera on blood glucose, serum ACE activity and lipid profile of alloxaninduced diabetic rats. Twenty-five apparently healthy male albino rats were divided into five groups of five ...

Pentacene field-effect transistors by in situ and real time electrical characterization: Comparison between purified and non-purified thin films

International Nuclear Information System (INIS)

Liu, Shun-Wei; Wen, Je-Min; Lee, Chih-Chien; Su, Wei-Cheng; Wang, Wei-Lun; Chen, Ho-Chien; Lin, Chun-Feng

2013-01-01

We present an electrical characterization of the organic field-effect transistor with purified and non-purified pentacene by using in situ and real time measurements. The field-effect phenomenon was observed at the thickness of 1.5 nm (approximately one monolayer of pentacene) for purified pentacene, as compared to 3.0 nm for the non-purified counterpart. Moreover, the hole mobility is improved from 0.13 to 0.23 cm 2 /V s after the sublimation process to purify the pentacene. With atomic force microscopic measurements, the purified pentacene thin film exhibits a larger grain size and film coverage, resulting in better crystallinity of the thin film structure due to the absence of the impurities. This is further confirmed by X-ray diffraction patterns, which show higher intensities for the purified pentacene. - Highlights: • We present in-situ characterization for pentacene field-effect transistors. • The hole mobility is improved after the sublimation process to purify the pentacene. • Purified pentacene thin film exhibits a larger grain size and film coverage. • Hole mobility of pentacene is improved from 0.13 to 0.23 cm 2 /V s. • The discontinuity of grain boundary may cause the shift of threshold voltage

Pentacene field-effect transistors by in situ and real time electrical characterization: Comparison between purified and non-purified thin films

Energy Technology Data Exchange (ETDEWEB)

Liu, Shun-Wei, E-mail: swliu@mail.mcut.edu.tw [Department of Electronic Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan, ROC (China); Wen, Je-Min; Lee, Chih-Chien; Su, Wei-Cheng; Wang, Wei-Lun; Chen, Ho-Chien [Department of Electronic Engineering, National Taiwan University of Science and Technology, Taipei, 10607 Taiwan, ROC (China); Lin, Chun-Feng [Department of Electronic Engineering, Ming Chi University of Technology, New Taipei City 24301, Taiwan, ROC (China)

2013-05-01

We present an electrical characterization of the organic field-effect transistor with purified and non-purified pentacene by using in situ and real time measurements. The field-effect phenomenon was observed at the thickness of 1.5 nm (approximately one monolayer of pentacene) for purified pentacene, as compared to 3.0 nm for the non-purified counterpart. Moreover, the hole mobility is improved from 0.13 to 0.23 cm{sup 2}/V s after the sublimation process to purify the pentacene. With atomic force microscopic measurements, the purified pentacene thin film exhibits a larger grain size and film coverage, resulting in better crystallinity of the thin film structure due to the absence of the impurities. This is further confirmed by X-ray diffraction patterns, which show higher intensities for the purified pentacene. - Highlights: • We present in-situ characterization for pentacene field-effect transistors. • The hole mobility is improved after the sublimation process to purify the pentacene. • Purified pentacene thin film exhibits a larger grain size and film coverage. • Hole mobility of pentacene is improved from 0.13 to 0.23 cm{sup 2}/V s. • The discontinuity of grain boundary may cause the shift of threshold voltage.

Effects of fertigation with purified urban wastewater on soil and pepper plant (Capsicum annuum L.) production, fruit quality and pollutant contents

Energy Technology Data Exchange (ETDEWEB)

Garcia-Delgado, C.; Eymar, E.; Contreras, J. I.; Segura, M. L.

2012-11-01

The effects, in greenhouse conditions, of Purified Urban Wastewater (PW) from Almeria (Spain), in the fertigation of pepper (Capsicum annuum L.) on sandy mulch soil, were evaluated. Primary, secondary (active sludges) and tertiary (Chlorination + ozonation) purification treatments were applied to wastewater. Irrigation treatments applied were PW, natural Ground Water (GW), Fertilizer PW (FPW) and Fertilizer GW (FGW). The vegetal biomass, yield and fruit quality were controlled. Heavy metals (Cr, Cd, Pb, Ni, Mn, Cu and Zn), arsenic (As) and Polycyclic Aromatic Hydrocarbons (PAH) in water, soil, leaf, and fruit were analysed. The PW presented heavy metal, As and PAH contents acceptable for its use in drip irrigation. In the soil, fertigated with PW, the concentration of heavy metals and As did not increase, whilst the PAH concentration decreased. The PW treatment supplied enough nutrients to obtain yield and fruit quality equal to that of GW with fertilization. A significant saving on N, P and K fertilizers (37%, 66% and 12% respectively) was achieved by using PW. The Cd, Pb and As contents of the fruit did not show risk for human consumption. The total PAH concentrations in the fruit were low, the highest of which was phenathrene, with no carcinogenic signification. (Author) 39 refs.

Directed modification of L-LcLDH1, an L-lactate dehydrogenase from Lactobacillus casei, to improve its specific activity and catalytic efficiency towards phenylpyruvic acid.

Science.gov (United States)

Li, Jian-Fang; Li, Xue-Qing; Liu, Yan; Yuan, Feng-Jiao; Zhang, Ting; Wu, Min-Chen; Zhang, Ji-Ru

2018-05-22

To improve the specific activity and catalytic efficiency of L-LcLDH1, an NADH-dependent allosteric L-lactate dehydrogenase from L. casei, towards phenylpyruvic acid (PPA), its directed modification was conducted based on the semi-rational design. The three variant genes, Lcldh1 Q88R , Lcldh1 I229A and Lcldh1 T235G , were constructed by whole-plasmid PCR as designed theoretically, and expressed in E. coli BL21(DE3), respectively. The purified mutant, L-LcLDH1 Q88R or L-LcLDH1 I229A , displayed the specific activity of 451.5 or 512.4 U/mg towards PPA, by which the asymmetric reduction of PPA afforded L-phenyllactic acid (PLA) with an enantiomeric excess (ee p ) more than 99%. Their catalytic efficiencies (k cat /K m ) without D-fructose-1,6-diphosphate (D-FDP) were 4.8- and 5.2-fold that of L-LcLDH1. Additionally, the k cat /K m values of L-LcLDH1 Q88R and L-LcLDH1 I229A with D-FDP were 168.4- and 8.5-fold higher than those of the same enzymes without D-FDP, respectively. The analysis of catalytic mechanisms by molecular docking (MD) simulation indicated that substituting I229 in L-LcLDH1 with Ala enlarges the space of substrate-binding pocket, and that the replacement of Q88 with Arg makes the inlet of pocket larger than that of L-LcLDH1. Copyright © 2018 Elsevier B.V. All rights reserved.

Antioxidant Activity of Purified Active Peptide Derived from Spirulina platensis Enzymatic Hydrolysates

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Nur Maulida Safitri

2017-08-01

Full Text Available The aim of this study is to isolate the antioxidative peptide from Spirulina platensis. Peptide was obtained by proteolytic digestion, ultrafiltration, fractionation by RP-HPLC, identified by LC-MS/MS—MASCOT Distiller and measured its antioxidant activity by DPPH (2.2-Diphenyl-1-picrylhydrazyl assay. Results showed that thermolysin was the most effective enzyme to digest this algae. The active peptide Phe-Ser-Glu-Ser-Ser-Ala-Pro-Glu-Gln-His-Tyr (m/z 1281.51 was identified and synthetized, which exhibited 45.98 ± 1.7% at concentration 128.15 µg/mL. Therefore, S. platensis is indicated as a potential therapeutic source for combating oxidative stress.

Proteomic analysis of purified coronavirus infectious bronchitis virus particles

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Shu Dingming

2010-06-01

Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

Apolipoprotein L1 confers pH-switchable ion permeability to phospholipid vesicles.

Science.gov (United States)

Bruno, Jonathan; Pozzi, Nicola; Oliva, Jonathan; Edwards, John C

2017-11-03

Apolipoprotein L1 (ApoL1) is a human serum protein conferring resistance to African trypanosomes, and certain ApoL1 variants increase susceptibility to some progressive kidney diseases. ApoL1 has been hypothesized to function like a pore-forming colicin and has been reported to have permeability effects on both intracellular and plasma membranes. Here, to gain insight into how ApoL1 may function in vivo , we used vesicle-based ion permeability, direct membrane association, and intrinsic fluorescence to study the activities of purified recombinant ApoL1. We found that ApoL1 confers chloride-selective permeability to preformed phospholipid vesicles and that this selectivity is strongly pH-sensitive, with maximal activity at pH 5 and little activity above pH 7. When ApoL1 and lipid were allowed to interact at low pH and were then brought to neutral pH, chloride permeability was suppressed, and potassium permeability was activated. Both chloride and potassium permeability linearly correlated with the mass of ApoL1 in the reaction mixture, and both exhibited lipid selectivity, requiring the presence of negatively charged lipids for activity. Potassium, but not chloride, permease activity required the presence of calcium ions in both the association and activation steps. Direct assessment of ApoL1-lipid associations confirmed that ApoL1 stably associates with phospholipid vesicles, requiring low pH and the presence of negatively charged phospholipids for maximal binding. Intrinsic fluorescence of ApoL1 supported the presence of a significant structural transition when ApoL1 is mixed with lipids at low pH. This pH-switchable ion-selective permeability may explain the different effects of ApoL1 reported in intracellular and plasma membrane environments. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and characteristics of a novel bacteriocin produced by Enterococcus faecalis L11 isolated from Chinese traditional fermented cucumber.

Science.gov (United States)

Gao, Yurong; Li, Benling; Li, Dapeng; Zhang, Liyuan

2016-05-01

To purify and characterize a novel bacteriocin with broad inhibitory spectrum produced by an isolate of Enterococcus faecalis from Chinese fermented cucumber. E. faecalis L11 produced a bacteriocin with antimicrobial activity against both Escherichia coli and Staphylococcus aureus. The amino acid sequence of the purified bacteriocin, enterocin L11, was assayed by Edman degradation method. It differs from other class II bacteriocins and exhibited a broad antimicrobial activity against not only Gram-positive bacteria, including Bacillus subtilis, S. aureus, Listeria monocytogenes, Sarcina flava, Lactobacillus acidophilus, L. plantarum, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus and Streptococcus thermophilus, but also some Gram-negative bacteria including Salmonella typhimurium, E. coli and Shigella flexneri. Enterocin L11 retained 91 % of its activity after holding at 121 °C for 30 min. It was also resistant to acids and alkalis. Enterocin L11 is a novel broad-spectrum Class II bacteriocin produced by E. faecalis L11, and may have potential as a food biopreservative.

Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity

DEFF Research Database (Denmark)

Winther, Jakob R.; Sørensen, P

1991-01-01

The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium...... folding pathway. The relatively large number of charged amino acid residues and a high theoretical potential for alpha-helix formation in the carboxypeptidase Y propeptide suggest a structural similarity to a number of other propeptides and heat shock proteins....

Influence of lysozyme complexation with purified Aldrich humic acid on lysozyme activity

NARCIS (Netherlands)

Li, Y.; Tan, W.F.; Wang, M.X.; Liu, F.; Weng, L.P.; Norde, W.; Koopal, L.K.

2012-01-01

Humic acid is an important component of dissolved organic matter and in two previous papers it has been shown that purified Aldrich humic acid (PAHA) forms strong complexes with the oppositely charged protein lysozyme (LSZ). The complexation and aggregation of enzymes with humic acids may lead to

Methods for Purifying Enzymes for Mycoremediation

Science.gov (United States)

Cullings, Kenneth W. (Inventor); DeSimone, Julia C. (Inventor); Paavola, Chad D. (Inventor)

2014-01-01

A process for purifying laccase from an ectomycorrhizal fruiting body is disclosed. The process includes steps of homogenization, sonication, centrifugation, filtration, affinity chromatography, ion exchange chromatography, and gel filtration. Purified laccase can also be separated into isomers.

Evaluation of Antimicrobial Activity of Probiotic Lactobacillus Strains against Growth and Urease Activity of Proteus spp.

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Leila Goudarzi

2017-10-01

Full Text Available Background:    Nowadays, the use of probiotic bacteria for the prevention and treatment of urinary tract infections is growing. Lactobacillus, as probiotic bacterial genus, is well known for its benefits for the human health.Methods:      The effects of partially purified antimicrobial compounds (bacteriocins and biosurfactants of Lactobacillus strains was assessed and their capacity to in vitro inhibit growth and urease production of various strains of Proteus spp, was studied. Inhibition of the urease production of Proteus spp. at sub-MIC levels was screened using spectrophotometry method.  Results:   Results revealed that semi-purified bacteriocins of L. acidophilus and L. plantarum showed a greater inhibitory activity on the bacterial urease, compared to biosurfactants of L. rhamnosus, L. casei and L. fermentum (P < 0.05.Conclusion:    It can be concluded that bacteriocins may affect Proteus pathogenesis by inhibition of the bacterial urease activity and therefore eliminate the stone formation by these bacteria.

GABA(A) Increases Calcium in Subventricular Zone Astrocyte-Like Cells Through L- and T-Type Voltage-Gated Calcium Channels

DEFF Research Database (Denmark)

Young, Stephanie Z; Platel, Jean-Claude; Nielsen, Jakob V

2010-01-01

In the adult neurogenic subventricular zone (SVZ), the behavior of astrocyte-like cells and some of their functions depend on changes in intracellular Ca(2+) levels and tonic GABA(A) receptor activation. However, it is unknown whether, and if so how, GABA(A) receptor activity regulates...... intracellular Ca(2+) dynamics in SVZ astrocytes. To monitor Ca(2+) activity selectively in astrocyte-like cells, we used two lines of transgenic mice expressing either GFP fused to a Gq-coupled receptor or DsRed under the human glial fibrillary acidic protein (hGFAP) promoter. GABA(A) receptor activation...... induced Ca(2+) increases in 40-50% of SVZ astrocytes. GABA(A)-induced Ca(2+) increases were prevented with nifedipine and mibefradil, blockers of L- and T-type voltage-gated calcium channels (VGCC). The L-type Ca(2+) channel activator BayK 8644 increased the percentage of GABA(A)-responding astrocyte...

In-gel detection of esterase-like albumin activity: Characterization of esterase-free sera albumin and its putative role as non-invasive biomarker of hepatic fibrosis

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Areeba Ahmad

2017-07-01

Full Text Available Albumin is a globular and un-glycosylated multifunctional plasma protein and thus correlated with several human diseases. Owing to esterase contamination, albumin levels are usually misleading. In this study, we propose methodical accuracy for albumin estimation taking healthy and fibrotic rats. Liver fibrosis in rats was generated by N′-Nitrosodimethylamine (NDMA (10 mg/kg body weight within three weeks followed by its confirmation through H&E and immunohistochemical staining for α-SMA expression. Animal sera were screened by native polyacrylamide gel electrophoresis (native-PAGE (7.5%. In-gel esterase-like albumin activity was detected using α- and β-naphthyl acetate (5.58 × 10−3 mM; pH 7.5 as substrate. Sera albumin was purified from unstained PA gel-slices through electroelution. Subsequent to conformation of albumin purity by its molecular weight determination using SDS–PAGE (10% and peptide mass fingerprinting by MALDI-TOF-MS, samples were treated with different concentrations of urea. Urea-treated albumins were screened for esterase activity, conformational change and, albumin levels by immunoblotting. Our results demonstrate that esterase-like albumin activity in rat sera albumin is located in domain-III. The esterase-like activity remains detectable up to 4 M urea, which diminishes with increasing urea concentrations. Further, immunoblotting of urea-treated albumin samples displays a significant decline in purified protein bands, indicating hypoalbuminemia during hepatic fibrosis in rats. In conclusion, the present approach of albumin separation and estimation is of potential interest and may be recommended for diagnostic purposes.

Evaluation of antioxidant and cytoprotective activities of Arnica montana L. and Artemisia absinthium L. ethanolic extracts

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Craciunescu Oana

2012-09-01

Full Text Available Abstract Background Arnica montana L. and Artemisia absinthium L. (Asteraceae are medicinal plants native to temperate regions of Europe, including Romania, traditionally used for treatment of skin wounds, bruises and contusions. In the present study, A. montana and A. absinthium ethanolic extracts were evaluated for their chemical composition, antioxidant activity and protective effect against H2O2-induced oxidative stress in a mouse fibroblast-like NCTC cell line. Results A. absinthium extract showed a higher antioxidant capacity than A. montana extract as Trolox equivalent antioxidant capacity, Oxygen radical absorbance capacity and 2,2-diphenyl-1-picrylhydrazyl free radical-scavenging activity, in correlation with its flavonoids and phenolic acids content. Both plant extracts had significant effects on the growth of NCTC cells in the range of 10–100 mg/L A. montana and 10–500 mg/L A. absinthium. They also protected fibroblast cells against hydrogen peroxide-induced oxidative damage, at the same doses. The best protection was observed in cell pre-treatment with 10 mg/L A. montana and 10–300 mg/L A. absinthium, respectively, as determined by Neutral red and lactate dehydrogenase assays. In addition, cell pre-treatment with plant extracts, at these concentrations, prevented morphological changes induced by hydrogen peroxide. Flow-cytometry analysis showed that pre-treatment with A. montana and A. absinthium extracts restored the proportion of cells in each phase of the cell cycle. Conclusions A. montana and A. absinthium extracts, rich in flavonoids and phenolic acids, showed a good antioxidant activity and cytoprotective effect against oxidative damage in fibroblast-like cells. These results provide scientific support for the traditional use of A. montana and A. absinthium in treatment of skin disorders.

High specific activity N-Acetyl-3{sup H}-{alpha}-Aspartyl- L-Glutamic at micro mole scale; Sintesis de N-Acetil-3{sup H}- {alpha} -Aspartil-Glutamico a escala de Micromoles

Energy Technology Data Exchange (ETDEWEB)

Suarez, C

1984-07-01

High specific activity N-Acetyl-3{sup H}- {alpha} -Aspartyl-I-Glutamic acid at micro mole scale in prepared acetylating L- {alpha} -Aspartyl-L-glutamic with 3{sup H}-acetic anhydride in re distilled toluene. The product le purified through cationic and anionic columns. The radiochemical purity as determined by thin-layer chromatography is greater then 99% at the time preparation. (Author) 5 refs.

Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag.

Science.gov (United States)

Bataille, Laure; Dieryck, Wilfrid; Hocquellet, Agnès; Cabanne, Charlotte; Bathany, Katell; Lecommandoux, Sébastien; Garbay, Bertrand; Garanger, Elisabeth

2015-06-01

Elastin-like polypeptides (ELPs) are biodegradable polymers with interesting physico-chemical properties for biomedical and biotechnological applications. The recombinant expression of hydrophobic elastin-like polypeptides is often difficult because they possess low transition temperatures, and therefore form aggregates at sub-ambient temperatures. To circumvent this difficulty, we expressed in Escherichia coli three hydrophobic ELPs (VPGIG)n with variable lengths (n=20, 40, and 60) in fusion with the maltose-binding protein (MBP). Fusion proteins were soluble and yields of purified MBP-ELP ranged between 66 and 127mg/L culture. After digestion of the fusion proteins by enterokinase, the ELP moiety was purified by using inverse transition cycling. The purified fraction containing ELP40 was slightly contaminated by traces of undigested fusion protein. Purification of ELP60 was impaired because of co-purification of the MBP tag during inverse transition cycling. ELP20 was successfully purified to homogeneity, as assessed by gel electrophoresis and mass spectrometry analyses. The transition temperature of ELP20 was measured at 15.4°C in low salt buffer. In conclusion, this method can be used to produce hydrophobic ELP of low molecular mass. Copyright © 2015 Elsevier Inc. All rights reserved.

Effect of preparation conditions on the characteristics and photocatalytic activity of TiO{sub 2}/purified diatomite composite photocatalysts

Energy Technology Data Exchange (ETDEWEB)

Sun, Zhiming, E-mail: szmcumtb@hotmail.com; Hu, Zhibo; Yan, Yang; Zheng, Shuilin, E-mail: shuilinzh@sina.com

2014-09-30

Highlights: • TiO{sub 2}/purified diatomite composites were synthesized under different conditions. • The optimum preparation conditions of composites were obtained. • The obtained photocatalyst showed good photocatalytic activity. • The dispersity and grain size of loaded TiO{sub 2} NPs are the critical factors. - Abstract: TiO{sub 2}/purified diatomite composite materials were prepared through a modified hydrolysis-deposition method under low temperature using titanium tetrachloride as precursor combined with a calcination crystallization process. The microstructure and crystalline phases of the obtained composites prepared under different preparation conditions were characterized by high resolution scanning electron microscope (SEM) and X-ray diffraction (XRD), respectively. The photocatalytic performance of TiO{sub 2}/purified diatomite composites was evaluated by Rhodamine B as the target pollutant under UV irradiation, and the optimum preparation conditions of composites were obtained. The TiO{sub 2} crystal form in composites prepared under optimum conditions was anatase, the grain size of which was 34.12 nm. The relationships between structure and property of composite materials were analyzed and discussed. It is indicated that the TiO{sub 2} nanoparticles uniformly dispersed on the surface of diatoms, and the photocatalytic performance of the composite materials was mainly determined by the dispersity and grain size of loaded TiO{sub 2} nanoparticles.

Biological and Histological Studies of Purified Product from Streptomyces janthinus M7 Metabolites

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Tawfik Zahira S.

2015-02-01

Full Text Available Fifteen clinical samples were taken out from patients suffering cancer, these patients being under the treatment with radio- and/or chemotherapy. The samples were used for the isolation of bacterial cells surrounding tumor; the samples were collected from Center of Cancer Therapy, Ain Shams University, Cairo, Egypt. The clinical bacterial isolates were purified and identified according to Bergey's manual of determinative bacteriology ninth edition (1994. The bacterial isolates were found to be Klebsiella oxytoca m1; Enterobacter cancerogenus m2; P. aeruginosa m3; Citrobacter diversus m4; Enterobacter agglomerans m5; Klebsiella oxytoca m6; Enterobacter dissolvens m7; Serratia fonticola m8; Escherichia coli m9; Citrobacter freundii m10; Staphylococcus aureus m11; Escherichia coli m12; P. aeruginosa m13; Staphylococcus aureus m14; and Bacillus cereus m15. In the present study both primary and secondary screening methods were used to screen the antibacterial activity of St. janthinus M7 against fifteen clinical bacterial isolates. The St. janthinus M7 showed an increase in antibacterial activity against all the tested human bacterial pathogens. In this study Gamma irradiation at dose levels (0.5 and 1.5 kGy was used for the enhancement of the antibacterial activity of Streptomyces strain against the clinical isolates. Several commercial antibiotic discs (Doxorubicin, Augmentin, Norfloxacin, Ofloxacin, Oxacillin, and Cefazolin were used for comparing their antimicrobial activity with purified product. The results declared a significant increase in the antibacterial activity in most cases. The physiochemical properties of the purified product were carried out for determination of Rf, empirical formula, M.W, and chemical structure of product and then analyzed by thin layer chromatography, elemental analysis, UV, Mass, and NMR. The result exhibited brown color, one spot, Rf (0.76, M.W (473, while it recorded 270 nm in UV region and the calculated

Production of active pigment epithelium-derived factor inE. coli.

Science.gov (United States)

Zhang, Tao; Guan, Ming; Lu, Yuan

2005-03-01

Human pigment epithelium-derived factor (PEDF), a neurotrophic factor, is the most potent natural inhibitor of angiogenesis. To produce the active PEDF, the gene coding for the human PEDF protein was expressed in E. coli. The rPEDF protein was expressed at 457 mg l-1 as a soluble protein. The yield of purified GST fusion protein was 14 mg l-1. Purified rPEDF inhibited tube formation in endothelial cells.

Production, characterization, and immobilization of partially purified surfactant-detergent and alkali-thermostable protease from newly isolated Aeromonas caviae.

Science.gov (United States)

Datta, Sumitra; Menon, Gopalakrishnan; Varughese, Bincy

2017-04-21

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0-11.0) and moderate salinity (0-5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128 U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl 2  · 2H 2 O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42 hr of incubation at 37°C. It was active over broad range of pH (7.0-12.0), temperature (15-100°C), and salinity (0-9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0-12.0 and salinities of 0-5% NaCl for 48 hr. Cu 2+ , Mn 2+ , Co 2+ , and Ca 2+ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant-detergent-stable partially purified extracellular protease from A. caviae.

RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

Science.gov (United States)

Sato, Shinya; Miyazono, Sadaharu; Tachibanaki, Shuji; Kawamura, Satoru

2015-01-30

Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

In vitro and in vivo antioxidant activity of a fructan from the roots of Arctium lappa L.

Science.gov (United States)

Liu, Wei; Wang, Jiajia; Zhang, Zhenzhen; Xu, Jinnan; Xie, Zhuohong; Slavin, Margaret; Gao, Xiangdong

2014-04-01

To explore new antioxidant resource from food, a water-soluble polysaccharide (ALP1) was extracted and purified from the roots of Arctium lappa L. (A. lappa L.) through hot water extraction followed by ethanol precipitation, ion-exchange chromatography and gel filtration. The antioxidant activity of ALP1 was then evaluated in vitro and in vivo. ALP1 was characterized as a fructan composed of fructose and glucose in the ratio of 13.0:1.0, with an average molecular weight of 4600 Da. The linkages in ALP1 were →1)-Fruf-(2→, Fruf-(2→ and Glcp-(1→. In vitro antioxidant assays demonstrated that ALP1 possessed moderate ABTS(+) scavenging activity, strong hydroxyl radical scavenging activity and strong ferrous ion chelating activity. In in vivo antioxidant assays, ALP1 administration significantly enhanced antioxidant enzyme activities and total antioxidant capacity, as well as decreased the levels of malondialdehyde (MDA) in both the serum and liver of aging mice. These results suggest that ALP1 has potential as a novel natural antioxidant in food industry and pharmaceuticals. Copyright © 2014 Elsevier B.V. All rights reserved.

TsAg5, a Taenia solium cysticercus protein with a marginal trypsin-like activity in the diagnosis of human neurocysticercosis.

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Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko

2011-12-01

Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.

In vitro effects of PCDDs/Fs on NK-like cell activity of Eisenia andrei earthworms

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Hayet Belmeskine

2012-02-01

Full Text Available In this study, we assessed in vitro the effects of PCDD/Fs on the NK-like cell activity in Eisenia andrei earthworms using flow cytometry for analysis. NK-like coelomocytes isolated from E. andrei and used as effectors were exposed to various concentrations of PCDDs/Fs mixture, C1 (6.25x10-3 ng 2378- TCDD/mL, C2 (12.5x10-3 ng 2378-TCDD/mL and C3 (25x10-3 ng 2378-TCDD/mL, before adding them to human tumoral cells (K562 used as targets. We evaluated the percentage of targets lysed by Nk-like cells. The results showed a significant stimulation of the NKlike activity at C3 when PCDD/Fs were not removed from effectors before contact with targets, while no effects were noted when the effectors were washed (PCDD/Fs removed or fixed. Assessment of the viability of the targets (K562, exposed alone and separately from effectors, to the three concentrations of PCDD/Fs, C1, C2 and C3, showed that all these concentrations were cytotoxic for K562. Results suggest that PCDD/Fs concentrations tested in this assay may be considered too low to induce suppressive effects on the immune function such as the NK-like activity in E. andrei earthworms.

Characterization of L-asparaginase from marine-derived Aspergillus niger AKV-MKBU, its antiproliferative activity and bench scale production using industrial waste.

Science.gov (United States)

Vala, Anjana K; Sachaniya, Bhumi; Dudhagara, Dushyant; Panseriya, Haresh Z; Gosai, Haren; Rawal, Rakesh; Dave, Bharti P

2018-03-01

L-asparaginase (LA), an enzyme with anticancer activities, produced by marine-derived Aspergillus niger was subjected to purification and characterization. The purified enzyme was observed to have molecular weight ∼90KDa. The enzyme retained activity over a wide range of pH, i.e. pH 4-10. The enzyme was quite stable in temperature range 20-40°C. Tween 80 and Triton X-100 were observed to enhance LA activity while inhibition of LA activity was observed in presence of heavy metals. The values for K m was found to be 0.8141 mM and V max was 6.228μM/mg/min. The enzyme exhibited noteworthy antiproliferative activity against various cancer cell lines tested. Successful bench scale production (in 5L bioreacator) of LA using groundnut oil cake as low cost substrate has also been carried out. Copyright © 2017 Elsevier B.V. All rights reserved.

Hydrogen purifier module with membrane support

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A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

2012-07-24

A hydrogen purifier utilizing a hydrogen-permeable membrane to purify hydrogen from mixed gases containing hydrogen is disclosed. Improved mechanical support for the permeable membrane is described, enabling forward or reverse differential pressurization of the membrane, which further stabilizes the membrane from wrinkling upon hydrogen uptake.

Activity of Renshaw cells during locomotor-like rhythmic activity in the isolated spinal cord of neonatal mice

DEFF Research Database (Denmark)

Nishimaru, Hiroshi; Restrepo, Carlos E.; Kiehn, Ole

2006-01-01

% of the recorded RCs fired in-phase with the ipsilateral L2 flexor-related rhythm, whereas the rest fired in the extensor phase. Each population of RCs fired throughout the corresponding locomotor phase. All RCs received both excitatory and inhibitory synaptic inputs during the locomotor-like rhythmic activity...

PURIFICATION AND PROPERTIES OF A FUNGAL L-ASPARAGINASE FROM TRICHODERMA VIRIDE PERS: SF GREY

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Lynette Lincoln

2015-02-01

Full Text Available A potent L-asparaginase-producing Trichoderma viride Pers: SF Grey was screened from the marine soil with the objective of studying the enzyme properties. The maximum enzyme production occurred on the third day at pH 6.5 and 37 °C when 0.5% L-asparagine supplemented with 0.5% peptone and 0.6% maltose. The enzyme was purified to homogeneity with a specific activity of 78.2 U.mg-1 and a molecular weight of 99 ± 1 kDa. It exhibited maximum activity at pH 7.0 and 37 °C. It was inhibited by Fe2+, Fe3+, Co2+ and Mn2+ but induced by Mg2+ and Na+. N-ethylemaleimide and phenylmethylsulphonylfluoride did not alter the enzyme activity, but strongly inhibited by ethylenediaminetetraacetate. L-asparaginase showed high affinity for L-asparagine with a Km of 2.56 μM. Thin layer chromatography confirmed the hydrolysis of L-asparagine. As the purified and characterized L-asparaginase of Trichoderma viride showed a good scavenging activity and reduced acrylamide level in potato products, it can further serve as an antileukemic protein and an acrylamide mitigation agent in heat-treated food stuffs rich in carbohydrates, respectively.

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass.

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Lee, Young-Sam; Gregory, Mark T; Yang, Wei

2014-02-25

DNA polymerase ζ (Pol ζ) is a eukaryotic B-family DNA polymerase that specializes in translesion synthesis and is essential for normal embryogenesis. At a minimum, Pol ζ consists of a catalytic subunit Rev3 and an accessory subunit Rev7. Mammalian Rev3 contains >3,000 residues and is twice as large as the yeast homolog. To date, no vertebrate Pol ζ has been purified for biochemical characterization. Here we report purification of a series of human Rev3 deletion constructs expressed in HEK293 cells and identification of a minimally catalytically active human Pol ζ variant. With a tagged form of an active Pol ζ variant, we isolated two additional accessory subunits of human Pol ζ, PolD2 and PolD3. The purified four-subunit Pol ζ4 (Rev3-Rev7-PolD2-PolD3) is much more efficient and more processive at bypassing a 1,2-intrastrand d(GpG)-cisplatin cross-link than the two-subunit Pol ζ2 (Rev3-Rev7). We show that complete bypass of cisplatin lesions requires Pol η to insert dCTP opposite the 3' guanine and Pol ζ4 to extend the primers.

Brassica juncea nitric oxide synthase like activity is stimulated by PKC activators and calcium suggesting modulation by PKC-like kinase.

Science.gov (United States)

Talwar, Pooja Saigal; Gupta, Ravi; Maurya, Arun Kumar; Deswal, Renu

2012-11-01

Nitric oxide (NO) is an important signaling molecule having varied physiological and regulatory roles in biological systems. The fact that nitric oxide synthase (NOS) is responsible for NO generation in animals, prompted major search for a similar enzyme in plants. Arginine dependent NOS like activity (BjNOSla) was detected in Brassica juncea seedlings using oxyhemoglobin and citrulline assays. BjNOSla showed 25% activation by NADPH (0.4 mM) and 40% by calcium (0.4 mM) but the activity was flavin mononucleotide (FMN), flavin dinucleotide (FAD) and calmodulin (CaM) independent. Pharmacological approach using mammalian NOS inhibitors, NBT (300 μM) and l-NAME (5 mM), showed significant inhibition (100% and 67% respectively) supporting that the BjNOSla operates via the oxidative pathway. Most of the BjNOSla activity (80%) was confined to shoot while root showed only 20% activity. Localization studies by NADPH-diaphorase and DAF-2DA staining showed the presence of BjNOSla in guard cells. Kinetic analysis showed positive cooperativity with calcium as reflected by a decreased K(m) (∼13%) and almost two fold increase in V(max). PMA (438 nM), a kinase activator, activated BjNOSla ∼1.9 fold while its inactive analog 4αPDD was ineffective. Calcium and PMA activated the enzyme to ∼3 folds. Interestingly, 1,2-DG6 (2.5 μM) and PS (1 μM) with calcium activated the enzyme activity to ∼7 fold. A significant inhibition of BjNOSla by PKC inhibitors-staurosporine (∼90%) and calphostin-C (∼40%), further supports involvement of PKC-like kinase. The activity was also enhanced by abiotic stress conditions (7-46%). All these findings suggest that BjNOSla generates NO via oxidative pathway and is probably regulated by phosphorylation. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Removal efficiency of water purifier and adsorbent for iodine, cesium, strontium, barium and zirconium in drinking water.

Science.gov (United States)

Sato, Itaru; Kudo, Hiroaki; Tsuda, Shuji

2011-01-01

The severe incident of Fukushima Daiichi Nuclear Power Station has caused radioactive contamination of environment including drinking water. Radioactive iodine, cesium, strontium, barium and zirconium are hazardous fission products because of the high yield and/or relatively long half-life. In the present study, 4 pot-type water purifiers and several adsorbents were examined for the removal effects on these elements from drinking water. Iodide, iodate, cesium and barium were removed by all water purifiers with efficiencies about 85%, 40%, 75-90% and higher than 85%, respectively. These efficiencies lasted for 200 l, which is near the recommended limits for use of filter cartridges, without decay. Strontium was removed with initial efficiencies from 70% to 100%, but the efficiencies were slightly decreased by use. Zirconium was removed by two models, but hardly removed by the other models. Synthetic zeolite A4 efficiently removed cesium, strontium and barium, but had no effect on iodine and zirconium. Natural zeolite, mordenite, removed cesium with an efficiency as high as zeolite A4, but the removal efficiencies for strontium and barium were far less than those of zeolite A4. Activated carbon had little removal effects on these elements. In case of radioactive contamination of tap water, water purifiers may be available for convenient decontamination of drinking water in the home.

The role of genetic variation in TCF7L2 and KCNJ11, dietary intake, and physical activity on fasting plasma glucagon-like peptide-1 in male adolescents

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Harry Freitag Luglio

2018-01-01

Full Text Available Background Transcription factor 7-like 2 (TCF7L2 and potassium voltage-gated channel subfamily j member 11 (KCNJ11 gene polymorphisms have been associated with type 2 diabetes mellitus (T2DM via regulation of insulin production. Ingested nutrients induce glucagon-like peptide-1 (GLP-1, which in turn induces insulin secretion. Objective To evaluate the relationship between TCF7L2 and KCNJ11 gene polymorphism, dietary intake, and physical activity on fasting plasma GLP-1 in normal male adolescents. Methods This observational study with a cross-sectional design included 54 male adolescents selected from high schools in Yogyakarta, Indonesia. Interviews were done to collect data on energy intake and physical activity. The GLP-1 and insulin levels were measured from fasting blood plasma. The TCF7L2 and KCNJ11 gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP. Results Fasting GLP-1 was positively correlated with energy intake (r=0.276; P=0.047, but not with physical activity (r=0.011; P=0.936. The GLP-1 concentration was not associated with TCF7L2 and KCNJ11 gene polymorphisms (all P>0.05. In subjects with an EE genotype (KCNJ11, GLP-1 was not correlated with insulin (r=-0.036; P=0.435. However, in subjects with an EK genotype (KCNJ11, GLP-1 was positively correlated with insulin (r=0.394; P=0.026. Conclusion GLP-1 concentration is positively correlated with body weight. Among male adolescents with a genetic variation in KCNJ11 (EK genotype, there is a significant correlation between GLP-1 and insulin signalling.

Characterization of purified Sindbis virus nsP4 RNA-dependent RNA polymerase activity in vitro

International Nuclear Information System (INIS)

Rubach, Jon K.; Wasik, Brian R.; Rupp, Jonathan C.; Kuhn, Richard J.; Hardy, Richard W.; Smith, Janet L.

2009-01-01

The Sindbis virus RNA-dependent RNA polymerase (nsP4) is responsible for the replication of the viral RNA genome. In infected cells, nsP4 is localized in a replication complex along with the other viral non-structural proteins. nsP4 has been difficult to homogenously purify from infected cells due to its interactions with the other replication proteins and the fact that its N-terminal residue, a tyrosine, causes the protein to be rapidly turned over in cells. We report the successful expression and purification of Sindbis nsP4 in a bacterial system, in which nsP4 is expressed as an N-terminal SUMO fusion protein. After purification the SUMO tag is removed, resulting in the isolation of full-length nsP4 possessing the authentic N-terminal tyrosine. This purified enzyme is able to produce minus-strand RNA de novo from plus-strand templates, as well as terminally add adenosine residues to the 3' end of an RNA substrate. In the presence of the partially processed viral replicase polyprotein, P123, purified nsP4 is able to synthesize discrete template length minus-strand RNA products. Mutations in the 3' CSE or poly(A) tail of viral template RNA prevent RNA synthesis by the replicase complex containing purified nsP4, consistent with previously reported template requirements for minus-strand RNA synthesis. Optimal reaction conditions were determined by investigating the effects of time, pH, and the concentrations of nsP4, P123 and magnesium on the synthesis of RNA

Purifying capability, enzyme activity, and nitrification potentials in December in integrated vertical flow constructed wetland with earthworms and different substrates.

