Immunomodulatory activity of purified arabinoxylans from finger millet (Eleusine coracana, v. Indaf 15) bran.

PubMed

Savitha Prashanth, M R; Shruthi, R R; Muralikrishna, G

2015-09-01

Biological activities of alkali extracted (Barium hydroxide: BE-480 kDa, Potassium hydroxide: KE1-1080 and KE2-40 kDa), purified arabinoxylans (AX) from the finger millet bran varying in their molecular weight, phenolic acid content, arabinose to xylose ratios were evaluated for their immune-stimulatory activities using murine lymphocytes and peritoneal exudate macrophages. All three purified AX displayed significant (p < 0.001) mitogenic activity and activation of macrophages including phagocytosis. Among these BE has shown higher enhancing lymphocyte proliferation (>2 fold) and macrophage phagocytosis than KE1 and KE2. The above results clearly documented that the immunostimulatory activity of arabinoxylans is directly proportional to the amount of ferulic acid content (0.11 mg/100 g), whereas molecular weight as well as arabinose/xylose ratio, did not have any bearing. Purified AX from the finger millet bran can be explored as a potent natural immunomodulator.

Antioxidant Activity of Oxygen Evolving Enhancer Protein 1 Purified from Capsosiphon fulvescens.

PubMed

Kim, Eun-Young; Choi, Youn Hee; Lee, Jung Im; Kim, In-Hye; Nam, Taek-Jeong

2015-06-01

This study was conducted to determine the antioxidant activity of a protein purified from Capsosiphon fulvescens. The purification steps included sodium acetate (pH 6) extraction and diethylaminoethyl-cellulose, reversed phase Shodex C4P-50 column chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis indicated that the molecular weight of the purified protein was 33 kDa. The N-terminus and partial peptide amino acid sequence of this protein was identical to the sequence of oxygen evolving enhancer (OEE) 1 protein. The antioxidant activity of the OEE 1 was determined in vitro using a scavenging test with 4 types of reactive oxygen species (ROS), including the 2,2-diphenyl-1-picrylhydrazyl radical, hydroxyl radical, superoxide anion, and hydrogen peroxide (H2 O2 ). OEE 1 had higher H2 O2 scavenging activity, which proved to be the result of enzymatic antioxidants rather than nonenzymatic antioxidants. In addition, OEE 1 showed less H2 O2 -mediated ROS formation in HepG2 cells. In conclusion, this study demonstrates that OEE 1 purified from C. fulvescens is an excellent antioxidant. © 2015 Institute of Food Technologists®

An AC-5 cathepsin B-like protease purified from Haemonchus contortus excretory secretory products shows protective antigen potential for lambs

PubMed Central

De Vries, Erik; Bakker, Nicole; Krijgsveld, Jeroen; Knox, Dave P.; Heck, Albert J.R.; Yatsuda, Ana Patricia

2009-01-01

The immunogenic properties of cysteine proteases obtained from excretory/secretory products (ES) of Haemonchus contortus were investigated with a fraction purified with a recombinant H. contortus cystatin affinity column. The enrichment of H. contortus ES for cysteine protease was confirmed with substrate SDS-PAGE gels since the cystatin-binding fraction activity was three times higher than total ES, despite representing only 3% of total ES. This activity was inhibited by a specific cysteine protease inhibitor (E64) and by recombinant cystatin. The one-dimensional profile of the cystatin-binding fraction displayed a single band with a molecular mass of 43 kDa. Mass spectrometry showed this to be AC-5, a cathepsin B-like cysteine protease which had not been identified in ES products of H. contortus before. The cystatin binding fraction was tested as an immunogen in lambs which were vaccinated three times (week 0, 2.5 and 5), challenged with 10 000 L3 H. contortus (week 6) before necropsy and compared to unvaccinated challenge controls and another group given total ES (n = 10 per group). The group vaccinated with cystatin-binding proteins showed 36% and 32% mean worm burden and eggs per gram of faeces (EPG) reductions, respectively, compared to the controls but total ES was almost without effect. After challenge the cystatin-binding proteins induced significantly higher local and systemic ES specific IgA and IgG responses. PMID:19401141

Antioxidant and ACE Inhibitory Bioactive Peptides Purified from Egg Yolk Proteins

PubMed Central

Yousr, Marwa; Howell, Nazlin

2015-01-01

Protein by-products from the extraction of lecithin from egg yolk can be converted into value-added products, such as bioactive hydrolysates and peptides that have potential health enhancing antioxidant, and antihypertensive properties. In this study, the antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of peptides isolated and purified from egg yolk protein were investigated. Defatted egg yolk was hydrolyzed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration to produce egg yolk gel filtration fractions (EYGF). Of these, two fractions, EYGF-23 and EYGF-33, effectively inhibited the peroxides and thiobarbituric acid reactive substance (TBARS) in an oxidizing linoleic acid model system. The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging and ferrous chelation. The presence of hydrophobic amino acids such as tyrosine (Y) and tryptophan (W), in sequences identified by LC-MS as WYGPD (EYGF-23) and KLSDW (EYGF-33), contributed to the antioxidant activity and were not significantly different from the synthetic BHA antioxidant. A third fraction (EYGF-56) was also purified from egg yolk protein by gel filtration and exhibited high ACE inhibitory activity (69%) and IC50 value (3.35 mg/mL). The SDNRNQGY peptide (10 mg/mL) had ACE inhibitory activity, which was not significantly different from that of the positive control captopril (0.5 mg/mL). In addition, YPSPV in (EYGF-33) (10 mg/mL) had higher ACE inhibitory activity compared with captopril. These findings indicated a substantial potential for producing valuable peptides with antioxidant and ACE inhibitory activity from egg yolk. PMID:26690134

Antioxidant and ACE Inhibitory Bioactive Peptides Purified from Egg Yolk Proteins.

PubMed

Yousr, Marwa; Howell, Nazlin

2015-12-07

Protein by-products from the extraction of lecithin from egg yolk can be converted into value-added products, such as bioactive hydrolysates and peptides that have potential health enhancing antioxidant, and antihypertensive properties. In this study, the antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of peptides isolated and purified from egg yolk protein were investigated. Defatted egg yolk was hydrolyzed using pepsin and pancreatin and sequentially fractionated by ultrafiltration, followed by gel filtration to produce egg yolk gel filtration fractions (EYGF). Of these, two fractions, EYGF-23 and EYGF-33, effectively inhibited the peroxides and thiobarbituric acid reactive substance (TBARS) in an oxidizing linoleic acid model system. The antioxidant mechanism involved superoxide anion and hydroxyl radicals scavenging and ferrous chelation. The presence of hydrophobic amino acids such as tyrosine (Y) and tryptophan (W), in sequences identified by LC-MS as WYGPD (EYGF-23) and KLSDW (EYGF-33), contributed to the antioxidant activity and were not significantly different from the synthetic BHA antioxidant. A third fraction (EYGF-56) was also purified from egg yolk protein by gel filtration and exhibited high ACE inhibitory activity (69%) and IC50 value (3.35 mg/mL). The SDNRNQGY peptide (10 mg/mL) had ACE inhibitory activity, which was not significantly different from that of the positive control captopril (0.5 mg/mL). In addition, YPSPV in (EYGF-33) (10 mg/mL) had higher ACE inhibitory activity compared with captopril. These findings indicated a substantial potential for producing valuable peptides with antioxidant and ACE inhibitory activity from egg yolk.

Stability Test of Partially Purified Bromelain from Pineapple (Ananas comosus (L.) Merr) Core Extract in Artificial Stomach Fluid

NASA Astrophysics Data System (ADS)

Setiasih, S.; Adimas, A. Ch. D.; Dzikria, V.; Hudiyono, S.

2018-01-01

This study aimed to isolate and purify bromelain from pineapple core (Ananascomosus (L.) Merr) accompanied by a stability test of its enzyme activity in artificial gastric juice. Purification steps start with fractionation by a precipitation method were carried out stepwise using several concentration of ammonium sulfate salt, followed by dialysis prosess and ion exchange chromatography on DEAE-cellulose column. Each step of purification produced an increasing specific activity in enzyme fraction, starting with crude extract, respectively: 0.276 U/mg; 14.591 U/mg; and 16.05 U/mg. Bromelain fraction with the highest level of purity was obtained in 50-80% ammonium sulphate fraction after dialyzed in the amount of 58.15 times compared to the crude extract. Further purification of the enzyme by DEAE-cellulose column produced bromelain which had a purity level 160-fold compared to crude enzyme. The result of bromelain stability test in artificial stomach juice by milk clotting units assay bromelain fraction have proteolytic activity in clotting milk substrate. Exposing bromelain fraction in artificial stomach juice which gave the highest core bromelain proteolytic activity was achieved at estimated volume of 0.4-0.5 mL. Exposure in a period of reaction time to artificial stomach juice that contained pepsin showed relatively stable proteolytic activity in the first 4 hours.

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

PubMed

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.

Helodermatine, a kallikrein-like, hypotensive enzyme from the venom of Heloderma horridum horridum (Mexican beaded lizard)

PubMed Central

1986-01-01

We have purified and characterized the major N-benzoyl-L-arginine ethyl ester hydrolase from the venom of Heloderma horridum horridum. The enzyme belongs to the serine proteinase family, and its activity vs. peptide amide substrates and human high-molecular-weight kininogen suggests a similarity to the family of kallikreins. This interpretation is corroborated by its reactivity with the natural inhibitors soybean trypsin inhibitor and Kunitz-type bovine pancreatic trypsin inhibitor (aprotinin). Injection of the enzyme (2-16 micrograms/kg) into anesthetized rabbits leads to a rapid dose-dependent transient decrease of the arterial blood pressure. Like glandular kallikrein it specifically converts single-chain tissue type plasminogen activator into its double chain form. In contrast to other kallikrein-like enzymes from snake venoms it shows no thrombin-like or plasminogen activator activity. The enzyme is a single-chain glycoprotein (Mr 63,000). The N-terminal sequence revealed significant homology to pig pancreatic kallikrein and to kallikrein like enzymes from Crotalus atrox and Crotalus adamanteus venom. This enzyme, which we name Helodermatine, is the first purified from Sauria with kallikrein-like properties. PMID:3537191

Specific activity and isotope abundances of strontium in purified strontium-82

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

Fitzsimmons, J. M.; Medvedev, D. G.; Mausner, L. F.

2015-11-12

A linear accelerator was used to irradiate a rubidium chloride target with protons to produce strontium-82 (Sr-82), and the Sr-82 was purified by ion exchange chromatography. The amount of strontium associated with the purified Sr-82 was determined by either: ICP-OES or method B which consisted of a summation of strontium quantified by gamma spectroscopy and ICP-MS. The summation method agreed within 10% to the ICP-OES for the total mass of strontium and the subsequent specific activities were determined to be 0.25–0.52 TBq mg -1. Method B was used to determine the isotope abundances by weight% of the purified Sr-82, andmore » the abundances were: Sr-82 (10–20.7%), Sr-83 (0–0.05%), Sr-84 (35–48.5%), Sr-85 (16–25%), Sr-86 (12.5–23%), Sr-87 (0%), and Sr-88 (0–10%). The purified strontium contained mass amounts of Sr-82, Sr-84, Sr-85, Sr-86, and Sr-88 in abundances not associated with natural abundance, and 90% of the strontium was produced by the proton irradiation. A comparison of ICP-OES and method B for the analysis of Sr-82 indicated analysis by ICP-OES would be easier to determine total mass of strontium and comply with regulatory requirements. An ICP-OES analytical method for Sr-82 analysis was established and validated according to regulatory guidelines.« less

Cylindrospermopsin inhibits growth and modulates protease activity in the aquatic plants Lemna minor L. and Wolffia arrhiza (L.) Horkel.

PubMed

Jámbrik, Katalin; Máthé, C; Vasas, G; Bácsi, I; Surányi, G; Gonda, S; Borbély, G; M-Hamvas, Márta

2010-01-01

The toxic effects of cylindrospermopsin (cyanobacterial toxin) on animals have been examined extensively, but little research has focused on their effects on plants. In this study cylindrospermopsin (CYN) caused alterations of growth, soluble protein content and protease enzyme activity were studied on two aquatic plants Lemna minor and Wolffia arrhiza in short-term (5 days) experiments. For the treatments we used CYN containing crude extracts of Aphanizomenon ovalisporum (BGSD-423) and purified CYN as well. The maximal inhibitory effects on fresh weight of L. minor and W. arrhiza caused by crude extract were 60% and 54%, respectively, while the maximum inhibitory effects were 30% and 43% in the case of purified CYN at 20 μg ml(-1) CYN content of culture medium. In CYN-treated plants the concentration of soluble protein showed mild increases, especially in W. arrhiza. Protease isoenzyme activity gels showed significant alterations of enzyme activities under the influence of CYN. Several isoenzymes were far more active and new ones appeared in CYN-treated plants. Treatments with cyanobacterial crude extract caused stronger effects than the purified cyanobacterial toxins used in equivalent CYN concentrations.

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

PubMed

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

PubMed Central

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

Purification and characterization of a novel and robust L-asparaginase having low-glutaminase activity from Bacillus licheniformis: in vitro evaluation of anti-cancerous properties.

PubMed

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 2.68×10(3) 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.

In vitro thrombolytic activity of purified streptokinase extracted from Streptococcus equinus VIT_VB2 isolated from bovine milk.

PubMed

Babu, Vaishnavi; Subathra Devi, C

2015-01-01

Streptokinase (SK) is an extracellular enzyme secreted by various strains of β-hemolytic Streptococci. The main focus of the current study is to evaluate the in vitro thrombolytic activity of purified SK extracted from Streptococcus equinus VIT_VB2 (Accession no. JX406835) isolated from milk sample. The growth rate of S. equinus VIT_VB2 strain was studied with pH and biomass content which has positive significant effect on enzyme yield. A temperature of 10 °C and pH of 6 was found to be optimum for maximum SK activity. The specific activity of the purified SK produced by VIT_VB2 strain was found to be 6,585 IU mg(-1). The molecular mass of the enzyme was determined as 47 kDa by SDS-PAGE. In vitro thrombolytic activity of purified SK was determined using synthetic chromogenic substrate S-2251, the activity of the purified enzyme was found to be 6,330 ± 2.2 IU. The purity of SK was compared with standard SK by HPLC. This is the first report which reveals the SK activity of S. equinus isolated from milk sample.

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

PubMed Central

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

The structure and mechanism of stem bromelain. Evaluation of the homogeneity of purified stem bromelain, determination of the molecular weight and kinetic analysis of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester

PubMed Central

Wharton, Christopher W.

1974-01-01

1. Purified stem bromelain (EC 3.4.22.4) was eluted from Sephadex G-100 as a single peak. The specific activity across the elution peak was approximately constant towards p-nitrophenyl hippurate but increased with elution volume with N2-benzoyl-l-arginine ethyl ester as substrate. 2. The apparent molecular weight, determined by elution analysis on Sephadex G-100, is 22500±1500, an anomalously low value. 3. Purified stem bromelain was eluted from CM-cellulose CM-32 as a single peak and behaved as a single species during column electrophoresis on Sephadex G-100. 4. Purified stem bromelain migrates as a single band during polyacrylamide-gel electrophoresis under a wide variety of conditions. 5. The molecular weight determined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate is 28500±1000. 6. Sedimentation-velocity and equilibrium-ultracentrifugation experiments, under a variety of conditions, indicate that bromelain is an apparently homogeneous single peptide chain of mol.wt. 28400±1400. 7. The N-terminal amino acid composition is 0.64±0.04mol of valine and 0.36±0.04mol of alanine per mol of enzyme of mol.wt. 28500. (The amino acid recovery of the cyanate N-terminal amino acid analysis was standardized by inclusion of carbamoyl-norleucine at the cyclization stage.) 8. The pH-dependence of the Michaelis parameters of the bromelain-catalysed hydrolysis of N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester was determined. 9. The magnitude and pH-dependence of the Michaelis parameters have been interpreted in terms of the mechanism of the enzyme. 10. The enzyme is able to bind N-benzyloxycarbonyl-l-phenylalanyl-l-serine methyl ester relatively strongly but seems unable to make use of the binding energy to promote catalysis. PMID:4462742

Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations.

PubMed

Koistinen, Hannu; Hautala, Laura; Koli, Katri; Stenman, Ulf-Håkan

2015-12-01

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies.

Absence of TGF-β Receptor Activation by Highly Purified hCG Preparations

PubMed Central

Hautala, Laura; Koli, Katri; Stenman, Ulf-Håkan

2015-01-01

Recently, several LH/human chorionic gonadotropin (hCG) receptor-independent activities for hCG have been described, including activation of the TGF-β receptor (TGFβR) by hyperglycosylated hCG and stimulation of trophoblast invasion. Because the hCG concentrations used in these studies have been rather high, reflecting physiological hCG levels in pregnancy, even a minor contamination with growth factors, which act at very low concentrations, may be significant. Several commercial hCG preparations have been found to contain significant amounts of epidermal growth factor (EGF), which we also confirmed here. Furthermore, we found that some hCG preparations also contain significant amounts of TGF-β1. These hCG preparations were able to activate ERK1/2 in JEG-3 choriocarcinoma cells or TGFβR in mink lung epithelial cells transfected with a reporter gene for TGFβR activation. No such activation was found with highly purified hCG or its free β-subunit (hCGβ), irrespective of whether they were hyperglycosylated or not. Taken together, our results suggest that the growth factor contaminations in the hCG preparations can cause activation of TGFβR and, at least in JEG-3 cells, MAPK signaling. This highlights the importance to carefully control for potential contaminations and that highly purified hCG preparations have to be used for biological studies. PMID:26495869

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

PubMed

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 < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of 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 < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive 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.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3).

PubMed

Fernández, María Belén; Daleo, Gustavo Raúl; Guevara, María Gabriela

2015-01-01

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

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

PubMed

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.

Activity of physalins purified from Physalis angulata in in vitro and in vivo models of cutaneous leishmaniasis.

PubMed

Guimarães, Elisalva T; Lima, Milena S; Santos, Luana A; Ribeiro, Ivone M; Tomassini, Therezinha B C; Ribeiro dos Santos, Ricardo; dos Santos, Washington L C; Soares, Milena B P

2009-07-01

We have previously demonstrated the immunomodulatory effects of physalins, secosteroids purified from Physalis angulata. Here we investigate the antileishmanial activity of physalins in vitro and in vivo in a model of cutaneous leishmaniasis. The antileishmanial activity of physalins B, D and F was tested in Leishmania-infected macrophage cultures. For the in vivo studies, BALB/c mice were infected with Leishmania amazonensis subcutaneously in the ear pinna and treated with physalin F by topical administration. Physalins B and F were able to reduce the percentage of Leishmania-infected macrophages and the intracellular parasite number in vitro at concentrations non-cytotoxic to macrophages. More importantly, topical treatment with physalin F significantly reduced the lesion size, the parasite load and histopathological alterations in BALB/c mice infected with L. amazonensis. Our results demonstrate the potent antileishmanial activity of physalins, especially physalin F, and suggest these molecules as the basis for the development of new therapeutic options for cutaneous leishmaniasis.

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

PubMed

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.

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

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-20

... antidumping duty orders on purified carboxymethylcellulose from Finland and the Netherlands would likely lead... Netherlands would likely lead to continuation or recurrence of dumping and, therefore, notified the U.S... revocation of the antidumping duty orders on purified CMC from Finland and the Netherlands would likely lead...

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

PubMed

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.