Science.gov (United States)

Xu, Defu; Gu, Jiaru; Li, Yingxue; Zhang, Yu; Howard, Alan; Guan, Yidong; Li, Jiuhai; Xu, Hui

2016-01-01

The response of purifying capability, enzyme activity, nitrification potentials, and total number of bacteria in the rhizosphere in December to wetland plants, substrates, and earthworms was investigated in integrated vertical flow constructed wetlands (IVFCW). The removal efficiency of total nitrogen (TN), NH4-N, chemical oxygen demand (COD), and total phosphorus (TP) was increased when earthworms were added into IVFCW. A significantly average removal efficiency of N in IVFCW that employed river sand as substrate and in IVFCW that employed a mixture of river sand and Qing sand as substrate was not found. However, the average removal efficiency of P was higher in IVFCW with a mixture of river sand and Qing sand as substrate than in IVFCW with river sand as substrate. Invertase activity in December was higher in IVFCW that used a mixture of river sand and Qing sand as substrate than in IVFCW which used only river sand as substrate. However, urease activity, nitrification potential, and total number of bacteria in December was higher in IVFCW that employed river sand as substrate than in IVFCW with a mixture of river sand and Qing sand as substrate. The addition of earthworms into the integrated vertical flow constructed wetland increased the above-ground biomass, enzyme activity (catalase, urease, and invertase), nitrification potentials, and total number of bacteria in December. The above-ground biomass of wetland plants was significantly positively correlated with urease and nitrification potentials (p earthworms into IVFCW increased enzyme activity and nitrification potentials in December, which resulted in improving purifying capability.

Catalytic Properties and Immobilization Studies of Catalase from Malva sylvestris L.

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G. Arabaci

2013-01-01

Full Text Available Catalase was partially purified from Malva sylvestris L. and immobilized onto chitosan. Then, its catalytic properties were investigated. (NH42SO4 precipitation and dialysis were performed in the extracted enzyme. Further purification was performed with sephadex G-200 column. Kinetic studies of the purified enzyme activity were measured and characterized. The inhibitory effects of KCN, NaN3, CuSO4, and EDTA on M. sylvestris L. catalase activity were observed except NaCl. Furthermore, M. sylvestris L. catalase was immobilized covalently with glutaraldehyde onto chitosan particles. The pH and temperature optima as well as the changes in the kinetics (Km, Vmax of the immobilized and free M. sylvestris L. catalase were determined. The Km value for immobilized catalase (23.4 mM was higher than that of free enzyme (17.6 mM. Optimum temperature was observed higher than that of the free enzyme. The optimum pH was the same for both free and immobilized catalases (pH 7.50. Immobilized catalase showed higher storage and thermal stabilities than free catalases. Free catalase lost all its activity within 60 days whereas immobilized catalase lost 45% of its activity during the same incubation period at 4°C. The remaining immobilized catalase activity was about 70% after 8 cycles of batch operations.

C. pneumoniae CdsL regulates CdsN ATPase activity, and disruption with a peptide mimetic prevents bacterial invasion

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Chris Blair Stone

2011-02-01

Full Text Available Chlamydiae are obligate intracellular pathogens that likely require type III secretion (T3S to invade cells and replicate intracellulary within a cytoplasmic vacuole called an inclusion body. C. pneumoniae possess a YscL ortholog, CdsL, that has been shown to interact with the T3S ATPase (CdsN. In this report we demonstrate that CdsL down-regulates CdsN enzymatic activity in a dose-dependent manner. Using PepScan epitope mapping we identified two separate binding domains to which CdsL binds viz. CdsN 221-229 and CdsN265-270. We confirmed the binding domains using a pull-down assay and showed that GST-CdsN221-270, which encompasses these peptides, co-purified with His-CdsL. Next, we used orthology modeling based on the crystal structure of a T3S ATPase ortholog from E. coli, EscN, to map the binding domains on the predicted three dimensional structure of CdsN. The CdsL binding domains mapped to the catalytic domain of the ATPase, one in the central channel of the ATPase hexamer and one on the outer face. Since peptide mimetics have been used to disrupt essential protein interactions of the chlamydial T3S system and inhibit T3S-mediated invasion of HeLa cells, we hypothesized that if CdsL – CdsN binding is essential for regulating T3S then a CdsN peptide mimetic could be used to potentially block T3S and Chlamydial invasion. Treatment of EBs with a CdsN peptide mimetic inhibited C. pneumoniae invasion into HeLa cells in a dose-dependent fashion. This report represents the first use of Pepscan technology to identify binding domains for specific T3S proteins viz. CdsL on the ATPase, CdsN, and demonstrates that peptide mimetics can be used as anti-virulence factors to block bacterial invasion.

21 CFR 880.6710 - Medical ultraviolet water purifier.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet water purifier. 880.6710... Miscellaneous Devices § 880.6710 Medical ultraviolet water purifier. (a) Identification. A medical ultraviolet water purifier is a device intended for medical purposes that is used to destroy bacteria in water by...

Aluminum Silicate Nanotube Modification of Cotton-Like Siloxane-poly(L-lactic acid-vaterite Composites

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Daiheon Lee

2013-01-01

Full Text Available In our earlier work, a cotton-like biodegradable composite, consisting of poly(L-lactic acid with siloxane-containing vaterite, has been prepared by electrospinning. In the present work, the fibers skeleton of the cotton-like composites was modified successfully with imogolite, which is hydrophilic and biocompatible, via a dip process using ethanol diluted solution to improve the cellular initial attachment. Almost no change in the fiber morphology after the surface modification was observed. The surface-modified composite showed the similar calcium and silicate ions releasabilities, for activating the osteoblasts, as an unmodified one. Cell culture tests showed that the initial adhesion of murine osteoblast-like cells on the surface of the fibers was enhanced by surface modification.

Purification and Characterization of Recombinant Vaccinia L1R Protein from Escherichia coli

Science.gov (United States)

2016-08-01

RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI 1. INTRODUCTION 1.1 Background Vaccinia virus (VACV) is the active component of the...the preparation of the recombinant VACV L1R protein fragment by denaturing , refolding, and purifying material expressed into inclusion bodies in...PURIFICATION AND CHARACTERIZATION OF RECOMBINANT VACCINIA L1R PROTEIN FROM ESCHERICHIA COLI ECBC-TR-1370

Fabrication of Simple Indoor Air Haze Purifier using Domestic Discarded Substances and Its Haze Removal Performance

Science.gov (United States)

Wang, Zhou; Cao, Haoshu; Zhao, Shuang

2018-01-01

Based on the concept of circular economy, discarded plastic bottles stuffed with discarded cotton, clothing and sofa cushion were used as pre-filter to remove big particles (dust and coal dust) in air and 4 L tap water in discarded plastic bottle was worked as an absorbing medium to dissolve the water soluble ions in air (SO4 2-, NO3-, NH4+, Cl- and Ca2+). Moreover, the internet control design was used in this homemade indoor air haze purifier to achieve the performance of remote control and intelligent management. The experimental results showed that this indoor air haze purifier can effectively reduce the level of indoor air haze and the air quality after 20 minutes treatment is higher than that of two commercial well-known air haze purifier

Production of L-allose and D-talose from L-psicose and D-tagatose by L-ribose isomerase.

Science.gov (United States)

Terami, Yuji; Uechi, Keiko; Nomura, Saki; Okamoto, Naoki; Morimoto, Kenji; Takata, Goro

2015-01-01

L-ribose isomerase (L-RI) from Cellulomonas parahominis MB426 can convert L-psicose and D-tagatose to L-allose and D-talose, respectively. Partially purified recombinant L-RI from Escherichia coli JM109 was immobilized on DIAION HPA25L resin and then utilized to produce L-allose and D-talose. Conversion reaction was performed with the reaction mixture containing 10% L-psicose or D-tagatose and immobilized L-RI at 40 °C. At equilibrium state, the yield of L-allose and D-talose was 35.0% and 13.0%, respectively. Immobilized enzyme could convert L-psicose to L-allose without remarkable decrease in the enzyme activity over 7 times use and D-tagatose to D-talose over 37 times use. After separation and concentration, the mixture solution of L-allose and D-talose was concentrated up to 70% and crystallized by keeping at 4 °C. L-Allose and d-talose crystals were collected from the syrup by filtration. The final yield was 23.0% L-allose and 7.30% D-talose that were obtained from L-psicose and D-tagatose, respectively.

Withania somnifera (Ashwagandha): a Novel Source of L-asparaginase

Institute of Scientific and Technical Information of China (English)

Vishal P. Oza; Shraddha D.Trivedi; Pritesh P.Parmar; R.B.Subramanian

2009-01-01

Different parts of plant species belonging to Solanaceae and Fabaceae families were screened for L-asparaginase enzyme (E.C.3.5.1.1.). Among 34 plant species screened for L-asparaginase enzyme, Withania somnifera L. Was identified as a potential source of the enzyme on the basis of high specific activity of the enzyme. The enzyme was purified and characterized from W. Somnifera, a popular medicinal plant in South East Asia and Southern Europe. Purification was carried out by a combination of protein precipitation with ammonium sulfate as well as Sephadex-gel filtration. The purified enzyme is a homodimer, with a molecular mass of 72±0.5 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresisand size exclusion chromatography. The enzyme has a pH optimum of 8.5 and an optimum temperature of 37℃. The Km value for the enzyme is 6.1×10-2 mmol/L. This is the first report for L-asparaginase from W. Somnifera, a traditionally used Indian medicinal plant.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

Energy Technology Data Exchange (ETDEWEB)

Takahashi, Hitoshi; Akazawa, Daisuke [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Kato, Takanobu; Date, Tomoko [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Shirakura, Masayuki [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Toray Industries, Inc., Kanagawa (Japan); Nakamura, Noriko; Mochizuki, Hidenori [Toray Industries, Inc., Kanagawa (Japan); Tanaka-Kaneko, Keiko; Sata, Tetsutaro [Department of Pathology, National Institute of Infectious Diseases, Tokyo (Japan); Tanaka, Yasuhito [Department of Clinical Molecular Informative Medicine, Nagoya City University Graduate School of Medicine, Nagoya (Japan); Mizokami, Masashi [Research Center for Hepatitis and Immunology, Kohnodai Hospital, International Medical Center of Japan, Chiba (Japan); Suzuki, Tetsuro [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Wakita, Takaji, E-mail: wakita@nih.go.jp [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan)

2010-05-14

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope

International Nuclear Information System (INIS)

Takahashi, Hitoshi; Akazawa, Daisuke; Kato, Takanobu; Date, Tomoko; Shirakura, Masayuki; Nakamura, Noriko; Mochizuki, Hidenori; Tanaka-Kaneko, Keiko; Sata, Tetsutaro; Tanaka, Yasuhito; Mizokami, Masashi; Suzuki, Tetsuro; Wakita, Takaji

2010-01-01

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Bovine pancreatic trypsin inhibitor immobilized onto sepharose as a new strategy to purify a thermostable alkaline peptidase from cobia (Rachycentron canadum) processing waste.

Science.gov (United States)

França, Renata Cristina da Penha; Assis, Caio Rodrigo Dias; Santos, Juliana Ferreira; Torquato, Ricardo José Soares; Tanaka, Aparecida Sadae; Hirata, Izaura Yoshico; Assis, Diego Magno; Juliano, Maria Aparecida; Cavalli, Ronaldo Olivera; Carvalho, Luiz Bezerra de; Bezerra, Ranilson Souza

2016-10-15

A thermostable alkaline peptidase was purified from the processing waste of cobia (Rachycentron canadum) using bovine pancreatic trypsin inhibitor (BPTI) immobilized onto Sepharose. The purified enzyme had an apparent molecular mass of 24kDa by both sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry. Its optimal temperature and pH were 50°C and 8.5, respectively. The enzyme was thermostable until 55°C and its activity was strongly inhibited by the classic trypsin inhibitors N-ρ-tosyl-l-lysine chloromethyl ketone (TLCK) and benzamidine. BPTI column allowed at least 15 assays without loss of efficacy. The purified enzyme was identified as a trypsin and the N-terminal amino acid sequence of this trypsin was IVGGYECTPHSQAHQVSLNSGYHFC, which was highly homologous to trypsin from cold water fish species. Using Nα-benzoyl-dl-arginine ρ-nitroanilide hydrochloride (BApNA) as substrate, the apparent km value of the purified trypsin was 0.38mM, kcat value was 3.14s(-1), and kcat/km was 8.26s(-1)mM(-1). The catalytic proficiency of the purified enzyme was 2.75×10(12)M(-1) showing higher affinity for the substrate at the transition state than other fish trypsin. The activation energy (AE) of the BApNA hydrolysis catalyzed by this enzyme was estimated to be 11.93kcalmol(-1) while the resulting rate enhancement of this reaction was found to be approximately in a range from 10(9) to 10(10)-fold evidencing its efficiency in comparison to other trypsin. This new purification strategy showed to be appropriate to obtain an alkaline peptidase from cobia processing waste with high purification degree. According with N-terminal homology and kinetic parameters, R. canadum trypsin may gathers desirable properties of psychrophilic and thermostable enzymes. Copyright © 2016 Elsevier B.V. All rights reserved.

Energy levels and radiative rates for transitions in B-like to F-like Xe ions (Xe L-XLVI)

International Nuclear Information System (INIS)

Aggarwal, K.M.; Keenan, F.P.; Lawson, K.D.

2010-01-01

Energy levels, radiative rates, oscillator strengths, line strengths, and lifetimes have been calculated for transitions in B-like to F-like Xe ions, Xe L-XLVI. For the calculations, a fully relativistic GRASP code has been adopted, and results are reported for all electric dipole, electric quadrupole, magnetic dipole, and magnetic quadrupole transitions among the lowest 125, 236, 272, 226, and 113 levels of Xe L, Xe XLIX, Xe XLVIII, Xe XLVII, and Xe XLVI, respectively, belonging to the n ≤ 3 configurations.

Bavachin from Psoralea corylifolia Improves Insulin-Dependent Glucose Uptake through Insulin Signaling and AMPK Activation in 3T3-L1 Adipocytes

Directory of Open Access Journals (Sweden)

Hyejin Lee

2016-04-01

Full Text Available The fruit of Psoralea corylifolia L. (Fabaceae (PC, known as “Bo-Gol-Zhee” in Korea has been used as traditional medicine. Ethanol and aqueous extracts of PC have an anti-hyperglycemic effect by increasing plasma insulin levels and decreasing blood glucose and total plasma cholesterol levels in type 2 diabetic rats. In this study, we purified six compounds from PC and investigated their anti-diabetic effect. Among the purified compounds, bavachin most potently accumulated lipids during adipocyte differentiation. Intracellular lipid accumulation was measured by Oil Red-O (ORO cell staining to investigate the effect of compounds on adipogenesis. Consistently, bavachin activated gene expression of adipogenic transcriptional factors, proliferator-activated receptorγ (PPARγ and CCAAT/enhancer binding protein-α (C/EBPα. Bavachin also increased adiponectin expression and secretion in adipocytes. Moreover, bavachin increased insulin-induced glucose uptake by differentiated adipocytes and myoblasts. In differentiated adipocytes, we found that bavachin enhanced glucose uptake via glucose transporter 4 (GLUT4 translocation by activating the Akt and 5′AMP-activated protein kinase (AMPK pathway in the presence or absence of insulin. These results suggest that bavachin from Psoralea corylifolia might have therapeutic potential for type 2 diabetes by activating insulin signaling pathways.

[Expression, purification and immunogenicity of human papillomavirus type 11 virus-like particles from Escherichia coli].

Science.gov (United States)

Yan, Chunyan; Li, Shaowei; Wang, Jin; Wei, Minxi; Huang, Bo; Zhuang, Yudi; Li, Zhongyi; Pan, Huirong; Zhang, Jun; Xia, Ningshao

2009-11-01

To produce human papillomavirus type 11 virus-like particles (HPV11 VLPs) from Escherichia coli and to investigate its immunogenicity and type cross neutralization nature. We expressed the major capsid protein of HPV11 (HPV11-L1) in Escherichia coli ER2566 in non fusion fashion and purified by amino sulfate precipitation, ion-exchange chromatography and hydrophobic interaction chromatography, sequentially. Then we removed the reductant DTT to have the purified HPV11-L1 self-assemble into VLPs in vitro. We investigated the morphology of these VLPs with dynamic light scattering and transmission electron microscopy. We assayed the immunogenicity of the resultant HPV11 VLPs by vaccinations on mice and evaluated by HPV6/11/16/18 pseudovirion neutralization cell models. We expressed HPV11 L1 in Escherichia coli with two forms, soluble and inclusion body. The soluble HPV11 L1 with over 95% purity can self assemble to VLPs in high efficiency. Morphologically, these VLPs were globular, homogeneous and with a diameter of - 50 nm, which is quite similar with native HPV11 virions. The half effective dosage (ED50) of HPV11 VLPs is 0.031 microg, and the maximum titer of neutralizing antibody elicited is averaged to 10(6). The cross neutralization activity (against HPV6/16/18) of the anti-HPV11 serum was found to have exact correlation to the inter-type homology in amino acid alignment. We can provide HPV11 VLPs with highly immunogenicity from prokaryote expression system, which may pave a new way for research and development of prophylactic vaccine for HPV11.

Antibody-dependent cellular cytotoxicity (ADCC) activity of a novel anti-PD-L1 antibody avelumab (MSB0010718C) on human tumor cells

Science.gov (United States)

Fantini, Massimo; Heery, Christopher R.; Gulley, James L.; Tsang, Kwong Yok; Schlom, Jeffrey

2015-01-01

Several anti-PD1/PD-L1 monoclonal antibodies (MAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these MAbs is to inhibit PD1 on immune cells interacting with PD-L1 on tumor cells. These MAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective MAb-mediated cancer therapies. A fully human anti-PD-L1 MAb would potentially be able to block PD-L1/PD1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 MAb. The studies reported here demonstrate (a) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (b) IFNγ can enhance tumor cell PD-L1 expression and in some cases enhance ADCC tumor cell lysis; (c) purified NK cells are potent effectors for avelumab; (d) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (e) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (f) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 MAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. PMID:26014098

Enhancing production and cytotoxic activity of polymeric soluble FasL-based chimeric proteins by concomitant expression of soluble FasL.

Directory of Open Access Journals (Sweden)

Aurore Morello

Full Text Available Membrane FasL is the natural trigger of Fas-mediated apoptosis. A soluble homotrimeric counterpart (sFasL also exists which is very weakly active, and needs oligomerization beyond its trimeric state to induce apoptosis. We recently generated a soluble FasL chimera by fusing the immunoglobulin-like domain of the leukemia inhibitory factor receptor gp190 to the extracellular region of human FasL, which enabled spontaneous dodecameric homotypic polymerization of FasL. This polymeric soluble human FasL (pFasL displayed anti-tumoral activity in vitro and in vivo without systemic cytotoxicity in mouse. In the present work, we focused on the improvement of pFasL, with two complementary objectives. First, we developed more complex pFasL-based chimeras that contained a cell-targeting module. Secondly, we attempted to improve the production and/or the specific activity of pFasL and of the cell-targeting chimeras. We designed two chimeras by fusing to pFasL the extracellular portions of the HLA-A2 molecule or of a human gamma-delta TCR, and analyzed the consequences of co-expressing these molecules or pFasL together with sFasL on their heterotopic cell production. This strategy significantly enhanced the production of pFasL and of the two chimeras, as well as the cytotoxic activity of the two chimeras but not of pFasL. These results provide the proof of concept for an optimization of FasL-based chimeric proteins for a therapeutic use.

Respirators: Air Purifying, Self-Study, Course 40723

Energy Technology Data Exchange (ETDEWEB)

Chochoms, Michael [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

2017-12-21

Respirators: Air Purifying Self-Study (COURSE 40723) is designed for Los Alamos National Laboratory (LANL) workers, support services subcontractors, and other LANL subcontractors who work under the LANL Respiratory Protection Program (RPP). This course also meets the air-purifying respirators (APRs) retraining requirement.

Bitistatin-functionalized fluorescent nanodiamond particles specifically bind to purified human platelet integrin receptor αIIbβ3 and activated platelets

Directory of Open Access Journals (Sweden)

Marcinkiewicz C

2017-05-01

Full Text Available Cezary Marcinkiewicz,1,2 Jonathan A Gerstenhaber,1 Mark Sternberg,2 Peter I Lelkes,1 Giora Feuerstein1,2 1Department of Bioengineering, College of Engineering, Temple University, Philadelphia, 2Debina Diagnostic, Inc., Newton Square, PA, USA Abstract: Thromboembolic events (TEE underwrite key causes of death in developed countries. While advanced imaging technologies such as computed tomography scans serve to diagnose blood clots during acute cardiovascular events, no such technology is available in routine primary care for TEE risk assessment. Here, we describe an imaging platform technology based on bioengineered fluorescent nanodiamond particles (F-NDPs functionalized with bitistatin (Bit, a disintegrin that specifically binds to the αIIbβ3 integrin, platelet fibrinogen receptor (PFR on activated platelets. Covalent linkage of purified Bit to F-NDP was concentration-dependent and saturable, as validated by enzyme-linked immunosorbent assay using specific anti-Bit antibodies. F-NDP–Bit interacted with purified PFR, either in immobilized or soluble form. Lotrafiban, a nonpeptide, αIIbβ3 receptor antagonist, specifically blocked F-NDP–Bit–PFR complex formation. Moreover, F-NDP–Bit specifically binds to activated platelets incorporated into a clot generated by thrombin-activated rat platelet-rich plasma (PRP. Our results suggest that engineered F-NDP–Bit particles could serve as noninvasive, “real-time” optical diagnostics for clots present in blood vessels. Keywords: carbon nanoparticles, blood clots, imaging, platelet fibrinogen receptor, fluorescence, disintegrin, thromboembolic complications, thrombosis

Characterization of the Entire Cystatin Gene Family in Barley and Their Target Cathepsin L-Like Cysteine-Proteases, Partners in the Hordein Mobilization during Seed Germination1[W

Science.gov (United States)

Martinez, Manuel; Cambra, Ines; Carrillo, Laura; Diaz-Mendoza, Mercedes; Diaz, Isabel

2009-01-01

Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds. PMID:19759340

Enzymatic conversion of D-galactose to D-tagatose: cloning, overexpression and characterization of L-arabinose isomerase from Pediococcus pentosaceus PC-5.

Science.gov (United States)

Men, Yan; Zhu, Yueming; Zhang, Lili; Kang, Zhenkui; Izumori, Ken; Sun, Yuanxia; Ma, Yanhe

2014-01-01

The gene encoding L-arabinose isomerase from food-grade strain Pediococcus pentosaceus PC-5 was cloned and overexpressed in Escherichia coli. The recombinant protein was purified and characterized. It was optimally active at 50 °C and pH 6.0. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its maximal activity evaluated at 0.6 mM Mn(2+) or 0.8 mM Co(2+). Interestingly, this enzyme was distinguished from other L-AIs, it could not use L-arabinose as its substrate. In addition, a three-dimensional structure of L-AI was built by homology modeling and L-arabinose and D-galactose were docked into the active site pocket of PPAI model to explain the interaction between L-AI and its substrate. The purified P. pentosaceus PC-5 L-AI converted D-galactose into D-tagatose with a high conversion rate of 52% after 24 h at 50 °C, suggesting its excellent potential in D-tagatose production. Crown Copyright © 2013. Published by Elsevier GmbH. All rights reserved.

Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

Directory of Open Access Journals (Sweden)

Sakuko Ueshima

2010-01-01

Full Text Available The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3 were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienylserine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

Neuropeptide Y binding sites in rat brain identified with purified neuropeptide Y-I125

International Nuclear Information System (INIS)

Walker, M.W.; Miller, R.J.

1986-01-01

Neuropeptide Y (NPY) is a widely distributed neuronally localized peptide with 36 amino acids, 5 of which are tyrosines. The authors wished to investigate the properties of specific receptors for NPY. They therefore labeled the tyrosines with I125 using chloramine T and then purified the peptide using HPLC. A single mono-iodinated species of NPY which yielded > 85% specific binding in rat forebrain synaptosomes was selected as the ligand for all subsequent experiments. A time course of binding showed that equilibrium conditions were reached in 60 minutes at 21 0 C. Scatchard plots revealed a single class of binding sites with a Kd and a Bmax of 3 x 10-10 M and 28 pmol/mg, respectively. Competition binding with unlabeled NPY showed 50% displacement of bound ligand at 1 x 10-10 M NPY. Competition binding with rat pancreatic polypeptide (RPP), a homologous peptide possessing little NPY-like activity, showed 50% displacement of bound ligand at 2 x 10 -7 M RPP. No binding was observed on F-11 or PC12 neuronal cell lines, or on HSWP fibroblast cells. They conclude that NPY-I125 purified to homogeneity with HPLC is a highly selective ligand for NPY receptor sites. They are currently investigating such sites in brain, gut, and other tissues

Biochemical Properties and Mechanism of Action of Enterocin LD3 Purified from Enterococcus hirae LD3.

Science.gov (United States)

Gupta, Aabha; Tiwari, Santosh Kumar; Netrebov, Victoria; Chikindas, Michael L

2016-09-01

Enterocin LD3 was purified using activity-guided multistep chromatography techniques such as cation-exchange and gel-filtration chromatography. The preparation's purity was tested using reverse-phase ultra-performance liquid chromatography. The specific activity was tested to be 187.5 AU µg(-1) with 13-fold purification. Purified enterocin LD3 was heat stable up to 121 °C (at 15 psi pressure) and pH 2-6. The activity was lost in the presence of papain, reduced by proteinase K, pepsin and trypsin, but was unaffected by amylase and lipase, suggesting proteinaceous nature of the compound and no role of carbohydrate and lipid moieties in the activity. MALDI-TOF/MS analysis of purified enterocin LD3 resolved m/z 4114.6, and N-terminal amino acid sequence was found to be H2NQGGQANQ-COOH suggesting a new bacteriocin. Dissipation of membrane potential, loss of internal ATP and bactericidal effect were recorded when indicator strain Micrococcus luteus was treated with enterocin LD3. It inhibited Gram-positive and Gram-negative bacteria including human pathogens such as Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Salmonella typhi, Shigella flexneri, Listeria monocytogenes, Escherichia coli O157:H7, E. coli (urogenic, a clinical isolate) and Vibrio sp. These properties of purified enterocin LD3 suggest its applications as a food biopreservative and as an alternative to clinical antibiotics.

Antibody-Dependent Cellular Cytotoxicity Activity of a Novel Anti-PD-L1 Antibody Avelumab (MSB0010718C) on Human Tumor Cells.

Science.gov (United States)

Boyerinas, Benjamin; Jochems, Caroline; Fantini, Massimo; Heery, Christopher R; Gulley, James L; Tsang, Kwong Yok; Schlom, Jeffrey

2015-10-01

Several anti-PD-1/PD-L1 monoclonal antibodies (mAb) are currently providing evidence of clinical benefit in subsets of cancer patients. The mode of action of these mAbs is to inhibit PD-1 on immune cells interacting with PD-L1 on tumor cells. These mAbs are either designed or engineered to eliminate antibody-dependent cell-mediated cytotoxicity (ADCC), which, however, has been implicated as an important mechanism in several highly effective mAb-mediated cancer therapies. A fully human anti-PD-L1 mAb would potentially be able to block PD-1/PD-L1 interactions and also mediate the ADCC lysis of tumor cells. MSB0010718C (designated avelumab) is a fully human IgG1 anti-PD-L1 mAb. The studies reported here demonstrate (i) the ability of avelumab to lyse a range of human tumor cells in the presence of PBMC or NK effectors; (ii) IFNγ can enhance tumor cell PD-L1 expression and, in some cases, enhance ADCC tumor cell lysis; (iii) purified NK cells are potent effectors for avelumab; (iv) similar levels of avelumab-mediated ADCC lysis of tumor cells are seen using purified NK as effectors from either healthy donors or cancer patients; (v) very low levels of avelumab-mediated lysis are seen using whole PBMCs as targets; this finding complements results seen in analyses of PBMC subsets of patients receiving avelumab; and (vi) the addition of IL12 to NK cells greatly enhances avelumab-mediated ADCC. These studies thus provide an additional mode of action for an anti-PD-L1 mAb and support the rationale for further studies to enhance avelumab-mediated ADCC activity. ©2015 American Association for Cancer Research.

Binding of human papilloma virus L1 virus-like particles to dendritic cells is mediated through heparan sulfates and induces immune activation

NARCIS (Netherlands)

de Witte, Lot; Zoughlami, Younes; Aengeneyndt, Birgit; David, Guido; van Kooyk, Yvette; Gissmann, Lutz; Geijtenbeek, Teunis B. H.

2007-01-01

Immunization using human papilloma virus (HPV)-L1 virus-like particles (VLPs) induces a robust and effective immune response, which has recently resulted in the implementation of the HPV-L1 VLP vaccination in health programs. However, during infection, HPV can escape immune surveillance leading to

Biomass derived graphene-like activated and non-activated porous ...

Indian Academy of Sciences (India)

Graphene-like activated and non-activated carbon nanostructures were synthesized from various natural sources like sugar, rice husk and jute. These carbon nanostructures were characterized using SEM, FTIR and Raman spectroscopy, surface area and thermogravimetric analysis. The electrochemical studies of these ...

Purification and characterization of camel liver L-asparaginase ...

African Journals Online (AJOL)

L-asparaginase from camel liver was isolated and purified by heat denaturation followed by QAE-Sephadex A-50 column chromatography and SP-Sepharose column chromatography. The purified camel liver L-asparaginase had a molecular weight of 180 kDa (consistent with a homotetramer) and a pI value of 8.6.

Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity.

Science.gov (United States)

dos Santos, Alda E; Kuster, Ricardo M; Yamamoto, Kristie A; Salles, Tiago S; Campos, Renata; de Meneses, Marcelo D F; Soares, Márcia R; Ferreira, Davis

2014-03-28

The arthropod-borne Mayaro virus (MAYV) causes 'Mayaro fever', a disease of medical significance, primarily affecting individuals in permanent contact with forested areas in tropical South America. Recently, MAYV has attracted attention due to its likely urbanization. Currently, there are no licensed drugs against most mosquito-transmitted viruses. Here, we investigated the in vitro anti-MAYV activity of the flavonoids quercetin and its derivatives from the Brazilian shrub Bauhinia longifolia (Bong.) Steud. Flavonoids were purified by chromatographic fractionation from leaf extracts of B. longifolia and chemically identified as quercetin and quercetin glycosides using spectroscopic techniques. Cytotoxicity of purified flavonoids and of EtOAc- and n-BuOH-containing flavonoid mixtures was measured by the dye-uptake assay while their antiviral activity was evaluated by a virus yield inhibition assay. The following flavonoids were purified from B. longifolia leaves: non-glycosylated quercetin and its glycosides guaijaverin, quercitrin, isoquercitrin, and hyperin. EtOAc and n-BuOH fractions containing these flavonoids demonstrated the highest antiviral activity of all tested substances, while quercetin had the highest antiviral activity amongst purified flavonoids. Quercetin, EtOAc, or n-BuOH fractions inhibited MAYV production by more than 90% at 25 μg/mL, displaying a stronger antiviral effect than the licensed antiviral ribavirin. A mixture of the isomers isoquercitrin and hyperin had a modest antiviral effect (IC90 = 104.9), while guaijaverin and quercitrin did not show significant antiviral activity. B. longifolia is a good source of flavonoids with anti-Mayaro virus activity. This is the first report of the activity of quercetin and its derivatives against an alphavirus.

31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

International Nuclear Information System (INIS)

Kim, Hyun Won

2011-01-01

Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

Monoterpene limonene induces brown fat-like phenotype in 3T3-L1 white adipocytes.

Science.gov (United States)

Lone, Jameel; Yun, Jong Won

2016-05-15

Several dietary compounds that are able to induce the brown fat-like phenotype in white adipocytes have been considered for treatment of obesity due to their ability to increase energy expenditure. Here, we report that limonene induces the brown fat-like phenotype in 3T3-L1 adipocytes by increasing expression of brown adipocyte-specific genes and proteins. Limonene-induced browning in white adipocytes was investigated by determining expression levels of brown fat-specific genes and proteins by real-time RT-PCR, immunoblot analysis, and immunocytochemical staining. Limonene enhanced mitochondrial biogenesis, as evidenced by increased mitochondrial content and immunofluorescent intensity. Limonene also significantly elevated protein levels of HSL, PLIN, p-AMPK, p-ACC, ACO, COX4, CPT1, and CYT C, suggesting its possible role in enhancement of lipolysis and lipid catabolism. Increased expression of PRDM16, UCP1, C/EBPβ, and other brown fat-specific markers by limonene was possibly mediated by activation of β3-adnergenic receptor (β3-AR), as inhibition of β3-AR inhibited up-regulation of brown fat-specific markers. Similarly, limonene-mediated activation of ERK and up-regulation of key brown adipocyte specific markers were eliminated by treatment with ERK antagonist. Taken together, these results suggest that limonene induces browning of 3T3-L1 adipocytes via activation of β3-AR and the ERK signaling pathway. In conclusion, our findings suggest that limonene plays a dual modulatory role in induction of the brown adipocyte-like phenotype as well as promotion of lipid metabolism and thus may have potential therapeutic implications for treatment of obesity. Copyright © 2016 Elsevier Inc. All rights reserved.

Synthesis, structure, and glutathione peroxidase-like activity of amino acid containing ebselen analogues and diaryl diselenides.

Science.gov (United States)

Selvakumar, Karuthapandi; Shah, Poonam; Singh, Harkesh B; Butcher, Ray J

2011-11-04

The synthesis of some ebselen analogues and diaryl diselenides, which have amino acid functions as an intramolecularly coordinating group (Se···O) has been achieved by the DCC coupling procedure. The reaction of 2,2'-diselanediylbis(5-tert-butylisophthalic acid) or the activated ester tetrakis(2,5-dioxopyrrolidin-1-yl) 2,2'-diselanediylbis(5-tert-butylisophthalate) with different C-protected amino acids (Gly, L-Phe, L-Ala, and L-Trp) afforded the corresponding ebselen analogues. The used precursor diselenides have been found to undergo facile intramolecular cyclization during the amide bond formation reaction. In contrast, the DCC coupling of 2,2'-diselanediyldibenzoic acid with C-protected amino acids (Gly, L/D-Ala and L-Phe) affords the corresponding amide derivatives and not the ebselen analogues. Some of the representative compounds have been structurally characterized by single-crystal X-ray crystallography. The glutathione peroxidase (GPx)-like activities of the ebselen analogues and the diaryl diselenides have been evaluated by using the coupled reductase assay method. Intramolecularly stabilized ebselen analogues show slightly higher maximal velocity (V(max)) than ebselen. However, they do not show any GPx-like activity at low GSH concentrations at which ebselen and related diselenides are active. This could be attributed to the peroxide-mediated intramolecular cyclization of the corresponding selenenyl sulfide and diaryl diselenide intermediates generated during the catalytic cycle. Interestingly, the diaryl diselenides with alanine (L,L or D,D) amide moieties showed excellent catalytic efficiency (k(cat)/K(M)) with low K(M) values in comparison to the other compounds. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phytochemical profile, aldose reductase inhibitory, and antioxidant activities of Indian traditional medicinal Coccinia grandis (L.) fruit extract.

Science.gov (United States)

Kondhare, Dasharath; Lade, Harshad

2017-12-01

Coccinia grandis (L.) fruits (CGFs) are commonly used for culinary purposes and has several therapeutic applications in the Southeast Asia. The aim of this work was to evaluate phytochemical profile, aldose reductase inhibitory (ARI), and antioxidant activities of CGF extract. The CGFs were extracted with different solvents including petroleum ether, dichloromethane, acetone, methanol, and water. The highest yield of total extractable compounds (34.82%) and phenolic content (11.7 ± 0.43 mg of GAE/g dried extract) was found in methanol extract, whereas water extract showed the maximum content of total flavonoids (82.8 ± 7.8 mg QE/g dried extract). Gas chromatography-mass spectroscopy (GC-MS) analysis of methanol and water extract revealed the presence of flavonoids, phenolic compounds, alkaloids, and glycosides in the CGFs. Results of the in vitro ARI activity against partially purified bovine lens aldose reductase showed that methanol extract of CGFs exhibited 96.6% ARI activity at IC 50 value 6.12 µg/mL followed by water extract 89.1% with the IC 50 value 6.50 µg/mL. In addition, methanol and water extracts of CGF showed strong antioxidant activities including ABTS *+ scavenging, DPPH* scavenging, and hydroxyl radical scavenging. Our results suggest that high percentage of both flavonoids and phenolic contents in the CGFs are correlated with the ARI and antioxidant activities. The fruits of C. grandis are thus potential bifunctional agents with ARI and antioxidant activities that can be used for the prevention and management of DM and associated diseases.

Modulation of phenotypic and functional maturation of murine dendritic cells (DCs) by purified Achyranthes bidentata polysaccharide (ABP).

Science.gov (United States)

Zou, Yaxuan; Meng, Jingjuan; Chen, Wenna; Liu, Jingling; Li, Xuan; Li, Weiwei; Lu, Changlong; Shan, Fengping

2011-08-01

There are a large number of interactions at molecular and cellular levels between the plant polysaccharides and immune system. Plant polysaccharides present an interesting effects as immunomodulators, particularly in the induction of the cells both in innate and adaptive immune systems. Activation of DCs could improve antitumoral responses usually diminished in cancer patients, and natural adjuvants provide a possibility of inducing this activation. ABP is a purified polysaccharide isolated from Achyranthes bidentata, a traditional Chinese medicine (TCM). The aim of this study is to investigate modulation of phenotypic and functional maturation of murine DCs by ABP. Both phenotypic and functional activities were assessed with use of conventional scanning electronic microscopy (SEM) for the morphology of the DC, transmitted electron microscopy (TEM) for intracellular lysosomes inside the DC, cellular immunohistochemistry for phagocytosis by the DCs, flow cytometry (FCM) for the changes in key surface molecules, bio-assay for the activity of acidic phosphatases (ACP), and ELISA for the production of pro-inflammatory cytokine IL-12. In fact, we found that purified ABP induced phenotypic maturation revealed by increased expression of CD86, CD40, and MHC II. Functional experiments showed the down-regulation of ACP inside DCs (which occurs when phagocytosis of DCs is decreased, and antigen presentation increased with maturation). Finally, ABP increased the production of IL-12. These data reveal that ABP promotes effective activation of murine DCs. This adjuvant-like activity may have therapeutic applications in clinical settings where immune responses need boosting. It is therefore concluded that ABP can exert positive modulation to murine DCs. Copyright © 2011 Elsevier B.V. All rights reserved.

Identification of a novel splice variant of human PD-L1 mRNA encoding an isoform-lacking Igv-like domain.

Science.gov (United States)

He, Xian-hui; Xu, Li-hui; Liu, Yi

2005-04-01

To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon? encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intracellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

Subpopulations in purified platelets adhering on glass.

Science.gov (United States)

Donati, Alessia; Gupta, Swati; Reviakine, Ilya

2016-06-22

Understanding how platelet activation is regulated is important in the context of cardiovascular disorders and their management with antiplatelet therapy. Recent evidence points to different platelet subpopulations performing different functions. In particular, procoagulant and aggregating subpopulations have been reported in the literature in platelets treated with the GPVI agonists. How the formation of platelet subpopulations upon activation is regulated remains unclear. Here, it is shown that procoagulant and aggregating platelet subpopulations arise spontaneously upon adhesion of purified platelets on clean glass surfaces. Calcium ionophore treatment of the adhering platelets resulted in one platelet population expressing both the procoagulant and the adherent population markers phosphatidylserine and the activated form of GPIIb/IIIa, while all of the platelets expressed CD62P independently of the ionophore treatment. Therefore, all platelets have the capacity to express all three activation markers. It is concluded that platelet subpopulations observed in various studies reflect the dynamics of the platelet activation process.

PRESERVATIVE POTENTIAL OF PURIFIED BACTERIOCIN PRODUCED FROM BREVIBACILLUS BORSTELENSIS AG1 ISOLATED FROM MARCHA – A TRADITIONAL WINE STARTER CULTURE CAKE IN TOMATO PASTE

Directory of Open Access Journals (Sweden)

Anupama Gupta

2015-04-01

Full Text Available Purified bacteriocin produced from Brevibacillus borstelensis AG1 isolated from Marcha a local wine starter herbal cake, was used to enhance the shelf life of tomato paste. Preservative effect of purified bacteriocin was studied for nine days in tomato paste inoculated with food borne pathogens and was compared to commercial biopreservative – nisin and chemical preservative – sodium benzoate. The indicator strains i.e. Listeria monocytogenes MTCC839, Bacillus subtilis CRI and Clostridium perfringens MTCC1739 were used at the amount 8.16, 8.13 and 8.18 log CFU/ml. Viable cells were counted periodically and a consistent reduction in number of viable cells of each tested pathogen was observed. It was found antagonistic against L. monocytogenes MTCC839, B. subtilis CRI and C. perfringes MTCC1739 which are the most challengeable and food borne pathogens found in processed vegetables products. Purified bacteriocin was found active over a wide pH range i.e. 3.0 to 11.0 and was able to withstand temperature up to 100oC. It showed a better preservative potential by reducing pathogenic load of the tested strains (by 2.02, 2.05 and 2.02 log cycles (CFU/ml of L. monocytogenes MTCC839, B. subtilis CRI and C. perfringes MTCC1739, respectively in tomato paste as compared to control (without bacteriocin. This proves efficiency of bacteriocin produced by B. borstelensis AG1 as biopreservative to enhance the safety and shelf life of acidic foods.

Investigating organic molecules responsible of auxin-like activity of humic acid fraction extracted from vermicompost

Energy Technology Data Exchange (ETDEWEB)

Scaglia, Barbara, E-mail: barbara.scaglia@unimi.it [Gruppo Ricicla Labs – DiSAA, Università degli Studi di Milano, Via Celoria 2 (Italy); Nunes, Ramom Rachide; Rezende, Maria Olímpia Oliveira [Laboratório de Química Ambiental, Universidade de São Paulo, Instituto de Química de São Carlos, Avenida Trabalhador São Carlense, 400, São Carlos (Brazil); Tambone, Fulvia [Gruppo Ricicla Labs – DiSAA, Università degli Studi di Milano, Via Celoria 2 (Italy); Adani, Fabrizio, E-mail: fabrizio.adani@unimi.it [Gruppo Ricicla Labs – DiSAA, Università degli Studi di Milano, Via Celoria 2 (Italy)

2016-08-15

This work studied the auxin-like activity of humic acids (HA) obtained from vermicomposts produced using leather wastes plus cattle dung at different maturation stages (fresh, stable and mature). Bioassays were performed by testing HA concentrations in the range of 100–6000 mg carbon L{sup −1}. {sup 13}C CPMAS-NMR and GC–MS instrumental methods were used to assess the effect of biological processes and starting organic mixtures on HA composition. Not all HAs showed IAA-like activity and in general, IAA-like activity increased with the length of the vermicomposting process. The presence of leather wastes was not necessary to produce the auxin-like activity of HA, since HA extracted from a mix of cattle manure and sawdust, where no leather waste was added, showed IAA-like activity as well. CPMAS {sup 13}CNMR revealed that HAs were similar independently of the mix used and that the humification process involved the increasing concentration of pre-existing alkali soluble fractions in the biomass. GC/MS allowed the identification of the molecules involved in IAA-like effects: carboxylic acids and amino acids. The concentration of active molecules, rather than their simple presence in HA, determined the bio-stimulating effect, and a good linear regression between auxin-like activity and active stimulating molecules concentration was found (R{sup 2} = − 0.85; p < 0.01, n = 6). - Highlights: • Vermicomposting converts waste into organic fertilizer. • Vermicomposts can have biostimulating effect for the presence of hormone-like molecules. • Auxine-like activity was associated to the vermicompost humic acid fraction (HA). • HA carboxylic acids and amino acids, were reported to act as auxin-like molecules. • A linear regression was found between molecules and auxin-like activity.

Methods for purifying carbon materials

Science.gov (United States)

Dailly, Anne [Pasadena, CA; Ahn, Channing [Pasadena, CA; Yazami, Rachid [Los Angeles, CA; Fultz, Brent T [Pasadena, CA

2009-05-26

Methods of purifying samples are provided that are capable of removing carbonaceous and noncarbonaceous impurities from a sample containing a carbon material having a selected structure. Purification methods are provided for removing residual metal catalyst particles enclosed in multilayer carbonaceous impurities in samples generate by catalytic synthesis methods. Purification methods are provided wherein carbonaceous impurities in a sample are at least partially exfoliated, thereby facilitating subsequent removal of carbonaceous and noncarbonaceous impurities from the sample. Methods of purifying carbon nanotube-containing samples are provided wherein an intercalant is added to the sample and subsequently reacted with an exfoliation initiator to achieve exfoliation of carbonaceous impurities.

Trypsin from unicorn leatherjacket (Aluterus monoceros) pyloric caeca: purification and its use for preparation of fish protein hydrolysate with antioxidative activity.

Science.gov (United States)

Zamani, Abbas; Benjakul, Soottawat

2016-02-01

Fish proteases, especially trypsin, could be used to prepare fish protein hydrolysates with antioxidative activities. In this study, trypsin from the pyloric caeca of unicorn leatherjacket was purified by ammonium sulfate precipitation and soybean trypsin inhibitor (SBTI)-Sepharose 4B affinity chromatography. Hydrolysate from Indian mackerel protein isolate with different degrees of hydrolysis (20, 30 and 40% DH) was prepared using the purified trypsin, and antioxidative activities (1,1-diphenyl-2-picrylhydrazyl and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical-scavenging activities, ferric-reducing antioxidant power and ferrous-chelating activity) of the hydrolysate were determined. Trypsin was purified 26.43-fold with a yield of 13.43%. The purified trypsin had a molecular weight (MW) of 23.5 kDa and optimal activity at pH 8.0 and 55 °C. It displayed high stability in the pH range of 6.0-11.0 and was thermally stable up to 50 °C. Both SBTI (0.05 mmol L(-1)) and N-p-tosyl-L-lysine-chloromethylketone (5 mmol L(-1)) completely inhibited trypsin activity. Antioxidative activities of the hydrolysate from Indian mackerel protein isolate increased with increasing DH up to 40% (P unicorn leatherjacket pyloric caeca was identified as trypsin based on its ability to hydrolyze a specific synthetic substrate and the response to specific trypsin inhibitors. The purified trypsin could hydrolyze Indian mackerel protein isolate, and the resulting hydrolysate exhibited antioxidative activity depending on its DH. © 2015 Society of Chemical Industry.

Confirmation of antibodies against L-tryptophan-like epitope in ...

African Journals Online (AJOL)

Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic. ... number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions. Key words: Tryptophan ...

Complete amino acid sequences of the ribosomal proteins L25, L29 and L31 from the archaebacterium Halobacterium marismortui.

Science.gov (United States)

Hatakeyama, T; Kimura, M

1988-03-15

Ribosomal proteins were extracted from 50S ribosomal subunits of the archaebacterium Halobacterium marismortui by decreasing the concentration of Mg2+ and K+, and the proteins were separated and purified by ion-exchange column chromatography on DEAE-cellulose. Ten proteins were purified to homogeneity and three of these proteins were subjected to sequence analysis. The complete amino acid sequences of the ribosomal proteins L25, L29 and L31 were established by analyses of the peptides obtained by enzymatic digestion with trypsin, Staphylococcus aureus protease, chymotrypsin and lysylendopeptidase. Proteins L25, L29 and L31 consist of 84, 115 and 95 amino acid residues with the molecular masses of 9472 Da, 12293 Da and 10418 Da respectively. A comparison of their sequences with those of other large-ribosomal-subunit proteins from other organisms revealed that protein L25 from H. marismortui is homologous to protein L23 from Escherichia coli (34.6%), Bacillus stearothermophilus (41.8%), and tobacco chloroplasts (16.3%) as well as to protein L25 from yeast (38.0%). Proteins L29 and L31 do not appear to be homologous to any other ribosomal proteins whose structures are so far known.

Antigenic activity of concentrated purified cultural rabies vaciine inactivated by gamma-rays

Energy Technology Data Exchange (ETDEWEB)

Morogova, V M; Krutilina, D.V.; Latypova, R G; Dulina, A V; Nigamov, F N; Pogrebnyak, E M [Ufimskij Inst. Vaktsin i Syvorotok; Sanitarno-Ehpidemiologicheskaya Stantsiya, Ufimskaya Gorodskaya [USSR

1978-12-01

The ability to stimulate the production of virus-neutralizing antibodies and the reactivity of concentrated, purified and gamma-inactivated cultural antirabic vaccine from the Vnukovo-32 strain (35-38th passages) were studied in experiments with humans and animals. After two intramuscular immunizations (2 ml) at 21- or 23-day intervals, this preparation of the vaccine yielded antibody titers (both in humans and in animals) not lower than those obtained after a full course of immunization with the cultural or cerebral antirabic vaccine.

DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

Science.gov (United States)

Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

2018-01-01

Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

Study of hot corrosion of flakes of non purified graphite and of purified graphite

International Nuclear Information System (INIS)

Boule, Michel

1967-01-01

The author reports the study of hot corrosion of the Ticonderoga graphite. He reports the study of the defects of graphite flakes (structure defects due to impurities), the dosing of these impurities, and then their removal by purification. Flakes have then been oxidised by means of a specially designed apparatus. Based on photographs taken by optical and electronic microscopy, the author compares the oxidation features obtained in dry air and in humid air, between purified and non purified flakes. He also reports the study of the evolution of oxidation with respect to the initial rate of impurities, and the study of the evolution of oxidation features in humid air during oxidation. All these comparisons are made while taking the oxidation rate into account [fr

Isolation of a urinary digitalis-like factor indistinguishable from digoxin

Energy Technology Data Exchange (ETDEWEB)

Goto, A.; Ishiguro, T.; Yamada, K.; Ishii, M.; Yoshioka, M.; Eguchi, C.; Shimora, M.; Sugimoto, T. (Univ. of Tokyo (Japan))

1990-12-31

A digitalis-like factor has been purified to apparent homogeneity from human urine based on the inhibitory effect on ({sup 3}H) ouabain binding to intact human erythrocytes. The purification scheme involved large scale adsorption followed by preparative, semipreparative and analytical high-performance liquid chromatography. The purified material showed a prominent digoxin-like immunoreactivity. The behaviour of the isolated substance was identical to that of authentic digoxin in three high-performance liquid chromatography and three thin-layer chromatography systems. Moreover, fast atom bombardment mass spectrum and proton nuclear magnetic resonance spectrum suggested that the purified material may be indistinguishable from digoxin.

Light aging of reactive fuels purified by various methods

Energy Technology Data Exchange (ETDEWEB)

Khodzhaeva, M G; Burtyshev, N Ya; Molodozhenyuk, T B; Ryabovda, N D

1976-01-01

A study of the effect of uv-radiation on aging of Fergana fuel TS-1 has been extended to the uv-effect on alkali-purified fuels (e.g., Krasnovodsk, Omsk, and Orsk TS-1), on hydro-purified (Syzran T-8, Syzran T-7, and Novokuybyshev T-7) and on adsorption-purified Fergana TS-1. The PRK-4 lamp was employed. Aging criteria were formation of insoluble gums, soluble gums separable on silicagel, acidity, and optical density. Fuels purified in the same manner aged practically identically; after 6 months storage the greatest gum formation was seen in the fuels Orsk TS-1 and Syzran T-8. 3 references, 1 figure, 1 table.

α-Glucosidase inhibitory activities of fatty acids purified from the internal organ of sea cucumber Stichopus japonicas.

Science.gov (United States)

Nguyen, T H; Kim, S M

2015-04-01

α-Glucosidase inhibitory activities of the various solvent fractions (n-hexane, CHCl3 , EtOAc, BuOH, and water) of sea cucumber internal organ were investigated. 1,3-Dipalmitolein (1) and cis-9-octadecenoic acid (2) with potent α-glucosidase inhibitory activity were purified from the n-hexane fraction of sea cucumber internal organ. IC50 values of compounds 1 and 2 were 4.45 and 14.87 μM against Saccharomyces cerevisiae α-glucosidase. These compounds mildly inhibited rat-intestinal α-glucosidase. In addition, both compounds showed a mixed competitive inhibition against S. cerevisiae α-glucosidase and were very stable at pH 2 up to 60 min. The KI values of compounds 1 and 2 were 0.48 and 1.24 μM, respectively. Therefore, the internal organ of sea cucumber might be a potential new source of α-glucosidase inhibitors suitably used for prevention of obesity and diabetes mellitus. © 2015 Institute of Food Technologists®

Differentiation of purified malignant B cells induced by PMA or by activated normal T cells

NARCIS (Netherlands)

van Kooten, C.; Rensink, I.; Aarden, L.; van Oers, R.

1993-01-01

We studied the in vitro differentiation (immunoglobulin production) of purified malignant B cells of 21 patients with different B-cell malignancies, including chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HCL) and non-Hodgkin lymphoma (NHL). Direct

L-tyrosine and L-DOPA as hormone-like regulators of melanocytes functions

Science.gov (United States)

Slominski, Andrzej; Zmijewski, Michal; Pawelek, John

2011-01-01

Summary Evidence reveals that L-tyrosine and L-DOPA, besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as it would regulate the melanocyte functions through activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate older theory proposing that receptors for amino-acid derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates. PMID:21834848

Dietary n-3 PUFA affect TcR-mediated activation of purified murine T cells and accessory cell function in co-cultures

Science.gov (United States)

CHAPKIN, R S; ARRINGTON, J L; APANASOVICH, T V; CARROLL, R J; MCMURRAY, D N

2002-01-01

Diets enriched in n-3 polyunsaturated fatty acids (PUFA) suppress several functions of murine splenic T cells by acting directly on the T cells and/or indirectly on accessory cells. In this study, the relative contribution of highly purified populations of the two cell types to the dietary suppression of T cell function was examined. Mice were fed diets containing different levels of n-3 PUFA; safflower oil (SAF; control containing no n-3 PUFA), fish oil (FO) at 2% and 4%, or 1% purified docosahexaenoic acid (DHA) for 2 weeks. Purified (>90%) T cells were obtained from the spleen, and accessory cells (>95% adherent, esterase-positive) were obtained by peritoneal lavage. Purified T cells or accessory cells from each diet group were co-cultured with the alternative cell type from every other diet group, yielding a total of 16 different co-culture combinations. The T cells were stimulated with either concanavalin A (ConA) or antibodies to the T cell receptor (TcR)/CD3 complex and the costimulatory molecule CD28 (αCD3/αCD28), and proliferation was measured after four days. Suppression of T cell proliferation in the co-cultures was dependent upon the dose of dietary n-3 PUFA fed to mice from which the T cells were derived, irrespective of the dietary treatment of accessory cell donors. The greatest dietary effect was seen in mice consuming the DHA diet (P = 0·034 in the anova; P = 0·0053 in the Trend Test), and was observed with direct stimulation of the T cell receptor and CD28 costimulatory ligand, but not with ConA. A significant dietary effect was also contributed accessory cells (P = 0·033 in the Trend Test). We conclude that dietary n-3 PUFA affect TcR-mediated by T cell activation by both direct and indirect (accessory cell) mechanisms. PMID:12296847

DMPD: Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17502339 Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage...May 14. (.png) (.svg) (.html) (.csml) Show Crosstalk among Jak-STAT, Toll-like receptor, and ITAM-dependent pathways inmacrophage...T, Toll-like receptor, and ITAM-dependent pathways inmacrophage activation. Authors Hu X, Chen J, Wang L, Iv

Measurement of carbon activity in sodium by Fe-Mn 20% alloy, and by strainless austenitic steel 304L and 316L

International Nuclear Information System (INIS)

Oberlin, C.; Saint Paul, P.; Baque, P.; Champeix, L.

1980-01-01

Precise knowledge of carbon activity in sodium used as coolant in fast breeder reactors, is essential for continuous survey of carburization-decarburization processes. Carbon activity can be periodically surveyed by measuring the carbon concentration or by hot trap like metal alloy strip placed in sodium loop. In fact, in equilibrium, activity of carbon in sodium is equal to the activity in metal alloy. Thus if the relation between concentration of carbon and it activity in the alloy is known, it is possible to estimate the activity of carbon in sodium. Materials to be used should have high solubility in carbon at the needed temperature. They should quickly attain equilibrium with sodium and they should not contain impurities that can affect the results. Materials chosen according to these criteria were Fe-Mn 20%, stainless austenitic steel AISI 304L and 316L

Pyroelectric energy harvesting using Olsen cycles in purified and porous poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] thin films

International Nuclear Information System (INIS)

Navid, Ashcon; Pilon, Laurent

2011-01-01

This paper is concerned with the direct conversion of heat into electricity using pyroelectric materials. The Olsen (or Ericsson) cycle was experimentally performed on three different types of 60/40 poly(vinylidene fluoride-trifluoroethylene) [P(VDF-TrFE)] copolymer samples, namely commercial, purified, and porous films. This cycle consists of two isoelectric field and two isothermal processes. The commercial and purified films were about 50 µm thick and produced a maximum energy density of 521 J l −1 and 426 J l −1 per cycle, respectively. This was achieved by successively dipping the films in cold and hot silicone oil baths at 25 and 110 °C under low and high applied electric fields of about 200 and 500 kV cm −1 , respectively. The 11 µm thick porous films achieved a maximum energy density of 188 J l −1 per cycle between 25 and 100 °C and electric field between 200 and 400 kV cm −1 . The performance of the purified and porous films suffered from their lower electrical resistivity and electric breakdown compared with commercial thin films. However, the energy densities of all 60/40 P(VDF-TrFE) films considered matched or exceeded those reported recently for 0.9Pb(Mg 1/3 Nb 2/3 )O 3 –0.10PbTiO 3 (0.9PMN–0.1PT) (186 J l −1 ) and Pb(Zn 1/3 Nb 2/3 ) 0.955 Ti 0.045 O 3 (243 J l −1 ) bulk ceramics. Furthermore, the results are discussed in light of recently proposed figures of merit for energy harvesting applications

Isoforms of purified methyltransferase from human blood platelets ...

African Journals Online (AJOL)

... purification from normal human blood platelets have not been investigated, hence, the aim of this study was to purify, characterise the enzyme from human blood platelets and determine its possible role in phospholipid transmethylation. The plasma membranes were purified by velocity and sucrose gradient centrifugation ...

Phytochemical Analysis and Biological Activities of Cola nitida Bark

Directory of Open Access Journals (Sweden)

Durand Dah-Nouvlessounon

2015-01-01

Full Text Available Kola nut is chewed in many West African cultures and is used ceremonially. The aim of this study is to investigate some biological effects of Cola nitida’s bark after phytochemical screening. The bark was collected, dried, and then powdered for the phytochemical screening and extractions. Ethanol and ethyl acetate extracts of C. nitida were used in this study. The antibacterial activity was tested on ten reference strains and 28 meat isolated Staphylococcus strains by disc diffusion method. The antifungal activity of three fungal strains was determined on the Potato-Dextrose Agar medium mixed with the appropriate extract. The antioxidant activity was determined by DPPH and ABTS methods. Our data revealed the presence of various potent phytochemicals. For the reference and meat isolated strains, the inhibitory diameter zone was from 17.5±0.7 mm (C. albicans to 9.5±0.7 mm (P. vulgaris. The MIC ranged from 0.312 mg/mL to 5.000 mg/mL and the MBC from 0.625 mg/mL to >20 mg/mL. The highest antifungal activity was observed with F. verticillioides and the lowest one with P. citrinum. The two extracts have an excellent reducing free radical activity. The killing effect of A. salina larvae was perceptible at 1.04 mg/mL. The purified extracts of Cola nitida’s bark can be used to hold meat products and also like phytomedicine.

Identification of a trypsin-like site associated with acetylcholinesterase by affinity labelling with (/sup 3/H)diisopropyl fluorophosphate

Energy Technology Data Exchange (ETDEWEB)

Small, D.H.; Chubb, I.W.

1988-07-01

In addition to its ability to hydrolyze acetylcholine, purified eel acetylcholinesterase possesses a trypsin-like endopeptidase activity. The tryptic activity is associated with a serine residue at a site that is distinct from the esteratic site. To label both the esteratic and tryptic sites, the enzyme was incubated with the serine hydrolase inhibitor (/sup 3/H)diisopropyl fluorophosphate. This compound labelled the protein in a biphasic manner, with both slow and rapid labelling kinetics. The time course of the rapid phase was similar to the time course of inactivation of the esteratic activity. The time course of the slow phase was similar to the time course of inactivation of the tryptic activity. Labelling of the nonesteratic site was inhibited by the trypsin inhibitor N alpha-p-tosyl-L-lysine chloromethyl ketone. The total number of sites labelled by (/sup 3/H)diisopropyl fluorophosphate on eel acetylcholinesterase was 2.6 mol/280,000 g protein, whereas the number of tryptic sites was less (0.52 mol/280,000 g). The results suggest that a subpopulation of acetylcholinesterase molecules may possess tryptic activity. Extensive chromatography of the purified enzyme by ion-exchange and gel filtration failed to separate the labelled tryptic component from acetylcholinesterase. On sodium dodecyl sulfate-polyacrylamide gels, the labelled tryptic component comigrated with a polypeptide of 50,000 molecular weight, which is a major proteolytic digestion product derived from the intact acetylcholinesterase monomer. Because of its localization in many noncholinergic peptide-containing cells, acetylcholinesterase could act as a neuropeptide processing enzyme in these cells.

The effects of doxycycline and micronized purified flavonoid fraction on human vein wall remodeling are not hypoxia-inducible factor pathway-dependent.

Science.gov (United States)

Lim, Chung Sim; Kiriakidis, Serafim; Paleolog, Ewa M; Davies, Alun H

2012-10-01

Doxycycline and micronized purified flavonoid fraction (MPFF) modulate vein wall remodeling that may be associated with hypoxia in varicose veins (VVs), vein graft stenosis, and deep venous thrombosis. We recently reported that in vitro exposure of non-VV (NVVs) and VVs to hypoxic conditions activates the hypoxia-inducible factor (HIF) pathway. This study investigated the in vitro effects of doxycycline and MPFF on the HIF pathway in hypoxic NVVs and VVs. Six NVVs and six VVs obtained from surgery were used to prepare vein organ cultures, which were exposed to hypoxia (1% O(2)), with and without MPFF (10(-5) mol/L) or doxycycline (5 μg/mL) for 16 hours. The veins were analyzed for HIF-1α, HIF-2α, and their target gene expression, with real-time polymerase chain reaction and Western blot. The differences between gene expressions were tested with one-way analysis of variance with repeated measures, followed by the Dunnett test for multiple comparisons. P factor, B-cell lymphoma 2/adenovirus E1B 19-kDa protein-interacting protein 3, prolyl hydroxylase domain-2, and prolyl hydroxylase domain-3, was not significantly altered in NVVs and VVs exposed to hypoxia and treated with doxycycline or MPFF compared with those untreated. Doxycycline and MPFF at a concentration corresponding to a therapeutic dose do not alter the activation of the HIF pathway in NVV and VV organ cultures exposed to hypoxia. Our findings suggest vein wall remodeling actions in NVVs and VVs are likely not HIF-dependent. Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch.

Science.gov (United States)

Unban, Kridsada; Kanpiengjai, Apinun; Takata, Goro; Uechi, Keiko; Lee, Wen-Chien; Khanongnuch, Chartchai

2017-09-01

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112 kDa with optimal pH value and temperature measured of 7.0 and 40 °C, respectively. It was stable at a pH range of 6.0-7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba 2+ , Al 3+ , and Co 2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2 g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10 g/L cassava starch as the sole carbon source when cultivated at 37 °C for 48 h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150 g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

Reproducible in vitro regeneration system for purifying sugarcane ...

African Journals Online (AJOL)

This procedure may be considered as one of the best ever published report on regeneration from in vitro grown plants to purify clones without subjecting the plants to field conditions and harvesting the mature cane. This technique was used to purify transgenic sugarcane plants carrying Bacillus thuringiensis gene.

YodL and YisK Possess Shape-Modifying Activities That Are Suppressed by Mutations in Bacillus subtilis mreB and mbl.

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Duan, Yi; Sperber, Anthony M; Herman, Jennifer K

2016-08-01

Many bacteria utilize actin-like proteins to direct peptidoglycan (PG) synthesis. MreB and MreB-like proteins are thought to act as scaffolds, guiding the localization and activity of key PG-synthesizing proteins during cell elongation. Despite their critical role in viability and cell shape maintenance, very little is known about how the activity of MreB family proteins is regulated. Using a Bacillus subtilis misexpression screen, we identified two genes, yodL and yisK, that when misexpressed lead to loss of cell width control and cell lysis. Expression analysis suggested that yodL and yisK are previously uncharacterized Spo0A-regulated genes, and consistent with these observations, a ΔyodL ΔyisK mutant exhibited reduced sporulation efficiency. Suppressors resistant to YodL's killing activity occurred primarily in mreB mutants and resulted in amino acid substitutions at the interface between MreB and the highly conserved morphogenic protein RodZ, whereas suppressors resistant to YisK occurred primarily in mbl mutants and mapped to Mbl's predicted ATP-binding pocket. YodL's shape-altering activity appears to require MreB, as a ΔmreB mutant was resistant to the effects of YodL but not YisK. Similarly, YisK appears to require Mbl, as a Δmbl mutant was resistant to the cell-widening effects of YisK but not of YodL. Collectively, our results suggest that YodL and YisK likely modulate MreB and Mbl activity, possibly during the early stages of sporulation. The peptidoglycan (PG) component of the cell envelope confers structural rigidity to bacteria and protects them from osmotic pressure. MreB and MreB-like proteins are thought to act as scaffolds for PG synthesis and are essential in bacteria exhibiting nonpolar growth. Despite the critical role of MreB-like proteins, we lack mechanistic insight into how their activities are regulated. Here, we describe the discovery of two B. subtilis proteins, YodL and YisK, which modulate MreB and Mbl activities. Our data suggest

Purifying hydrocarbon oils

Energy Technology Data Exchange (ETDEWEB)

Rostin, H

1938-08-11

A process is described for continuously purifying hydrocarbon oils consisting in conducting the vapors of the same at a temperature of 300 to 400/sup 0/C over the oelitic ore minette together with reducing gases in presence of steam the proportion of the reducing gases and steam being such that the sulfur of the hydrocarbons escapes from the reaction chamber in the form of sulfuretted hydrogen without permanent sulfide of iron being formed.

Niemann-Pick C1 like 1 gene expression is down-regulated by LXR activators in the intestine

International Nuclear Information System (INIS)

Duval, Caroline; Touche, Veronique; Tailleux, Anne; Fruchart, Jean-Charles; Fievet, Catherine; Clavey, Veronique; Staels, Bart; Lestavel, Sophie

2006-01-01

Niemann-Pick C1 like 1 (NPC1L1) is a protein critical for intestinal cholesterol absorption. The nuclear receptors peroxisome proliferator-activated receptor alpha (PPARα) and liver X receptors (LXRα and LXRβ) are major regulators of cholesterol homeostasis and their activation results in a reduced absorption of intestinal cholesterol. The goal of this study was to define the role of PPARα and LXR nuclear receptors in the regulation of NPC1L1 gene expression. We show that LXR activators down-regulate NPC1L1 mRNA levels in the human enterocyte cell line Caco-2/TC7, whereas PPARα ligands have no effect. Furthermore, NPC1L1 mRNA levels are decreased in vivo, in duodenum of mice treated with the LXR agonist T0901317. In conclusion, the present study identifies NPC1L1 as a novel LXR target gene further supporting a crucial role of LXR in intestinal cholesterol homeostasis

Structural and functional comparisons and production of recombinant crustacean hyperglycemic hormone (CHH) and CHH-like peptides from the mud crab Scylla olivacea.

Science.gov (United States)

Chang, Chih-Chun; Tsai, Kuo-Wei; Hsiao, Nai-Wan; Chang, Cheng-Yen; Lin, Chih-Lung; Watson, R Douglas; Lee, Chi-Ying

2010-05-15

Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are presumably alternatively spliced gene products. In this study, Sco-CHH and Sco-CHH-L were isolated from the tissues using high performance liquid chromatography. Identity of the native peptides was confirmed using mass spectrometric (MS) analyses of purified materials and of trypsin-digested peptide fragments. Additionally, characterizations using circular dichroism (CD) spectrometry revealed that the 2 peptides have similar CD spectral profiles, showing they are composed mainly of alpha-helices, and are similarly thermo-stable with a melting temperature of 74-75 degrees C. Results of bioassays indicated that Sco-CHH exerted hyperglycemic and molt-inhibiting activity, whereas Sco-CHH-L did not. Further, recombinant Sco-CHH-Gly (rSco-CHH-Gly, a glycine extended Sco-CHH) and Sco-CHH-L (rSco-CHH-L) were produced using an Escherichia coli expression system, refolded, and purified. rSco-CHH-Gly was further alpha-amidated at the C-terminal end to produce rSco-CHH. MS analyses of enzyme-digested peptide fragments of rSco-CHH-Gly and rSco-CHH-L showed that the two peptides share a common disulfide bond pattern: C7-C43, C23-C39, and C26-C52. Circular dichroism analyses and hyperglycemic assay revealed that rSco-CHH and rSco-CHH-L resemble their native counterparts, in terms of CD spectral profiles, melting curve profiles, and biological activity. rSco-CHH-Gly has a lower alpha-helical content (32%) than rSco-CHH (47%), a structural deviation that may be responsible for the significant decrease in the biological activity of rSco-CHH-Gly. Finally, modeled structure of Sco-CHH and Sco-CHH-L indicated that they are similarly folded, each with an N-terminal tail region and 4 alpha-helices. Putative surface residues located in corresponding positions of Sco-CHH and Sco-CHH-L but with side chains of different properties

Antibodies induced by vaccination with purified chick embryo cell culture vaccine (PCECV) cross-neutralize non-classical bat lyssavirus strains.

Science.gov (United States)

Malerczyk, Claudius; Selhorst, Thomas; Tordo, Noël; Moore, Susan; Müller, Thomas

2009-08-27

Tissue-culture vaccines like purified chick embryo cell vaccine (PCECV) have been shown to provide protection against classical rabies virus (RABV) via pre-exposure or post-exposure prophylaxis. A cross-neutralization study was conducted using a panel of 100 human sera, to determine, to what extent after vaccination with PCECV protection exists against non-classical bat lyssavirus strains like European bat lyssavirus (EBLV) type 1 and 2 and Australian bat lyssavirus (ABLV). Virus neutralizing antibody (VNA) concentrations against the rabies virus variants CVS-11, ABLV, EBLV-1 and EBLV-2 were determined by using a modified rapid fluorescent focus inhibition test. For ABLV and EBLV-2, the comparison to CVS-11 revealed almost identical results (100% adequate VNA concentrations >or=0.5 IU/mL; correlation coefficient r(2)=0.69 and 0.77, respectively), while for EBLV-1 more scattering was observed (97% adequate VNA concentrations; r(2)=0.50). In conclusion, vaccination with PCECV produces adequate VNA concentrations against classical RABV as well as non-classical lyssavirus strains ABLV, EBLV-1, and EBLV-2.

Characterization of purified and Xerogel immobilized Novel Lignin Peroxidase produced from Trametes versicolor IBL-04 using solid state medium of Corncobs

Directory of Open Access Journals (Sweden)

Asgher Muhammad

2012-08-01

Full Text Available Abstract Background Cost-effective production of industrially important enzymes is a key for their successful exploitation on industrial scale. Keeping in view the extensive industrial applications of lignin peroxidase (LiP, this study was performed to purify and characterize the LiP from an indigenous strain of Trametes versicolor IBL-04. Xerogel matrix enzyme immobilization technique was applied to improve the kinetic and thermo-stability characteristics of LiP to fulfil the requirements of the modern enzyme consumer sector of biotechnology. Results A novel LiP was isolated from an indigenous T. versicolor IBL-04 strain. T. versicolor IBL-04 was cultured in solid state fermentation (SSF medium of corn cobs and maximum LiP activity of 592 ± 6 U/mL was recorded after five days of incubation under optimum culture conditions. The crude LiP was 3.3-fold purified with specific activity of 553 U/mg after passing through the DEAE-cellulose and Sephadex-G-100 chromatography columns. The purified LiP exhibited a relatively low molecular weight (30 kDa homogenous single band on native and SDS-PAGE. The LiP was immobilized by entrapping in xerogel matrix of trimethoxysilane (TMOS and proplytetramethoxysilane (PTMS and maximum immobilization efficiency of 88.6% was achieved. The free and immobilized LiPs were characterized and the results showed that the free and immobilized LiPs had optimum pH 6 and 5 while optimum temperatures were 60°C and 80°C, respectively. Immobilization was found to enhance the activity and thermo-stability potential of LiP significantly and immobilized LiP remained stable over broad pH and temperature range as compare to free enzyme. Kinetic constants Km and Vmax were 70 and 56 μM and 588 and 417 U/mg for the free and immobilized LiPs, respectively. Activity of this novel extra thermo-stable LiP was stimulated to variable extents by Cu2+, Mn2+ and Fe2+ whereas, Cystein, EDTA and Ag+ showed inhibitory effects

Linagliptin potentiates the effect of l-dopa on the behavioural, biochemical and immunohistochemical changes in experimentally-induced Parkinsonism: Role of toll-like receptor 4, TGF-β1, NF-κB and glucagon-like peptide 1.

Science.gov (United States)

Kabel, Ahmed M; Omar, Mohamed S; Alhadhrami, A; Alharthi, Salman S; Alrobaian, Majed M

2018-05-01

Our aim was to assess the effect of different doses of linagliptin with or without l-dopa/Carbidopa on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced parkinsonism in mice. Eighty Balb/c mice were divided into 8 equal groups: Control; MPTP; MPTP + l-dopa/Carbidopa; MPTP + linagliptin 3 mg/kg/day; MPTP + linagliptin 10 mg/kg/day; MPTP + Carboxymethyl cellulose; MPTP + l-dopa/Carbidopa + linagliptin 3 mg/kg/day and MPTP + l-dopa/Carbidopa + linagliptin 10 mg/kg/day. Striatal dopamine, tumor necrosis factor alpha (TNF-α), interleukin 10 (IL-10), transforming growth factor beta 1 (TGF-β1), toll-like receptor 4 (TLR4), antioxidant enzymes, adenosine triphosphate (ATP), glucagon-like peptide-1 (GLP-1), receptors of advanced glycation end products (RAGE), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heme oxygenase-1 (HO-1), mitochondrial complex I activity, catalepsy and total swim scores were measured. Also, the substantia nigra was subjected to immunohistochemical examination. The combination of l-dopa/Carbidopa and linagliptin in a dose-dependent manner resulted in significant improvement of the behavioural changes, striatal dopamine, antioxidant parameters, Nrf2/HO-1 content, GLP-1, ATP and mitochondrial complex I activity with significant decrease in striatal RAGE, TGF-β1, TNF-α, IL-10, TLR4 and alleviated the immunohistochemical changes better than the groups that received either l-dopa/Carbidopa or linagliptin alone. The combination of l-dopa/Carbidopa and linagliptin might represent a promising therapeutic modality for management of parkinsonism. Copyright © 2018 Elsevier Inc. All rights reserved.