PubMed

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

2016-09-08

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(-1)min(-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.

Effects of high pressure processing on activity and structure of soluble acid invertase in mango pulp, crude extract, purified form and model systems.

PubMed

Li, Renjie; Wang, Yongtao; Ling, Jiangang; Liao, Xiaojun

2017-09-15

The effects of high pressure processing (HPP) on the activity of soluble acid invertase (SAI) in mango pulp, crude extract, purified SAI and purified SAI in model systems (pectin, bovine serum albumin (BSA), sugars and pH 3-7) were investigated. The activity of SAI in mango pulp was increased after HPP, and that in crude extract stayed unchanged. The activity of purified SAI was decreased after HPP at 45 and 50°C. Pectin exhibited a concentration-dependent protection for purified SAI against HPP at 50°C/600MPa for 30min. Pectin that had an esterification degree (DE) of 85% exhibited a greater protection than pectin that had a DE of 20-34%. BSA, acidic pH (3-6) and sucrose also exhibited protection for purified SAI against HPP. HPP at 50°C/600MPa for 30min disrupted the secondary structure and tertiary structure of purified SAI, but no aggregation of purified SAI was observed after HPP. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hypoglycemic effect of cooked Lupinus mutabilis and its purified alkaloids in subjects with type-2 diabetes.

PubMed

Baldeón, M E; Castro, J; Villacrés, E; Narváez, L; Fornasini, M

2012-01-01

Developing countries are experiencing an epidemic of chronic non-communicable chronic diseases with high socio-economic costs. Studies of traditional foods with beneficial health properties could contribute to diminish these problems. Legumes rich in proteins like Lupinus mutabilis decreases blood glucose and improves insulin sensitivity in animals and humans. We report the results of a phase II clinical trial conducted to assess the role of cooked L. mutabilis and its purified alkaloids on blood glucose and insulin in volunteers with diabetes. Results indicate that consumption of cooked L. mutabilis or its purified alkaloids decreased blood glucose and insulin levels. The decreases in serum glucose concentrations from base line to 90 minutes were statistically significant within both treatment groups; however, there were not differences between groups. Serum insulin levels were also decreased in both groups however the differences were not statistically significant. None of the volunteers in either group presented side effects.

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

PubMed

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.

Purified polysaccharides of Geoffroea spinosa barks have anticoagulant and antithrombotic activities devoid of hemorrhagic risks.

PubMed

Souza, Racquel O S; Assreuy, Ana M S; Madeira, Juliana C; Chagas, Francisco D S; Parreiras, Luane A; Santos, Gustavo R C; Mourão, Paulo A S; Pereira, Maria G

2015-06-25

Polysaccharides were extracted from the barks of Geoffroea spinosa, purified using anion exchange chromatography and characterized by chemical and methylation analysis, complemented by infrared and NMR spectroscopies. These polysaccharides were tested for their anticoagulant, antithrombotic and antiplatelet activities and also for their effects on bleeding. Unfractionated polysaccharide contains low levels of protein and high levels of carbohydrate (including hexuronic acid). The purified polysaccharides (fractions FII and FIII) are composed of arabinose (Ara), rhamnose (Rha), hexuronic acid, small amounts of galactose, but no sulfate ester. They have highly complex structure, which was partially characterized. NMR and methylation analysis indicate that the polysaccharides have a core of α-Rhap and branches of 5-linked α-Araf. Residues of 4-linked α-GalpA are also found in the structure. The unfractionated (TPL) and fraction FIII, but not fractions FI and FII, prolonged the activated partial thromboplastin time (aPTT). TPL, FII and FIII inhibited the platelet aggregation induced by ADP. More significantly, both unfractionated and purified fractions exhibited potent antithrombotic effect (31-60%) and the fractions did not modify the bleeding tendency. These plant polysaccharides could be alternative source of new anticoagulant, antiplatelet and antithrombotic compounds devoid of the undesirable risk of hemorrhage. Copyright © 2015 Elsevier Ltd. All rights reserved.

76 FR 29194 - Purified Carboxymethylcellulose From Mexico and Sweden: Revocation of Antidumping Duty Orders

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-20

... carboxymethylcellulose from Mexico and Sweden would not be likely to lead to continuation or recurrence of material... orders on purified CMC from Mexico and Sweden would be likely to lead to the continuation or recurrence... purified CMC from Mexico and Sweden would not be likely to lead to continuation or recurrence of material...

Preparation of purified fractions for polysaccharides from Monetaria moneta Linnaeus and comparison their characteristics and antioxidant activities.

PubMed

Yuan, Jun; Chen, Shuxian; Wang, Liping; Xu, Tingting; Shi, Xu; Jing, Yi; Zhang, Haijiang; Huang, Yange; Xu, Ying; Li, Dong; Chen, Xing; Chen, Jianhui; Xiong, Qingping

2018-03-01

The aim of this paper was to prepare purified fractions of polysaccharides from Monetaria moneta Linnaeus and further compare their characteristics and antioxidant activities. Firstly, three novel purified fractions, named MM-P1, MM-P2 and MM-P3, were successfully prepared by a DEAE-Sepharose fast-flow column. Then, their characteristics were compared using chemical testing, FT-IR, GC and HPGPC. The results suggested that MM-P3 had higher molecular weights than MM-P1 and MM-P2. MM-P1 was consisted of glucose, MM-P2 was consisted of glucose and xylose, and MM-P3 was comprised of glucose, xylose and mannose. Differed from MM-P1 and MM-P2, MM-P3 had sulfuric radical and uronic acid groups. Finally, their antioxidant activities were also compared. We found that MM-P3 exhibited better antioxidant bioactivities than MM-P1 and MM-P2. The data demonstrated that three purified fractions derived from different adsorption capacity of DEAE-Sepharose fast-flow column possessed different structural characteristics and antioxidant activity. Copyright © 2017 Elsevier B.V. All rights reserved.

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

PubMed

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.

PubMed

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.

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum.

PubMed

Buckley, Seamus J; Collins, Patrick J; O'Connor, Brendan F

2004-07-01

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.

PROCESS OF PURIFYING URANIUM

DOEpatents

Seaborg, G.T.; Orlemann, E.F.; Jensen, L.H.

1958-12-23

A method of obtaining substantially pure uranium from a uranium composition contaminated with light element impurities such as sodium, magnesium, beryllium, and the like is described. An acidic aqueous solution containing tetravalent uranium is treated with a soluble molybdate to form insoluble uranous molybdate which is removed. This material after washing is dissolved in concentrated nitric acid to obtaln a uranyl nitrate solution from which highly purified uranium is obtained by extraction with ether.

L1 stimulation of human glioma cell motility correlates with FAK activation

PubMed Central

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A.; Boulos, Magdy I.; Kappes, John C.

2011-01-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass

L1 stimulation of human glioma cell motility correlates with FAK activation.

PubMed

Yang, Muhua; Li, Yupei; Chilukuri, Kalyani; Brady, Owen A; Boulos, Magdy I; Kappes, John C; Galileo, Deni S

2011-10-01

The neural adhesion/recognition protein L1 (L1CAM; CD171) has been shown or implicated to function in stimulation of cell motility in several cancer types, including high-grade gliomas. Our previous work demonstrated the expression and function of L1 protein in stimulation of cell motility in rat glioma cells. However, the mechanism of this stimulation is still unclear. This study further investigated the function of L1 and L1 proteolysis in human glioblastoma multiforme (GBM) cell migration and invasion, as well as the mechanism of this stimulation. L1 mRNA was found to be present in human T98G GBM cell line but not in U-118 MG grade III human glioma cell line. L1 protein expression, proteolysis, and release were found in T98G cells and human surgical GBM cells by Western blotting. Exosome-like vesicles released by T98G cells were purified and contained full-length L1. In a scratch assay, T98G cells that migrated into the denuded scratch area exhibited upregulation of ADAM10 protease expression coincident with loss of surface L1. GBM surgical specimen cells exhibited a similar loss of cell surface L1 when xenografted into the chick embryo brain. When lentivirally introduced shRNA was used to attenuate L1 expression, such T98G/shL1 cells exhibited significantly decreased cell motility by time lapse microscopy in our quantitative Super Scratch assay. These cells also showed a decrease in FAK activity and exhibited increased focal complexes. L1 binding integrins which activate FAK were found in T98G and U-118 MG cells. Addition of L1 ectodomain-containing media (1) rescued the decreased cell motility of T98G/shL1 cells and (2) increased cell motility of U-118 MG cells but (3) did not further increase T98G cell motility. Injection of L1-attenuated T98G/shL1 cells into embryonic chick brains resulted in the absence of detectable invasion compared to control cells which invaded brain tissue. These studies support a mechanism where glioma cells at the edge of a cell mass

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

PubMed

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

PubMed

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.

Characterization of Purified Staphylococcal Lipase1

PubMed Central

Vadehra, D. V.; Harmon, L. G.

1967-01-01

Purified staphylococcal lipase had an optimal pH of 8.3 for activity at 37 C, and an optimal temperature of 45 C at pH 8.0. During storage, the enzyme lost less than 10% of the activity over a period of 21 days at 4 and -23 C. The enzyme retained 93% of the activity when heated for 30 min at 50 C and was 95% destroyed in 30 min at 70 C. The purified lipase was capable of hydrolyzing a variety of natural fats and oils. However, the enzyme was three times more active on nonhydrogenated soybean oil than on hydrogenated soybean oil with an iodine value of <3.0. The enzyme was also capable of hydrolyzing fatty acids on the α, β, and α′ positions of a synthetic mixed triglyceride. In general, the presence of oxidizing agents increased the activity and the presence of reducing agents decreased the activity of the lipase enzyme. PMID:6035042

Gas stream purifier

NASA Technical Reports Server (NTRS)

Adam, Steven J.

1994-01-01

A gas stream purifier has been developed that is capable of removing corrosive acid, base, solvent, organic, inorganic, and water vapors as well as particulates from an inert mixed gas stream using only solid scrubbing agents. This small, lightweight purifier has demonstrated the ability to remove contaminants from an inert gas stream with a greater than 99 percent removal efficiency. The Gas Stream Purifier has outstanding market and sales potential in manufacturing, laboratory and science industries, medical, automotive, or any commercial industry where pollution, contamination, or gas stream purification is a concern. The purifier was developed under NASA contract NAS9-18200 Schedule A for use in the international Space Station. A patent application for the Gas Stream Purifier is currently on file with the United States Patent and Trademark Office.

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.

PubMed

Shammas, Masood A; Qazi, Aamer; Batchu, Ramesh B; Bertheau, Robert C; Wong, Jason Y Y; Rao, Manjula Y; Prasad, Madhu; Chanda, Diptiman; Ponnazhagan, Selvarangan; Anderson, Kenneth C; Steffes, Christopher P; Munshi, Nikhil C; De Vivo, Immaculata; Beer, David G; Gryaznov, Sergei; Weaver, Donald W; Goyal, Raj K

2008-08-01

The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

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

PubMed

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.

Global vascular expression of murine CD34, a sialomucin-like endothelial ligand for L-selectin.

PubMed

Baumhueter, S; Dybdal, N; Kyle, C; Lasky, L A

1994-10-15

Extravasation of leukocytes into organized lymphoid tissues and into sites of inflammation is critical to immune surveillance. Leukocyte migration to peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN) and Peyer's patches (PP) depends on L-selectin, which recognizes carbohydrate-bearing, sialomucin-like endothelial cell surface glycoproteins. Two of these ligands have been identified at the molecular level. One is the potentially soluble mucin, GlyCAM 1, which is almost exclusively produced by high endothelial venules (HEV) of PLN and MLN. The second HEV ligand for L-selectin is the membrane-bound sialomucin CD34. Historically, this molecule has been successfully used to purify human pluripotent bone marrow stem cells, and limited data suggest that human CD34 is present on the vascular endothelium of several organs. Here we describe a comprehensive analysis of the vascular expression of CD34 in murine tissues using a highly specific antimurine CD34 polyclonal antibody. CD34 was detected on vessels in all organs examined and was expressed during pancreatic and skin inflammatory episodes. A subset of HEV-like vessels in the inflamed pancreas of nonobese diabetic (NOD) mice are positive for both CD34 and GlyCAM 1, and bind to an L-selectin/immunoglobulin G (IgG) chimeric probe. Finally, we found that CD34 is present on vessels of deafferentiated PLN, despite the fact that these vessels are no longer able to interact with L-selectin or support lymphocyte binding in vitro or trafficking in vivo. Our data suggest that the regulation of posttranslational carbohydrate modifications of CD34 is critical in determining its capability to act as an L-selectin ligand. Based on its ubiquitous expression, we propose that an appropriately glycosylated form of vascular CD34 may act as a ligand for L-selectin-mediated leukocyte trafficking to both lymphoid and nonlymphoid sites.

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

PubMed

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.

Guine pig liver L-asparaginase. Separation, purification, and intracellular localisation of two distinct enzymatic activities.

PubMed

Rogez, J C; Plaquet, R; Biserte, G

1975-12-18

Two distinct L-asparaginase (EC 3.5.1.1) activities were detected in guinea pig liver: Asparaginase 1 and Asparaginase 2. Asparaginase 1 has been purified 272 fold from the crude homogenate; its molecular weight was evaluated by gel filtration to be about 150 000. The purified preparation was shown to be homogeneous by cellulose acetate strip and polyacrylamide disc-gel electrophoresis. Asparaginase 2 has been purified 63.5 fold from the crude homogenate. Its molecular weight was evaluated by gel filtration to be about 21 500. Cellulose acetate strip electrophoresis demonstrated two bands, one of which corresponded to Asparaginase 1 and the other to Asparaginase 2. Cellular fractionation in the ultracentrifuge, showed Asparaginase 1 to be present only in the cytosol fraction. Asparaginase 2 which was unstable at 105 000 X g seemed mostly localized in the mitochondria and secondarily in the cytoplasmic fraction.

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

PubMed

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.

Regulation of L-phenylalanine ammonia-lyase from Rhizoctonia solani.

PubMed Central

Kalghatgi, K K; Subba Rao, P V

1976-01-01

Maximal levels of L-henylalanine ammonia-lyase activity were observed when the mycelial felts of Rhizoctonia solani were grown for 4.5 days on Byrde synthetic medium containing 3.5% glucose and 0.3% L-phenylalanine, Differential centrifugation studies have indicated that the enzyme is localized in the soluble fraction. The time course of induction of L-phenylalanine ammonia-lyase activity by L-phenylalanine showed a lag period of 1 to 1.5 h and reached a maximum around 4 to 6 h after the addition of the inducer to the medium. L-Phenylalanine, L-tyrosine, and L-tryptophan were nearly equally efficient inducers of the enzyme. D-Phenylalanine was as efficient as the L-isomer, whereas D-tyrosine was a poor inducer. Light, gibberellic acid, indole 3-acetic acid, and kinetin had no effect on the induction of L-phenylalanine ammonia-lyase activity. Cycloheximide did not inhibit the uptake of amino acids by the mycelia but completely blocked the incorporation of radioactive amino acids into soluble proteins and the development of L-phenylalanine ammonia-lyase activity. Actinomycin D inhibited both the incorporation of 32P into ribonucleic acid and the enzyme activity. Conclusive evidence for de novo synthesis of L-phenylalanine ammonia-lyase was obtained by the incorporation of radioactive amino acids into the enzyme. Electrophoretic analysis of the purified preparation showed a single protein band that coincided with radioactivity and L-phenylalanine ammonia-lyase activity. Glucose and intermediates of the tricarboxylic acid cycle, like citric acid, alpha-ketoglutaric acid, and succinic acid, and the metabolites of L-phenylalanine, like o-coumaric acid, o-hydroxyphenylacetic acid, and protocatechuic acid, significantly repressed L-phenylalanine ammonia-lyase activity. The observed repression was not relieved by cyclic adenosine 5'-triphosphate. Images PMID:1262311

Purifying Nanomaterials

NASA Technical Reports Server (NTRS)

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.

PubMed

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.

Unique properties of arginase purified from camel liver cytosol.

PubMed

Maharem, Tahany M; Zahran, Walid E; Hassan, Rasha E; Abdel Fattah, Mohamed M

2018-03-01

Arginase (ARG) is an enzyme involved in urea cycle, where it catalyzes the hydrolysis of L-arginine into L-ornithine and urea. Since there is no information about the isolation and purification of ARG from camel liver, this investigation was designed to purify and characterize ARG from camel liver and compare its molecular and kinetic properties with that reported from other species. Camel liver arginase (CL-ARG) was purified to homogeneity using heat denaturation followed by ammonium sulphate precipitation with a combination of DEAE-cellulose, SP-Sepharose and Sephadex G 100-120 chromatography columns. The specific activity of CL-ARG was increased to 18,485 units/mg proteins with 23.5-fold purification over crude homogenate. It was observed that CL-ARG showed a similarity with other species such as behaviour on DEAE-cellulose column, kinetics of inhibition, necessity for metal ions as cofactor, and alkaline optimum pH. On the contrary, CL-ARG differed in its molecular weight (180kDa), oligomeric protein structure, slightly neutral-alkaline pI value (7.7), K m value (7.1mM), optimum pH (9, 10.7), and higher optimum temperature (70°C). In conclusion, this study investigated the properties of CL-ARG via a simple and reproducible purification procedure and provided valuable information for its production from available source in Egypt for medical and industrial purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

[Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis].

PubMed

Liao, Chengshui; Wang, Xiaoli; Tian, Wenjing; Zhang, Mengke; Zhang, Chunjie; Li, Yinju; Wu, Tingcai; Cheng, Xiangchao

2017-08-25

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.

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

PubMed

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.

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

PubMed Central

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 (%T660nm = 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. PMID:24948919

Antibacterial Activity of AI-Hemocidin 2, a Novel N-Terminal Peptide of Hemoglobin Purified from Arca inflata.

PubMed

Li, Chunlei; Zhu, Jianhua; Wang, Yanqing; Chen, Yuyan; Song, Liyan; Zheng, Weiming; Li, Jingjing; Yu, Rongmin

2017-06-29

The continued emergence of antibiotic resistant bacteria in recent years is of great concern. The search for new classes of antibacterial agents has expanded to non-traditional sources such as shellfish. An antibacterial subunit of hemoglobin (Hb-I) was purified from the mantle of Arca inflata by phosphate extraction and ion exchange chromatography. A novel antibacterial peptide, AI-hemocidin 2, derived from Hb-I, was discovered using bioinformatics analysis. It displayed antibacterial activity across a broad spectrum of microorganisms, including several Gram-positive and Gram-negative bacteria, with minimal inhibitory concentration (MIC) values ranging from 37.5 to 300 μg/mL, and it exhibited minimal hemolytic or cytotoxic activities. The antibacterial activity of AI-hemocidin 2 was thermostable (25-100 °C) and pH resistant (pH 3-10). The cellular integrity was determined by flow cytometry. AI-hemocidin 2 was capable of permeating the cellular membrane. Changes in the cell morphology were observed with a scanning electron microscope. Circular dichroism spectra suggested that AI-hemocidin 2 formed an α-helix structure in the membrane mimetic environment. The results indicated that the anti-bacterial mechanism for AI-hemocidin 2 occurred through disrupting the cell membrane. AI-hemocidin 2 might be a potential candidate for tackling antibiotic resistant bacteria.