Purification and antibacterial activities of an L-amino acid oxidase from king cobra (Ophiophagus hannah venom

Directory of Open Access Journals (Sweden)

CS Phua

2012-01-01

Full Text Available Some constituents of snake venom have been found to display a variety of biological activities. The antibacterial property of snake venom, in particular, has gathered increasing scientific interest due to antibiotic resistance. In the present study, king cobra venom was screened against three strains of Staphylococcus aureus [including methicillin-resistant Staphylococcus aureus (MRSA], three other species of gram-positive bacteria and six gram-negative bacteria. King cobra venom was active against all the 12 bacteria tested, and was most effective against Staphylococcus spp. (S. aureus and S. epidermidis. Subsequently, an antibacterial protein from king cobra venom was purified by gel filtration, anion exchange and heparin chromatography. Mass spectrometry analysis confirmed that the protein was king cobra L-amino acid oxidase (Oh-LAAO. SDS-PAGE showed that the protein has an estimated molecular weight of 68 kDa and 70 kDa under reducing and non-reducing conditions, respectively. The minimum inhibitory concentrations (MIC of Oh-LAAO for all the 12 bacteria were obtained using radial diffusion assay method. Oh-LAAO had the lowest MIC value of 7.5 µg/mL against S. aureus ATCC 25923 and ATCC 29213, MRSA ATCC 43300, and S. epidermidis ATCC 12228. Therefore, the LAAO enzyme from king cobra venom may be useful as an antimicrobial agent.

Alkaline pH activates the transport activity of GLUT1 in L929 fibroblast cells.

Science.gov (United States)

Gunnink, Stephen M; Kerk, Samuel A; Kuiper, Benjamin D; Alabi, Ola D; Kuipers, David P; Praamsma, Riemer C; Wrobel, Kathryn E; Louters, Larry L

2014-04-01

The widely expressed mammalian glucose transporter, GLUT1, can be acutely activated in L929 fibroblast cells by a variety of conditions, including glucose deprivation, or treatment with various respiration inhibitors. Known thiol reactive compounds including phenylarsine oxide and nitroxyl are the fastest acting stimulators of glucose uptake, implicating cysteine biochemistry as critical to the acute activation of GLUT1. In this study, we report that in L929 cells glucose uptake increases 6-fold as the pH of the uptake solution is increased from 6 to 9 with the half-maximal activation at pH 7.5; consistent with the pKa of cysteine residues. This pH effect is essentially blocked by the pretreatment of the cells with either iodoacetamide or cinnamaldehyde, compounds that form covalent adducts with reduced cysteine residues. In addition, the activation by alkaline pH is not additive at pH 8 with known thiol reactive activators such as phenylarsine oxide or hydroxylamine. Kinetic analysis in L929 cells at pH 7 and 8 indicate that alkaline conditions both increases the Vmax and decreases the Km of transport. This is consistent with the observation that pH activation is additive to methylene blue, which activates uptake by increasing the Vmax, as well as to berberine, which activates uptake by decreasing the Km. This suggests that cysteine biochemistry is utilized in both methylene blue and berberine activation of glucose uptake. In contrast a pH increase from 7 to 8 in HCLE cells does not further activate glucose uptake. HCLE cells have a 25-fold higher basal glucose uptake rate than L929 cells and the lack of a pH effect suggests that the cysteine biochemistry has already occurred in HCLE cells. The data are consistent with pH having a complex mechanism of action, but one likely mediated by cysteine biochemistry. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Method and device for feeding purified water to a pressure vessel

International Nuclear Information System (INIS)

Hirato, Miharu.

1982-01-01

Purpose: To prevent thermal wear at the junction of feedwater pipes and purified water pipes, as well as maintain the function of the purified water feeding system by stopping the introduction of purified water to the heated water feeding system and introducing purified water to the recycling water system upon transient operation or start-up. Constitution: Since a feedwater heater does not function well during transient operation or upon start-up, the temperature of heated water flowing through the feedwater pipe is reduced to produce a temperature difference relative to the set temperature for the purified water feeding system. The temperature difference is detected by a temperature sensor and, when it arrives at a predetermined difference, an operation valve is switched to interrupt the feed of the purified water to the heated water feeding system and it is sent to a water recycling system. Then, the purified water is sent from the water recycling system by way of the discharge portion to the inside of a pressure vessel. Thus, since only the heated water flows to the junction between the cleaned water pipes and the heated water pipes, neither shocks are generated nor the performance of the purified water feeding system is impaired. (Moriyama, K.)

Role of Purified Anthocyanins in Improving Cardiometabolic Risk Factors in Chinese Men and Women with Prediabetes or Early Untreated Diabetes-A Randomized Controlled Trial.

Science.gov (United States)

Yang, Liping; Ling, Wenhua; Yang, Yan; Chen, Yuming; Tian, Zezhong; Du, Zhicheng; Chen, Jianying; Xie, Yuanling; Liu, Zhaomin; Yang, Lili

2017-10-10

Objective: In vitro and animal studies suggest that purified anthocyanins have favorable effects on metabolic profiles, but clinical trials have reported inconsistent findings. Furthermore, no study has been specifically conducted among individuals with prediabetes. The aim of this study was to investigate whether purified anthocyanins could improve cardiometabolic risk factors in Chinese adults with early untreated hyperglycemia. Research Design and Methods: This was a 12-week randomized, double-blind, placebo-controlled trial. A total of 160 participants aged 40-75 years with prediabetes or early untreated diabetes were randomly allocated to receive either purified anthocyanins (320 mg/day, n = 80) or placebo ( n = 80) of identical appearance. A three-hour oral glucose tolerance test (OGTT) was performed, and cardiometabolic biomarkers (glycated hemoglobin A1c (HbA1c), fasting and postprandial glucose, insulin, C-peptide, and lipids) were measured at baseline and at the end of the trial. Results: A total of 138 subjects completed the protocol. Compared with placebo, purified anthocyanins moderately reduced HbA1c (-0.14%, 95% CI: -0.23~-0.04%; p = 0.005), low-density lipoprotein-c (LDL-c) (-0.2 mmol/L, 95% CI: -0.38~-0.01, p = 0.04), apolipoprotein A-1 (apo A1) (0.09 g/L, 95% CI: 0.02~0.17; p = 0.02), and apolipoprotein B (apo B) (-0.07 g/L, 95% CI: -0.13~-0.01; p = 0.01) according to intention-to-treat analysis. Subgroup analyses suggested that purified anthocyanins were more effective at improving glycemic control, insulin sensitivity, and lipids among patients with elevated metabolic markers. Conclusions: The 12-week randomized controlled trials (RCT) in Chinese adults with prediabetes or early untreated diabetes indicated that purified anthocyanins favorably affected glycemic control and lipid profile. Future studies of a longer duration that explore the dose-response relationship among patients with cardiometabolic disorders are needed to confirm our findings.

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

Energy Technology Data Exchange (ETDEWEB)

Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri (Turkey); Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey); Mus Alparslan University Faculty of Sciences, Department of Moleculer Biology, 49250-Mus (Turkey); Özdemir, Hasan, E-mail: hozdemir@atauni.edu.tr [Ataturk University, Science Faculty, Department of Chemistry, 25240-Erzurum (Turkey)

2016-04-18

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC{sub 50} values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

Science.gov (United States)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-04-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC50 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Determining inhibition effects of some aromatic compounds on peroxidase enzyme purified from white and red cabbage

International Nuclear Information System (INIS)

Öztekin, Aykut; Almaz, Züleyha; Özdemir, Hasan

2016-01-01

Peroxidases (E.C.1.11.1.7) catalyze the one electron oxidation of wide range of substrates. They are used in synthesis reaction, removal of peroxide from industrial wastes, clinical biochemistry and immunoassays. In this study, the white cabbage (Brassica Oleracea var. capitata f. alba) and red cabbage (Brassica oleracea L. var. capitata f. rubra) peroxidase enzymes were purified for investigation of inhibitory effect of some aromatic compounds on these enzymes. IC_5_0 values and Ki constants were calculated for the molecules of 6-Amino nicotinic hydrazide, 6-Amino-5-bromo nicotinic hydrazide, 2-Amino-5-hydroxy benzohydrazide, 4-Amino-3-hydroxy benzohydrazide on purified enzymes and inhibition type of these molecules were determined. (This research was supported by Ataturk University. Project Number: BAP-2015/98).

Purified blueberry anthocyanins and blueberry juice alter development of obesity in mice fed an obesogenic high-fat diet.

Science.gov (United States)

Prior, Ronald L; E Wilkes, Samuel; R Rogers, Theodore; Khanal, Ramesh C; Wu, Xianli; Howard, Luke R

2010-04-14

Male C57BL/6J mice (25 days of age) were fed either a low-fat diet (10% kcal from fat) (LF) or a high-fat diet (45% kcal from fat) (HF45) for a period of 72 days. Blueberry juice or purified blueberry anthocyanins (0.2 or 1.0 mg/mL) in the drinking water were included in LF or HF45 treatments. Sucrose was added to the drinking water of one treatment to test if the sugars in blueberry juice would affect development of obesity. Total body weights (g) and body fat (%) were higher and body lean tissue (%) was lower in the HF45 fed mice compared to the LF fed mice after 72 days, but in mice fed HF45 diet plus blueberry juice or blueberry anthocyanins (0.2 mg/mL), body fat (%) was not different from those mice fed the LF diet. Anthocyanins (ACNs) decreased retroperitoneal and epididymal adipose tissue weights. Fasting serum glucose concentrations were higher in mice fed the HF45 diet. However, it was reduced to LF levels in mice fed the HF45 diet plus 0.2 mg of ACNs/mL in the drinking water, but not with blueberry juice. beta cell function (HOMA-BCF) score was lowered with HF45 feeding but returned to normal levels in mice fed the HF45 diet plus purified ACNs (0.2 mg/mL). Serum leptin was elevated in mice fed HF45 diet, and feeding either blueberry juice or purified ACNs (0.2 mg/mL) decreased serum leptin levels relative to HF45 control. Sucrose in drinking water, when consumption was restricted to the volume of juice consumed, produced lower serum leptin and insulin levels, leptin/fat, and retroperitoneal and total fat (% BW). Blueberry juice was not as effective as the low dose of anthocyanins in the drinking water in preventing obesity. Additional studies are needed to determine factors responsible for the differing responses of blueberry juice and whole blueberry in preventing the development of obesity.

Improved Chemotherapeutic Activity by Morus alba Fruits through Immune Response of Toll-Like Receptor 4

Directory of Open Access Journals (Sweden)

Bo Yoon Chang

2015-10-01

Full Text Available Morus alba L. fruits have long been used in traditional medicine by many cultures. Their medicinal attributes include cardiovascular, hepatoprotective, neuroprotective and immunomodulatory actions. However, their mechanism of macrophage activation and anti-cancer effects remain unclear. The present study investigated the molecular mechanisms of immune stimulation and improved chemotherapeutic effect of M. alba L. fruit extract (MFE. MFE stimulated the production of cytokines, nitric oxide (NO and tumor necrosis factor-α (TNF-α and tumoricidal properties of macrophages. MFE activated macrophages through the mitogen-activated protein kinase (MAPKinase and nuclear factor-κB (NF-κB signaling pathways downstream from toll-like receptor (TLR 4. MFE was shown to exhibit cytotoxicity of CT26 cells via the activated macrophages, even though MFE did not directly affect CT26 cells. In a xenograft mouse model, MFE significantly enhanced anti-cancer activity combined with 5-fluorouracil and markedly promoted splenocyte proliferation, natural killer (NK cell activity, cytotoxic T lymphocyte (CTL activity and IFN-γ production. Immunoglobulin G (IgG antibody levels were significantly increased. These results indicate the indirect anti-cancer activity of MFE through improved immune response mediated by TLR4 signaling. M. alba L. fruit extract might be a potential anti-tumor immunomodulatory candidate chemotherapy agent.

Improved Chemotherapeutic Activity by Morus alba Fruits through Immune Response of Toll-Like Receptor 4.

Science.gov (United States)

Chang, Bo Yoon; Kim, Seon Beom; Lee, Mi Kyeong; Park, Hyun; Kim, Sung Yeon

2015-10-13

Morus alba L. fruits have long been used in traditional medicine by many cultures. Their medicinal attributes include cardiovascular, hepatoprotective, neuroprotective and immunomodulatory actions. However, their mechanism of macrophage activation and anti-cancer effects remain unclear. The present study investigated the molecular mechanisms of immune stimulation and improved chemotherapeutic effect of M. alba L. fruit extract (MFE). MFE stimulated the production of cytokines, nitric oxide (NO) and tumor necrosis factor-α (TNF-α) and tumoricidal properties of macrophages. MFE activated macrophages through the mitogen-activated protein kinase (MAPKinase) and nuclear factor-κB (NF-κB) signaling pathways downstream from toll-like receptor (TLR) 4. MFE was shown to exhibit cytotoxicity of CT26 cells via the activated macrophages, even though MFE did not directly affect CT26 cells. In a xenograft mouse model, MFE significantly enhanced anti-cancer activity combined with 5-fluorouracil and markedly promoted splenocyte proliferation, natural killer (NK) cell activity, cytotoxic T lymphocyte (CTL) activity and IFN-γ production. Immunoglobulin G (IgG) antibody levels were significantly increased. These results indicate the indirect anti-cancer activity of MFE through improved immune response mediated by TLR4 signaling. M. alba L. fruit extract might be a potential anti-tumor immunomodulatory candidate chemotherapy agent.

Characterization of a purified nicotinic receptor from rat brain by using idiotypic and anti-idiotypic antibodies

International Nuclear Information System (INIS)

Abood, L.G.; Langone, J.J.; Bjercke, R.; Lu, X.; Banerjee, S.

1987-01-01

The availability of an anti-nicotine monoclonal antibody has made it possible to further establish the nature of the nicotine recognition proteins purified from rat brain by affinity chromatography and to provide a highly sensitive assay for determining [ 3 H]nicotine binding to the purified material. An enantiomeric analogue of nicotine. (-)-6-hydroxymethylnicotine, was used to prepare the affinity column. In addition, with the use of an anti-idiotypic monoclonal antibody, it was confirmed that the recognition site for nicotine resides on a protein complex composed of two components with molecular masses of 62 and 57 kDa. It was also demonstrated that the same two proteins could be purified by immunoaffinity chromatography with the use of an anti-idiotypic monoclonal antibody. With the use of the anti-nicotine antibody to measure [ 3 H]nicotine binding, the purified material was shown to bind 250 pmol/mg of protein. By utilizing a procedure in which the purified receptor protein was conjugated to membranes by disulfide bonds, a binding activity of 80 pmol/mg was obtained. With the availability of sterospecific monoclonal antibodies to (-)-nicotine as well as monoclonal anti-idiotypic antibodies derived when the anti-nicotine antibodies were used as immunogens, additional procedures became available for the further characterization of the purified nicotine receptor and examining its (-)-[ 3 H]nicotine-binding characteristics

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

Energy Technology Data Exchange (ETDEWEB)

Lorenzato, Annalisa; Biolatti, Marta [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Delogu, Giuseppe [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Capobianco, Giampiero [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Farace, Cristiano [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco [Department of Surgical, Microsurgical and Medical Sciences, University of Sassari, Sassari (Italy); Madeddu, Roberto [Department of Biomedical Sciences-Histology, University of Sassari, Sassari (Italy); National Institute of Biostructures and Biosystems, Rome (Italy); Olivero, Martina [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy); Di Renzo, Maria Flavia, E-mail: mariaflavia.direnzo@unito.it [Department of Oncology, University of Torino School of Medicine, Torino (Italy); Institute for Cancer Research at Candiolo, Candiolo, Torino (Italy)

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells

International Nuclear Information System (INIS)

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-01-01

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. - highlights: • CSE1L is a key player in nucleocytoplasmic traffic by forming complex with Ran. • AKT phosphorylates RanBP3 that regulates the nucleocytoplasmic gradient of Ran. • The activated oncogenic AKT drives the nuclear accumulation of CSE1L. • CSE1L in the nucleus up-regulates genes conveying pro-oncogenic signals. • CSE1L might contribute to tumor progression driven by the activated oncogenic AKT

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

Science.gov (United States)

Lorenzato, Annalisa; Biolatti, Marta; Delogu, Giuseppe; Capobianco, Giampiero; Farace, Cristiano; Dessole, Salvatore; Cossu, Antonio; Tanda, Francesco; Madeddu, Roberto; Olivero, Martina; Di Renzo, Maria Flavia

2013-10-15

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals. © 2013 Elsevier Inc. All rights reserved.

The purified ATPase from chromaffin granule membranes is an anion-dependent proton pump.

Science.gov (United States)

Moriyama, Y; Nelson, N

1987-07-05

The proton-ATPase of chromaffin granules was purified so as to maintain its proton-pumping activity when reconstituted into phospholipid vesicles. The purification procedure involved solubilization with polyoxyethylene 9 lauryl ether, hydroxylapatite column, precipitation by ammonium sulfate, and glycerol gradient centrifugation. The protease inhibitor mixture used in previous studies inhibited the proton-pumping activity of the enzyme; therefore, the protein was stabilized by pepstatin A and leupeptin. The enzyme was purified at least 50-fold with respect to both ATPase and proton-pumping activity. The ATP-dependent proton uptake activity of the reconstituted enzyme was absolutely dependent on the presence of Cl- or Br- outside the vesicles, whereas sulfate, acetate, formate, nitrate, and thiocyanate were inhibitory. Sulfate inhibition seems to be due to competition with Cl- on the anion-binding site outside the vesicles, whereas nitrate and thiocyanate inhibited only from the internal side. As with the inhibition by N-ethylmaleimide, the proton-pumping activity was much more sensitive to nitrate than the ATPase activity. About 20 mM nitrate were sufficient for 90% inhibition of the proton-pumping activity while 100 mM inhibited only 50% of the ATPase activity both in situ and in the reconstituted enzyme. The possible regulatory effect of anions on the ATP-dependent proton uptake in secretory granules is discussed.

Insights into signal transduction by a hybrid FixL: Denaturation study of on and off states of a multi-domain oxygen sensor.

Science.gov (United States)

Guimarães, Wellinson G; Gondim, Ana C S; Costa, Pedro Mikael da Silva; Gilles-Gonzalez, Marie-Alda; Lopes, Luiz G F; Carepo, Marta S P; Sousa, Eduardo H S

2017-07-01

FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(Fe III ) and inactive cyanomet-FixL (Fe III -CN - ) form was monitored by UV-visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV-visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔG H2O were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8kJmol -1 ). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2kJmol -1 ). For the three-state mechanism exhibited by fluorescence, the ΔG H2O for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors. Copyright © 2017 Elsevier Inc. All rights reserved.

Biological Activities of Libidibia (Caesalpinia) ferrea var. parvifolia (Mart. ex Tul.) L. P. Queiroz Pod Preparations.

Science.gov (United States)

Freitas, A C C; Ximenes, N C A; Aguiar, J S; Nascimento, S C; Lins, T U L; Magalhães, L R; Coelho, L C B B; Carneiro-da-Cunha, M G; Gonçalves-Silva, T; Correia, M T S

2012-01-01

Libidibia ferrea has been used in folk medicine throughout Brazil, and this study evaluated the biological activities of crude extract (CE) as well as a partially purified fraction (F80) obtained from its pods. Results from the MTT assay revealed that only F80 inhibited NCI-H292 cell growth; however, neither CE nor F80 reduced HEp-2 cell growth or sarcoma 180 tumor weight with the in vivo assay. Acute oral toxicity of the extract and fraction was evaluated following the steps of Guideline 423, using female mice; LD(50) for both preparations was determined as 2,500 mg/kg body weight. CE and F80 promoted a reduction of the leukocyte number and nitrite level in inflammatory exudates when the anti-inflammatory assay (carrageenan-induced peritonitis) was performed. CE and F80 inhibited writhing regarding antinociceptive activity (acetic acid-induced writhing response in mice). In conclusion, CE and F80 have no significant cytotoxic or antitumor activities in cell lines showing low toxicity and no action against tumors in vivo. Both preparations revealed anti-inflammatory and antinociceptive activities, corroborating the pharmacological basis of L. ferrea for ethnomedical use.

Assay for the pattern recognition molecule collectin liver 1 (CL-L1)

DEFF Research Database (Denmark)

Axelgaard, Esben; Jensenius, Jens Christian; Thiel, Steffen

Collectin liver 1 (also termed collectin 10 and CL-L1) is a C-type lectin that functions as a pattern recognition molecule (PRM) in the innate immune system1. We have produced antibodies against CL-L1 and have developed a sandwich-type time-resolved immuno-fluorometric assay (TRIFMA...... to co-purify with MASPs, possibly rendering it a role in complement. CL-L1 showed binding activity towards mannose-TSK beads in a Ca2+-dependent manner. This binding could be inhibited by mannose and glucose, but not by galactose, indicating that CL-L1 binds via its carbohydrate-recognition domain (CRD)....

Purification and properties of acid phosphatase from Avena elatior L. seeds

Directory of Open Access Journals (Sweden)

E. Wieczorek

2015-01-01

Full Text Available Acid phosphatase F1 from Avena elatior seeds was isolated and partially purified by means of alcohol precepitation, DEAE-, CM-column chromatography, Sephadex G-150, Sephadex G-200 and Sepharose 4B - gel filtration. The enzyme was stable at 50°C, pH 5.1. The pH optimum for phosphatase activity was 4.2. Fluoride, Zn2+, molybdate were effective inhibitors. EDTA and l, 10-phenanthroline activated the enzyme.

Antisense myb inhibition of purified erythroid progenitors in development and differentiation is linked to cycling activity and expression of DNA polymerase alpha

International Nuclear Information System (INIS)

Valtieri, M.; Venturelli, D.; Care, A.; Fossati, C.; Pelosi, E.; Labbaye, C.; Mattia, G.; Gewirtz, A.M.; Calabretta, B.; Peschle, C.

1991-01-01

These studies aimed to determine the expression and functional role of c-myb in erythroid progenitors with different cycling activities. In the first series of experiments the erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E) populations from adult peripheral blood (PB), bone marrow (BM), and embryonic-fetal liver (FL) were treated with either c-myb antisense oligomers or 3H-thymidine (3H-TdR). A direct correlation was always observed between the inhibitory effect of anti-myb oligomers and the level of cycling activity. Thus, the inhibitory effect of antisense c-myb on the number of BFU-E colonies was 28.3% +/- 15.8% in PB, 53.4% +/- 9.3% in BM, and 68.2% +/- 24.5% in FL. Both adult and embryonic CFU-E were markedly inhibited. Using purified PB progenitors, we observed a similar pattern, although with slightly lower inhibitory effects. In the 3H-TdR suicide assay the killing index of BFU-E was 8.9% +/- 4.2% in PB, 29.4% +/- 6.5% in BM, and 40.1% +/- 9.6% in FL. The values for adult and embryonic CFU-E were 55.7% +/- 7.9% and 60.98% +/- 6.6%, respectively. We then investigated the kinetics of c-myb mRNA level during the erythroid differentiation of purified adult PB and FL BFU-E, as evaluated in liquid-phase culture by reverse transcription-polymerase chain reaction. Adult erythroid precursors showed a gradual increase of c-myb mRNA from day 4 through day 8 of culture and a sharp decrease at later times, whereas the expression of c-myb mRNA and protein in differentiation embryonic precursors peaked 2 days earlier. In both cases, c-myb mRNA level peaked at the CFU-E stage of differentiation. Finally, highly purified adult PB BFU-E were stimulated into cycling by a 3-day treatment with interleukin-3 in liquid phase: both the sensitivity to c-myb antisense oligomers and the 3H-TdR suicide index showed a gradual, strictly parallel increase

Acquiring native-like intonation in Dutch and Spanish : Comparing the L1 and L2 of native speakers and second language learners

NARCIS (Netherlands)

van Maastricht, L.J.; Swerts, M.G.J.; Krahmer, E.J.

2013-01-01

ACQUIRING NATIVE-LIKE INTONATION IN DUTCH AND SPANISH Comparing the L1 and L2 of native speakers and second language learners Introduction Learning more about the interaction between the native language (L1) and the target language (L2) has been the aim of many studies on second language acquisition

Purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) pulp.

Science.gov (United States)

Yang, C P; Fujita, S; Ashrafuzzaman, M; Nakamura, N; Hayashi, N

2000-07-01

Polyphenol oxidase (EC 1.10.3.1, PPO) in the pulp of banana (Musa sapientum L.) was purified to 636-fold with a recovery of 3.0%, using dopamine as substrate. The purified enzyme exhibited a clear single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. The molecular weight of the enzyme was estimated to be about 41000 and 42000 by gel filtration and SDS-PAGE, respectively. The enzyme quickly oxidized dopamine, and its K(m) value for dopamine was 2.8 mM. The optimum pH was at 6.5, and the enzyme activity was stable in the range of pH 5-11 at 5 degrees C for 48 h. The enzyme had an optimum temperature of 30 degrees C and was stable even after a heat treatment at 70 degrees C for 30 min. The enzyme activity was completely inhibited by L-ascorbic acid, cysteine, sodium diethyldithiocarbamate, and potassium cyanide. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

Measurement of superoxide dismutase-like activity in peel and pulp of apple from Anshan acres

Science.gov (United States)

Zhao, Lijuan

2018-04-01

Pyrogallol autoxidation method was used for measurement of SOD-like activity. They are 259.56 u/g and 148.78 u/g in peel and pulp of apple from Anshan acres. The proper-conditions for this measurement was determined through experiment as following: detection wavelength of spectrophotometer 325nm, buffer system Tris-HAc, concentration of Tris-HAc solution 50 mmoL and pH8.2. The inhibition rate of SOD-L again pyrogallol antioxidation are 92% and 90% in peel and pulp of apple.

Purifying hydrocarbons

Energy Technology Data Exchange (ETDEWEB)

Demoulins, H D; Garner, F H

1923-02-07

Hydrocarbon distillates, including natural gases and vapors produced by cracking hydrocarbon oils, are desulfurized etc. by treating the vapor with an aqueous alkaline solution of an oxidizing agent. The hydrocarbons may be previously purified by sulfuric acid. In examples aqueous solutions of sodium or calcium hydrochlorite containing 1.5 to 5.0 grams per liter of available chlorine and sufficient alkali to give an excess of 0.1 percent in the spent reagent are preheated to the temperature of the vapor, and either sprayed or atomized into the vapors near the outlet of the dephlegmator or fractionating tower, or passed in countercurrent to the vapors through one or a series of scrubbers.

Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways.

Science.gov (United States)

Xie, Song-Zi; Hao, Ran; Zha, Xue-Qiang; Pan, Li-Hua; Liu, Jian; Luo, Jian-Ping

2016-08-01

The present work aimed at investigating the pattern recognition receptor (PRR) and immunostimulatory mechanism of a purified Dendrobium huoshanense polysaccharide (DHP). We found that DHP could bind to the surface of macrophages and stimulate macrophages to secrete NO, TNF-α and IL-1β. To unravel the mechanism for the binding of DHP to macrophages, flow cytometry, confocal laser-scanning microscopy, affinity electrophoresis, SDS-PAGE and western blotting were employed to verify the type of PRR responsible for the recognition of DHP by RAW264.7 macrophages and peritoneal macrophages of C3H/HeN and C3H/HeJ macrophages. Results showed that toll-like receptor 4 (TLR4) was an essential receptor for macrophages to directly bind DHP. Further, the phosphorylation of ERK, JNK, Akt and p38 were observed to be time-dependently promoted by DHP, as well as the nuclear translocation of NF-κB p65. These results suggest that DHP activates macrophages via its direct binding to TLR4 to trigger TLR4 signaling pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biological activities of Curcuma longa L.

Directory of Open Access Journals (Sweden)

Araújo CAC

2001-01-01

Full Text Available There are several data in the literature indicating a great variety of pharmacological activities of Curcuma longa L. (Zingiberaceae, which exhibit anti-inflammatory, anti-human immunodeficiency virus, anti-bacteria, antioxidant effects and nematocidal activities. Curcumin is a major component in Curcuma longa L., being responsible for its biological actions. Other extracts of this plant has been showing potency too. In vitro, curcumin exhibits anti-parasitic, antispasmodic, anti-inflammatory and gastrointestinal effects; and also inhibits carcinogenesis and cancer growth. In vivo, there are experiments showing the anti-parasitic, anti-inflammatory potency of curcumin and extracts of C. longa L. by parenteral and oral application in animal models. In this present work we make an overview of the pharmacological activities of C. longa L., showing its importance.

Purified deoxynivalenol or feed restriction reduces mortality in rainbow trout, Oncorhynchus mykiss (Walbaum), with experimental bacterial coldwater disease but biologically relevant concentrations of deoxynivalenol do not impair the growth of Flavobacterium psychrophilum.

Science.gov (United States)

Ryerse, I A; Hooft, J M; Bureau, D P; Hayes, M A; Lumsden, J S

2015-09-01

Diets containing deoxynivalenol (DON) were fed to rainbow trout Oncorhynchus mykiss (Walbaum) for 4 weeks followed by experimental infection (intraperitoneal) with Flavobacterium psychrophilum (4.1 × 10(6) colony-forming units [CFU] mL(-1) ). Mortality of rainbow trout fed either 6.4 mg kg(-1) DON or trout pair-fed the control diet was significantly reduced (P trout fed the control diet to apparent satiation (trout were fed one of three experimental diets; a control diet, a diet produced with corn naturally contaminated with DON (3.3 mg kg(-1) DON) or a diet containing purified DON (3.8 mg kg(-1) ); however, these fish were not experimentally infected. The presence of DON resulted in significant reduction (P trout fed diets containing purified DON (3.8 mg kg(-1) ) was significantly higher (P < 0.05) at 35 day post-exposure compared with controls. The antimicrobial activity of DON was examined by subjecting F. psychrophilum in vitro to serial dilutions of the chemical. Complete inhibition occurred at a concentration of 75 mg L(-1) DON, but no effect was observed below this concentration (0-30 mg L(-1) ). © 2014 John Wiley & Sons Ltd.

Antifungal activity of acetone extracts from Punica granatum L., Quercus suber L. and Vicia faba L.

Science.gov (United States)

Akroum, S

2017-03-01

Human and animal mycoses become more frequent and more resistant to traditional treatments. In this work, we tested the in vitro antifungal activity of acetonic extracts of Punica granatum L., Quercus suber L. and Vicia faba L. against seven pathogen fungi and the in vivo antifungal activity against Candida albicans and Trichophyton mentagrophytes. The phytochemical screening was also carried out and showed that the extracts contained mainly proanthocyanidins. Other polyphenols were also present but in low quantity. The acetone extract of V. faba L. gave a good in vitro inhibition of yeasts and was the most active for treating candidiasis in mice. It decreased the percentage of mortality with only 20μg. But the in vivo antifungal activity of this extract on T. mentagrophytes was low. It only showed a small diminution of crusting and erythema after the administration of 100μg. On the contrary, the acetone extracts of P. granatum L. had a poor activity against yeasts and a better one against moulds. It gave the best in vivo antifungal activity against T. mentagrophytes by healing animals with 40μg. The extract of P. granatum L. gave also an interesting in vivo antifungal activity against T. mentagrophytes with an active dose of 80μg. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

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Chin-Soon Chee

2014-01-01

Full Text Available Glutathione transferases (GST were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW of 23 kDa. 2-dimensional (2-D gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5 and GST2 (pI 6.2 with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase and F0KKB0 (glutathione S-transferase III of Acinetobacter calcoaceticus strain PHEA-2, respectively.

Characterization of Affinity-Purified Isoforms of Acinetobacter calcoaceticus Y1 Glutathione Transferases

Science.gov (United States)

Chee, Chin-Soon; Tan, Irene Kit-Ping; Alias, Zazali

2014-01-01

Glutathione transferases (GST) were purified from locally isolated bacteria, Acinetobacter calcoaceticus Y1, by glutathione-affinity chromatography and anion exchange, and their substrate specificities were investigated. SDS-polyacrylamide gel electrophoresis revealed that the purified GST resolved into a single band with a molecular weight (MW) of 23 kDa. 2-dimensional (2-D) gel electrophoresis showed the presence of two isoforms, GST1 (pI 4.5) and GST2 (pI 6.2) with identical MW. GST1 was reactive towards ethacrynic acid, hydrogen peroxide, 1-chloro-2,4-dinitrobenzene, and trans,trans-hepta-2,4-dienal while GST2 was active towards all substrates except hydrogen peroxide. This demonstrated that GST1 possessed peroxidase activity which was absent in GST2. This study also showed that only GST2 was able to conjugate GSH to isoproturon, a herbicide. GST1 and GST2 were suggested to be similar to F0KLY9 (putative glutathione S-transferase) and F0KKB0 (glutathione S-transferase III) of Acinetobacter calcoaceticus strain PHEA-2, respectively. PMID:24892084

Inhibition of purified enolases from oral bacteria by fluoride.

Science.gov (United States)

Guha-Chowdhury, N; Clark, A G; Sissons, C H

1997-04-01

Enolase activity in strains of oral streptococci previously has been found to be inhibited by 50% (Ki) by fluoride concentrations ranging from 50 to 300 microM or more in the presence of 0.5 to 1.0 mM inorganic phosphate ions. In this study, enolase was extracted and partly purified by a two-step process from five oral bacterial species and the effect of fluoride on the kinetics of enolase examined. The molecular weight of the putative enolase proteins was 46-48 kDa. The Vmax values ranged from 20 to 323 IU/mg and K(m) for glycerate-2-phosphate from 0.22 to 0.74 mM. Enolase activity was inhibited competitively by fluoride, with Ki values ranging from 16 to 54 microM in the presence of 5 mM inorganic phosphate ions. Ki values for phosphate ranged from 2 to 8 mM. The enolase from Streptococcus sanguis ATCC 10556 was more sensitive to fluoride (Ki = 16 +/- 2) than was enolase from Streptococcus salivarius ATCC 10575 (Ki = 19 +/- 2) or Streptococcus mutans NCTC 10449 (Ki = 40 +/- 4) and all three streptococcal strains were more sensitive to fluoride than either Actinomyces naeslundii WVU 627 (Ki = 46 +/- 6) or Lactobacillus rhamnosus ATCC 7469 (Ki = 54 +/- 6) enolases. The levels of fluoride found to inhibit the streptococcal enolases in this study are much lower than previously reported and are likely to be present in plaque, especially during acidogenesis, and could exert an anti-glycolytic effect.

Coagulation mechanism of salt solution-extracted active component in Moringa oleifera seeds.

Science.gov (United States)

Okuda, T; Baes, A U; Nishijima, W; Okada, M

2001-03-01

This study focuses on the coagulation mechanism by the purified coagulant solution (MOC-SC-PC) with the coagulation active component extracted from M. oleifera seeds using salt solution. The addition of MOC-SC-PC tap water formed insoluble matters. This formation was responsible for kaolin coagulation. On the other hand, insoluble matters were not formed when the MOC-SC-PC was added into distilled water. The formation was affected by Ca2+ or other bivalent cations which may connect each molecule of the active coagulation component in MOC-SC-PC and form a net-like structure. The coagulation mechanism of MOC-SC-PC seemed to be an enmeshment of Kaolin by the insoluble matters with the net-like structure. In case of Ca2+ ion (bivalent cations), at least 0.2 mM was necessary for coagulation at 0.3 mgC l-1 dose of MOC-SC-PC. Other coagulation mechanisms like compression of double layer, interparticle bridging or charge neutralization were not responsible for the coagulation by MOC-SC-PC.

Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

Science.gov (United States)

Shen, Jian-Dong; Cai, Qiu-Feng; Yan, Long-Jie; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Ke, Caihuan; Cao, Min-Jie

2015-12-01

Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone. Copyright © 2015 Elsevier Ltd. All rights reserved.