Antibacterial Activity of AI-Hemocidin 2, a Novel N-Terminal Peptide of Hemoglobin Purified from Arca inflata

PubMed Central

Li, Chunlei; Zhu, Jianhua; Wang, Yanqing; Chen, Yuyan; Song, Liyan; Zheng, Weiming; Li, Jingjing; Yu, Rongmin

2017-01-01

The continued emergence of antibiotic resistant bacteria in recent years is of great concern. The search for new classes of antibacterial agents has expanded to non-traditional sources such as shellfish. An antibacterial subunit of hemoglobin (Hb-I) was purified from the mantle of Arca inflata by phosphate extraction and ion exchange chromatography. A novel antibacterial peptide, AI-hemocidin 2, derived from Hb-I, was discovered using bioinformatics analysis. It displayed antibacterial activity across a broad spectrum of microorganisms, including several Gram-positive and Gram-negative bacteria, with minimal inhibitory concentration (MIC) values ranging from 37.5 to 300 μg/mL, and it exhibited minimal hemolytic or cytotoxic activities. The antibacterial activity of AI-hemocidin 2 was thermostable (25–100 °C) and pH resistant (pH 3–10). The cellular integrity was determined by flow cytometry. AI-hemocidin 2 was capable of permeating the cellular membrane. Changes in the cell morphology were observed with a scanning electron microscope. Circular dichroism spectra suggested that AI-hemocidin 2 formed an α-helix structure in the membrane mimetic environment. The results indicated that the anti-bacterial mechanism for AI-hemocidin 2 occurred through disrupting the cell membrane. AI-hemocidin 2 might be a potential candidate for tackling antibiotic resistant bacteria. PMID:28661457

Serine protease activity of Cur l 1 from Curvularia lunata augments Th2 response in mice.

PubMed

Tripathi, Prabhanshu; Kukreja, Neetu; Singh, B P; Arora, Naveen

2009-05-01

Studies with mite allergens demonstrated that proteolytic activity augments allergic airway inflammation. This knowledge is limited to few enzyme allergens. The objective of this study is to investigate the effect of serine protease Cur l 1 from Curvularia lunata in airway inflammation/hyper-responsiveness. Cur l 1 was purified and inactivated using a serine protease inhibitor. Balb/c mice were sensitized with enzymatically active Cur l 1 or C. lunata extract. Sensitized mice were given booster dose on day 14 with active or inactivated Cur l 1. Intranasal challenge was given on day 28, 29, and 30. Airway hyper-responsiveness was measured by plethysmography. Blood, bronchoalveolar lavage fluid (BALF), spleen, and lungs from mice were analyzed for cellular infiltration, immunoglobulins, and cytokine levels. Mice challenged with enzymatically active Cur l 1 demonstrated significantly higher airway inflammation than inactive Cur l 1 group mice (p < 0.01). There was a significant difference in serum IgE and IgG1 levels among mice immunized with active Cur l 1 and inactive Cur l 1 (p < 0.01). IL-4 and IL-5 were higher in BALF and splenocyte culture supernatant of active Cur l 1 than inactive Cur l 1 mice. Lung histology revealed increased eosinophil infiltration, goblet cell hyperplasia and mucus secretion in active group. Proteolytic activity of Cur l 1 plays an important role in airway inflammation and the inactivated Cur l 1 has potential to be explored for immunotherapy.

In vitro efficacy of latex and purified papain from Carica papaya against Strongyloides venezuelensis eggs and larvae.

PubMed

Moraes, Dayane; Levenhagen, Marcelo Arantes; Costa-Cruz, Julia Maria; Costa, Antônio Paulino da; Rodrigues, Rosângela Maria

2017-04-03

Latex from Carica papaya is rich in bioactive compounds, especially papain, which may help to control parasitic diseases. This study evaluated the efficacy of latex from C. papaya and purified papain against Strongyloides venezuelensis. The Egg Hatching Test (EHT) and the Larval Motility Test (LMT) using fresh and frozen latex (250mg/mL), lyophilized latex (34mg/mL), and purified papain (2.8 mg/mL) were performed. Albendazole (0.025 mg/mL) and ivermectin (316 ppm) were used as positive controls. EHT and LMT were carried out through the incubation of each solution with S. venezuelensis eggs or larvae (± 100 specimens), and results were analyzed after 48h (EHT) or 24, 48, and 72h (LMT). EHT showed that latex preparations at higher concentrations (1:10 to 1:100) resulted in partial or complete destruction of eggs and larvae inside the eggs. The result from the 1:1,000 dilution was similar to the positive control. LMT showed effectiveness in all the tested dilutions compared to negative controls. Purified papain showed a dose-dependent response in the EHT. Purified papain (2.8 mg/ mL) showed similar results to lyophilized latex at 1:1,000 in the EHT. Latex and purified papain from C. papaya were effective against S. venezuelensis eggs and larvae in vitro, suggesting their potential use as an alternative treatment for strongyloidiasis.

[Bacterial recombinant L-asparaginases: properties, structure and anti-proliferative activity].

PubMed

Sokolov, N N; Eldarov, M A; Pokrovskaya, M V; Aleksandrova, S S; Abakumova, O Yu; Podobed, O V; Melik-Nubarov, N S; Kudryashova, E V; Grishin, D V; Archakov, A I

2015-01-01

For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemia in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases as Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA) and Rhodospirillum rubrum (RrA); special attention is paid to isolation of purified enzymes and their crystallization, modification by chitosan/polyethylene, physicochemical, kinetic and structural properties characterization, and the study of the cytotoxic or anti-proliferative activity of new recombinant L-asparaginases on cell cultures in vitro. The resultant recombinant L-asparaginases (EwA, YpA, HpA и RrA) exhibit reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available L-asparaginase EcA and represent practical interest in respect to creation, on their basis, new effective antineoplastic remedies. Further prospects of researches on bacterial L-asparaginases are associated with development of analogs of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes of the protein structure using genetic engineering, development of chito-PEGylation for receiving L-asparaginase preparations with improved pharmacokinetic characteristics.

Purification and characterization of a novel fibrinolytic α chymotrypsin like serine metalloprotease from the edible mushroom, Lyophyllum shimeji.

PubMed

Moon, Sung-Min; Kim, Jae-Sung; Kim, Heung-Joong; Choi, Mi Suk; Park, Bo Ram; Kim, Su-Gwan; Ahn, Hoon; Chun, Hong Sung; Shin, Yong Kook; Kim, Jong-Jin; Kim, Do Kyung; Lee, Sook-Young; Seo, Young-Woo; Kim, Yong Hwan; Kim, Chun Sung

2014-05-01

A novel fibrinolytic enzyme was purified from Lyophyllum shimeji, a popular edible mushroom in Asia. The enzyme was purified using combination of anion exchange chromatography on a Mono Q 5/5 column and size exclusion gel filtration chromatography on Superdex 200 100/300 column. This purification protocol resulted 80.9-fold purification of the enzyme and a final yield of 5.7%. The molecular weight of the purified enzyme was estimated to be 21 kDa by SDS-PAGE and size exclusion gel filtration. The N-terminal amino acid sequence was found to be ITFQSASP, which is dissimilar from that of known fibrinolytic enzymes. The purified enzyme was a neutral protease with an optimal reaction pH and temperature of 8.0 and 37°C, respectively. Enzymatic activity was inhibited by Cu(2+) and Co(2+). It was also significantly inhibited by PMSF and TPCK. Furthermore, it was found to exhibit a higher specificity for S-7388, a well-known chymotrypsin chromogenic substrate, indicating chymotrypsin like serine metalloprotease. The relative fibrinolytic activity of 5 μg purified enzyme have two fold more activity than 1 unit/ml of plasmin on fibrin plate. Furthermore, purified enzyme preferentially hydrolyzed the Aα-chain followed by the Bβ- and γ-chain of fibrinogen, which is precursor of fibrin. Therefore, these data suggests that the fibrinolytic enzyme derived from edible mushroom, L. shimeji, might be useful for thrombolytic therapy and preventing thrombotic disease. Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A chymotrypsin-like proteinase from the midgut of Tenebrio molitor larvae.

PubMed

Elpidina, E N; Tsybina, T A; Dunaevsky, Y E; Belozersky, M A; Zhuzhikov, D P; Oppert, B

2005-08-01

A chymotrypsin-like proteinase was isolated from the posterior midgut of larvae of the yellow mealworm, Tenebrio molitor, by ion-exchange and gel filtration chromatography. The enzyme, TmC1, was purified to homogeneity as determined by SDS-PAGE and postelectrophoretic activity detection. TmC1 had a molecular mass of 23.0 kDa, pI of 8.4, a pH optimum of 9.5, and the optimal temperature for activity was 51 degrees C. The proteinase displayed high stability at temperatures below 43 degrees C and in the pH range 6.5-11.2, which is inclusive of the pH of the posterior and middle midgut. The enzyme hydrolyzed long chymotrypsin peptide substrates SucAAPFpNA, SucAAPLpNA and GlpAALpNA and did not hydrolyze short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SucAAPFpNA, with k(cat app) 36.5 s(-1) and K(m) 1.59 mM. However, the enzyme had a lower K(m) for SucAAPLpNA, 0.5 mM. Phenylmethylsulfonyl fluoride (PMSF) was an effective inhibitor of TmC1, and the proteinase was not inhibited by either tosyl-l-phenylalanine chloromethyl ketone (TPCK) or N(alpha)-tosyl-l-lysine chloromethyl ketone (TLCK). However, the activity of TmC1 was reduced with sulfhydryl reagents. Several plant and insect proteinaceous proteinase inhibitors were active against the purified enzyme, the most effective being Kunitz soybean trypsin inhibitor (STI). The N-terminal sequence of the enzyme was IISGSAASKGQFPWQ, which was up to 67% similar to other insect chymotrypsin-like proteinases and 47% similar to mammalian chymotrypsin A. The amino acid composition of TmC1 differed significantly from previously isolated T. molitor enzymes.

Adult Schistosoma mansoni express cathepsin L proteinase activity.

PubMed

Smith, A M; Dalton, J P; Clough, K A; Kilbane, C L; Harrop, S A; Hole, N; Brindley, P J

1994-09-01

This report presents the deduced amino acid sequence of a novel cathepsin L proteinase from Schistosoma mansoni, and describes cathepsin L-like activity in extracts of adult schistosomes. Using consensus primers specific for cysteine proteinases, gene fragments were amplified from adult S. mansoni cDNA by PCR and cloned. One of these fragments showed marked identity to Sm31, the cathepsin B cysteine proteinase of adult S. mansoni, whereas another differed from Sm31 and was employed as a probe to isolate two cDNAs from an adult S. mansoni gene library. Together these cDNAs encoded a novel preprocathepsin L of 319 amino acids; this zymogen is predicted to be processed in vivo into a mature, active cathepsin L proteinase of 215 amino acids. Closest homologies were with cathepsins L from rat, mouse, and chicken (46-47% identity). Southern hybridization analysis suggested that only one or a few copies of the gene was present per genome, demonstrated that its locus was distinct from that of Sm31, and that a homologous sequence was present in Schistosoma japonicum. Because these results indicated that schistosomes expressed a cathepsin L proteinase, extracts of adult S. mansoni were examined for acidic, cysteine proteinase activity. Based on rates of cleavage of peptidyl substrates employed to discriminate between classes of cysteine proteinases, namely cathepsin L (Z-phe-arg-AMC), cathepsin B (Z-arg-arg-AMC) and cathepsin H (Bz-arg-AMC), the extracts were found to contain vigorous cathepsin L-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro efficacy of latex and purified papain from Carica papaya against Strongyloides venezuelensis eggs and larvae

PubMed Central

Moraes, Dayane; Levenhagen, Marcelo Arantes; Costa-Cruz, Julia Maria; da Costa, Antônio Paulino; Rodrigues, Rosângela Maria

2017-01-01

ABSTRACT Latex from Carica papaya is rich in bioactive compounds, especially papain, which may help to control parasitic diseases. This study evaluated the efficacy of latex from C. papaya and purified papain against Strongyloides venezuelensis. The Egg Hatching Test (EHT) and the Larval Motility Test (LMT) using fresh and frozen latex (250mg/mL), lyophilized latex (34mg/mL), and purified papain (2.8 mg/mL) were performed. Albendazole (0.025 mg/mL) and ivermectin (316 ppm) were used as positive controls. EHT and LMT were carried out through the incubation of each solution with S. venezuelensis eggs or larvae (± 100 specimens), and results were analyzed after 48h (EHT) or 24, 48, and 72h (LMT). EHT showed that latex preparations at higher concentrations (1:10 to 1:100) resulted in partial or complete destruction of eggs and larvae inside the eggs. The result from the 1:1,000 dilution was similar to the positive control. LMT showed effectiveness in all the tested dilutions compared to negative controls. Purified papain showed a dose-dependent response in the EHT. Purified papain (2.8 mg/ mL) showed similar results to lyophilized latex at 1:1,000 in the EHT. Latex and purified papain from C. papaya were effective against S. venezuelensis eggs and larvae in vitro, suggesting their potential use as an alternative treatment for strongyloidiasis. PMID:28380118

Effects of anti-inflammatory and anti-rheumatic drugs on the activities of purified and membrane-bound Na+/K+ adenosine triphosphatase

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

Chan, M.K.; Minta, J.O.

1985-08-01

The authors have examined the effects of anti-inflammatory and anti-rheumatic drugs on membrane-bound and purified Na /K -ATPase activity in vitro. Only the gold-containing compounds (gold sodium thiomalate and auranofin) were found to inhibit the enzyme activity in a dose-dependent manner. Sodium thiomalate and triethylphosphine, the ligand compounds for gold sodium thiomalate and auranofin, respectively, had no effect on ATPase activity. The antagonistic properties was abolished by preincubation of the gold compounds with dithiothreitol. Lineweaver-Burke analysis of the inhibitions of purified ATPase by the gold compounds was found to follow uncompetitive kinetics. Inhibition of ATPase by gold may cause disruptionmore » of transmembrane cation transport and thus result in impairment of several metabolic processes and cellular functions.« less

AtFXG1, an Arabidopsis gene encoding alpha-L-fucosidase active against fucosylated xyloglucan oligosaccharides.

PubMed

de La Torre, Francisco; Sampedro, Javier; Zarra, Ignacio; Revilla, Gloria

2002-01-01

An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium. The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.

Purified discord and multipartite entanglement

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

Brown, Eric G.; Webster, Eric J.; Martín-Martínez, Eduardo, E-mail: emmfis@gmail.com

2013-10-15

We study bipartite quantum discord as a manifestation of a multipartite entanglement structure in the tripartite purified system. In particular, we find that bipartite quantum discord requires the presence of both bipartite and tripartite entanglement in the purification. This allows one to understand the asymmetry of quantum discord, D(A,B)≠D(B,A) in terms of entanglement monogamy. As instructive special cases, we study discord for qubits and Gaussian states in detail. As a result of this we shed new light on a counterintuitive property of Gaussian states: the presence of classical correlations necessarily requires the presence of quantum correlations. Finally, our results alsomore » shed new light on a protocol for remote activation of entanglement by a third party. -- Highlights: •Bipartite quantum discord as a manifestation of multipartite entanglement. •Relevance of quantum discord as a utilizable resource for quantum info. tasks. •Quantum discord manifests itself in entanglement in the purified state. •Relation between asymmetry of discord and entanglement monogamy. •Protocol for remote activation of entanglement by a third party.« less

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration*

PubMed Central

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W.

2014-01-01

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions. PMID:24532791

Conserved loop cysteines of vitamin K epoxide reductase complex subunit 1-like 1 (VKORC1L1) are involved in its active site regeneration.

PubMed

Tie, Jian-Ke; Jin, Da-Yun; Stafford, Darrel W

2014-03-28

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1's active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1's overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1's active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.

Dynamic properties of biologically active synthetic heparin-like hexasaccharides.

PubMed

Angulo, Jesús; Hricovíni, Milos; Gairi, Margarida; Guerrini, Marco; de Paz, José Luis; Ojeda, Rafael; Martín-Lomas, Manuel; Nieto, Pedro M

2005-10-01

A complete study of the dynamics of two synthetic heparin-like hexasaccharides, D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (1) and -->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHAc-6-SO4-alpha-(1-->4)-L-IdoA-alpha-(1-->4)-D-GlcNHSO3-alpha-(1-->4)-L-IdoA-2-SO4-alpha-1-->iPr (2), has been performed using 13C-nuclear magnetic resonance (NMR) relaxation parameters, T1, T2, and heteronuclear nuclear Overhauser effect (NOEs). Compound 1 is constituted from sequences corresponding to the major polysaccharide heparin region, while compound 2 contains a sequence never found in natural heparin. They differ from each other only in sulphation patterns, and are capable of stimulating fibroblast growth factors (FGFs)-1 induced mitogenesis. Both oligosaccharides exhibit a remarkable anisotropic overall motion in solution as revealed by their anisotropic ratios (tau /tau||), 4.0 and 3.0 respectively. This is a characteristic behaviour of natural glycosaminoglycans (GAG) which has also been observed for the antithrombin (AT) binding pentasaccharide D-GlcNHSO3-6-SO4-alpha-(1-->4)-D-GlcA-beta-(1-->4)-D-GlcNHSO3-(3,6-SO4)-alpha-(1-->4)-L-IdoA-2-SO4-alpha-(1-->4)-D-GlcNHSO3-6-SO4-alpha-1-->Me (3) (Hricovíni, M., Guerrini, M., Torri, G., Piani, S., and Ungarelli, F. (1995) Conformational analysis of heparin epoxide in aqueous solution. An NMR relaxation study. Carbohydr. Res., 277, 11-23). The motional properties observed for 1 and 2 provide additional support to the suitability of these compounds as heparin models in agreement with previous structural (de Paz, J.L., Angulo, J., Lassaletta, J.M., Nieto, P.M., Redondo-Horcajo, M., Lozano, R.M., Jiménez-Gallego, G., and Martín-Lomas, M. (2001) The activation of fibroblast growth factors by heparin: synthesis, structure and biological activity of heparin-like oligosaccharides. Chembiochem, 2, 673-685; Ojeda, R

Influence of purified multiwalled carbon nanotubes on the mechanical and morphological behavior in poly (L-lactic acid) matrix.

PubMed

Leal, C V; Martinez, D S T; Más, B A; Alves, O L; Duek, E A R

2016-06-01

Poly (L-latic acid) (PLLA) is a bioresorbable polymer widely used as a biomaterial, but its fragility can limit its use. An alternative is to produce polymer nanocomposites, which can enhance the mechanical properties of polymeric matrix, resulting in a material with differentiated properties. In this work, PLLA based nanocomposites containing 0.25, 0.5 and 1.0wt% of purified multiwalled carbon nanotubes (p-MWCNTs) were prepared by the solvent casting method. The morphology and mechanical properties results show an improvement in strain at break for 0.25 and 0.5wt% p-MWCNTs and an increase in stiffness and elastic modulus for all compositions. Nanocomposites presented a p-MWCNTs agglomeration; however, there was a good stress transfer between PLLA and p-MWCNTs, which was confirmed by the increase in the hardness and elastic modulus. Atomic force microscopy analysis indicated an increase in roughness after nanotube addition. The in vitro biological study showed that PLLA/p-MWCNTs nanocomposites are cytocompatible with osteoblasts cells. The capacity of PLLA nanocomposites to stimulate osteogenesis was investigated by alkaline phosphatase (ALP) activity assay. Higher ALP activity was found on osteoblasts cultured on nanocomposites with 0.25 and 0.5wt% p-MWCNT compared to neat PLLA, confirming that PLLA cytocompatibility was improved on these compositions. Finally, our results showed that by a simple and inexpensive solvent casting method, it is possible to manufacture biofunctional nanocomposites devices with potential for orthopedic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Biocompatibility selenium nanoparticles with an intrinsic oxidase-like activity

NASA Astrophysics Data System (ADS)

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.