3,3',5-Triiodo-L-thyronine-like activity in effluents from domestic sewage treatment plants detected by in vitro and in vivo bioassays

International Nuclear Information System (INIS)

Murata, Tomonori; Yamauchi, Kiyoshi

2008-01-01

Thyroid system-disrupting activity in effluents from municipal domestic sewage treatment plants was detected using three in vitro assays and one in vivo assay. Contaminants in the effluents were extracted by solid-phase extraction (SPE) and eluted stepwise with different organic solvents. The majority of the thyroid system-disrupting activity was detected in the dichloromethane/methanol (1/1) fraction after SPE in all three in vitro assays: competitive assays of 3,3',5-[ 125 I]triiodo-L-thyronine ([ 125 I]T 3 ) binding to the plasma protein transthyretin (TTR assay) and thyroid hormone receptor (TR assay) and T 3 -dependent luciferase assay (Luc assay). Subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) of the dichloromethane/methanol (1/1) fraction separated contaminants potent in the TR and Luc assays from those potent in the TTR assay. The contaminants potent in the TR and Luc assays were also potent in an in vivo short-term gene expression assay in Xenopus laevis (Tadpole assay). The present study demonstrated that the effluents from domestic sewage treatment plants contain contaminants with T 3 -like activity of ∼ 10 -10 M T 3 -equivalent concentration (T 3 EQ) and that the TR and Luc assays are powerful in vitro bioassays for detecting thyroid system-disrupting activity in effluents. The availability and applicability of these bioassays for screening contaminants with thyroid system-disrupting activity in the water environment are discussed

Oriented gold ripple-like structures on poly-L-lactic acid

Energy Technology Data Exchange (ETDEWEB)

Juřík, Petr, E-mail: petr.jurik@gmail.com [Department of Solid State Engineering, Institute of Chemical Technology, Prague (Czech Republic); Slepička, Petr [Department of Solid State Engineering, Institute of Chemical Technology, Prague (Czech Republic); Mistrík, Jan; Janíček, Petr [Department of Applied Physics and Mathematics, University of Pardubice (Czech Republic); Rimpelová, Silvie [Department of Biochemistry and Microbiology, Institute of Chemical Technology, Prague (Czech Republic); Kolská, Zdeňka [Faculty of Science, University of J. E. Purkyně, Ústí nad Labem (Czech Republic); Švorčík, Václav [Department of Solid State Engineering, Institute of Chemical Technology, Prague (Czech Republic)

2014-12-01

Highlights: • We prepared oriented ripple-like structures on polymer surface with thin gold layer. • These structures show preferential orientation over large areas which is unusual for heat induced wrinkling. • Significant electrical and optical anisotropy was observed. • Zeta-potential, XPS, goniometry and ellipsometry suggest formation of gold lines separated by polymer gaps. • Increase in cell growth in comparison with poly-styrene mock was observed. - Abstract: In this paper chemical, morphological, electrical and biological properties of poly-L-lactic acid thin films with gold nanolayers were studied. These samples were examined as-sputtered and annealed at glass transition temperature. Morphological changes of poly-L-lactic films introduced by annealing were studied by means of atomic force microscopy. This method showed formation of oriented ripple-like structures on the surface of the film. X-ray photoelectron spectroscopy, goniometry, ellipsometry, sheet resistance measurement and electrokinetic analysis were used to determine distribution of gold on the surface. Combined data suggests that these ripple-like structures were formed by gold lines with insulating polymer gaps in between. These lines show preferential orientation over large areas and under proper conditions offer simple way to form electrically anisotropic material on large scale. Also cytocompatibility was studied showing increased cell adhesion and proliferation of mouse embryonic fibroblasts offering another use for these easily formed structures.

Oriented gold ripple-like structures on poly-L-lactic acid

International Nuclear Information System (INIS)

Juřík, Petr; Slepička, Petr; Mistrík, Jan; Janíček, Petr; Rimpelová, Silvie; Kolská, Zdeňka; Švorčík, Václav

2014-01-01

Highlights: • We prepared oriented ripple-like structures on polymer surface with thin gold layer. • These structures show preferential orientation over large areas which is unusual for heat induced wrinkling. • Significant electrical and optical anisotropy was observed. • Zeta-potential, XPS, goniometry and ellipsometry suggest formation of gold lines separated by polymer gaps. • Increase in cell growth in comparison with poly-styrene mock was observed. - Abstract: In this paper chemical, morphological, electrical and biological properties of poly-L-lactic acid thin films with gold nanolayers were studied. These samples were examined as-sputtered and annealed at glass transition temperature. Morphological changes of poly-L-lactic films introduced by annealing were studied by means of atomic force microscopy. This method showed formation of oriented ripple-like structures on the surface of the film. X-ray photoelectron spectroscopy, goniometry, ellipsometry, sheet resistance measurement and electrokinetic analysis were used to determine distribution of gold on the surface. Combined data suggests that these ripple-like structures were formed by gold lines with insulating polymer gaps in between. These lines show preferential orientation over large areas and under proper conditions offer simple way to form electrically anisotropic material on large scale. Also cytocompatibility was studied showing increased cell adhesion and proliferation of mouse embryonic fibroblasts offering another use for these easily formed structures

Isolation and Purification of an Early Pregnancy Factor–Like Molecule from Culture Supernatants Obtained from Lymphocytes of Pregnant Women

OpenAIRE

Aranha, Clara; Natraj, Usha; Iyer, K. S.; Shahani, Savitri

1998-01-01

Purpose:Our purpose was to determine whether lymphocytes synthesize proteins during pregnancy, to observe whether one of the proteins synthesized has early pregnancy factor (EPF)–like activity and to isolate and purify this molecule from culture supernatants obtained from stimulated lymphocytes of pregnant women.

Characterization and purification of polyphenol oxidase from artichoke (Cynara scolymus L.).

Science.gov (United States)

Dogan, Serap; Turan, Yusuf; Ertürk, Hatibe; Arslan, Oktay

2005-02-09

In this study, the polyphenol oxidase (PPO) of artichoke (Cynara scolymus L.) was first purified by a combination of (NH(4))(2)SO(4) precipitation, dialysis, and a Sepharose 4B-L-tyrosine-p-aminobenzoic acid affinity column. At the end of purification, 43-fold purification was achieved. The purified enzyme migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Polyacrylamide gel electrophoresis indicated that PPO had a 57 kDa molecular mass. Second, the contents of total phenolic and protein of artichoke head extracts were determined. The total phenolic content of artichoke head was determined spectrophotometrically according to the Folin-Ciocalteu procedure and was found to be 425 mg 100 g(-1) on a fresh weight basis. Protein content was determined according to Bradford method. Third, the effects of substrate specificity, pH, temperature, and heat inactivation were investigated on the activity of PPO purified from artichoke. The enzyme showed activity to 4-methylcatechol, pyrogallol, catechol, and L-dopa. No activity was detected toward L-tyrosine, resorsinol, and p-cresol. According to V(max)/K(m) values, 4-methylcatechol (1393 EU min(-1) mM(-1)) was the best substrate, followed by pyrogallol (1220 EU min(-1) mM(-1)), catechol (697 EU min(-1) mM(-1)), and L-dopa (102 EU min(-1) mM(-1)). The optimum pH values for PPO were 5.0, 8.0, and 7.0 using 4-methylcatechol, pyrogallol, and catechol as substrate, respectively. It was found that optimum temperatures were dependent on the substrates studied. The enzyme activity decreased due to heat denaturation of the enzyme with increasing temperature and inactivation time for 4-methylcatechol and pyrogallol substrates. However, all inactivation experiments for catechol showed that the activity of artichoke PPO increased with mild heating, reached a maximum, and then decreased with time. Finally, inhibition of artichoke PPO was investigated with inhibitors such as L-cysteine, EDTA, ascorbic

Improved detection of a staphylococcal infection by monomeric and protein A-purified polyclonal human immunoglobulin

International Nuclear Information System (INIS)

Calame, W.

1993-01-01

The present study was undertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99m Tc-labelled monomeric human immunoglobulin (m-Ig), 99m Tc-labelled, protein A-purified, human immunoglobulin (A-IG) and 99m Tc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99m Tc-labelled Igs bound to bacteria in vitro: The percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-to-non-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purified Ig yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99m Tc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99m Tc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein A-purified monomeric Ig was the highest of all tracer agents tested. (orig.)

In vitro effects of policosanol (Saccharum officinarum L wax alcohols on the 5-lipooxygenase enzyme

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Yohani Pérez Guerra

Full Text Available Introduction: policosanol, a mixture of high molecular weight aliphatic alcohols purified from sugarcane with octacosanol as the main component, shows cholesterol-lowering and antiplatelet effects in addition to an inhibitory effect on type I cicloxygenase. Objective: to determine whether policosanol may inhibit 5-LOX enzyme activity in vitro. Methods: effects on 5-LOX enzyme activities were assessed in rat blood polymorphonuclear leukocytes. Vehicle or Policosanol suspensions (0.6 to 6 000 µg/mL were added to tubes containing the reaction mix and then absorbance changes at 234 nm were measured. Results: added Policosanol inhibited in vitro 5-LOX activity by 30 %, which was not a significant figure but depended on the concentration(r= 0.992; p< 0.05; it was 1 250 µg/mL. Conclusions: policosanol did not significantly inhibit 5-LOX enzyme activity in rat PMNL preparations, so that it does not seem to be a dual inhibitor of COX and-LOX enzymes. This result differs from that found for beeswax alcohols and underlines the different effects of the mixtures of long-chain fatty alcohols purified from the sugarcane and the beeswax.

Induction, purification, and characterization of two extracellular alpha-L-arabinofuranosidases from Fusarium oxysporum

DEFF Research Database (Denmark)

Panagiotou, Gianni; Topakas, E.; Economou, L.

2003-01-01

In the presence of L-arabinose as sole carbon source, Fusarium oxysporum produces two alpha-L-arabinofuranosidases (ABFs) named ABF1 and ABF2, with molecular masses of 200 and 180 kDa, respectively. The two F. oxysporum proteins have been purified to homogeneity. The purified enzymes are composed...

Human immunodeficiency virus-like particles activate multiple types of immune cells

International Nuclear Information System (INIS)

Sailaja, Gangadhara; Skountzou, Ioanna; Quan, Fu-Shi; Compans, Richard W.; Kang, Sang-Moo

2007-01-01

The rapid spread of human immunodeficiency virus (HIV) worldwide makes it a high priority to develop an effective vaccine. Since live attenuated or inactivated HIV is not likely to be approved as a vaccine due to safety concerns, HIV virus like particles (VLPs) offer an attractive alternative because they are safe due to the lack of a viral genome. Although HIV VLPs have been shown to induce humoral and cellular immune responses, it is important to understand the mechanisms by which they induce such responses and to improve their immunogenicity. We generated HIV VLPs, and VLPs containing Flt3 ligand (FL), a dendritic cell growth factor, to target VLPs to dendritic cells, and investigated the roles of these VLPs in the initiation of adaptive immune responses in vitro and in vivo. We found that HIV-1 VLPs induced maturation of dendritic cells and monocyte/macrophage populations in vitro and in vivo, with enhanced expression of maturation markers and cytokines. Dendritic cells pulsed with VLPs induced activation of splenocytes resulting in increased production of cytokines. VLPs containing FL were found to increase dendritic cells and monocyte/macrophage populations in the spleen when administered to mice. Administration of VLPs induced acute activation of multiple types of cells including T and B cells as indicated by enhanced expression of the early activation marker CD69 and down-regulation of the homing receptor CD62L. VLPs containing FL were an effective form of antigen in activating immune cells via dendritic cells, and immunization with HIV VLPs containing FL resulted in enhanced T helper type 2-like immune responses

Hydrothermal growth of photoelectrochemically active titanium dioxide cauliflower-like nanostructures

International Nuclear Information System (INIS)

Pawar, Sachin A.; Devan, R.S.; Patil, D.S.; Burungale, V.V.; Bhat, T.S.; Mali, S.S.; Shin, S.W.; Ae, J.E.; Hong, C.K.; Ma, Y.R.; Kim, J.H.; Patil, P.S.

2014-01-01

Hierarchical titanium dioxide nanostructures have been synthesized by a simple and cost-effective hydrothermal deposition method onto the conducting glass substrates. In order to study the effect of titanium tetrachloride precursor quantity on the growth of TiO 2 ; the thin films of TiO 2 have been synthesized with the variations in the TiCl 4 from 0.4 mL to 1.0 mL at the interval of 0.2 mL. These films are characterized for their optical, structural, compositional, morphological properties using UV-vis spectrophotometer, Photoluminescence, X-ray Diffraction, High resolution transmission electron microscopy, X-ray Photoelectron Spectroscopy and Field Emission Scanning Electron Microscopy techniques. The optical band gap energy is found to increase from 2.74 to 3.06 eV with the increase in TiCl 4 quantities exhibiting a blue shift. XRD patterns show the formation of polycrystalline TiO 2 with the tetragonal crystal structure possessing rutile phase. Rise in the TiCl 4 quantity leads to the decrease in the particle size. The chemical composition and valence states of the constituent elements were analysed by XPS. FESEM images showed the formation of cauliflower-like structure at the highest TiCl 4 precursor quantity. The films were photoelectrochemically active with the maximum current density of 202 μA/cm 2 for the sample prepared at 1.0 mL

Structures of a putative RNA 5-methyluridine methyltransferase, Thermus thermophilus TTHA1280, and its complex with S-adenosyl-l-homocysteine

International Nuclear Information System (INIS)

Pioszak, Augen A.; Murayama, Kazutaka; Nakagawa, Noriko; Ebihara, Akio; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

2005-01-01

Three structures of a putative RNA 5-methyluridine methyltransferase from T. thermophilus, including its complex with S-adenosyl-l-homocysteine, are presented. The structures reveal the mode of cofactor binding, architecture of the putative active site, and the presence of a deep cleft adjacent to the active site that may bind RNA. The Thermus thermophilus hypothetical protein TTHA1280 belongs to a family of predicted S-adenosyl-l-methionine (AdoMet) dependent RNA methyltransferases (MTases) present in many bacterial and archaeal species. Inspection of amino-acid sequence motifs common to class I Rossmann-fold-like MTases suggested a specific role as an RNA 5-methyluridine MTase. Selenomethionine (SeMet) labelled and native versions of the protein were expressed, purified and crystallized. Two crystal forms of the SeMet-labelled apoprotein were obtained: SeMet-ApoI and SeMet-ApoII. Cocrystallization of the native protein with S-adenosyl-l-homocysteine (AdoHcy) yielded a third crystal form, Native-AdoHcy. The SeMet-ApoI structure was solved by the multiple anomalous dispersion method and refined at 2.55 Å resolution. The SeMet-ApoII and Native-AdoHcy structures were solved by molecular replacement and refined at 1.80 and 2.60 Å, respectively. TTHA1280 formed a homodimer in the crystals and in solution. Each subunit folds into a three-domain structure composed of a small N-terminal PUA domain, a central α/β-domain and a C-terminal Rossmann-fold-like MTase domain. The three domains form an overall clamp-like shape, with the putative active site facing a deep cleft. The architecture of the active site is consistent with specific recognition of uridine and catalysis of methyl transfer to the 5-carbon position. The cleft is suitable in size and charge distribution for binding single-stranded RNA.

Anxiolytic-like and sedative effects of Hydrocotyle umbellata L., Araliaceae, extract in mice

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Fábio F. Rocha

2011-02-01

Full Text Available The plant Hydrocotyle umbellata L., Araliaceae (water pennywort, is widely used in Brazilian folk medicine to reduce anxiety. This work investigates the anxiolytic-like effects of the ethanol extract from H. umbellata subterraneous parts as well as the extract's other putative central nervous system effects that could justify its common use. Oral dosing of the extract (0.3 and 1 g/kg clearly showed an anxiolytic-like profile in the elevated plus maze test where it increased the percentage of entries into and the time spent in the open arms of the maze. In the marble-burying test, the extract induced anxiolytic-like effects only at a dose of 1 g/kg, which also causes mild sedative properties in other models. The sedated state was characterized by a slight reduction in spontaneous exploratory activity during the open field test and a potentiating of pentobarbital-induced hypnosis. No signs of motor impairment were detected in the rota rod or chimney tests. The extract did not show antidepressant properties in mice as assessed by the forced swimming test. These results support the use of H. umbellata in Brazilian folk medicine as an anxiolytic and contribute to the scientific knowledge of this possible phytotherapeutic resource.

A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L

International Nuclear Information System (INIS)

Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis

2006-01-01

The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form

Gene structure of the pregnancy-associated glycoprotein-like (PAG-L) in the Eurasian beaver (Castor fiber L.).

Science.gov (United States)

Lipka, Aleksandra; Majewska, Marta; Panasiewicz, Grzegorz; Bieniek-Kobuszewska, Martyna; Szafranska, Bozena

2017-09-01

The pregnancy-associated glycoprotein-like family (PAG-L) is a large group of chorionic products, expressed in the pre-placental trophoblast and later in the post-implantational chorionic epithelium, and are involved in proper placenta development and embryo-maternal interaction in eutherians. This study describes identification of the PAG-L family in the genome of the Eurasian beaver (Castor fiber L.), named CfPAG-L. We identified 7657 bp of the CfPAG-L gDNA sequence (Acc. No. KX377932), encompassing nine exons (1-9) and eight introns (A-H). The length of the CfPAG-L exons (59-200 bp) was equivalently similar to the only known counterparts of bPAG1, bPAG2, and pPAG2. The length of the CfPAG-L introns ranged 288-1937 bp and was completely different from previously known PAG introns. The exonic CfPAG-L regions revealed 50.3-72.9% homology with equivalent segments of bPAG1 and pPAG2 structure. The intronic CfPAG-L regions alignments revealed a lack of homology. Within the entire CfPAG-L gene, 31 potential single nucleotide variants (SNV: 7 transversions and 24 transitions) were predicted. The identified exonic polymorphic loci did not affect the amino acid sequence of the CfPAG-L polypeptide precursor. This is the first report describing the CfPAG-L gene sequence, structural organization, and SNVs in the Eurasian beaver, one of the largest rodents.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a)

DEFF Research Database (Denmark)

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brondsted

2017-01-01

SPCA1a displays a higher apparent Ca2+ affinity and lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linole-/oleamide and phosphatidyl ethanolamine inhibit, whereas phosphatidic acid and sphingomyelin enhance SPCA1a...... activity. Moreover, SPCA1a is blocked by μM concentrations of commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid (CPA) and 2,5-di-tert-butyl hydroquinone (BHQ). Since tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a...

Oxidation of aromatic alcohols by purified methanol dehydrogenase from Methylosinus trichosporium.

OpenAIRE

Mountfort, D O

1990-01-01

Methanol dehydrogenase was found to be present in subcellular preparations of methanol-grown Methylosinus trichosporium and occurred almost wholly in the soluble fraction of the cell. The enzyme, purified by DEAE-Sephadex and Sephadex G-100 chromatography, showed broad specificity toward different substrates and oxidized the aromatic alcohols benzyl, vanillyl, and veratryl alcohols in addition to a range of aliphatic primary alcohols. No enzyme activity was found toward the corresponding alde...

Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema

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Tonon Jair

2013-01-01

Full Text Available Abstract Background Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD. Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner. Methods We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20 and 40 mg/mL (E40 papain. Results The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p Conclusions Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of oxidative stress in a severity-dependent manner.

Production of rabbit antibodies against purified Glucose oxidase

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Muhammad Anjum Zia

2012-02-01

Full Text Available Glucose oxidase is an active oxygen species generating enzyme produced from Aspergillus niger grown in submerged fermentation. Disintegration of the mycelium resulted in high glucose oxidase activity that was subjected to ammonium sulfate precipitation at 60-85% saturation rates that resulted to 6.14 U mg -1 specific activity. Purification of enzyme by anion exchange column (DEAE-Cellulose resulted into 22.53 U mg-1 specific activity and 10.27 fold purification. This was applied to sephadex G-200 column for gel filtration chromatography. It was observed that enzyme achieved 59.37 U mg-1of specific activity with 27.08 fold purity and 64.36% recovery. Purified glucose oxidase was injected into rabbits through intravenous route, to raise the glucose oxidase antibodies. After 30 days incubation period, the rabbits were slaughtered and serum was separated from blood. The antibodies were isolated by ammonium sulfate precipitation and confirmed by agar gel precipitation test. This could be a convenient and low cost alternate assay for the estimation of glucose oxidase in biological fluids. Moreover, such antibodies against the said enzyme could be used in various therapeutic and diagnostic applications.

CaV1.3 L-type Ca2+ channels modulate depression-like behaviour in mice independent of deaf phenotype.

Science.gov (United States)

Busquet, Perrine; Nguyen, Ngoc Khoi; Schmid, Eduard; Tanimoto, Naoyuki; Seeliger, Mathias W; Ben-Yosef, Tamar; Mizuno, Fengxia; Akopian, Abram; Striessnig, Jörg; Singewald, Nicolas

2010-05-01

Mounting evidence suggests that voltage-gated L-type Ca2+ channels can modulate affective behaviour. We therefore explored the role of CaV1.3 L-type Ca2+ channels in depression- and anxiety-like behaviours using CaV1.3-deficient mice (CaV1.3-/-). We showed that CaV1.3-/- mice displayed less immobility in the forced swim test as well as in the tail suspension test, indicating an antidepressant-like phenotype. Locomotor activity in the home cage or a novel open-field test was not influenced. In the elevated plus maze (EPM), CaV1.3-/- mice entered the open arms more frequently and spent more time there indicating an anxiolytic-like phenotype which was, however, not supported in the stress-induced hyperthermia test. By performing parallel experiments in Claudin 14 knockout mice (Cldn14-/-), which like CaV1.3-/- mice are congenitally deaf, an influence of deafness on the antidepressant-like phenotype could be ruled out. On the other hand, a similar EPM behaviour indicative of an anxiolytic phenotype was also found in the Cldn14-/- animals. Using electroretinography and visual behavioural tasks we demonstrated that at least in mice, CaV1.3 channels do not significantly contribute to visual function. However, marked morphological changes were revealed in synaptic ribbons in the outer plexiform layer of CaV1.3-/- retinas by immunohistochemistry suggesting a possible role of this channel type in structural plasticity at the ribbon synapse. Taken together, our findings indicate that CaV1.3 L-type Ca2+ channels modulate depression-like behaviour but are not essential for visual function. The findings raise the possibility that selective modulation of CaV1.3 channels could be a promising new therapeutic concept for the treatment of mood disorders.

Purification of Escherichia coli L-asparaginase mutants by a native polyacrylamide gel electrophoresis.

Science.gov (United States)

Wei, Yujun; Chen, Jianhua; Jia, Ruibo; Wang, Min; Wu, Wutong

2008-07-01

The antigenicity of L-asaparaginase (L-ASP) has been problematic for the treatment of leukemia for many years. In order to establish a relationship between the antigenic epitope of L-asparaginase and its antigenicity, several L-asparaginase mutants (mL-ASPs) are constructed and expressed. To effectively purify these enzyme mutants for further investigation, a native preparative polyacrylamide gel electrophoresis is developed. The simplicity and reproducibility of this approach permits the purification of different mutants from the crude enzyme extracts, with a sufficient activity to perform immunological and biological studies. Furthermore, the newly developed method is efficient and cost-effective compared with other methods, such as column chromatography and affinity chromatography. As a result, the enzyme mutants with specific activity of 300 approximately 400 U/mg are obtained by the single-step purification with a high degree of purity.

Steroidogenesis in amlodipine treated purified Leydig cells

Energy Technology Data Exchange (ETDEWEB)

Latif, Rabia, E-mail: rabialatif08@hotmail.com [Department of Physiology, Army Medical College, National University of Sciences and Technology, Islamabad (Pakistan); Lodhi, Ghulam Mustafa, E-mail: drmustafa786@gmail.com [Department of Physiology, Wah Medical College, Wah (Pakistan); Hameed, Waqas, E-mail: waqham@hotmail.com [Department of Physiology, Rehman Medical College, Peshawar (Pakistan); Aslam, Muhammad, E-mail: professormaslam@yahoo.com [Department of Physiology, Shifa College of Medicine, Islamabad (Pakistan)

2012-01-01

Drugs have been shown to adversely affect male fertility and recently anti-hypertensive drugs were added to the list. The anti-fertility effects of amlodipine, a calcium channel blocker, are well-illustrated in in vivo experiments but lack an in vitro proof. The present study was designed to experimentally elucidate the effects of amlodipine on Leydig cell steroidogenesis and intracellular calcium in vitro. Leydig cells of Sprague–Dawley rats were isolated and purified by Percoll. Cells were incubated for 3 h with/without amlodipine in the presence/absence of LH, dbcAMP, Pregnenolone and 25-Hydroxycholesterol. Cytosolic calcium was measured in purified Leydig cells by fluorometric technique. The results showed significantly reduced (P < 0.05) steroidogenesis and intracellular calcium in amlodipine exposed rats. The site of amlodipine induced steroidogenic inhibition seems to be prior to the formation of Pregnenolone at the level of StAR protein. -- Highlights: ► Inhibition of steroidogenesis in isolated and purified Leydig cells by amlodipine. ► Site of inhibition was before Pregnenolone formation, at the level of StAR protein. ► Inhibition of LH stimulated rise in cytosolic calcium by amlodipine.

Biological activities and medicinal properties of Cajanus cajan (L Millsp.

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Dilipkumar Pal

2011-01-01

Full Text Available Cajanus cajan (L Millsp. (Sanskrit: Adhaki, Hindi: Arhar, English: Pigeon pea, Bengali: Tur (family: Fabaceae is the most important grain legume crop of rain-fed agriculture in semi-arid tropics. It is both a food crop and a cover/forage crop with high levels of proteins and important amino acids like methionine, lysine and tryptophan. During the last few decades extensive studies have been carried out regarding the chemistry of C. cajan and considerable progress has been achieved regarding its biological activities and medicinal applications. This review article gives an overview on the biological activities of the compounds isolated, pharmacological actions and clinical studies of C. cajan extracts apart from its general details.

Biological activities and medicinal properties of Cajanus cajan (L) Millsp.

Science.gov (United States)

Pal, Dilipkumar; Mishra, Pragya; Sachan, Neetu; Ghosh, Ashoke K

2011-10-01

Cajanus cajan (L) Millsp. (Sanskrit: Adhaki, Hindi: Arhar, English: Pigeon pea, Bengali: Tur) (family: Fabaceae) is the most important grain legume crop of rain-fed agriculture in semi-arid tropics. It is both a food crop and a cover/forage crop with high levels of proteins and important amino acids like methionine, lysine and tryptophan. During the last few decades extensive studies have been carried out regarding the chemistry of C. cajan and considerable progress has been achieved regarding its biological activities and medicinal applications. This review article gives an overview on the biological activities of the compounds isolated, pharmacological actions and clinical studies of C. cajan extracts apart from its general details.

Immunomodulatory Effects of the Mycosporine-Like Amino Acids Shinorine and Porphyra-334

OpenAIRE

Kathrin Becker; Anja Hartmann; Markus Ganzera; Dietmar Fuchs; Johanna M. Gostner

2016-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porp...

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

OpenAIRE

Lewis, R E; Czech, M P

1987-01-01

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for ...

Colorimetric assay of copper ions based on the inhibition of peroxidase-like activity of MoS2 nanosheets

Science.gov (United States)

Chen, Huan; Li, Zhihong; Liu, Xueting; Zhong, Jianhai; Lin, Tianran; Guo, Liangqia; Fu, Fengfu

2017-10-01

The peroxidase-like catalytic activity of MoS2 nanomaterials has been utilized for colorimetric bioassays and medical diagnostics. However, the application of peroxidase-like catalytic activity of MoS2 nanomaterials in environmental analysis was seldom explored. Herein, copper ions were found to inhibit the peroxidase-like catalytic activity of MoS2 nanosheets, which can catalyze the oxidation of 3, 3‧, 5, 5‧-tetramethylbenzidine by H2O2 to produce a colorimetric product. Based on this finding, a simple sensitive colorimetric method for the detection of copper ions was developed. In the presence of copper ions, the absorbance and color of the solution decreased with the increasing concentration of copper ions. The color of the solution can be used to semi-quantitative on-site assay of copper ions by naked eyes. A linear relationship between the absorbance and the concentration of copper ions was observed in the range of 0.4-4.0 μmol L- 1 with a detection limit of 92 nmol L- 1, which was much lower than the maximum contaminant level of copper in drinking water legislated by the Environmental Protection Agency of USA and the World Health Organization. The method was applied to detect copper ions in environmental water samples with satisfactory results.

Efeitos de níveis de ácido L-glutâmico e de vitamina K da dieta sobre a atividade de alfa-amilase em frangos de corte Effects of dietary levels of L-glutamic acid and vitamin K in the activity of alpha-amylase of chicks

Directory of Open Access Journals (Sweden)

Flavia Escapini Fanchiotti

2005-04-01

Full Text Available Foram investigados os efeitos nutricionais de dois níveis de ácido L-glutâmico (L-Glu combinados com quatro níveis de vitamina K (Vit K sobre a atividade de alfa-amilase no quimo e pâncreas de aves de corte. Frangos de corte machos de um dia foram criados em baterias aquecidas e alimentados, à vontade, com dietas contendo todos L-aminoácidos essenciais, minerais e vitaminas (exceto Vit K até os 14 dias de idade. O experimento foi realizado em esquema fatorial, em delineamento inteiramente casualizado 2x4, com quatro repetições de oito aves cada. A dieta básica foi suplementada com 6,25 e 12,5% de L-Glu combinados com 0,02; 0,2; 20,0 e 200,0 mg de Vit K/kg de ração. Efeitos significativos de L-Glu e Vit K foram observados no quimo. A atividade específica máxima (1.005,78±245,25 UI/mg de proteína foi observada nas aves alimentadas com 6,25% de L-Glu e 20,0 mg de Vit K. Houve redução da atividade com a suplementação de 12,5% de L-Glu. No pâncreas, não houve interação entre L-Glu e Vit K, todavia, foi observado efeito de L-Glu sobre as atividades relativas, expressas em UI/g de tecido e UI/100 g de peso corporal. Os resultados sugerem que a associação entre L-Glu e Vit K interfere na atividade enzimática de alfa-amilase em aves de corte submetidas a dietas purificadas. Os resultados indicam que o nível de 12,5% de L-Glu associado aos diferentes níveis de vitamina K reduziu a atividade enzimática.The effects of two levels of L-glutamic acid (L-Glu combined with four levels of vitamin K (Vit K were studied with the objective of evaluating the activities of alpha-amylase in the chyme and pancreas of chicks. Day-old male broilers were reared in eletrically heated batteries, fed with purified amino acids diets, minerals and vitamins (except Vit K at the requirement levels, for 14 days. Feed and water were provided ad libitum. The experimental design was a factorial 2x4 with four replicates with eight chicks each The

A role for serotonin in the antidepressant activity of NG-Nitro-L-arginine, in the rat forced swimming test.

Science.gov (United States)

Gigliucci, Valentina; Buckley, Kathleen Niamh; Nunan, John; O'Shea, Karen; Harkin, Andrew

2010-02-01

The present study determined regional serotonin (5-HT) synthesis and metabolism changes associated with the nitric oxide synthase (NOS) inhibitor N(G)-nitro-L-arginine (L-NA) and the influence of 5-HT receptor blockade in the antidepressant-like actions of L-NA in the forced swimming test (FST). Regional effects of L-NA (5,10 and 20mg/kg i.p.) on tryptophan hydroxylase (TPH) activity, the rate limiting enzyme for 5-HT synthesis, were determined by measuring accumulation of the transient intermediate 5-hydoxytryptophan (5-HTP) following in vivo administration of the amino acid decarboxylase inhibitor, NSD 1015 (100mg/kg). L-NA (5-20mg/kg) dose dependently increased 5-HTP accumulation, particularly in the amygdaloid cortex, following exposure to the FST. L-NA also provoked an increase in regional brain 5-HIAA concentrations and in the 5-HIAA:5-HT metabolism ratio. Co-treatment with NSD-1015 failed to consistently modify the antidepressant-like effects of L-NA in the FST. Sub-active doses of L-NA (1mg/kg) and the 5-HT re-uptake inhibitor fluoxetine (2.5mg/kg) acted synergistically to increase swimming in the test. Co-treatment with the non-selective 5-HT receptor antagonist metergoline (1, 2 and 4mg/kg), attenuated the L-NA (20mg/kg)-induced reduction in immobility and increase in swimming behaviours. Metergoline alone however provoked an increase in immobility and reduction in swimming behaviours in the test. A similar response was obtained following co-treatment with the preferential 5-HT(2A) receptor antagonist ketanserin (5mg/kg) and the 5-HT(2C) receptor antagonist RO-430440 (5mg/kg). Co-treatment with the 5-HT(1A) receptor antagonist WAY 100635 (0.3mg/kg) or the 5-HT(1B) receptor antagonist GR 127935 (4mg/kg) failed to influence the antidepressant-like activity of L-NA. Taken together these data provide further support for a role for 5-HT in the antidepressant-like properties of NOS inhibitors. Copyright 2009 Elsevier Inc. All rights reserved.

An Artificial Channel Experiment for Purifying Drainage Water Containing Arsenic by Using Eleocharis acicularis

Science.gov (United States)

Okazaki, Kenji; Yamazaki, Shusaku; Kurahashi, Toshiyuki; Sakakibara, Masayuki

2017-06-01

This paper reports the results of an artificial channel experiment in which water containing arsenic was purified by using Eleocharis acicularis. The experiment was conducted to investigate the feasibility of phytoremediation by Eleocharis acicularis in civil engineering projects. In the experiment, 15 m2 of Eleocharis acicularis mats were laid in an artificial channel. Three sessions of artificial flow were implemented by leading 100.0 L of river water containing 0.234 mg/L of arsenic into the channel each time. The arsenic concentration of the leachate from the channel was analyzed. As the results of experiment, the arsenic concentrations of the leachate for the three sessions were 0.045 mg/L, 0.133 mg/L, and 0.249 mg/L. This shows that the arsenic concentration decreased during the first two sessions, whose flow totaled 200 L. The arsenic concentrations in the Eleocharis acicularis were 0.87 mg/kg, 1.01 mg/kg, and 4.16 mg/kg, which show that the plant absorbs arsenic. Moreover, it was found that the amount of sample water was reduced through evapotranspiration from the plant and the artificial channel.

Development of a biogas purifier for rural areas in Japan

Energy Technology Data Exchange (ETDEWEB)

Kimura, Y.; Hinata, T. [Hokkaido Central Agricultural Experiment Station, Hokkaido (Japan); Yasui, S. [Zukosha Co. Ltd., Obihiro, Hokkaido (Japan); Noguchi, N. [Hokkaido Univ., Sapporo, Hokkaido (Japan); Tsukamoto, T. [IHI Shibaura. Co. Ltd., Obihiro, Hokkaido (Japan); Imai, T. [Green Plan Co. Ltd., Sapporo, Hokkaido (Japan); Kanai, M. [Air Water Co. Ltd, Sakai, Osaka (Japan); Matsuda, Z. [Hokuren Agricultural Research Center, Sapporo, Hokkaido (Japan)

2010-07-01

Although the biogas that is currently produced for dairy farms in Japan is a carbon-neutral energy, its use is restricted to farming areas only because there is no effective method of transporting unused biogas. There is a need for establishing practical methods for biogas removal from operating systems. In this study, a gas separation membrane was used in order to modify biogas to city gas 12A specifications, and to develop a biogas purifier equipped with a device to fill high pressure purified gas into cylinders to be taken outside the farming area. The objective was to expand the use of biogas produced from stand-alone gas plants. The amount of purified gas produced at a newly created refining-compression-filling (RCF) facility was approximately 97.0 Nm{sup 3}/day, for a raw material amount of about 216.0 Nm{sup 3}/day. The heat quantity of the purified gas was 38.9 MJ/Nm{sup 3}, which was within city gas 12A specifications. A total of 14.3 cylinders were filled each day with the manufactured purified gas. Test calculations along with a simulation exercise revealed that it would be possible to provide purified gas to approximately 6 per cent of common residences in a town in northern Japan. It was concluded that the newly created RCF facility allowed the modification of carbon-neutral biogas to conform to city gas 12A specifications, and allowed the transport of this gas out of the farming area.

Method for purifying bidentate organophosphorus compounds

International Nuclear Information System (INIS)

Schulz, W.W.

1977-01-01

Bidentate organophosphorus compounds useful for extracting actinide elements from acidic nuclear waste solutions are purified of undesirable acidic impurities by contacting the compounds with ethylene glycol which preferentially extracts the impurities found in technical grade bidentate compounds

Analgesic and cytotoxic activity of Acorus calamus L., Kigelia pinnata L., Mangifera indica L. and Tabernaemontana divaricata L.

Science.gov (United States)

Khan, Mohammad Ahad Ali; Islam, Mohammad Torequl

2012-04-01

The aim of the study was to evaluate analgesic and cytotoxic activity of Acorus calamus L., Kigelia pinnata L., Mangifera indica L., Tabernaemontana divaricata L. extracts by using acetic acid-induced writhing method in mice and brine shrimp lethality assay. The ethanolic extracts of the plants were obtained by simple maceration method and were subjected to standardization by using pharmacognostical and phytochemical screening methods, which were followed by acetic acid writhing and brine shrimp lethality test methods. Dose selection was made on the basis of acute oral toxicity study (10-1000 mg/kg body weight). In analgesic test, M. indica L. extract produced 28.16% and 22.02% writhing protection at the doses of 250 and 500 mg/kg body weight in mice, respectively. While the T. divaricata L. extract produced 22.02% and 33.93%, K. pinnata L. extract produced 11.55% and 47.29% and A. calamus L. extract produced 15.16% and 54.51% of writhing protection at the same doses. The percent mortality (mean ± SD) was found to be 58.7 ± 25.22, 56.25 ± 22.88, 52.50 ± 24.37, and 61.25 ± 26.66 with M. indica L., T. divaricata L., K. pinnata L., and A. calamus L., respectively. And the LC(50) and LC(90) values were found to be 100 and 300 μg/mL for M. indica L. and that were (200 and 350 μg/mL), (100 and 350 μg/mL) and (50 and 300 μg/mL) for T. divaricata L., K. pinnata L., and A. calamus L., respectively. Thus it can be concluded that bark of M. indica L., leaves of T. divaricata L., bark of K. pinnata L., and roots of A. calamus L. have significant analgesic and cytotoxic activity and can be preferred in the treatment of pain and tumor.

Analgesic and cytotoxic activity of Acorus calamus L., Kigelia pinnata L., Mangifera indica L. and Tabernaemontana divaricata L.