α-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds

PubMed Central

Hata, Keishi; Tanaka, Mika; Tsumuraya, Yoichi; Hashimoto, Yohichi

1992-01-01

An α-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl α-l-arabinofuranoside and p-nitrophenyl β-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding α-l-arabinopyranoside and β-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg2+, Ag+, l-arabino-(1→4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off α-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of α-l-arabinofuranosidase and β-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety. Images Figure 2 PMID:16652973

Damage-associated molecular pattern activated Toll-like receptor 4 signalling modulates blood pressure in L-NAME-induced hypertension.

PubMed

Sollinger, Daniel; Eißler, Ruth; Lorenz, Steffen; Strand, Susanne; Chmielewski, Stefan; Aoqui, Cristiane; Schmaderer, Christoph; Bluyssen, Hans; Zicha, Josef; Witzke, Oliver; Scherer, Elias; Lutz, Jens; Heemann, Uwe; Baumann, Marcus

2014-03-01

Recent publications have shed new light on the role of the adaptive and innate immune system in the pathogenesis of hypertension. However, there are limited data whether receptors of the innate immune system may influence blood pressure. Toll-like receptor 4 (TLR4), a pattern recognition receptor, is a key component of the innate immune system, which is activated by exogenous and endogenous ligands. Hypertension is associated with end-organ damage and thus might lead to the release of damage-associated molecular patterns (DAMPs), which are endogenous activators of TLR4 receptors. The present study aimed to elucidate whether TLR4 signalling is able to modulate vascular contractility in an experimental model of hypertension thus contributing to blood pressure regulation. NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension was blunted in TLR4(-/-) when compared with wild-type mice. Treatment with l-NAME was associated with a release of DAMPs, leading to reactive oxygen species production of smooth muscle cells in a TLR4-dependent manner. As oxidative stress leads to an impaired function of the NO-sGC-cyclic GMP (cGMP) pathway, we were able to demonstrate that TLR4(-/-) was protected from sGC inactivation. Consequently, arterial contractility was reduced in TLR4(-/-). Cell damage-associated TLR4 signalling might act as a direct mediator of vascular contractility providing a molecular link between inflammation and hypertension.

Inhibitory effects of purified antibody against α-1 repeat (117-137) on Na(+)-Ca(2+) exchange and L-type Ca(2+) currents in rat cardiomyocytes.

PubMed

Feng, Qi-Long; Wu, Dong-Mei; Cui, Xiang-Li; Zhao, Hua-Chen; Lin, Yuan-Yuan; Zhao, Lu-Ying; Wu, Bo-Wei

2010-10-25

Considering that α-1 repeat region may be involved in the ion binding and translocation of Na(+)-Ca(2+) exchanger (NCX), it is possible that the antibodies against NCX α-1 repeat may have a crucial action on NCX activity. The aim of the present study is to investigate the effect of antibody against α-1 repeat (117-137), designated as α-1(117-137), on NCX activity. The antibody against the synthesized α-1(117-137) was prepared and affinity-purified. Whole-cell patch clamp technique was used to study the change of Na(+)-Ca(2+) exchange current (I(Na/Ca)) in adult rat cardiomyocytes. To evaluate the functional specificity of this antibody, its effects on L-type Ca(2+) current (I(Ca,L)), voltage-gated Na(+) current (I(Na)) and delayed rectifier K(+) current (I(K)) were also observed. The amino acid sequences of α-1(117-137) in NCX and residues 1 076-1 096 within L-type Ca(2+) channel were compared using EMBOSS Pairwise Alignment Algorithms. The results showed that outward and inward I(Na/Ca) were decreased by the antibody against α-1(117-137) dose-dependently in the concentration range from 10 to 160 nmol/L, with IC(50) values of 18.9 nmol/L and 22.4 nmol/L, respectively. Meanwhile, the antibody also decreased I(Ca,L) in a concentration-dependent manner with IC(50) of 22.7 nmol/L. No obvious effects of the antibody on I(Na) and I(K) were observed. Moreover, comparison of the amino acid sequences showed there was 23.8% sequence similarity between NCX α-1(117-137) and residues 1 076-1 096 within L-type Ca(2+) channel. These results suggest that antibody against α-1(117-137) is a blocking antibody to NCX and can also decrease I(Ca,L) in a concentration-dependent manner, while it does not have obvious effects on I(Na) and I(K).

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides.

PubMed

Heikal, Adam; Box, Karl; Rothnie, Alice; Storm, Janet; Callaghan, Richard; Allen, Marcus

2009-02-01

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at -80 degrees C. For example, at 20 degrees C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.

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

NASA Technical Reports Server (NTRS)

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.

Comparative study of disinfectants for use in low-cost gravity driven household water purifiers.

PubMed

Patil, Rajshree A; Kausley, Shankar B; Balkunde, Pradeep L; Malhotra, Chetan P

2013-09-01

Point-of-use (POU) gravity-driven household water purifiers have been proven to be a simple, low-cost and effective intervention for reducing the impact of waterborne diseases in developing countries. The goal of this study was to compare commonly used water disinfectants for their feasibility of adoption in low-cost POU water purifiers. The potency of each candidate disinfectant was evaluated by conducting a batch disinfection study for estimating the concentration of disinfectant needed to inactivate a given concentration of the bacterial strain Escherichia coli ATCC 11229. Based on the concentration of disinfectant required, the size, weight and cost of a model purifier employing that disinfectant were estimated. Model purifiers based on different disinfectants were compared and disinfectants which resulted in the most safe, compact and inexpensive purifiers were identified. Purifiers based on bromine, tincture iodine, calcium hypochlorite and sodium dichloroisocyanurate were found to be most efficient, cost effective and compact with replacement parts costing US$3.60-6.00 for every 3,000 L of water purified and are thus expected to present the most attractive value proposition to end users.

l-Asparaginase from Proteus vulgaris1

PubMed Central

Tosa, Tetsuya; Sano, Ryujiro; Yamamoto, Kozo; Nakamura, Masatoshi; Ando, Katsuko; Chibata, Ichiro

1971-01-01

To produce an immunologically and enzymologically new type of l-asparaginase, 108 strains of bacteria were screened for enzyme production. As a result, 13 bacteria belonging to the genera Alcaligenes, Bacterium, and Proteus were found to produce l-asparaginases in high levels. Among these l-asparaginases, partially purified l-asparaginases from B. cadaveris and P. vulgaris showed antitumor activity. A partially purified l-asparaginase preparation of P. vulgaris did not react with the antibody of Escherichia colil-asparaginase on the Ouchterlony agar plate. Culture conditions for the production of l-asparaginase by P. vulgaris were investigated in detail. The enzyme was produced in high yields when cells were grown aerobically in a medium containing sodium fumarate and corn steep liquor. The addition of glucose or ammonium ion to the medium, however, resulted in depressed production of l-asparaginase. Under the optimum conditions, 3,700 international units of l-asparaginase was obtained from 1 liter of culture medium. Images PMID:5000866

Polar Constituents and Biological Activity of the Berry-Like Fruits from Hypericum androsaemum L.

PubMed Central

Caprioli, Giovanni; Alunno, Alessia; Beghelli, Daniela; Bianco, Armandodoriano; Bramucci, Massimo; Frezza, Claudio; Iannarelli, Romilde; Papa, Fabrizio; Quassinti, Luana; Sagratini, Gianni; Tirillini, Bruno; Venditti, Alessandro; Vittori, Sauro; Maggi, Filippo

2016-01-01

Hypericum androsaemum, also known as Tutsan, is a small evergreen shrub common in the Mediterranean basin where it is traditionally used as diuretic and hepatoprotective herbal drug. This plant possesses the peculiarity to produce fleshy and berry-like fruits that ripen from red to shiny black. In the present work, the chemical constituents of methanolic extracts and infusions of red and black fruits were analyzed by HPLC, and correlated with their antioxidant properties which were evaluated by the DPPH, β-Carotene/linoleic acid, and hypochlorous acid tests. In addition, the red pigment of the fruit was isolated by column chromatography and structurally elucidated by NMR. Results showed that H. androsaemum fruits contain high amounts of shikimic and chlorogenic acids, while their color was given by a tetraoxygenated-type xanthone, reported for the first time in Hypericum species. The red berries infusion gave the highest content of total phenolic compounds, DPPH, and hypochlorous acid scavenging activity, and β-carotene bleaching. Cytotoxicity of the berries extracts on three human tumor cell lines (malignant melanoma, breast adenocarcinoma, and colon carcinoma) was evaluated by MTT assay, and relevant inhibition on colon carcinoma cells (IC50 value of 8.4 μg/mL) was found. Finally, the effects of red berries extract on the immune system were evaluated by peripheral blood mononuclear cell (PBMC) proliferation assay that revealed a strong stimulation on lymphocytes at low doses (0.4–6 μg/mL). PMID:26973675

Effects of cyanobacterial biomass and purified microcystins on malformations in Xenopus laevis: teratogenesis assay (FETAX).