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Mohammad Ahad Ali Khan

2012-01-01

Full Text Available Objectives: The aim of the study was to evaluate analgesic and cytotoxic activity of Acorus calamus L., Kigelia pinnata L., Mangifera indica L., Tabernaemontana divaricata L. extracts by using acetic acid-induced writhing method in mice and brine shrimp lethality assay. Materials and Methods: The ethanolic extracts of the plants were obtained by simple maceration method and were subjected to standardization by using pharmacognostical and phytochemical screening methods, which were followed by acetic acid writhing and brine shrimp lethality test methods. Dose selection was made on the basis of acute oral toxicity study (10-1000 mg/kg body weight. Results and Conclusion: In analgesic test, M. indica L. extract produced 28.16% and 22.02% writhing protection at the doses of 250 and 500 mg/kg body weight in mice, respectively. While the T. divaricata L. extract produced 22.02% and 33.93%, K. pinnata L. extract produced 11.55% and 47.29% and A. calamus L. extract produced 15.16% and 54.51% of writhing protection at the same doses. The percent mortality (mean ± SD was found to be 58.7 ± 25.22, 56.25 ± 22.88, 52.50 ± 24.37, and 61.25 ± 26.66 with M. indica L., T. divaricata L., K. pinnata L., and A. calamus L., respectively. And the LC 50 and LC 90 values were found to be 100 and 300 μg/mL for M. indica L. and that were (200 and 350 μg/mL, (100 and 350 μg/mL and (50 and 300 μg/mL for T. divaricata L., K. pinnata L., and A. calamus L., respectively. Thus it can be concluded that bark of M. indica L., leaves of T. divaricata L., bark of K. pinnata L., and roots of A. calamus L. have significant analgesic and cytotoxic activity and can be preferred in the treatment of pain and tumor.

Construction of Escherichia coli Mutant with Decreased Endotoxic Activity by Modifying Lipid A Structure

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Qiong Liu

2015-05-01

Full Text Available Escherichia coli BL21 (DE3 and its derivatives are widely used for the production of recombinant proteins, but these purified proteins are always contaminated with lipopolysaccharide (LPS. LPS is recognized by the toll-like receptor 4 and myeloid differentiation factor 2 complex of mammalian immune cells and leads to release of pro-inflammatory cytokines. It is a vital step to remove LPS from the proteins before use for therapeutic purpose. In this study, we constructed BL21 (DE3 ∆msbB28 ∆pagP38 mutant, which produces a penta-acylated LPS with reduced endotoxicity. The plasmids harboring pagL and/or lpxE were then introduced into this mutant to further modify the LPS. The new strain (S004 carrying plasmid pQK004 (pagL and lpxE produced mono-phosphoryated tetra-acylated lipid A, which induces markedly less production of tumor necrosis factor-α in the RAW264.7 and IL-12 in the THP1, but still retains ability to produce recombinant proteins. This study provides a strategy to decrease endotoxic activity of recombinant proteins purified from E. coli BL21 backgrounds and a feasible approach to modify lipid A structure for alternative purposes such as mono-phosphoryl lipid A (MPL as vaccine adjuvants.

Process for purifying zirconium sponge

International Nuclear Information System (INIS)

Abodishish, H.A.M.; Kimball, L.S.

1992-01-01

This patent describes a Kroll reduction process wherein a zirconium sponge contaminated with unreacted magnesium and by-product magnesium chloride is produced as a regulus, a process for purifying the zirconium sponge. It comprises: distilling magnesium and magnesium chloride from: a regulus containing a zirconium sponge and magnesium and magnesium chloride at a temperature above about 800 degrees C and at an absolute pressure less than about 10 mmHg in a distillation vessel to purify the zirconium sponge; condensing the magnesium and the magnesium chloride distilled from the zirconium sponge in a condenser; and then backfilling the vessel containing the zirconium sponge and the condenser containing the magnesium and the magnesium chloride with a gas; recirculating the gas between the vessel and the condenser to cool the zirconium sponge from above about 800 degrees C to below about 300 degrees C; and cooling the recirculating gas in the condenser containing the condensed magnesium and the condensed magnesium chloride as the gas cools the zirconium sponge to below about 300 degrees C

Measurement of Ozone Emission and Particle Removal Rates from Portable Air Purifiers

Science.gov (United States)

Mang, Stephen A.; Walser, Maggie L.; Nizkorodov, Sergey A.; Laux, John M.

2009-01-01

Portable air purifiers are popular consumer items, especially in areas with poor air quality. Unfortunately, most users of these air purifiers have minimal understanding of the factors affecting their efficiency in typical indoor settings. Emission of the air pollutant ozone (O[subscript 3]) by certain air purifiers is of particular concern. In an…

Easy and Rapid Purification of Highly Active Nisin

NARCIS (Netherlands)

Abts, André; Mavaro, Antonino; Stindt, Jan; Bakkes, Patrick J.; Metzger, Sabine; Driessen, Arnold J.M.; Smits, Sander H.J.; Schmitt, Lutz

2011-01-01

Nisin is an antimicrobial peptide produced and secreted by several L. lactis strains and is specifically active against Gram-positive bacteria. In previous studies, nisin was purified via cation exchange chromatography at low pH employing a single-step elution using 1M NaCl. Here, we describe an

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Directory of Open Access Journals (Sweden)

Yingying Cai

Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

Science.gov (United States)

Cai, Yingying; Liu, Yuting; Culhane, Kelly J; DeVree, Brian T; Yang, Yang; Sunahara, Roger K; Yan, Elsa C Y

2017-01-01

Family B G protein-coupled receptors (GPCRs) play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1) receptor (GLP1R), whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL) particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

The thermodynamic signature of ligand binding to histone deacetylase-like amidohydrolases is most sensitive to the flexibility in the L2-loop lining the active site pocket.

Science.gov (United States)

Meyners, Christian; Krämer, Andreas; Yildiz, Özkan; Meyer-Almes, Franz-Josef

2017-07-01

The analysis of the thermodynamic driving forces of ligand-protein binding has been suggested to be a key component for the selection and optimization of active compounds into drug candidates. The binding enthalpy as deduced from isothermal titration calorimetry (ITC) is usually interpreted assuming single-step binding of a ligand to one conformation of the target protein. Although successful in many cases, these assumptions are oversimplified approximations of the reality with flexible proteins and complicated binding mechanism in many if not most cases. The relationship between protein flexibility and thermodynamic signature of ligand binding is largely understudied. Directed mutagenesis, X-ray crystallography, enzyme kinetics and ITC methods were combined to dissect the influence of loop flexibility on the thermodynamics and mechanism of ligand binding to histone deacetylase (HDAC)-like amidohydrolases. The general ligand-protein binding mechanism comprises an energetically demanding gate opening step followed by physical binding. Increased flexibility of the L2-loop in HDAC-like amidohydrolases facilitates access of ligands to the binding pocket resulting in predominantly enthalpy-driven complex formation. The study provides evidence for the great importance of flexibility adjacent to the active site channel for the mechanism and observed thermodynamic driving forces of molecular recognition in HDAC like enzymes. The flexibility or malleability in regions adjacent to binding pockets should be given more attention when designing better drug candidates. The presented case study also suggests that the observed binding enthalpy of protein-ligand systems should be interpreted with caution, since more complicated binding mechanisms may obscure the significance regarding potential drug likeness. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

International Nuclear Information System (INIS)

Lai Yuping; Peng Yifei; Zuo Yi; Li Jun; Huang Jing; Wang Linfa; Wu Zirong

2005-01-01

Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 μM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both α-helix and β-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L

Purified water quality study

International Nuclear Information System (INIS)

Spinka, H.; Jackowski, P.

2000-01-01

Argonne National Laboratory (HEP) is examining the use of purified water for the detection medium in cosmic ray sensors. These sensors are to be deployed in a remote location in Argentina. The purpose of this study is to provide information and preliminary analysis of available water treatment options and associated costs. This information, along with the technical requirements of the sensors, will allow the project team to determine the required water quality to meet the overall project goals

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

Science.gov (United States)

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-10-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

Impaired CD23 and CD62L expression and tissue inhibitors of metalloproteinases secretion by eosinophils in adults with atopic dermatitis.

Science.gov (United States)

de Oliveira Titz, T; Orfali, R L; de Lollo, C; Dos Santos, V G; da Silva Duarte, A J; Sato, M N; Aoki, V

2016-12-01

Eosinophils are multifunctional, polymorphonuclear leucocytes that secrete proteins within cytoplasmic granules, such as cytokines, chemokines, metalloproteinases (MMPs) and metalloproteinases tissue inhibitors (TIMPs). Although eosinophilia is a hallmark of atopic dermatitis (AD), several functional aspects of eosinophils remain unknown. We aimed to evaluate the phenotype and functional response of eosinophils under staphylococcal enterotoxin B (SEB) and Toll-like receptor (TLR)-2/6 (FSL-1) stimulation in the secretion of CCL5, MMPs and TIMPs in adults with AD. Forty-one adult patients with AD and 45 healthy controls enrolled for the study. Phenotype of eosinophils from granulocytes of peripheral blood was analysed by flow cytometry. We performed evaluation of CCL5 (cytometric bead array), MMP and TIMP (ELISA) secretion, in culture supernatants of purified eosinophils stimulated with SEB or TLR2/6 agonist (FSL-1). We found a higher frequency of LIN1 - CCR3 + eosinophils, and decreased expression of CD23 and CD62L receptors in eosinophils of AD patients. There was no difference in MMP and TIMP serum levels between the evaluated groups. However, we detected decreased basal levels of TIMP-1, TIMP-2 and CCL5 in culture supernatants from purified, unstimulated eosinophils from AD patients. In adults with AD, phenotypical features of eosinophils reveal decreased expression of early activation and L-selectin receptors. Regarding the functional profile of purified eosinophils related to tissue remodelling in atopic dermatitis, innate immune stimulation (TLR2/6 agonist and SEB) did not affect the ratio of MMP/TIMPs secretion in AD. Our findings reinforce the potential breakdown in tissue remodelling process mediated by eosinophils in AD. © 2016 European Academy of Dermatology and Venereology.

Purifying oils

Energy Technology Data Exchange (ETDEWEB)

1930-04-15

Gasoline, lamp oils, and lubricating or other mineral or shale oils are refined by contacting the vapor with a hot aqueous solution of salts of zinc, cadmium, or mercury, or mixtures thereof which may contain 0-5-3-0 percent of oxide or hydroxide in solution or suspension. Chlorides, bromides, iodides, sulfates, nitrates, and sulfonates of benzol, toluol, xylol, and petroleum are specified. Washing with a solution of sodium or potassium hydroxide or carbonate of calcium hydroxide may follow. The oil may first be purified by sulfuric acid or other known agent, or afterwards caustic alkali and sulfuric acid. The Specification as open to inspection under Sect. 91 (3) (a) describes also the use of salts of copper, iron, chromium, manganese, aluminum, nickel, or cobalt, with or without their oxides or hydroxides. This subject-matter does not appear in the Specification as accepted.

The antitussive activity of polysaccharides from Althaea officinalis l., var. Robusta, Arctium lappa L., var. Herkules, and Prunus persica L., Batsch.

Science.gov (United States)

Sutovska, M; Nosalova, G; Franova, S; Kardosova, A

2007-01-01

The therapy of pathological type of cough presents serious medical problem. The aim of experiments was to investigate polysaccacharide influence on experimentally induced cough. The purified and/or modified polysaccharides from the flowers and plants, characterized by chemical composition and molecular properties were subjected to tests for antitussive activity on cough, induced mechanically in conscious cats of both sexes. The results revealed that the tested polysaccharides exhibited statistically significant cough-suppressing activity, which was noticeably higher than that of the non-narcotic drug used in clinical practice to treat coughing. The most expressive antitussive activity was observed with the polysaccharide from marsh mallow, containing the highest proportion of the uronic acid constituent. Negative influence of the tested compounds on expectoration was negligible when compared to that of codeine. Antitussive activity of various plant polysaccharides was confirmed. These results allow ranging them among prospective antitussive agents (Tab. 2, Fig. 6, Ref. 15) Full Text (Free, PDF) www.bmj.sk.

L-shell dielectronic recombination for 0-like ions

International Nuclear Information System (INIS)

Omar, G.; Semedal, R.; Shahin, F.; El-Sherbini, T.H.

2007-01-01

In electron-Ion (e/I) collisions, a free electron may be captured by a positively charged ion having bound electrons with a simultaneous, excitation. This radiationless capture, most probably, creates resonance (d) states. These d- states may stabilize by emission of radiation (x-rays). This two-step process is known as Dielectronic recombination (D R). At high incident-electron energy, D R dominates over the radiative recombination (R R) and three- body recombination (Tbr) processes. Thus, D R is one of the most effective recombination for ionisation balance in solar corona and artificial plasma . In addition, the D R rates are needed for the development of nuclear fusion plasma. Thus, D R is still an interesting process m both experimental and theoretical research. Previously we have done the D R rates for 0-like AL 5+ , Cl 9+ Ti 14+ and Zn 22+ ions with K-shell excitation. In this work, the D R rates are calculated for the same ions, but with L-shell excitation. It is found that, the peak values of the D R rates for L-shell excitation are 1000 times larger than that for K-shell excitation. This means that, D R process is the most efficient mechanism for outer-shell excitations. It is found also that, the Dr rates for L-shell excitation peak at Kt = 6 Ry, 22 Ry for AL 5+ and Ti 14+ ions respectively. These Kt values are much smaller than that for for K-shell excitation

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

Science.gov (United States)

de Sousa, Marylane; Manzo, Ricardo M; García, José L; Mammarella, Enrique J; Gonçalves, Luciana R B; Pessela, Benevides C

2017-12-06

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N -His-l-AI and C -His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C -His-l-AI was preferentially hexameric in solution, whereas N -His-l-AI was mainly monomeric. The specific activity of the N -His-l-AI at acidic pH was higher than that of C -His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg -1 , respectively. However, C -His-l-AI was more active and stable at alkaline pH than N -His-l-AI. N -His-l-AI follows a Michaelis-Menten kinetic, whereas C -His-l-AI fitted to a sigmoidal saturation curve.

Chromatographic decontamination of medium-activity waste concentrates

International Nuclear Information System (INIS)

Faubel, W.; Menzler, P.M.; Sameh, A.A.

1988-01-01

The chromatographic decontamination of a MAW concentrate was carried out in a laboratory plant in 1-l-batches in the following way: In order to purify the nitric MAW concentrate from its solid and organic contamination products, it is passed through a filter and an absorber (SM7) for organic species. Subsequently the purified solution runs on-line through all following columns. First the main activity carrier cesium ( 137 Cs, 134 Cs) is transferred to ammonium molybdate phosphate (AMP-1) by means of a newly developed fluidized bed process. In the further course, 125 Sb is separated on metal oxides (Sb 2 O 5 , MnO 2 ) and the three-valued actinides/lanthanides on an extraction-chromatographic CMPO column. Finally the remaining 106 Ru and 60 Co activities are separated on dimethylglyoximes (DMG) coated on active carbon. (orig./RB) [de

Functional reconstitution into liposomes of purified human RhCG ammonia channel.

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Isabelle Mouro-Chanteloup

Full Text Available BACKGROUND: Rh glycoproteins (RhAG, RhBG, RhCG are members of the Amt/Mep/Rh family which facilitate movement of ammonium across plasma membranes. Changes in ammonium transport activity following expression of Rh glycoproteins have been described in different heterologous systems such as yeasts, oocytes and eukaryotic cell lines. However, in these complex systems, a potential contribution of endogenous proteins to this function cannot be excluded. To demonstrate that Rh glycoproteins by themselves transport NH(3, human RhCG was purified to homogeneity and reconstituted into liposomes, giving new insights into its channel functional properties. METHODOLOGY/PRINCIPAL FINDINGS: An HA-tag introduced in the second extracellular loop of RhCG was used to purify to homogeneity the HA-tagged RhCG glycoprotein from detergent-solubilized recombinant HEK293E cells. Electron microscopy analysis of negatively stained purified RhCG-HA revealed, after image processing, homogeneous particles of 9 nm diameter with a trimeric protein structure. Reconstitution was performed with sphingomyelin, phosphatidylcholine and phosphatidic acid lipids in the presence of the C(12E(8 detergent which was subsequently removed by Biobeads. Control of protein incorporation was carried out by freeze-fracture electron microscopy. Particle density in liposomes was a function of the Lipid/Protein ratio. When compared to empty liposomes, ammonium permeability was increased two and three fold in RhCG-proteoliposomes, depending on the Lipid/Protein ratio (1/300 and 1/150, respectively. This strong NH(3 transport was reversibly inhibited by mercuric and copper salts and exhibited a low Arrhenius activation energy. CONCLUSIONS/SIGNIFICANCE: This study allowed the determination of ammonia permeability per RhCG monomer, showing that the apparent Punit(NH3 (around 1x10(-3 microm(3xs(-1 is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60x10

Effectiveness of air purifier on health outcomes and indoor particles in homes of children with allergic diseases in Fresno, California: A pilot study.

Science.gov (United States)

Park, Hye-Kyung; Cheng, Kai-Chung; Tetteh, Afua O; Hildemann, Lynn M; Nadeau, Kari C

2017-05-01

Epidemiologic studies indicate that indoor air pollution is correlated with morbidity caused by allergic diseases. We evaluated the effectiveness of reducing the levels of indoor fine particulate matter <2.5 micrometer diameter (PM 2.5 ) in Fresno, California using air purifiers on health outcomes in children with asthma and/or allergic rhinitis. The active group (with air purifiers) and the control group consisted of eight houses each. Air purifiers were installed in the living rooms and bedrooms of the subjects in the active group during the entire 12-week study duration. Childhood asthma control test, peak flow rate monitoring, and nasal symptom scores were evaluated at weeks 0, 6, and 12. At 12 weeks, the active group showed a trend toward an improvement of childhood asthma control test scores and mean evening peak flow rates, whereas the control group showed deterioration in the same measures. Total and daytime nasal symptoms scores significantly reduced in the active group (p = 0.001 and p = 0.011, respectively). The average indoor PM 2.5 concentrations reduced by 43% (7.42 to 4.28 μg/m 3 ) in the active group (p = 0.001). Intervention with air purifiers reduces indoor PM 2.5 levels with significant improvements in nasal symptoms in children with allergic rhinitis in Fresno.

Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

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Wagner A S Judice

Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

Peroxidase-like activity of magnetoferritin

Czech Academy of Sciences Publication Activity Database

Melníková, V.; Pospíšková, K.; Mitróová, Z.; Kopčanský, P.; Šafařík, Ivo

2014-01-01

Roč. 181, 3-4 (2014), s. 295-301 ISSN 0026-3672 R&D Projects: GA MŠk(CZ) LD13021 Institutional support: RVO:67179843 Keywords : magnetoferritin * magnetic nanoparticles * peroxidase-like activity * hydrogen peroxide * oxidative stress Subject RIV: CE - Biochemistry Impact factor: 3.741, year: 2014

Impossibility criterion for obtaining pure entangled states from mixed states by purifying protocols

International Nuclear Information System (INIS)

Chen Pingxing; Liang Linmei; Li Chengzu; Huang Mingqiu

2002-01-01

Purifying noisy entanglement is a protocol that can increase the entanglement of a mixed state (as a source) at the expense of the entanglement of others (such as an ancilla) by collective measurement. A protocol with which one can get a pure entangled state from a mixed state is defined as purifying mixed states. We address a basic question: can one get a pure entangled state from a mixed state? We give a necessary and sufficient condition of purifying a mixed state by fit local operations and classical communication and show that for a class of source states and ancilla states in arbitrary bipartite systems purifying mixed states is impossible by finite rounds of purifying protocols. For 2x2 systems, it is proved that arbitrary states cannot be purified by individual measurement. The possible application and meaning of the conclusion are discussed

Studies on screening, isolation and purification of a fibrinolytic protease from an isolate (VK12) of Ganoderma lucidum and evaluation of its antithrombotic activity.

Science.gov (United States)

Kumaran, Sekar; Palani, Perumal; Nishanthi, Ramasami; Kaviyarasan, Venkatesan

2011-01-01

Antithrombotic activity of a protease purified from a medicinal mushroom, Ganoderma lucidum, has been evaluated platelet aggregation in vitro and pulmonary thrombosis in vivo. The purified protease exhibited concentration dependent inhibitory effects on platelet aggregation induced by ADP (adenosine diphosphate), with an IC(50) value of 2.4 mg/mL. The purified protease protected mice against thrombotic death or paralysis induced by collagen and epinephrine in a dose dependent manner when administered orally. It produced a significant inhibition of thrombotic death or paralysis at 60 µg/kg body weight, while aspirin produced a significant inhibition of thrombosis at 10-20 mg/kg body weight. The purified protease also has showed fibrinolytic activity and alters coagulation parameters such as activated partial thromboplastin time (APTT), and thrombin time (TT) in rat platelet. These results suggested that the antithrombotic activity of Ganoderma lucidum protease might be due to antiplatelet activity rather than anticoagulation activity.

Practical screening of purified cellobiohydrolases and endoglucanases with α-cellulose and specification of hydrodynamics

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Jäger Gernot

2010-08-01

Full Text Available Abstract Background It is important to generate biofuels and society must be weaned from its dependency on fossil fuels. In order to produce biofuels, lignocellulose is pretreated and the resulting cellulose is hydrolyzed by cellulases such as cellobiohydrolases (CBH and endoglucanases (EG. Until now, the biofuel industry has usually applied impractical celluloses to screen for cellulases capable of degrading naturally occurring, insoluble cellulose. This study investigates how these cellulases adsorb and hydrolyze insoluble α-cellulose − considered to be a more practical substrate which mimics the alkaline-pretreated biomass used in biorefineries. Moreover, this study investigates how hydrodynamics affects cellulase adsorption and activity onto α-cellulose. Results First, the cellulases CBH I, CBH II, EG I and EG II were purified from Trichoderma reesei and CBH I and EG I were utilized in order to study and model the adsorption isotherms (Langmuir and kinetics (pseudo-first-order. Second, the adsorption kinetics and cellulase activities were studied under different hydrodynamic conditions, including liquid mixing and particle suspension. Third, in order to compare α-cellulose with three typically used celluloses, the exact cellulase activities towards all four substrates were measured. It was found that, using α-cellulose, the adsorption models fitted to the experimental data and yielded parameters comparable to those for filter paper. Moreover, it was determined that higher shaking frequencies clearly improved the adsorption of cellulases onto α-cellulose and thus bolstered their activity. Complete suspension of α-cellulose particles was the optimal operating condition in order to ensure efficient cellulase adsorption and activity. Finally, all four purified cellulases displayed comparable activities only on insoluble α-cellulose. Conclusions α-Cellulose is an excellent substrate to screen for CBHs and EGs. This current investigation

cGMP and NHR signaling co-regulate expression of insulin-like peptides and developmental activation of infective larvae in Strongyloides stercoralis.

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Jonathan D Stoltzfus

2014-07-01

Full Text Available The infectious form of the parasitic nematode Strongyloides stercoralis is a developmentally arrested third-stage larva (L3i, which is morphologically similar to the developmentally arrested dauer larva in the free-living nematode Caenorhabditis elegans. We hypothesize that the molecular pathways regulating C. elegans dauer development also control L3i arrest and activation in S. stercoralis. This study aimed to determine the factors that regulate L3i activation, with a focus on G protein-coupled receptor-mediated regulation of cyclic guanosine monophosphate (cGMP pathway signaling, including its modulation of the insulin/IGF-1-like signaling (IIS pathway. We found that application of the membrane-permeable cGMP analog 8-bromo-cGMP potently activated development of S. stercoralis L3i, as measured by resumption of feeding, with 85.1 ± 2.2% of L3i feeding in 200 µM 8-bromo-cGMP in comparison to 0.6 ± 0.3% in the buffer diluent. Utilizing RNAseq, we examined L3i stimulated with DMEM, 8-bromo-cGMP, or the DAF-12 nuclear hormone receptor (NHR ligand Δ7-dafachronic acid (DA--a signaling pathway downstream of IIS in C. elegans. L3i stimulated with 8-bromo-cGMP up-regulated transcripts of the putative agonistic insulin-like peptide (ILP -encoding genes Ss-ilp-1 (20-fold and Ss-ilp-6 (11-fold in comparison to controls without stimulation. Surprisingly, we found that Δ7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM Δ7-DA-mediated activation (93.3 ± 1.1% L3i feeding can be blocked using this IIS inhibitor at 100 µM (7.6 ± 1.6% L3i feeding. To determine the tissues where promoters of ILP-encoding genes are active, we expressed promoter::egfp reporter constructs in transgenic S. stercoralis post-free-living larvae. Ss-ilp-1 and Ss-ilp-6 promoters are active in the hypodermis and neurons and the Ss-ilp-7 promoter is active in the

Mosquito adulticidal activity of the leaf extracts of Spondias mombin L. against Aedes aegypti L. and isolation of active principles.

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Ajaegbu, Elijah Eze; Danga, Simon Pierre Yinyang; Chijoke, Ikemefuna Uzochukwu; Okoye, Festus Basden Chiedu

2016-03-01

Aedes aegypti is a domestic mosquito and one of the primary vectors for dengue and yellow fever. Since, it is a vector of deadly diseases, its control becomes essential. Medicinal plants may be an alternative to adulticidal agents since they contain rich source of bioactive compounds. This study was designed to determine the adulticidal activity of Spondias mombin leaf methanol crude extract, n-hexane, dichloromethane and ethyl acetate fractions against female adults of Ae. aegypti mosquitoes and isolate active compound(s) responsible for the bioactivity. All leaf extract and fractions were evaluated for adulticidal activity against Ae. aegypti mosquitoes. Adult mortality was observed after 24 h of exposure. The dichloromethane fraction was further purified being the most active fraction using silica gel column chromatography and the active compounds were identified with the aid of HPLC and LC-ESI-MS/MS. The LC50 and LC90 were determined by Probit analysis. Dichloromethane fraction was the most effective fraction with LC50 value of 2172.815 μg/ml. Compounds identified were mainly ellagic acid and 1-O-Galloyl-6-O-luteoyl-α-D-glucose. The S. mombin leaf extracts and fractions proved to be a strong candidate for a natural, safe and stable adulticide, alternative to synthetic adulticide.

Purification and characterization of xylitol dehydrogenase with l-arabitol dehydrogenase activity from the newly isolated pentose-fermenting yeast Meyerozyma caribbica 5XY2.

Science.gov (United States)

Sukpipat, Wiphat; Komeda, Hidenobu; Prasertsan, Poonsuk; Asano, Yasuhisa

2017-01-01

Meyerozyma caribbica strain 5XY2, which was isolated from an alcohol fermentation starter in Thailand, was found to catabolize l-arabinose as well as d-glucose and d-xylose. The highest production amounts of ethanol from d-glucose, xylitol from d-xylose, and l-arabitol from l-arabinose were 0.45 g/g d-glucose, 0.60 g/g d-xylose, and 0.61 g/g l-arabinose with 21.7 g/L ethanol, 20.2 g/L xylitol, and 30.3 g/l l-arabitol, respectively. The enzyme with l-arabitol dehydrogenase (LAD) activity was purified from the strain and found to exhibit broad specificity to polyols, such as xylitol, d-sorbitol, ribitol, and l-arabitol. Xylitol was the preferred substrate with K m =16.1 mM and k cat /K m =67.0 min -1 mM -1 , while l-arabitol was also a substrate for the enzyme with K m =31.1 mM and k cat /K m =6.5 min -1  mM -1 . Therefore, this enzyme from M. caribbica was named xylitol dehydrogenase (McXDH). McXDH had an optimum temperature and pH at 40°C and 9.5, respectively. The McXDH gene included a coding sequence of 1086 bp encoding a putative 362 amino acid protein of 39 kDa with an apparent homopentamer structure. Native McXDH and recombinant McXDH exhibited relative activities toward l-arabitol of approximately 20% that toward xylitol, suggesting the applicability of this enzyme with the functions of XDH and LAD to the development of pentose-fermenting Saccharomyces cerevisiae. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

Science.gov (United States)

Kwon, Chang Woo; Yang, Hee; Yeo, SuBin; Park, Kyung-Min; Jeong, Ae Jin; Lee, Ki Won; Ye, Sang-Kyu; Chang, Pahn-Shick

2018-12-01

Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar K i value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).

Heterologous expression and characterization of Bacillus coagulans L-arabinose isomerase.

Science.gov (United States)

Zhou, Xingding; Wu, Jin Chuan

2012-05-01

Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure L: -lactic acid from both hexose and pentose sugars including L: -arabinose with high yield, titer and productivity under thermophilic conditions. The L: -arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn(2+) was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic pH than at alkaline pH. The kinetic studies showed that the K (m), V (max) and k (cat)/K (m) for the conversion of L: -arabinose were 106 mM, 84 U/mg and 34.5 mM(-1)min(-1), respectively. The equilibrium ratio of L: -arabinose to L: -ribulose was 78:22 under optimal conditions. L: -ribulose (97 g/L) was obtained from 500 g/l of L: -arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion of 19.4% and a production rate of 65 g L(-1) h(-1).

l-Cysteine-Assisted Synthesis of Urchin-Like γ-MnS and Its Lithium Storage Properties

Science.gov (United States)

Xu, Dan; Jiao, Ranran; Sun, Yuanwei; Sun, Dezhi; Zhang, Xianxi; Zeng, Suyuan; Di, Youying

2016-10-01

MnS has been attracting more and more attentions in the fields of lithium ion batteries (LIBs) because of its high energy density and low voltage potential. In this paper, we present a simple method for the preparation of urchin-like γ-MnS microstructures using l-cysteine and MnCl2 · 4H2O as the starting materials. The urchin-like γ-MnS microstructures exhibit excellent cycling stability (823.4 mA h g-1 at a current density of 500 mA g-1, after 1000 cycles). And the discharge voltage is about 0.75 V, making it a good candidate for the application as the anode material in LIBs. SEM, TEM, and XRD were employed to inspect the changes of the active materials during the electrochemical process, which clearly indicate that the structural pulverization and reformation of the γ-MnS microstructures play important roles for the maintenance of the electrochemical performance during the charge/discharge process.

Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

Science.gov (United States)

Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

2012-09-01

Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

Apoptotic cell death through inhibition of protein kinase CKII activity by 3,4-dihydroxybenzaldehyde purified from Xanthium strumarium.

Science.gov (United States)

Lee, Bang Hyo; Yoon, Soo-Hyun; Kim, Yun-Sook; Kim, Sang Kook; Moon, Byong Jo; Bae, Young-Seuk

2008-01-01

The CKII inhibitory compound was purified from the fruit of Xanthium strumarium by organic solvent extraction and silica gel chromatography. The inhibitory compound was identified as 3,4-dihydroxybenzaldehyde by analysis with FT-IR, FAB-Mass, EI-Mass, (1)H-NMR and (13)C-NMR. 3,4-dihydroxybenzaldehyde inhibited the phosphotransferase activity of CKII with IC(50) of about 783 microM. Steady-state studies revealed that the inhibitor acts as a competitive inhibitor with respect to the substrate ATP. A value of 138.6 microM was obtained for the apparent K(i). Concentration of 300 microM 3,4-dihydroxybenzaldehyde caused 50% growth inhibition of human cancer cell U937. 3,4-dihydroxybenzaldehyde-induced cell death was characterised with the cleavage of poly(ADP-ribose) polymerase and procaspase-3. Furthermore, the inhibitor induced the fragmentation of DNA into multiples of 180 bp, indicating that it triggered apoptosis. This induction of apoptosis by 3,4-dihydroxybenzaldehyde was also confirmed by using flow cytometry analysis. Since CKII is involved in cell proliferation and oncogenesis, these results suggest that 3,4-dihydroxybenzaldehyde may function by inhibiting oncogenic disease, at least in part, through the inhibition of CKII activity.

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel.

Science.gov (United States)

Yang, C P; Fujita, S; Kohno, K; Kusubayashi, A; Ashrafuzzaman, M; Hayashi, N

2001-03-01

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Science.gov (United States)

2011-01-01

Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we reported the purification and the

The acid-tolerant L-arabinose isomerase from the mesophilic Shewanella sp. ANA-3 is highly active at low temperatures

Directory of Open Access Journals (Sweden)

Rhimi Moez

2011-11-01

Full Text Available Abstract Background L-arabinose isomerases catalyse the isomerization of L-arabinose into L-ribulose at insight biological systems. At industrial scale of this enzyme is used for the bioconversion of D-galactose into D-tagatose which has many applications in pharmaceutical and agro-food industries. The isomerization reaction is thermodynamically equilibrated, and therefore the bioconversion rates is shifted towards tagatose when the temperature is increased. Moreover, to prevent secondary reactions it will be of interest to operate at low pH. The profitability of this D-tagatose production process is mainly related to the use of lactose as cheaper raw material. In many dairy products it will be interesting to produce D-tagatose during storage. This requires an efficient L-arabinose isomerase acting at low temperature and pH values. Results The gene encoding the L-arabinose isomerase from Shewanella sp. ANA-3 was cloned and overexpressed in Escherichia coli. The purified protein has a tetrameric arrangement composed by four identical 55 kDa subunits. The biochemical characterization of this enzyme showed that it was distinguishable by its maximal activity at low temperatures comprised between 15-35°C. Interestingly, this biocatalyst preserves more than 85% of its activity in a broad range of temperatures from 4.0 to 45°C. Shewanella sp. ANA-3 L-arabinose isomerase was also optimally active at pH 5.5-6.5 and maintained over 80% of its activity at large pH values from 4.0 to 8.5. Furthermore, this enzyme exhibited a weak requirement for metallic ions for its activity evaluated at 0.6 mM Mn2+. Stability studies showed that this protein is highly stable mainly at low temperature and pH values. Remarkably, T268K mutation clearly enhances the enzyme stability at low pH values. Use of this L-arabinose isomerase for D-tagatose production allows the achievement of attractive bioconversion rates of 16% at 4°C and 34% at 35°C. Conclusions Here we

Influence of population diversity on neurovirulence potential of plaque purified L-Zagreb variants.

Science.gov (United States)

Ivancic-Jelecki, Jelena; Forcic, Dubravko; Jagusic, Maja; Kosutic-Gulija, Tanja; Mazuran, Renata; Balija, Maja Lang; Isakov, Ofer; Shomron, Noam

2016-04-29

Despite continuing research efforts, determinants of mumps virus virulence are still largely unknown. One of consequences of this is difficulty in striking a balance between efficacy and safety of live attenuated mumps vaccines. Among mumps vaccine strains associated with occurrence of postvaccinal aseptic meningitis is L-Zagreb, developed by further attenuation of vaccine strain L-3. Starting from an archived L-Zagreb sample with suboptimal neuroattenuation score, we isolated different viral variants and compared their genetic and phenotypic properties, in investigation of neurovirulence markers. Six different L-Zagreb variants were isolated by plaque purification. Their neurovirulent status was determined by rat-based neurovirulence test; population structure was determined by deep sequencing. We isolated one well neuroattenuated viral variant, two marginally neuroattenuated, and three insufficiently neuroattenuated. No genetic markers of neurovirulence could be identified. None of variants had detectable amounts of defective interfering particles. Two characteristics set insufficiently neuroattenuated variants apart from less-neurovirulent ones: elevated variability level in regions 1293-3314, 5363-7773 and 9382-11657, and/or elevated number of mutations present in frequencies ≥ 1%. The most neurovirulent variants possessed both of these features. Distinctive heterogeneity profiles were obtained for insufficiently neuroattenuated L-Zagreb variants. No markers that would discriminate between marginally and well neuroattenuated variants were identified. The findings of this study may serve as a guideline during development of an improved L3/L-Zagreb vaccine strain. Copyright © 2016 Elsevier Ltd. All rights reserved.

Frequency dependence of p-mode frequency shifts induced by magnetic activity in Kepler solar-like stars

Science.gov (United States)

Salabert, D.; Régulo, C.; Pérez Hernández, F.; García, R. A.

2018-04-01

The variations of the frequencies of the low-degree acoustic oscillations in the Sun induced by magnetic activity show a dependence on radial order. The frequency shifts are observed to increase towards higher-order modes to reach a maximum of about 0.8 μHz over the 11-yr solar cycle. A comparable frequency dependence is also measured in two other main sequence solar-like stars, the F-star HD 49933, and the young 1 Gyr-old solar analog KIC 10644253, although with different amplitudes of the shifts of about 2 μHz and 0.5 μHz, respectively. Our objective here is to extend this analysis to stars with different masses, metallicities, and evolutionary stages. From an initial set of 87 Kepler solar-like oscillating stars with known individual p-mode frequencies, we identify five stars showing frequency shifts that can be considered reliable using selection criteria based on Monte Carlo simulations and on the photospheric magnetic activity proxy Sph. The frequency dependence of the frequency shifts of four of these stars could be measured for the l = 0 and l = 1 modes individually. Given the quality of the data, the results could indicate that a physical source of perturbation different from that in the Sun is dominating in this sample of solar-like stars.

Gastroprotective effects of arctigenin of Arctium lappa L. on a rat model of gastric ulcers

OpenAIRE

Li, Xiao-Mei; Miao, Yu; Su, Qin-Yong; Yao, Jing-Chun; Li, Hong-Hua; Zhang, Gui-Min

2016-01-01

In the present study, the gastroprotective effects of arctigenin of Fructus Arctii were evaluated and the possible underlying mechanisms of action were elucidated. Arctigenin (high-performance liquid chromatography purity, >99.0%) was isolated and purified from the seeds of Arctium lappa L. The anti-ulcerogenic activity of arctigenin against ulcers induced by absolute ethanol and acetic acid was evaluated in a Sprague-Dawley rat model. In addition, the antioxidant activity was assessed by mea...

Spectroscopic characterization of D-003 obtained from the sugar cane (Saccharum officinarum L.) wax

International Nuclear Information System (INIS)

Marrero Delange, David; Cora Medina, Miriam; Laguna Granja, Abilio; Gonzalez Canavaciolo Victor L

2013-01-01

D-003, an active pharmaceutical ingredient (API) purified from sugar cane (Saccharum officinarum L.) wax with cholesterol-lowering and antioxidant effects, is composed of a mixture of free saturated very long chain fatty acids (VLCFAs), each within specific relative concentration ranges as determined by the gas chromatography (GC). However, the spectroscopic characterization of D-003 had not been previously reported

Isolation and Preliminary Characterization of Proteinaceous Toxins with Insecticidal and Antibacterial Activities from Black Widow Spider (L. tredecimguttatus Eggs

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Qian Lei

2015-03-01

Full Text Available The eggs of black widow spider (L. tredecimguttatus have been demonstrated to be rich in toxic proteinaceous components. The study on such active components is of theoretical and practical importance. In the present work, using a combination of multiple biochemical and biological strategies, we isolated and characterized the proteinaceous components from the aqueous extract of the black widow spider eggs. After gel filtration of the egg extract, the resulting main protein and peptide peaks were further fractionated by ion exchange chromatography and reversed-phase high performance liquid chromatography. Two proteinaceous components, named latroeggtoxin-III and latroeggtoxin-IV, respectively, were purified to homogeneity. Latroeggtoxin-III was demonstrated to have a molecular weight of about 36 kDa. Activity analysis indicated that latroeggtoxin-III exhibited neurotoxicity against cockroaches but had no obvious effect on mice, suggesting that it is an insect-specific toxin. Latroeggtoxin-IV, with a molecular weight of 3.6 kDa, was shown to be a broad-spectrum antibacterial peptide, showing inhibitory activity against all five species of bacteria tested, with the highest activity against Staphylococcus aureus. Finally, the implications of the proteinaceous toxins in egg protection and their potential applications were analyzed and discussed.

dlk acts as a negative regulator of Notch1 activation through interactions with specific EGF-like repeats

International Nuclear Information System (INIS)

Baladron, Victoriano; Ruiz-Hidalgo, Maria Jose; Nueda, Maria Luisa; Diaz-Guerra, Maria Jose M.; Garcia-Ramirez, Jose Javier; Bonvini, Ezio; Gubina, Elena; Laborda, Jorge

2005-01-01

The protein dlk, encoded by the Dlk1 gene, belongs to the Notch epidermal growth factor (EGF)-like family of receptors and ligands, which participate in cell fate decisions during development. The molecular mechanisms by which dlk regulates cell differentiation remain unknown. By using the yeast two-hybrid system, we found that dlk interacts with Notch1 in a specific manner. Moreover, by using luciferase as a reporter gene under the control of a CSL/RBP-Jk/CBF-1-dependent promoter in the dlk-negative, Notch1-positive Balb/c 14 cell line, we found that addition of synthetic dlk EGF-like peptides to the culture medium or forced expression of dlk decreases endogenous Notch activity. Furthermore, the expression of the gene Hes-1, a target for Notch1 activation, diminishes in confluent Balb/c14 cells transfected with an expression construct encoding for the extracellular EGF-like region of dlk. The expression of Dlk1 and Notch1 increases in 3T3-L1 cells maintained in a confluent state for several days, which is associated with a concomitant decrease in Hes-1 expression. On the other hand, the decrease of Dlk1 expression in 3T3-L1 cells by antisense cDNA transfection is associated with an increase in Hes-1 expression. These results suggest that dlk functionally interacts in vivo with Notch1, which may lead to the regulation of differentiation processes modulated by Notch1 activation and signaling, including adipogenesis

Characterization of an L-arabinose isomerase from Bacillus thermoglucosidasius for D-tagatose production.

Science.gov (United States)

Seo, Myung-Ji

2013-01-01

L-Arabinose isomerase from Bacillus thermoglucosidasius KCTC 1828 (BTAI) was expressed in Escherichia coli. The optimal temperature and pH for the activity of the purified BTAI were 40 °C and pH 7.0. The Mn(2+) ion was an activator of BTAI activity. The kinetic parameters of BTAI for D-galactose were a K(m) of 175 mM and a k(cat)/K(m) of 2.8 mM(-1)min(-1). The conversion ratio by BTAI to D-tagatose reached 45.6% at 40 °C.

FORMULASI MIKROEMULSI EKSTRAK TERPURIFIKASI DAUN BAYAM MERAH (Amaranthus tricolor L. SEBAGAI SUPLEMEN ANTIOKSIDAN

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Dwi Lestari Handayani

2017-03-01

Full Text Available A research has conducted about the microemulsion formulation of purified extract of red spinach leaves (Amaranthus tricolor L. as an antioxidant supplement with the aim to be able to know the formula to form a microemulsion which meet the physical quality stability and determine antioxidant activity (IC50 of the preparation. Extracts prepared by maceration method using ethanol 96% and then later do the purification using solvent n-hexane and ethyl acetate, after it tested its antioxidant activity. Formula to form a microemulsion purified extract of red spinach leaves clear is to use virgin coconut oil (VCO by 15%, tween 80 for 40%, 35% glycerin and 10% distilled water. Physical stability test was conducted on the organoleptic test, measuring the diameter of globules, pH test, test and test viscosity centrifugation. Test of antioxidant activity in vitro using DPPH method and using Vitamin C as a positive control. Measurement data were statistically analyzed using paired samples T test. The test results of antioxidant activity microemulsion purified extracts of spinach leaves, red show IC50 values on day 1 was 1.83 ppm and the 28th day amounted to 3.71 ppm. While vitamin C microemulsion shows IC50 values on day 1 of 0.24 ppm and the 28th day of 2.51 ppm. Despite the decreased antioxidant activity, but each of the stocks included in the category of very powerful antioxidants.

Effects of purified lignin on rumen metabolism and growth performance of feedlot cattle

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Yuxi Wang

2017-03-01

Full Text Available Objective The objectives were to assess the effects of purified lignin from wheat straw (sodium hydroxide dehydrated lignin; SHDL on in vitro ruminal fermentation and on the growth performance of feedlot cattle. Methods In vitro experiments were conducted by incubating a timothy-alfalfa (50:50 forage mixture (48 h and barley grain (24 h with 0, 0.25, 0.5, 1.0, and 2.0 mg/mL of rumen fluid (equivalent to 0, 2, 4, 8, and 16 g SHDL/kg diet. Productions of CH4 and total gas, volatile fatty acids, ammonia, dry matter (DM disappearance (DMD and digestion of neutral detergent fiber (NDF or starch were measured. Sixty Hereford-Angus cross weaned steer calves were individually fed a typical barley silage-barley grain based total mixed ration and supplemented with SHDL at 0, 4, 8, and 16 g/kg DM for 70 (growing, 28 (transition, and 121 d (finishing period. Cattle were slaughtered at the end of the experiment and carcass traits were assessed. Results With forage, SHDL linearly (p<0.001 reduced 48-h in vitro DMD from 54.9% to 39.2%, NDF disappearance from 34.1% to 18.6% and the acetate: propionate ratio from 2.56 to 2.41, but linearly (p<0.001 increased CH4 production from 9.5 to 12.4 mL/100 mg DMD. With barley grain, SHDL linearly increased (p<0.001 24-h DMD from74.6% to 84.5%, but linearly (p<0.001 reduced CH4 production from 5.6 to 4.2 mL/100 mg DMD and NH3 accumulation from 9.15 to 4.49 μmol/mL. Supplementation of SHDL did not affect growth, but tended (p = 0.10 to linearly reduce feed intake, and quadratically increased (p = 0.059 feed efficiency during the finishing period. Addition of SHDL also tended (p = 0.098 to linearly increase the saleable meat yield of the carcass from 52.5% to 55.7%. Conclusion Purified lignin used as feed additive has potential to improve feed efficiency for finishing feedlot cattle and carcass quality.

Antifungal properties of wheat histones (H1-H4) and purified wheat histone H1

Science.gov (United States)

Wheat (Triticum sp.) histones H1, H2, H3, and H4 were extracted. H1 was further purified. Their activities against fungi with varying degrees of wheat pathogenicity were determined. They included Aspergillus flavus, A. fumigatus, A. niger, F. oxysporum, F. verticillioides, F. solani, F. graminearu...

Antibacterial activity of bacteriocin-like substance P34 on Listeria monocytogenes in chicken sausage

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Voltaire Sant'Anna

2013-12-01

Full Text Available The antimicrobial activity of the bacteriocin-like substance (BLS P34 against Listeria monocytogenes was investigated in chicken sausage. The BLS was applied to chicken sausages (256 AU g-1 previously inoculated with a suspension of 10² cfu g-1 of L. monocytogenes. BLS P34 inhibited the indicator microorganism in situ in all incubation times for up to 10 days at 5 °C. The effectiveness of BLS P34 was increased when it was added in combination with nisin. The bacteriocin was also tested in natural eatable natural bovine wrapping (salty semi-dried tripe against the same indicator microorganism, also showing inhibitory capability in vitro. BLS P34 showed potential to control L. monocytogenes in refrigerated meat products.

Metabolic control analysis of Aspergillus niger L-arabinose catabolism

NARCIS (Netherlands)

Groot, de M.J.L.; Prathumpai, W.; Visser, J.; Ruijter, G.J.G.

2005-01-01

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their

Lung injury-dependent oxidative status and chymotrypsin-like activity of skeletal muscles in hamsters with experimental emphysema.

Science.gov (United States)

Tonon, Jair; Cecchini, Alessandra Lourenço; Brunnquell, Cláudia Roberta; Bernardes, Sara Santos; Cecchini, Rubens; Guarnier, Flávia Alessandra

2013-01-23

Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD). Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner. We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20) and 40 mg/mL (E40) papain. The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p < 0.05) and E40 (52.27 ± 4.7; crossed the alveolar intercepts) groups. As compared to the control group, there was a reduction on body (171.6 ± 15.9 g) and muscle weight (251.87 ± 24.87 mg) in the E20 group (157.5 ± 10.3 mg and 230.12 ± 23.52 mg, for body and muscle weight, respectively), which was accentuated in the E40 group (137.4 ± 7.2 g and 197.87 ± 10.49 mg, for body and muscle weight, respectively). Additionally, the thiobarbituric acid reactive substances (TBARS), tert-butyl hydroperoxide-initiated chemiluminescence (CL), carbonylated proteins, and chymotrypsin-like proteolytic activity were elevated in the E40 group as compared to the E20 group (p < 0.05 for all comparisons). The severity of emphysema significantly correlated with the progressive increase in CL (r = -0.95), TBARS (r = -0.98), carbonyl proteins (r = -0.99), and chymotrypsin-like proteolytic activity (r = -0.90). Furthermore, augmentation of proteolytic activity correlated significantly with CL (r = 0.97), TBARS (r = 0.96), and carbonyl proteins (r = 0.91). Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of

Home drinking-water purifiers

International Nuclear Information System (INIS)

Pizzichini, Massimo; Pozio, Alfonso; Russo, Claudio

2005-01-01

To salve the widespread problem of contaminated drinking water, home purifiers are now sold in Italy as well as other countries. This article describes how these devices work, how safe they are to use and how safe the water they produce, in the broad context of regulations on drinking water and mineral water. A new device being developed by ENEA to treat municipal water and ground water could provide greater chemical and bacteriological safety. However, the appearance of these new systems makes it necessary to update existing regulations [it

Glutamine-Elicited Secretion of Glucagon-Like Peptide 1 Is Governed by an Activated Glutamate Dehydrogenase.

Science.gov (United States)

Andersson, Lotta E; Shcherbina, Liliya; Al-Majdoub, Mahmoud; Vishnu, Neelanjan; Arroyo, Claudia Balderas; Aste Carrara, Jonathan; Wollheim, Claes B; Fex, Malin; Mulder, Hindrik; Wierup, Nils; Spégel, Peter

2018-03-01

Glucagon-like peptide 1 (GLP-1), secreted from intestinal L cells, glucose dependently stimulates insulin secretion from β-cells. This glucose dependence prevents hypoglycemia, rendering GLP-1 analogs a useful and safe treatment modality in type 2 diabetes. Although the amino acid glutamine is a potent elicitor of GLP-1 secretion, the responsible mechanism remains unclear. We investigated how GLP-1 secretion is metabolically coupled in L cells (GLUTag) and in vivo in mice using the insulin-secreting cell line INS-1 832/13 as reference. A membrane-permeable glutamate analog (dimethylglutamate [DMG]), acting downstream of electrogenic transporters, elicited similar alterations in metabolism as glutamine in both cell lines. Both DMG and glutamine alone elicited GLP-1 secretion in GLUTag cells and in vivo, whereas activation of glutamate dehydrogenase (GDH) was required to stimulate insulin secretion from INS-1 832/13 cells. Pharmacological inhibition in vivo of GDH blocked secretion of GLP-1 in response to DMG. In conclusion, our results suggest that nonelectrogenic nutrient uptake and metabolism play an important role in L cell stimulus-secretion coupling. Metabolism of glutamine and related analogs by GDH in the L cell may explain why GLP-1 secretion, but not that of insulin, is activated by these secretagogues in vivo. © 2017 by the American Diabetes Association.

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain.

Science.gov (United States)

Hörnig, Christina; Albert, Dana; Fischer, Lutz; Hörnig, Michael; Rådmark, Olof; Steinhilber, Dieter; Werz, Oliver

2005-07-22

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.

Characterization of the molluscicidal activity of Bauhinia variegata and Mimusops elengi plant extracts against the fasciola vector Lymnaea acuminata.

Science.gov (United States)

Singh, Kanchan Lata; Singh, D K; Singh, Vinay Kumar

2012-01-01

The molluscicidal activity of Bauhinia variegata leaf and Mimusops elengi bark was studied against vector snail Lymnaea acuminata. The toxicity of both plants was time and concentration-dependent. Among organic extracts, ethanol extracts of both plants were more toxic. Toxicity of B. variegata leaf ethanolic extract (96h LC50- 14.4 mg/L) was more pronounced than M. elengi bark ethanolic extract (96h LC50-15.0 mg/L). The 24h LC50 of column purified fraction of B. variegata and M. elengi bark were 20.3 mg/L and 18.3 mg/L, respectively. Saponin and quercetin were characterized and identified as active molluscicidal component. Co-migration of saponin (Rf 0.48) and quercetin (Rf 0.52) with column purified bark of M. elengi and leaf of B. variegata on thin layer chromatography demonstrate same Rf value i.e. 0.48 and 0.52, respectively. The present study clearly indicates the possibility of using M. elengi and/or B. variegata as potent molluscicide.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis

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Marylane de Sousa

2017-12-01

Full Text Available l-Arabinose isomerase (EC 5.3.1.4 (l-AI from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg−1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Spectroscopic characterization of Simultaneous determination of Albendazol from the sugar cane (Saccharum officinarum L.) wax

International Nuclear Information System (INIS)

Marrero Delange, David; Cora Medina, Miriam; Laguna Granja, Abilio; Gonzalez Canavaciolo, Victor L

2013-01-01

D-003, an active pharmaceutical ingredient (API) purified from sugar cane (Saccharum officinarum L.) wax with cholesterol-lowering and antioxidant effects, is composed of a mixture of free saturated very long chain fatty acids (VLCFAs), each within specific relative concentration ranges as determined by the gas chromatography (GC). However, the spectroscopic characterization of D-003 had not been previously reported

A conserved residue of l-alanine dehydrogenase from Bacillus pseudofirmus, Lys-73, participates in the catalytic reaction through hydrogen bonding.

Science.gov (United States)

He, Guangzheng; Xu, Shujing; Wang, Shanshan; Zhang, Qing; Liu, Dong; Chen, Yuling; Ju, Jiansong; Zhao, Baohua

2018-03-01

A multiple protein sequence alignment of l-alanine dehydrogenases from different bacterial species revealed that five highly conserved amino acid residues Arg-15, Lys-73, Lys-75, His-96 and Asp-269 are potential catalytic residues of l-alanine dehydrogenase from Bacillus pseudofirmus OF4. In this study, recombinant OF4Ald and its mutants of five conserved residues were constructed, expressed in Escherichia coli, purified by His 6 -tag affinity column and gel filtration chromatography, structure homology modeling, and characterized. The purified protein OF4Ald displayed high specificity to l-alanine (15Umg -1 ) with an optimal temperature and pH of 40°C and 10.5, respectively. Enzymatic assay and activity staining in native gels showed that mutations at four conserved residue Arg-15, Lys-75, His-96 and Asp-269 (except residue Lys-73) resulted in a complete loss in enzymatic activity, which signified that these predicted active sites are indispensable for OF4Ald activity. In contrast, the mutant K73A resulted in 6-fold improvement in k cat /K m towards l-alanine as compared to the wild type protein. Further research of the residue Lys-73 substituted by various amino acids and structural modeling revealed that residue Lys-73 might be involved in the catalytic reaction of the enzyme by influencing the enzyme-substrate binding through the hydrogen-bonding interaction with conserved residue Lys-75. Copyright © 2017 Elsevier Inc. All rights reserved.

In vivo behavior of detergent-solubilized purified rabbit thrombomodulin on intravenous injection into rabbits

International Nuclear Information System (INIS)

Ehrlich, H.J.; Esmon, N.L.; Bang, N.U.

1990-01-01

Thrombomodulin is a thrombin endothelial cell membrane receptor. The thrombomodulin-thrombin complex rapidly activates protein C resulting in anticoagulant activity. We investigated the anticoagulant effects and pharmacokinetic behavior of detergent-solubilized purified rabbit thrombomodulin labeled with iodine 125 when intravenously injected into rabbits. Thrombomodulin half-life (t1/2) was determined by tracking the 125I-radiolabeled protein and the biologic activity as determined by the prolongation of the activated partial thromboplastin time (APTT) and thrombin clotting time (TCT). When 200 micrograms/kg 125I-thrombomodulin was injected into rabbits, the APTT and TCT were immediately prolonged, whereas no effect on the prothrombin time was seen. In vitro calibration curves enabled us to convert the prolongations of the clotting times into micrograms per milliliter thrombomodulin equivalents. The best fit (r greater than 0.99) for the disappearance curves was provided by a two-compartment model with mean t1/2 alpha (distribution phase) of 18 minutes for 125I, 12 minutes for APTT, and 20 minutes for TCT, and mean t1/2 beta (elimination phase) of 385 minutes for 125I, 460 for APTT, and 179 for TCT. The administration of two doses of endotoxin (50 micrograms/kg) 24 hours apart did not accelerate the turnover rate of 125I-thrombomodulin as measured by the disappearance of 125I from the circulation. Thus, detergent-solubilized purified thrombomodulin administered intravenously circulates in a biologically active form for appreciable time periods

Anti-inflammatory activity of hydroalcoholic extracts of Lavandula dentata L. and Lavandula stoechas L.

Science.gov (United States)

Algieri, Francesca; Rodriguez-Nogales, Alba; Vezza, Teresa; Garrido-Mesa, Jose; Garrido-Mesa, Natividad; Utrilla, M Pilar; González-Tejero, M Reyes; Casares-Porcel, Manuel; Molero-Mesa, Joaquin; Del Mar Contreras, Maria; Segura-Carretero, Antonio; Pérez-Palacio, José; Diaz, Caridad; Vergara, Noemí; Vicente, Francisca; Rodriguez-Cabezas, M Elena; Galvez, Julio

2016-08-22

Plants from genus Lavandula have been used as anti-inflammatory drugs in Mediterranean traditional medicine. Nowadays, there is a growing interest for complementary medicine, including herbal remedies, to treat inflammatory bowel disease (IBD). To test the anti-inflammatory properties of Lavandula dentata and Lavandula stoechas extracts in two inflammatory experimental models: TNBS model of rat colitis and the carrageenan-induced paw edema in mice, in order to mimic the intestinal conditions and the extra-intestinal manifestations of human IBD, respectively. The extracts were characterized through the qualitative HPLC analysis. Then, they were assayed in vitro and in vivo. In vitro studies were performed in BMDMs and CMT-93 epithelial cells with different concentrations of the extracts (ranging from 0.1 to 100µg/ml). The extracts were tested in vivo in the TNBS model of rat colitis (10 and 25mg/kg) and in the carrageenan-induced paw edema in mice (10, 25 and 100mg/kg). L. dentata and L. stoechas extracts displayed immunomodulatory properties in vitro down-regulating different mediators of inflammation like cytokines and nitric oxide. They also showed anti-inflammatory effects in the TNBS model of colitis as evidenced by reduced myeloperoxidase activity and increased total glutathione content, indicating a decrease of neutrophil infiltration and an improvement of the oxidative state. Besides, both extracts modulated the expression of pro-inflammatory cytokines and chemokines, and ameliorated the altered epithelial barrier function. They also displayed anti-inflammatory effects in the carrageenan-induced paw edema in mice, since a significant reduction of the paw thickness was observed. This was associated with a down-regulation of the expression of different inducible enzymes like MMP-9, iNOS and COX-2 and pro-inflammatory cytokines, all involved in the maintenance of the inflammatory condition. L. dentata and L. stoechas extracts showed intestinal anti

Robust sparse image reconstruction of radio interferometric observations with PURIFY

Science.gov (United States)

Pratley, Luke; McEwen, Jason D.; d'Avezac, Mayeul; Carrillo, Rafael E.; Onose, Alexandru; Wiaux, Yves

2018-01-01

Next-generation radio interferometers, such as the Square Kilometre Array, will revolutionize our understanding of the Universe through their unprecedented sensitivity and resolution. However, to realize these goals significant challenges in image and data processing need to be overcome. The standard methods in radio interferometry for reconstructing images, such as CLEAN, have served the community well over the last few decades and have survived largely because they are pragmatic. However, they produce reconstructed interferometric images that are limited in quality and scalability for big data. In this work, we apply and evaluate alternative interferometric reconstruction methods that make use of state-of-the-art sparse image reconstruction algorithms motivated by compressive sensing, which have been implemented in the PURIFY software package. In particular, we implement and apply the proximal alternating direction method of multipliers algorithm presented in a recent article. First, we assess the impact of the interpolation kernel used to perform gridding and degridding on sparse image reconstruction. We find that the Kaiser-Bessel interpolation kernel performs as well as prolate spheroidal wave functions while providing a computational saving and an analytic form. Secondly, we apply PURIFY to real interferometric observations from the Very Large Array and the Australia Telescope Compact Array and find that images recovered by PURIFY are of higher quality than those recovered by CLEAN. Thirdly, we discuss how PURIFY reconstructions exhibit additional advantages over those recovered by CLEAN. The latest version of PURIFY, with developments presented in this work, is made publicly available.

Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

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Michael P. Greenwood

2017-12-01

Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

Staphylococcal aureus Enterotoxin C and Enterotoxin-Like L Associated with Post-partum Mastitis

DEFF Research Database (Denmark)

Franck, Kristina T; Gumpert, Heidi; Olesen, Bente

2017-01-01

between isolates from the two outbreaks revealed a S. aureus pathogenicity island containing enterotoxin C and enterotoxin-like L only in isolates from outbreak 2. Enterotoxin C and enterotoxin-like L carrying S. aureus are associated with bovine mastitis and our findings indicate that these may also...... PVL and ACME negative. In outbreak 1, the isolates harbored SCCmec IVa and in outbreak 2 SCCmec V. The clinical presentation differed between the two outbreaks, as none of five MRSA positive mothers in outbreak 1 had mastitis vs. five of six MRSA positive mothers in outbreak 2 (p ....02). To investigate if whole-genome sequencing could identify virulence genes associated with mastitis, t015:ST45 isolates from Denmark (N = 101) were whole-genome sequenced. Sequence analysis confirmed two separate outbreaks with no sign of sustained spread into the community. Analysis of the accessory genome...

Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from 'Marcha'.

Science.gov (United States)

Sharma, Nivedita; Gupta, Anupama; Gautam, Neha

2014-01-01

In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative.

Adenosine triphosphate stimulates Aquifex aeolicus MutL endonuclease activity.

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Jerome Mauris

2009-09-01

Full Text Available Human PMS2 (hPMS2 homologues act to nick 5' and 3' to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn(++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X(2E(X(4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.We examined the effect ATP had on the Mn(++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6+/-0.08x10(-5 s(-1 and 4.2+/-0.3x10(-5 s(-1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X(2E(X(4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.ATP stimulated the Mn(++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X(2E(X(4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn(++ induced nicking activity.

Metabolism of rutin and poncirin by human intestinal microbiota and cloning of their metabolizing α-L-rhamnosidase from Bifidobacterium dentium.

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Bang, Seo-Hyeon; Hyun, Yang-Jin; Shim, Juwon; Hong, Sung-Woon; Kim, Dong-Hyun

2015-01-01

To understand the metabolism of flavonoid rhamnoglycosides by human intestinal microbiota, we measured the metabolic activity of rutin and poncirin (distributed in many functional foods and herbal medicine) by 100 human stool specimens. The average α-Lrhamnosidase activities on the p-nitrophenyl-α-L-rhamnopyranoside, rutin, and poncirin subtrates were 0.10 ± 0.07, 0.25 ± 0.08, and 0.15 ± 0.09 pmol/min/mg, respectively. To investigate the enzymatic properties, α-L-rhamnosidase-producing bacteria were isolated from the specimens, and the α-L-rhamnosidase gene was cloned from a selected organism, Bifidobacterium dentium, and expressed in E. coli. The cloned α-L-rhamnosidase gene contained a 2,673 bp sequcence encoding 890 amino acid residues. The cloned gene was expressed using the pET 26b(+) vector in E. coli BL21, and the expressed enzyme was purified using Ni(2+)-NTA and Q-HP column chromatography. The specific activity of the purified α-L-rhamnosidase was 23.3 μmol/min/mg. Of the tested natural product constituents, the cloned α-L-rhamnosidase hydrolyzed rutin most potently, followed by poncirin, naringin, and ginsenoside Re. However, it was unable to hydrolyze quercitrin. This is the first report describing the cloning, expression, and characterization of α-L-rhamnosidase, a flavonoid rhamnoglycosidemetabolizing enzyme, from bifidobacteria. Based on these findings, the α-L-rhamnosidase of intestinal bacteria such as B. dentium seem to be more effective in hydrolyzing (1-->6) bonds than (1-->2) bonds of rhamnoglycosides, and may play an important role in the metabolism and pharmacological effect of rhamnoglycosides.

Optimization, purification and characterization of recombinant L ...

African Journals Online (AJOL)

We studied optimal L-asparaginase sequence from GenBank accession number X12746 encoding for Lasparaginase from Erwinia chrysanthemi NCPPB1125. The expression level of recombinant Lasparaginase was determined as 78% of the total proteins. The purified L-asparaginase had a molecular mass of 37 kDa with ...

Exopolysaccharides from Pleurotus pulmonarius: Fermentation Optimization, Characterization and Antioxidant Activity

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Jin-Wen Shen

2013-01-01

Full Text Available Culture conditions for exopolysaccharide (EPS production by Pleurotus pulmonarius in submerged culture are optimized. The suggested medium composition was as follows: 60 g/L of xylose, 6 g/L of soy extract, 5 mM of KH2PO4 and 5 mM of MgSO4. Under the optimized culture conditions in a 5-litre stirred tank fermentor, the maximum concentration of EPS was 6.36 g/L. Furthermore, the morphological parameters (i.e. average diameter, circularity, roughness and compactness of the pellets and the broth viscosity are characterized. It has been proven that mycelial morphology and broth viscosity may be the critical parameters affecting the EPS yield. After deproteinization using Sevag method, a group of EPS (designated as fraction was obtained from the culture filtrates by gel filtration chromatography. FT-IR analysis of the purified EPS revealed prominent characteristic groups corresponding to polyhydric alcohols. GC analysis showed that the purified EPS were mainly composed of galactose and glucose. Furthermore, thermogravimetric analysis indicated that the degradation temperature of the purified EPS was 217 °C. Finally, the antioxidant activity of the EPS fraction was investigated and the relationship with molecular properties was discussed as well.

Immunomodulatory Effects of the Mycosporine-Like Amino Acids Shinorine and Porphyra-334

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Kathrin Becker

2016-06-01

Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porphyra sp. using chromatographic methods. The effect of MAAs on central signaling cascades, such as transcription factor nuclear factor kappa b (NF-κB activation, as well as tryptophan metabolism, was investigated in human myelomonocytic THP-1 and THP-1-Blue cells. Cells were exposed to the MAAs in the presence or absence of lipopolysaccharide (LPS. NF-κB activity and the activity of tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO-1 were used as readout. Compounds were tested in the concentration range from 12.5 to 200 µg/mL. Both MAAs were able to induce NF-κB activity in unstimulated THP-1-Blue cells, whereby the increase was dose-dependent and more pronounced with shinorine treatment. While shinorine also slightly superinduced NF-κB in LPS-stimulated cells, porphyra-334 reduced NF-κB activity in this inflammatory background. Modulation of tryptophan metabolism was moderate, suppressive in stimulated cells with the lower treatment concentration of both MAAs and with the unstimulated cells upon porphyra-334 treatment. Inflammatory pathways are affected by MAAs, but despite the structural similarity, diverse effects were observed.

Immunomodulatory Effects of the Mycosporine-Like Amino Acids Shinorine and Porphyra-334

Science.gov (United States)

Becker, Kathrin; Hartmann, Anja; Ganzera, Markus; Fuchs, Dietmar; Gostner, Johanna M.

2016-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porphyra sp. using chromatographic methods. The effect of MAAs on central signaling cascades, such as transcription factor nuclear factor kappa b (NF-κB) activation, as well as tryptophan metabolism, was investigated in human myelomonocytic THP-1 and THP-1-Blue cells. Cells were exposed to the MAAs in the presence or absence of lipopolysaccharide (LPS). NF-κB activity and the activity of tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO-1) were used as readout. Compounds were tested in the concentration range from 12.5 to 200 µg/mL. Both MAAs were able to induce NF-κB activity in unstimulated THP-1-Blue cells, whereby the increase was dose-dependent and more pronounced with shinorine treatment. While shinorine also slightly superinduced NF-κB in LPS-stimulated cells, porphyra-334 reduced NF-κB activity in this inflammatory background. Modulation of tryptophan metabolism was moderate, suppressive in stimulated cells with the lower treatment concentration of both MAAs and with the unstimulated cells upon porphyra-334 treatment. Inflammatory pathways are affected by MAAs, but despite the structural similarity, diverse effects were observed. PMID:27338421

In vitro and in vivo inhibitory effects of some fungicides on catalase produced and purified from white-rot fungus Phanerochaete chrysosporium.

Science.gov (United States)

Kavakçıoğlu, Berna; Tarhan, Leman

2014-10-01

In this study, in vitro and in vivo effects of some commonly used fungicides, antibiotics, and various chemicals on isolated and purified catalase from Phanerochaete chrysosporium were investigated. The catalase was purified 129.10-fold by using 60% ammonium sulfate and 60% ethanol precipitations, DEAE-cellulose anion exchange and Sephacryl-S-200 gel filtration chromatographies from P. chrysosporium growth in carbon- and nitrogen-limited medium for 12 days. The molecular weight of native purified catalase from P. chrysosporium was found to be 290 ± 10 kDa, and sodium dodecyl sulfate (SDS)-PAGE results indicated that enzyme consisted of four apparently identical subunits, with a molecular weight of 72.5 ± 2.5 kDa. Kinetic characterization studies showed that optimum pH and temperature, Km and Vmax values of the purified catalase which were stable in basic region and at comparatively high temperatures were 7.5, 30°C, 289.86 mM, and 250,000 U/mg, respectively. The activity of purified catalase from P. chrysosporium was significantly inhibited by dithiothreitol (DTT), 2-mercaptoethanol, iodoacetamide, EDTA, and sodium dodecyl sulfate (SDS). It was found that while antibiotics had no inhibitory effects, 45 ppm benomyl, 144 ppm captan, and 47.5 ppm chlorothalonil caused 14.52, 10.82, and 38.86% inhibition of purified catalase, respectively. The inhibition types of these three fungicides were found to be non-competitive inhibition with the Ki values of 1.158, 0.638, and 0.145 mM and IC50 values of 0.573, 0.158, 0.010 mM, respectively. The results of in vivo experiments also showed that benomyl, captan and chlorothalonil caused 15.25, 1.96, and 36.70% activity decreases after 24-h treatments compared to that of the control.

Detergent activation of the binding protein in the folate radioassay

International Nuclear Information System (INIS)

Hansen, S.I.; Holm, J.; Lyngbye, J.

1982-01-01

A minor cow's whey protein associated with β-lactoglobulin is used as binding protein in the competitive radioassay for serum and erythrocyte folate. Seeking to optimize the assay, we tested the performance of binder solutions of increasing purity. The folate binding protein was isolated from cow's whey by means of CM-Sepharose CL-6B cation-exchange chromatography, and further purified on a methotrexate-AH-Sepharose 4B affinity matrix. In contrast to β-lactoglobulin, the purified protein did not bind folate unless the detergents cetyltrimethylammonium (10 mmol/Ll) or Triton X-100 (1 g/L) were present. Such detergent activation was not needed in the presence of serum. There seems to be a striking analogy between these phenomena and the well-known reactivation of certain purified membrane-derived enzymes by surfactants

Identification of interleukin-8 converting enzyme as cathepsin L.

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Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

2003-06-26

IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

Effect of preparation conditions on the characteristics and photocatalytic activity of TiO2/purified diatomite composite photocatalysts

Science.gov (United States)

Sun, Zhiming; Hu, Zhibo; Yan, Yang; Zheng, Shuilin

2014-09-01

TiO2/purified diatomite composite materials were prepared through a modified hydrolysis-deposition method under low temperature using titanium tetrachloride as precursor combined with a calcination crystallization process. The microstructure and crystalline phases of the obtained composites prepared under different preparation conditions were characterized by high resolution scanning electron microscope (SEM) and X-ray diffraction (XRD), respectively. The photocatalytic performance of TiO2/purified diatomite composites was evaluated by Rhodamine B as the target pollutant under UV irradiation, and the optimum preparation conditions of composites were obtained. The TiO2 crystal form in composites prepared under optimum conditions was anatase, the grain size of which was 34.12 nm. The relationships between structure and property of composite materials were analyzed and discussed. It is indicated that the TiO2 nanoparticles uniformly dispersed on the surface of diatoms, and the photocatalytic performance of the composite materials was mainly determined by the dispersity and grain size of loaded TiO2 nanoparticles.

Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

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Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.

2008-01-01

The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and

Comparison of solubilized and purified plasma membrane and nuclear insulin receptors

International Nuclear Information System (INIS)

Wong, K.Y.; Hawley, D.; Vigneri, R.; Goldfine, I.D.

1988-01-01

Prior studies have detected biochemical and immunological differences between insulin receptors in plasma membranes and isolated nuclei. To further investigate these receptors, they were solubilized in Triton X-100 partially purified by wheat germ agglutinin-agarose chromatography. In these preparations, the nuclear and plasma membrane receptors had very similar pH optima (pH 8.0) and reactivities to a group of polyclonal antireceptor antibodies. Further, both membrane preparations had identical binding activities when labeled insulin was competed for by unlabeled insulin (50% inhibition at 800 pM). Next, nuclear and plasma membranes were solubilized and purified to homogeneity by wheat germ agglutinin-agarose and insulin-agarose chromatography. In both receptors, labeled insulin was covalently cross-linked to a protein of 130 kilodaltons representing the insulin receptor α subunit. When preparations of both receptors were incubated with insulin and then adenosine 5'-[γ- 32 P]triphosphate, a protein of 95 kilodaltons representing the insulin receptor β subunit was phosphorylated in a dose-dependent manner. These studies indicate, therefore, that solubilized plasma membrane and nuclear insulin receptors have similar structures and biochemical properties, and they suggest that they are the same (or very similar) proteins

Process for purifying graphite

International Nuclear Information System (INIS)

Clausius, R.A.

1985-01-01

A process for purifying graphite comprising: comminuting graphite containing mineral matter to liberate at least a portion of the graphite particles from the mineral matter; mixing the comminuted graphite particles containing mineral matter with water and hydrocarbon oil to form a fluid slurry; separating a water phase containing mineral matter and a hydrocarbon oil phase containing grahite particles; and separating the graphite particles from the hydrocarbon oil to obtain graphite particles reduced in mineral matter. Depending upon the purity of the graphite desired, steps of the process can be repeated one or more times to provide a progressively purer graphite

Home Air Purifiers Eradicate Harmful Pathogens

Science.gov (United States)

2014-01-01

Marshall Space Flight Center funded the University of Madison-Wisconsin to develop ethylene scrubbers to keep produce fresh in space. Akida Holdings of Jacksonville, Florida, licensed the technology and developed Airocide, an air purifier that can kill airborne pathogens. Previously designed for industrial spaces, there is now a specially designed unit for home use.

[Digestive utilization of purified cellulose in the rainbow trout (Salmo gairdneri) and the common carp (Cyprinus carpio)].

Science.gov (United States)

Bergot, F

1981-01-01

A semi-purified diet containing 22 p. 100 of a wood cellulose extract without lignin but still containing 22 p. 100 of hemicelluloses was distributed for one month to rainbow trout and common carp reared at 17 and 20 degrees C, respectively. The digestibility of the main dietary constituents was determined by an indirect method using chrome oxide as an inert tracer. The feces were recovered by a continuous automatic collector which rapidly removed them from the water, minimizing alteration by leaching. The cellulose content was estimated by the Weende (crude fiber) and the Van Soest (neutral detergent fiber and acid detergent fiber) methods. The digestibility coefficients obtained for trout as well as for carp indicate that cellulose and hemicelluloses were not digested. In both species, volatile fatty acid concentration in the different segments of the digestive tract was low (less than 10 mM/l). These results lead us to suggest that trout and carp cannot degrade purified cellulose.

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis

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Agna Cristina Guimarães

2018-02-01

Full Text Available BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121. Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1. OBJECTIVES We evaluated the lipophosphoglycans (LPGs of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector and Ph. papatasi (Old World, restrictive or specific vector. FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

Lower galactosylation levels of the Lipophosphoglycan from Leishmania (Leishmania) major-like strains affect interaction with Phlebotomus papatasi and Lutzomyia longipalpis.

Science.gov (United States)

Guimarães, Agna Cristina; Nogueira, Paula Monalisa; Silva, Soraia de Oliveira; Sadlova, Jovana; Pruzinova, Katerina; Hlavacova, Jana; Melo, Maria Norma; Soares, Rodrigo Pedro

2018-01-01

BACKGROUND Leishmania major is an Old World species causing cutaneous leishmaniasis and is transmitted by Phlebotomus papatasi and Phlebotomus duboscqi. In Brazil, two isolates from patients who never left the country were characterised as L. major-like (BH49 and BH121). Using molecular techniques, these isolates were indistinguishable from the L. major reference strain (FV1). OBJECTIVES We evaluated the lipophosphoglycans (LPGs) of the strains and their behaviour in Old and New World sand fly vectors. METHODS LPGs were purified, and repeat units were qualitatively evaluated by immunoblotting. Experimental in vivo infection with L. major-like strains was performed in Lutzomyia longipalpis (New World, permissive vector) and Ph. papatasi (Old World, restrictive or specific vector). FINDINGS The LPGs of both strains were devoid of arabinosylated side chains, whereas the LPG of strain BH49 was more galactosylated than that of strain BH121. All strains with different levels of galactosylation in their LPGs were able to infect both vectors, exhibiting colonisation of the stomodeal valve and metacyclogenesis. The BH121 strain (less galactosylated) exhibited lower infection intensity compared to BH49 and FV1 in both vectors. MAIN CONCLUSIONS Intraspecific variation in the LPG of L. major-like strains occur, and the different galactosylation levels affected interactions with the invertebrate host.

Peptide Fraction pOh2 Exerts Antiadipogenic Activity through Inhibition of C/EBP-α and PPAR-γ Expression in 3T3-L1 Adipocytes

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Thi Tuyet Nhung Nguyen

2017-01-01

Full Text Available Many studies have comprehensively examined the venom of Ophiophagus hannah snake. Its venom comprises different compounds exhibiting a wide range of pharmacological activities. In this investigation, four peptide fractions (PFs, ranging from 3 kDa to 10 kDa, isolated from the Vietnamese snake venom of O. hannah were separated by HPLC and investigated for their inhibitory activity on adipogenesis in 3T3-L1 adipocytes. The most effective PF was then further purified, generating two peptides, pOh1 and pOh2. Upon investigation of these two peptides on 3T3-L1 adipocytes, it was revealed that, at 10 μg/mL, pOh2 was able to inhibit the lipid accumulation in 3T3-L1 adipocytes by up to 56%, without affecting cell viability. Furthermore, the pOh2 downregulated the gene expression of important transcription factors C/EBP-α and PPAR-γ. In addition, aP2 and GPDH adipocyte-specific markers were also significantly reduced compared to untreated differentiated cells. Taken together, pOh2 inhibited the expression of key transcription factors C/EBP-α and PPAR-γ and their target genes, aP2 and GPDH, thereby blocking the adipocyte differentiation. In conclusion, this novel class of peptide might have potential for in vivo antiobesity effects.

A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

International Nuclear Information System (INIS)

Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

2006-01-01

PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

Purification, crystallization and preliminary X-ray analysis of l-sorbose reductase from Gluconobacter frateurii complexed with l-sorbose or NADPH

International Nuclear Information System (INIS)

Kubota, Keiko; Nagata, Koji; Miyazono, Ken-ichi; Toyama, Hirohide; Matsushita, Kazunobu; Tanokura, Masaru

2009-01-01

NADPH-dependent l-sorbose reductase from G. frateurii (SR) was expressed, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method. Crystals of the SR–l-sorbose complex and SR–NADPH complex diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. NADPH-dependent l-sorbose reductase (SR) from Gluconobacter frateurii was expressed in Escherichia coli, purified and crystallized with l-sorbose or NADPH using the sitting-drop vapour-diffusion method at 293 K. Crystals of the SR–l-sorbose complex and the SR–NADPH complex were obtained using reservoir solutions containing PEG 2000 or PEG 400 as precipitants and diffracted X-rays to 2.38 and 1.90 Å resolution, respectively. The crystal of the SR–l-sorbose complex belonged to space group C222 1 , with unit-cell parameters a = 124.2, b = 124.1, c = 60.8 Å. The crystal of the SR–NADPH complex belonged to space group P2 1 , with unit-cell parameters a = 124.3, b = 61.0, c = 124.5 Å, β = 89.99°. The crystals contained two and eight molecules, respectively, in the asymmetric unit

Characterization of Bacteriocin like inhibitory substance produced by a new Strain Brevibacillus borstelensis AG1 Isolated from ‘Marcha’

Science.gov (United States)

Sharma, Nivedita; Gupta, Anupama; Gautam, Neha

2014-01-01

In the present study, a bacterium isolated from Marcha- a herbal cake used as traditional starter culture to ferment local wine in North East India, was evaluated for bacteriocin like inhibitory substance production and was tested against six food borne/spoilage causing pathogens viz. Listeria monocytogenes MTCC 839, Bacillus subtilis MTCC 121, Clostridium perfringens MTCC 450, Staphylococcus aureus, Lactobacillus plantarum and Leuconostoc mesenteroides MTCC 107 by using bit/disc method followed by well diffusion method. The bacterial isolate was identified as Brevibacillus borstelensis on the basis of phenotypic, biochemical and molecular characteristics using 16Sr RNA gene technique. Bacteriocin like inhibitory substance produced by Brevibacillus borstelensis AG1 was purified by gel exclusion chromatography. The molecular mass of the Brevibacillus borstelensis AG1 was found to be 12 kDa. Purified bacteriocin like inhibitory substance of Brevibacillus borstelensis was further characterized by studying the effect of temperature, pH, proteolytic enzyme and stability. Bacteriocin like inhibitory substance was found to be thermostable upto 100 °C, active at neutral pH, sensitive to trypsin, and partially stable till third week of storage thus showing a bright prospective to be used as a potential food biopreservative. PMID:25477937

Mincle suppresses Toll-like receptor 4 activation.

Science.gov (United States)

Greco, Stephanie H; Mahmood, Syed Kashif; Vahle, Anne-Kristin; Ochi, Atsuo; Batel, Jennifer; Deutsch, Michael; Barilla, Rocky; Seifert, Lena; Pachter, H Leon; Daley, Donnele; Torres-Hernandez, Alejandro; Hundeyin, Mautin; Mani, Vishnu R; Miller, George

2016-07-01

Regulation of Toll-like receptor responses is critical for limiting tissue injury and autoimmunity in both sepsis and sterile inflammation. We found that Mincle, a C-type lectin receptor, regulates proinflammatory Toll-like receptor 4 signaling. Specifically, Mincle ligation diminishes Toll-like receptor 4-mediated inflammation, whereas Mincle deletion or knockdown results in marked hyperresponsiveness to lipopolysaccharide in vitro, as well as overwhelming lipopolysaccharide-mediated inflammation in vivo. Mechanistically, Mincle deletion does not up-regulate Toll-like receptor 4 expression or reduce interleukin 10 production after Toll-like receptor 4 ligation; however, Mincle deletion decreases production of the p38 mitogen-activated protein kinase-dependent inhibitory intermediate suppressor of cytokine signaling 1, A20, and ABIN3 and increases expression of the Toll-like receptor 4 coreceptor CD14. Blockade of CD14 mitigates the increased sensitivity of Mincle(-/-) leukocytes to Toll-like receptor 4 ligation. Collectively, we describe a major role for Mincle in suppressing Toll-like receptor 4 responses and implicate its importance in nonmycobacterial models of inflammation. © Society for Leukocyte Biology.

Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme.

Science.gov (United States)

Gervais, David; Foote, Nicholas

2014-10-01

The enzyme Erwinia chrysanthemi L-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s(-1)) when compared to the WT (k cat 565 s(-1)), which was further increased in the double mutant, with a k cat of 798 s(-1). Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.

Mine water purify from radium

International Nuclear Information System (INIS)

Lebecka, J.

1996-01-01

The article describes purification of radium containing water in coal mines. Author concludes that water purification is relatively simple and effective way to decrease environmental pollution caused by coal mining. The amount of radium disposed with type A radium water has been significantly decreased. The results of investigations show that it will be soon possible to purify also type B radium water. Article compares the amounts of radium disposed by coal mines in 1990, 1995 and forecast for 2000

Modulation of chaperone-like and membranolytic activities of major ...

Indian Academy of Sciences (India)

C Sudheer Kumar

2017-06-20

Jun 20, 2017 ... Keywords. Capacitation; membranolytic activity; molecular chaperone; oxidative stress ... also shown to extract phospholipids from the membrane resulting ..... Gulcin I 2006 Antioxidant and antiradical activities of L-carnitine.

Pharmacological evidences for DFK167-sensitive presenilin-independent gamma-secretase-like activity.

Science.gov (United States)

Sevalle, Jean; Ayral, Erwan; Hernandez, Jean-François; Martinez, Jean; Checler, Frédéric

2009-07-01

Amyloid-beta (Abeta) peptides production is thought to be a key event in the neurodegenerative process ultimately leading to Alzheimer's disease (AD) pathology. A bulk of studies concur to propose that the C-terminal moiety of Abeta is released from its precursor beta-amyloid precursor protein by a high molecular weight enzymatic complex referred to as gamma-secretase, that is composed of at least, nicastrin (NCT), Aph-1, Pen-2, and presenilins (PS) 1 or 2. They are thought to harbor the gamma-secretase catalytic activity. However, several lines of evidence suggest that additional gamma-secretase-like activities could potentially contribute to Abeta production. By means of a quenched fluorimetric substrate (JMV2660) mimicking the beta-amyloid precursor protein sequence targeted by gamma-secretase, we first show that as expected, this probe allows monitoring of an activity detectable in several cell systems including the neuronal cell line telencephalon specific murine neurons (TSM1). This activity is reduced by DFK167, N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), and LY68458, three inhibitors known to functionally interact with PS. Interestingly, JMV2660 but not the unrelated peptide JMV2692, inhibits Abeta production in an in vitrogamma-secretase assay as expected from a putative substrate competitor. This activity is enhanced by PS1 and PS2 mutations known to be responsible for familial forms of AD and reduced by aspartyl mutations inactivating PS or in cells devoid of PS or NCT. However, we clearly establish that residual JMV2660-hydrolysing activity could be recovered in PS- and NCT-deficient fibroblasts and that this activity remained inhibited by DFK167. Overall, our study describes the presence of a proteolytic activity displaying gamma-secretase-like properties but independent of PS and still blocked by DFK167, suggesting that the PS-dependent complex could not be the unique gamma-secretase activity responsible for Abeta

Spectrin-like proteins in plant nuclei

NARCIS (Netherlands)

Ruijter, de N.C.A.; Ketelaar, T.; Blumenthal, S.S.D.; Emons, A.M.C.; Schel, J.H.N.

2000-01-01

We analysed the presence and localization of spectrin-like proteins in nuclei of various plant tissues, using several anti-erythrocyte spectrin antibodies on isolated pea nuclei and nuclei in cells. Western blots of extracted purified pea nuclei show a cross-reactive pair of bands at 220–240 kDa,

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: metal activation, solvent isotope effects, and kinetic mechanism.

Science.gov (United States)

Born, T L; Zheng, R; Blanchard, J S

1998-07-21

Hydrolysis of N-succinyl-L,L-diaminopimelic acid by the dapE-encoded desuccinylase is required for the bacterial synthesis of lysine and meso-diaminopimelic acid. We have investigated the catalytic mechanism of the recombinant enzyme from Haemophilus influenzae. The desuccinylase was overexpressed in Escherichia coli and purified to homogeneity. Steady-state kinetic experiments verified that the enzyme is metal-dependent, with a Km for N-succinyl-L,L-diaminopimelic acid of 1.3 mM and a turnover number of 200 s-1 in the presence of zinc. The maximal velocity was independent of pH above 7 but decreased with a slope of 1 below pH 7. The pH dependence of V/K was bell-shaped with apparent pKs of 6.5 and 8.3. Both L,L- and D,L-diaminopimelic acid were competitive inhibitors of the substrate, but d,d-diaminopimelic acid was not. Solvent kinetic isotope effect studies yielded inverse isotope effects, with values for D2OV/K of 0.62 and D2OV of 0.78. Determination of metal stoichiometry by ICP-AES indicated one tightly bound metal ion, while sequence homologies suggest the presence of two metal binding sites. On the basis of these observations, we propose a chemical mechanism for this metalloenzyme, which has a number of important structurally defined homologues.

Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

Science.gov (United States)

Latty, Sarah Louise; Sakai, Jiro; Hopkins, Lee; Verstak, Brett; Paramo, Teresa; Berglund, Nils A; Cammorota, Eugenia; Cicuta, Pietro; Gay, Nicholas J; Bond, Peter J; Klenerman, David; Bryant, Clare E

2018-01-24

Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling. © 2018, Latty et al.

Aspidosperma (Apocynaceae plant cytotoxicity and activity towards malaria parasites. Part II: experimental studies withAspidosperma ramiflorum in vivo and in vitro

Directory of Open Access Journals (Sweden)

Anna CC Aguiar

2015-11-01

Full Text Available Several species of Aspidospermaplants are used to treat diseases in the tropics, including Aspidosperma ramiflorum, which acts against leishmaniasis, an activity that is experimentally confirmed. The species, known as guatambu-yellow, yellowperoba, coffee-peroba andmatiambu, grows in the Atlantic Forest of Brazil in the South to the Southeast regions. Through a guided biofractionation of A. ramiflorumextracts, the plant activity against Plasmodium falciparumwas evaluated in vitro for toxicity towards human hepatoma G2 cells, normal monkey kidney cells and nonimmortalised human monocytes isolated from peripheral blood. Six of the seven extracts tested were active at low doses (half-maximal drug inhibitory concentration < 3.8 µg/mL; the aqueous extract was inactive. Overall, the plant extracts and the purified compounds displayed low toxicity in vitro. A nonsoluble extract fraction and one purified alkaloid isositsirikine (compound 5 displayed high selectivity indexes (SI (= 56 and 113, respectively, whereas compounds 2 and 3 were toxic (SI < 10. The structure, activity and low toxicity of isositsirikine in vitro are described here for the first time in A. ramiflorum, but only the neutral and precipitate plant fractions were tested for activity, which caused up to 53% parasitaemia inhibition of Plasmodium bergheiin mice with blood-induced malaria. This plant species is likely to be useful in the further development of an antimalarial drug, but its pharmacological evaluation is still required.

Characterization of antioxidant and antimicrobial activities of nettle leaves (Urtica dioica L.

Directory of Open Access Journals (Sweden)

Kukrić Zoran Z.

2012-01-01

Full Text Available Samples of stinging nettle or common nettle (Urtica dioica L. were collected from the area of Banja Luka. To measure and evaluate the content of chlorophyll (a and b, carotenoids, and soluble proteins, as well as peroxidase activity (POD, EC 1.11.1.7., fresh nettle leaves of different developmental stages were used. Dried nettle leaves were used to obtain ethanol extract. The dry residue of ethanol extract was dissolved in methanol and the obtained solution was used to determine the content of total phenols, flavonoids, flavonols, as well as non-enzymatic antioxidant activity and antimicrobial activity. The non-enzymatic antioxidant activity was determined by different methods: FRAP, DPPH, and ABTS. The results were compared to those of standard substances like vitamin C, BHT, and BHA. Antimicrobial activity was screened by using macrodilution method. The obtained results showed insignificantly higher content of chlorophyll, carotenoids, and proteins in young nettle leaves as well as an increase in the soluble peroxidase activities. Native electrophoresis of the soluble fraction showed the presence of two peroxidase isophorms in the soluble protein fraction of nettle leaves. The total phenolic content in nettle extracts amounted to 208.37 mg GAE/gdw, the content of total flavonoids was 20.29 mg QE/gdw, and the content of total flavonols was 22.83 mg QE/gdw. The antioxidant activity determined by FRAP method was 7.50 mM Fe(II/gdw, whereas the antioxidant activity measured by using DPPH and ABTS methods, with IC50 values, were 31.38 and 23.55 μg mL-1, respectively. These results showed the weak and moderate antioxidant capacity of stinging nettle. Extract of Urtica dioica L. was tested for antibacterial acivity against various Grampositive and Gram-negative bacteria: Bacillus subtilis IP 5832, Lactobacillus plantarum 299v (Lp299v, Pseudomonas aeruginosa, and Escherichia coli isolated from food and Escherichia coli isolated from urine samples

ANTIMICROBIAL ACTIVITY OF TUSSILAGO FARFARA L.

Directory of Open Access Journals (Sweden)

Miroslava Kačániová

2013-02-01

Full Text Available In this study, ethanolic extracts of Tussilago farfara L. which had been described in herbal books, were screened for their antimicrobial activity. The following strains of bacteria for antimicrobial activity were used Escherichia coli CCM 3988, Serratia rubidea CCM 4684, Staphylococcus epidermis CC 4418, Lactobacillus rhamnosus CCM 1828, Pseudomonas aeroginosa CCM 1960 and Enterococcus raffinosus CCM 4216. The yeast strain used in this study was Saccharomyces cerevisiae CCM 8191 using disc diffusion method and microbroth dilution technique. The highest antibacterial activity of Tussilago farfara L. ethanolic extract was measured in Grampositive bacteria Lactobacillus rhamnosus (6.67±1.53 mm and lower in yeast Saccharomyces cerevisiae (1.67±0.58 mm with disc diffusion method used. The ethanolic extract present an important activity against Saccharomyces cerevisiae (MIC50=24 µg.ml-1; MIC90=25.69 µg.ml-1 and Serratia rubidaea (MIC=48.01 µg.ml-1; MIC90=51.26 µg.ml-1 with microbroth dilution technique used.

ACETYL-L-CARNITINE AFFECTS THE ELECTRICAL ACTIVITY OF MECHANOSENSORY NEURONS IN HIRUDO MEDICINALIS GANGLIA

Directory of Open Access Journals (Sweden)

Giovanna Traina

2017-04-01

Full Text Available Was previously discovered that in the leech Hirudo medicinalis, acetyl-l-carnitine (ALC affects forms of non-associative learning, such as sensitization and dishabituation, due to nociceptive stimulation of the dorsal skin in the swim induction behavioural paradigm, likely through modulating the activity of the mechanosensory tactile (T neurons, which initiate swimming. Since was found that ALC impaired sensitization and dishabituation, both of which are mediated by the neurotransmitter serotonin, the present study analyzed how ALC may interfere with the sensitizing response. Was already found that ALC reduced the activity of nociceptive (N neurons, which modulate T cell activity through serotonergic mediation.

Activation of the pacidamycin PacL adenylation domain by MbtH-like proteins.

Science.gov (United States)

Zhang, Wenjun; Heemstra, John R; Walsh, Christopher T; Imker, Heidi J

2010-11-23

Nonribosomal peptide synthetase (NRPS) assembly lines are major avenues for the biosynthesis of a vast array of peptidyl natural products. Several hundred bacterial NRPS gene clusters contain a small (∼70-residue) protein belonging to the MbtH family for which no function has been defined. Here we show that two strictly conserved Trp residues in MbtH-like proteins contribute to stimulation of amino acid adenylation in some NRPS modules. We also demonstrate that adenylation can be stimulated not only by cognate MbtH-like proteins but also by homologues from disparate natural product pathways.

Anxiolytic-like effect of Sonchus oleraceus L. in mice.

Science.gov (United States)

Cardoso Vilela, Fabiana; Soncini, Roseli; Giusti-Paiva, Alexandre

2009-07-15

Sonchus oleraceus L. has been used as a general tonic in Brazilian folk medicine. Nevertheless, available scientific information regarding this species is scarce; there are no reports related to its possible effect on the central nervous system. This study was conducted to establish the anxiolytic effect of extracts from the aerial parts of Sonchus oleraceus. This study evaluated the effect of hydroethanolic and dichloromethane extracts of Sonchus oleraceus in mice submitted to the elevated plus-maze and open-field tests. Clonazepam was used as the standard drug. In the elevated plus-maze test, the Sonchus oleraceus extracts increased the percentage of open arm entries (PSonchus oleraceus extract exerts an anxiolytic-like effect on mice.

Isolation, purification and characterization of antimicrobial protein from seedlings of Bauhinia purpurea L.

Science.gov (United States)

Sakthivel, Muthu; Palani, Perumal

2016-05-01

A novel antimicrobial protein was purified from the seedlings of Bauhinia purpurea by sequential procedures entailing ammonium sulfate precipitation, cation exchange chromatography, preparative native-PAGE and a yield of 2.7% was obtained from the crude extract. The purified antimicrobial protein appeared as a single protein band on SDS-PAGE with the molecular mass of 20.9 kDa. Purified antimicrobial protein exhibited a potent antimicrobial activity against both Gram-positive and Gram-negative bacteria. Analysis of the trypsin digested peptides of purified protein using the MALDI-TOF MS/MS resulted in the identification of 174 amino acids. The purified protein had an optimum of pH of 5.5 and was stable at 35 °C for exhibiting its maximal antibacterial activity. The addition of metal ions such as Mn(2+) and Ca(2+) to the purified protein enhanced the antimicrobial activity of purified protein. The MIC of purified protein against Bacillus cereus and Escherichia coli were 13 μg/ml and 15 μg/ml, respectively. The purified protein digested the peptidoglycan layer of bacteria which was visualized by TEM analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

Studies on a novel peptide isolated and purified from rat insulinoma tissue

Energy Technology Data Exchange (ETDEWEB)

Al-Akhras, G N

1987-01-01

Rat insulinoma peptide (RIP) which appears to be either a fragment of, or an altered rat C-peptide was isolated and purified by dialysis. The purity of this peptide was investigated using polyacrylamide gel electrophoresis with sodium dodecyl sulfate, isoelectric focusing, and high performance liquid chromatography. RIP may contain two peptides similar to each other but differing in their isoelectric points. The molecular weight of RIP was found to be 1982 daltons by fast atoms bombardment mass spectrometry giving a chain length of approximately 22 amino acid residues. From information obtained using radioimmunoassay employing antiserum R901, RIP appears to share a common C-terminus with rat C-peptide. A radioimmunoassay for RIP was developed using the purified RIP as immunogen and for standards and tracers. An indirect enzyme linked immunosorbent assay (ELISA) for rat insulinoma peptide was developed using purified RIP for immunogen and semi-purified RIP as a standard.

Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry (Morus alba L.) leaves.

Science.gov (United States)

Yuan, Qingxia; Xie, Yufeng; Wang, Wei; Yan, Yuhua; Ye, Hong; Jabbar, Saqib; Zeng, Xiaoxiong

2015-09-05

Extraction optimization, characterization and antioxidant activity in vitro of polysaccharides from mulberry leaves (MLP) were investigated in the present study. The optimal extraction conditions with an extraction yield of 10.0 ± 0.5% for MLP were determined as follows: extraction temperature 92 °C, extraction time 3.5h and ratio (v/w, mL/g) of extraction solvent (water) to raw material 34. Two purified fractions, MLP-3a and MLP-3b with molecular weights of 80.99 and 3.64 kDa, respectively, were obtained from crude MLP by chromatography of DEAE-Cellulose 52 and Sephadex G-100. Fourier transform-infrared spectroscopy revealed that crude MLP, MLP-3a and MLP-3b were acidic polysaccharides. Furthermore, crude MLP and MLP-3a had more complicated monosaccharide compositions, while MLP-3b had a relatively higher content of uronic acid. Crude MLP, MLP-3a and MLP-3b exhibited potent Fe(2+) chelating power and scavenging activities on 1,1-diphenyl-2-picrylhydrazyl, hydroxyl, superoxide and 2,2'-azinobis-(3-ethyl-benzothiazolin-6-sulfonic acid) radicals. The results suggested that MLP could be explored as natural antioxidant. Copyright © 2015 Elsevier Ltd. All rights reserved.

DsbA-L prevents obesity-induced inflammation and insulin resistance by suppressing the mtDNA release-activated cGAS-cGAMP-STING pathway.

Science.gov (United States)

Bai, Juli; Cervantes, Christopher; Liu, Juan; He, Sijia; Zhou, Haiyan; Zhang, Bilin; Cai, Huan; Yin, Dongqing; Hu, Derong; Li, Zhi; Chen, Hongzhi; Gao, Xiaoli; Wang, Fang; O'Connor, Jason C; Xu, Yong; Liu, Meilian; Dong, Lily Q; Liu, Feng

2017-11-14

Chronic inflammation in adipose tissue plays a key role in obesity-induced insulin resistance. However, the mechanisms underlying obesity-induced inflammation remain elusive. Here we show that obesity promotes mtDNA release into the cytosol, where it triggers inflammatory responses by activating the DNA-sensing cGAS-cGAMP-STING pathway. Fat-specific knockout of disulfide-bond A oxidoreductase-like protein (DsbA-L), a chaperone-like protein originally identified in the mitochondrial matrix, impaired mitochondrial function and promoted mtDNA release, leading to activation of the cGAS-cGAMP-STING pathway and inflammatory responses. Conversely, fat-specific overexpression of DsbA-L protected mice against high-fat diet-induced activation of the cGAS-cGAMP-STING pathway and inflammation. Taken together, we identify DsbA-L as a key molecule that maintains mitochondrial integrity. DsbA-L deficiency promotes inflammation and insulin resistance by activating the cGAS-cGAMP-STING pathway. Our study also reveals that, in addition to its well-characterized roles in innate immune surveillance, the cGAS-cGAMP-STING pathway plays an important role in mediating obesity-induced metabolic dysfunction.

Purification and characterization of ornithine transcarbamylase from pea (Pisum sativum L.)

Science.gov (United States)

Slocum, R. D.; Richardson, D. P.

1991-01-01

Pea (Pisum sativum) ornithine transcarbamylase (OTC) was purified to homogeneity from leaf homogenates in a single-step procedure, using delta-N-(phosphonacetyl)-L-ornithine-Sepharose 6B affinity chromatography. The 1581-fold purified OTC enzyme exhibited a specific activity of 139 micromoles citrulline per minute per milligram of protein at 37 degrees C, pH 8.5. Pea OTC represents approximately 0.05% of the total soluble protein in the leaf. The molecular weight of the native enzyme was approximately 108,200, as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single molecular weight band of 36,500 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the pea OTC is a trimer of identical subunits. The overall amino acid composition of pea OTC is similar to that found in other eukaryotic and prokaryotic OTCs, but the number of arginine residues is approximately twofold higher. The increased number of arginine residues probably accounts for the observed isoelectric point of 7.6 for the pea enzyme, which is considerably more basic than isoelectric point values that have been reported for other OTCs.

Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer.

LENUS (Irish Health Repository)

Sullivan, Shane

2012-02-01

Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

Inhibition of fumarate reductase in Leishmania major and L. donovani by chalcones

DEFF Research Database (Denmark)

Chen, M; Zhai, L; Christensen, S B

2001-01-01

Our previous studies have shown that chalcones exhibit potent antileishmanial and antimalarial activities in vitro and in vivo. Preliminary studies showed that these compounds destroyed the ultrastructure of Leishmania parasite mitochondria and inhibited the respiration and the activity...... of mitochondrial dehydrogenases of Leishmania parasites. The present study was designed to further investigate the mechanism of action of chalcones, focusing on the parasite respiratory chain. The data show that licochalcone A inhibited the activity of fumarate reductase (FRD) in the permeabilized Leishmania major...... promastigote and in the parasite mitochondria, and it also inhibited solubilized FRD and a purified FRD from L. donovani. Two other chalcones, 2,4-dimethoxy-4'-allyloxychalcone (24m4ac) and 2,4-dimethoxy-4'-butoxychalcone (24mbc), also exhibited inhibitory effects on the activity of solubilized FRD in L. major...

Cooling performance of R510A in domestic water purifiers

International Nuclear Information System (INIS)

Park, Ki Jung; Lee, Yo Han; Jung, Dong Soo

2010-01-01

Cooling performance of R510A is examined both numerically and experimentally in an effort to replace HFC134a in the refrigeration system of domestic water purifiers. Although the use of HFC134a is currently dominant, it is being phased out in Europe and most developed countries due to its high potential contribution to global warming. To solve this problem, cycle simulation and experimental measurements are conducted with a new refrigerant mixture of 88%RE170/12%R600a using actual domestic water purifiers. This mixture has been recently numbered and listed as R510A by ASHRAE. Test results show that, due to the small internal volume of the refrigeration system of the domestic water purifiers, system performance with R510A is greatly influenced by the amount of charge. With the optimum charge amount of 20 to 21 g, approximately 50% that of HFC134a, the energy consumption of R510A is 22.3% lower than that of HFC134a. The compressor discharge temperature of R510A is 3.7 .deg. C lower than that of HFC134a at the optimum charge. Overall, R510A, a new, long term, and environmentally safe refrigerant, is a good alternative for HFC134a. Furthermore, it requires only minor changes in the refrigeration system of the domestic water purifiers

A sensitive hydrogen peroxide sensor based on leaf-like silver

International Nuclear Information System (INIS)

Meng, Zuchao; Zhang, Mingyin; Zhang, Hongfang; Zheng, Jianbin

2014-01-01

A novel non-enzymatic hydrogen peroxide sensor based on leaf-like silver was constructed. The leaf-like silver was synthesized on the surface of L-cysteine (L-cys) by electrodeposition. Scanning electron microscopy and electrochemical techniques were used to characterize the leaf-like silver nanoparticles. The sensor showed high electrocatalytic activity towards the reduction of hydrogen peroxide. A wide linear range of 2.5–1.5 mM with a low detection limit of 0.7 µM was obtained. Excellent electrocatalytic activity, large surface-to-volume ratio and efficient electron transport properties of leaf-like silver have enabled stable and highly sensitive performance for the non-enzymatic hydrogen peroxide sensor. (paper)

An Enterotoxin-Like Binary Protein from Pseudomonas protegens with Potent Nematicidal Activity.

Science.gov (United States)

Wei, Jun-Zhi; Siehl, Daniel L; Hou, Zhenglin; Rosen, Barbara; Oral, Jarred; Taylor, Christopher G; Wu, Gusui

2017-10-01

Soil microbes are a major food source for free-living soil nematodes. It is known that certain soil bacteria have evolved systems to combat predation. We identified the nematode-antagonistic Pseudomonas protegens strain 15G2 from screening of microbes. Through protein purification we identified a binary protein, designated Pp-ANP, which is responsible for the nematicidal activity. This binary protein inhibits Caenorhabditis elegans growth and development by arresting larvae at the L1 stage and killing older-staged worms. The two subunits, Pp-ANP1a and Pp-ANP2a, are active when reconstituted from separate expression in Escherichia coli The binary toxin also shows strong nematicidal activity against three other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and Acrobeloides sp.), but we did not find any activity against insects and fungi under test conditions, indicating specificity for nematodes. Pp-ANP1a has no significant identity to any known proteins, while Pp-ANP2a shows ∼30% identity to E. coli heat-labile enterotoxin (LT) subunit A and cholera toxin (CT) subunit A. Protein modeling indicates that Pp-ANP2a is structurally similar to CT/LT and likely acts as an ADP-ribosyltransferase. Despite the similarity, Pp-ANP shows several characteristics distinct from CT/LT toxins. Our results indicate that Pp-ANP is a new enterotoxin-like binary toxin with potent and specific activity to nematodes. The potency and specificity of Pp-ANP suggest applications in controlling parasitic nematodes and open an avenue for further research on its mechanism of action and role in bacterium-nematode interaction. IMPORTANCE This study reports the discovery of a new enterotoxin-like binary protein, Pp-ANP, from a Pseudomonas protegens strain. Pp-ANP shows strong nematicidal activity against Caenorhabditis elegans larvae and older-staged worms. It also shows strong activity on other free-living nematodes ( Pristionchus pacificus , Panagrellus redivivus , and

Crystallization and preliminary X-ray crystallographic studies of Rab6A′(Q72L): a GTP-locked form

International Nuclear Information System (INIS)

Shin, Young-Cheul; Jang, Tae-Ho; Yoon, Jong Hwan; Jeon, Ju-Hong; Park, Hyun Ho

2012-01-01

Rab6A′(Q72L), a constitutively active GTP-binding form of Rab6A, was purified and crystallized. The crystals were found to belong to space group P22 1 2 1 , with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The crystals were obtained at 293 K and diffracted to a resolution of 1.9 Å. Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A′. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A′ and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A′(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A′(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22 1 2 1 with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules

Distribution and activity of a Dippu DH31-like peptide in the large milkweed bug Oncopeltus fasciatus.

Science.gov (United States)

Te Brugge, V A; Orchard, I

2008-02-01

The milkweed bug, Oncopeltus fasciatus, is a plant feeding hemipteran. While there has been much research done on the neurohormonal control of the post-feeding diuresis in the blood-feeding hemipteran, Rhodnius prolixus, little is known about the control of the post-feeding diuresis in O. fasciatus. One of the neurohormones that may play a role in this rapid diuresis belongs to the calcitonin-like diuretic hormone (DH31) family of insect peptides. In this study we demonstrate the presence of DH31-like immunoreactivity in the central nervous system (CNS) and gut of O. fasciatus 5th instars. As well, DH31-like material was quantified and partially purified from the CNS of 5th instar O. fasciatus using reversed-phase liquid chromatography (RPLC) and monitored with an enzyme-linked immunosorbent assay (ELISA). When tested on O. fasciatus 5th instar Malpighian tubules, DH31-like peptides significantly increased the rate of secretion over saline controls. The results suggest that there is a DH31-like peptide(s) present in the CNS of O. fasciatus and that this peptide may play a role in the control of Malpighian tubule secretion.

Evidence for mouse Th1- and Th2-like helper T cells in vivo. Selective reduction of Th1-like cells after total lymphoid irradiation

International Nuclear Information System (INIS)

Bass, H.; Mosmann, T.; Strober, S.

1989-01-01

Purified CD4+ BALB/c spleen T cells obtained 4-6 wk after total lymphoid irradiation (TLI) helped normal syngeneic B cells to produce a vigorous antibody response to TNP keyhole limpet hemocyanin in adoptive cell transfer experiments. However, the same cells failed to transfer delayed-type hypersensitivity to the adoptive hosts as measured by a foot pad swelling assay. In addition, purified CD4+ cells from TLI-treated mice were unable to induce graft vs. host disease in lethally irradiated allogeneic C57BL/Ka recipient mice. In response to mitogen stimulation, unfractionated spleen cells obtained from TLI mice secreted normal levels of IL-4 and IL-5, but markedly reduced levels of IL-2 and INF-gamma. A total of 229 CD4+ clones from spleen cells of both normal and TLI-treated mice were established, and the cytokine secretion pattern from each clone was analyzed. The results demonstrate that the ratio of Th1- and Th2-like clones in the spleens of normal BALB/c mice is 1:0.6, whereas the ratio in TLI mice is approximately 1:7. These results suggest that Th2-like cells recover rapidly (at approximately 4-6 wk) after TLI treatment and account for the early return of antibody helper activity and secretion of IL-4 and IL-5, but Th1-like cells recover more slowly (in approximately 3 mo) after irradiation, and this accounts for the deficit in cell-mediated immunity and the reduced amount of IL-2 and IFN-gamma secretion

Mathematical Modeling of Interacting Glucose-Sensing Mechanisms and Electrical Activity Underlying Glucagon-Like Peptide 1 Secretion.

Directory of Open Access Journals (Sweden)

Michela Riz

2015-12-01

Full Text Available Intestinal L-cells sense glucose and other nutrients, and in response release glucagon-like peptide 1 (GLP-1, peptide YY and other hormones with anti-diabetic and weight-reducing effects. The stimulus-secretion pathway in L-cells is still poorly understood, although it is known that GLP-1 secreting cells use sodium-glucose co-transporters (SGLT and ATP-sensitive K+-channels (K(ATP-channels to sense intestinal glucose levels. Electrical activity then transduces glucose sensing to Ca2+-stimulated exocytosis. This particular glucose-sensing arrangement with glucose triggering both a depolarizing SGLT current as well as leading to closure of the hyperpolarizing K(ATP current is of more general interest for our understanding of glucose-sensing cells. To dissect the interactions of these two glucose-sensing mechanisms, we build a mathematical model of electrical activity underlying GLP-1 secretion. Two sets of model parameters are presented: one set represents primary mouse colonic L-cells; the other set is based on data from the GLP-1 secreting GLUTag cell line. The model is then used to obtain insight into the differences in glucose-sensing between primary L-cells and GLUTag cells. Our results illuminate how the two glucose-sensing mechanisms interact, and suggest that the depolarizing effect of SGLT currents is modulated by K(ATP-channel activity. Based on our simulations, we propose that primary L-cells encode the glucose signal as changes in action potential amplitude, whereas GLUTag cells rely mainly on frequency modulation. The model should be useful for further basic, pharmacological and theoretical investigations of the cellular signals underlying endogenous GLP-1 and peptide YY release.

l-Arabinose Isomerase and d-Xylose Isomerase from Lactobacillus reuteri: Characterization, Coexpression in the Food Grade Host Lactobacillus plantarum, and Application in the Conversion of d-Galactose and d-Glucose

Science.gov (United States)

2014-01-01

The l-arabinose isomerase (l-AI) and the d-xylose isomerase (d-XI) encoding genes from Lactobacillus reuteri (DSMZ 17509) were cloned and overexpressed in Escherichia coli BL21 (DE3). The proteins were purified to homogeneity by one-step affinity chromatography and characterized biochemically. l-AI displayed maximum activity at 65 °C and pH 6.0, whereas d-XI showed maximum activity at 65 °C and pH 5.0. Both enzymes require divalent metal ions. The genes were also ligated into the inducible lactobacillal expression vectors pSIP409 and pSIP609, the latter containing a food grade auxotrophy marker instead of an antibiotic resistance marker, and the l-AI- and d-XI-encoding sequences/genes were coexpressed in the food grade host Lactobacillus plantarum. The recombinant enzymes were tested for applications in carbohydrate conversion reactions of industrial relevance. The purified l-AI converted d-galactose to d-tagatose with a maximum conversion rate of 35%, and the d-XI isomerized d-glucose to d-fructose with a maximum conversion rate of 48% at 60 °C. PMID:24443973

Highly selective water channel activity measured by voltage clamp: analysis of planar lipid bilayers reconstituted with purified AqpZ.

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