PubMed

Dvoráková, Dagmar; Dvoráková, Katerina; Bláha, Ludek; Marsálek, Blahoslav; Knotková, Zora

2002-12-01

Xenopus laevis (African clawed frog) embryos in a 96-h teratogenesis assay (FETAX) were exposed to 0-250 microg/L and 500 microg/L of purified microcystin-LR (MCYST-LR) for the estimation of lethality, as well as to equivalent concentrations of biomass containing MCYST-LR (natural water bloom dominated by Microcystis aeruginosa) and biomass without MCYST-LR (bloom dominated by Microcystis wesenbergii). The highest tested concentrations of purified MCYST-LR caused up to 30% lethality after a 96-h exposure, corresponding to a LC(25) of 380 microg/L. Cyanobacterial biomass containing MCYST-LR caused significant lethality up to 50% at the highest tested concentrations (300 mg/L, i.e., 250 microg/L of MCYST-LR). The estimated 96-h LC(25) values varied from 125 mg/L (biomass containing MCYST-LR) up to 232 mg/L (biomass without MCYST-LR). A statistically significant increase in the number of malformed embryos was observed after exposure to cyanobacterial samples. Purified MCYST-LR at and above 25 microg/L significantly increased the number of malformations, with 53% of surviving embryos malformed in the highest tested concentration, 250 microg/L (EC(25) = 27 microg/L). Exposure to the highest concentration of MCYST-LR containing biomass resulted in more than 60% of the embryos being malformed and an EC(25) of 52 mg/L (i.e., 43 microg of MCYST-LR/L). Cyanobacterial biomass with no natural microcystin also induced substantial malformations-about 50% aberrant embryos at the highest concentration, 300 mg/L (EC(25) = 75 mg/L). External additions of purified MCYST-LR to the biomass that was originally without microcystins resulted in a slight additional increase in the rate of malformations (80% at the highest concentration, 300 mg of biomass plus 250 microg of MCYST-LR per liter). A comparison of lethality and effects on malformations (teratogenic index, TI = LC(25)/EC(25)) showed that all samples had significant teratogenic potential in the FETAX assay (TI(MCYST-LR) = 14; TI

THE PREPARATION AND PROPERTIES OF HIGHLY PURIFIED ASCORBIC ACID OXIDASE

PubMed Central

Powers, Wendell H.; Lewis, Stanley; Dawson, Charles R.

1944-01-01

1. A method is described for the preparation of a highly purified ascorbic acid oxidase containing 0.24 per cent copper. 2. Using comparable activity measurements, this oxidase is about one and a half times as active on a dry weight basis as the hitherto most highly purified preparation described by Lovett-Janison and Nelson. The latter contained 0.15 per cent copper. 3. The oxidase activity is proportional to the copper content and the proportionality factor is the same as that reported by Lovett-Janison and Nelson. 4. When dialyzed free of salt, the blue concentrated oxidase solutions precipitate a dark green-blue protein which carries the activity. This may be prevented by keeping the concentrated solutions about 0.1 M in Na2HPO4. 5. When highly diluted for activity measurements the oxidase rapidly loses activity (irreversibly) previous to the measurement, unless the dilution is made with a dilute inert protein (gelatin) solution. Therefore activity values obtained using such gelatin-stabilized dilute solutions of the oxidase run considerably higher than values obtained by the Lovett-Janison and Nelson technique. 6. The effect of pH and substrate concentration on the activity of the purified oxidase in the presence and absence of inert protein was studied. PMID:19873382

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

PubMed

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.

PubMed

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.

AtFXG1, an Arabidopsis Gene Encoding α-l-Fucosidase Active against Fucosylated Xyloglucan Oligosaccharides1

PubMed Central

de la Torre, Francisco; Sampedro, Javier; Zarra, Ignacio; Revilla, Gloria

2002-01-01

An α-l-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an α-l-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an α-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known α-l-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the α-l-fucosidase activities secreted to the culture medium. The α-l-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2′-fucosyl-lactitol but not against p-nitrophenyl-α-l-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2′-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism. PMID:11788770

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

PubMed Central

Vyas, Sangita

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. PMID:27978542

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

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

Dick, Robert A.; Datta, Siddhartha A. K.; Nanda, Hirsh

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, whichmore » 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 results show that RSV Gag is highly flexible and can adopt

Pseudomonas aeruginosa arylsulfatase: a purified enzyme for the mild hydrolysis of steroid sulfates.

PubMed

Stevenson, Bradley J; Waller, Christopher C; Ma, Paul; Li, Kunkun; Cawley, Adam T; Ollis, David L; McLeod, Malcolm D

2015-10-01

The hydrolysis of sulfate ester conjugates is frequently required prior to analysis for a range of analytical techniques including gas chromatography-mass spectrometry (GC-MS). Sulfate hydrolysis may be achieved with commercial crude arylsulfatase enzyme preparations such as that derived from Helix pomatia but these contain additional enzyme activities such as glucuronidase, oxidase, and reductase that make them unsuitable for many analytical applications. Strong acid can also be used to hydrolyze sulfate esters but this can lead to analyte degradation or increased matrix interference. In this work, the heterologously expressed and purified arylsulfatase from Pseudomonas aeruginosa is shown to promote the mild enzyme-catalyzed hydrolysis of a range of steroid sulfates. The substrate scope of this P. aeruginosa arylsulfatase hydrolysis is compared with commercial crude enzyme preparations such as that derived from H. pomatia. A detailed kinetic comparison is reported for selected examples. Hydrolysis in a urine matrix is demonstrated for dehydroepiandrosterone 3-sulfate and epiandrosterone 3-sulfate. The purified P. aeruginosa arylsulfatase contains only sulfatase activity allowing for the selective hydrolysis of sulfate esters in the presence of glucuronide conjugates as demonstrated in the short three-step chemoenzymatic synthesis of 5α-androstane-3β,17β-diol 17-glucuronide (ADG, 1) from epiandrosterone 3-sulfate. The P. aeruginosa arylsulfatase is readily expressed and purified (0.9 g per L of culture) and thus provides a new and selective method for the hydrolysis of steroid sulfate esters in analytical sample preparation. Copyright © 2015 John Wiley & Sons, Ltd.

Thigmomorphogenesis: the relationship of mechanical perturbation to elicitor-like activity and ethylene production

NASA Technical Reports Server (NTRS)

Takahashi, H.; Jaffe, M. J.

1984-01-01

An extracellular solution obtained from bean (Phaseolus vulgaris L. cv. Resistant Cherokee Wax) stems induced phytoalexin-like substance and ethylene production in a soybean [Glycine max (L.) Merr. cv. Wayne] cotyledon bioassay. The elicitor-like activity for phytoalexin formation and ethylene production was increased by mechanical perturbation of bean stems. Moreover, the application of extracted or known elicitors to bean plants mimicked the effect of mechanical perturbation (i.e., inhibition of stem elongation and enhancement of radial growth). The effects of extract when applied exogenously, on elicitor-like activity in the bioassay as well as stem thickening were decreased by aminoethoxyvinylglycine, an inhibitor of ethylene biosynthesis. These results suggest that elicitor-like substances which are formed in response to mechanical perturbation contribute to the thigmomorphogenesis.

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

PubMed

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.

PubMed

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.

Proteasome 20S in multiple myeloma: comparison of concentration and chymotrypsin-like activity in plasma and serum.

PubMed

Romaniuk, Wioletta; Kalita, Joanna; Ostrowska, Halina; Kloczko, Janusz

2018-03-05

The ubiquitin-proteasome system is relevant in the pathobiology of many haematological malignancies, including multiple myeloma. The assessment of proteasome concentration and chymotrypsin-like (ChT-L) activity might constitute a new approach to diagnosis, prognosis and monitoring of anticancer treatment of patients with haematological malignancies and other diseases. The aim of our study was to determine which material, plasma or serum, is better for measuring chymotrypsin-like (ChT-L) activity and proteasome concentration. We analysed proteasome concentration and chymotrypsin-like (ChT-L) activity in 70 plasma and serum samples drawn from 28 patients at different treatment stages for multiple myeloma (MM) and 31 healthy volunteers. Proteasome ChT-L activity and concentration in multiple myeloma patients were significantly higher in plasma compared to serum. In this group we observed significant and positive correlations both between the plasma and serum proteasome ChT-L activity and plasma and serum proteasome concentration. The higher values of proteasome concentration and ChT-L activity in plasma than in serum and their better correlations with parameters of tumour load and prognosis suggest that plasma constitutes a better biological material for measuring ChT-L activity and proteasome concentration than serum in multiple myeloma patients.

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

NASA Astrophysics Data System (ADS)

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.

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation.

PubMed

Vasu, Prasanna; Savary, Brett J; Cameron, Randall G

2012-07-15

We purified a Carica papaya pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-exchange chromatographies and then purified by affinity chromatography using Sepharose-immobilized kiwi PME inhibitor protein to obtain a single electrophoretically homogeneous protein. The enzyme was purified 92-fold with 38% yield, providing a specific activity of 1200 U/mg. The molecular weight was determined to be 35,135 by MALDI-TOF-MS in linear mode. MALDI-TOF-MS peptide mass fingerprinting following trypsin digestion indicated CpL-PME represents a novel Carica PME isoform. The CpL-PME required salt for activity, and it showed a broad activity range (pH 6-9) and moderate thermostability (optimum ca. 70°C). A calcium-insensitive methylated lime pectin treated with CpL-PME to reduce degree of methylesterification by 6% converted the substrate to high calcium sensitivity, indicating a processive mode of action. These properties support further research to apply CpL-PME to tailor pectin nanostructure. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-05-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit.

A cherry protein and its gene, abundantly expressed in ripening fruit, have been identified as thaumatin-like.

PubMed Central

Fils-Lycaon, B R; Wiersma, P A; Eastwell, K C; Sautiere, P

1996-01-01

A 29-kD polypeptide is the most abundant soluble protein in ripe cherry fruit (Prunus avium L); accumulation begins at the onset of ripening as the fruit turns from yellow to red. This protein was extracted from ripe cherries and purified by size-exclusion and ion-exchange chromatography. Antibodies to the purified protein were used to screen a cDNA library from ripe cherries. Numerous recombinant plaques reacted positively with the antibodies; the DNA sequence of representative clones encoded a polypeptide of 245 amino acid residues. A signal peptide was indicated, and the predicted mature protein corresponded to the purified protein in size (23.3 kD, by mass spectrometry) and isoelectric point (4.2). A search of known protein sequences revealed a strong similarity between this polypeptide and the thaumatin family of pathogenesis-related proteins. The cherry thaumatin-like protein does not have a sweet taste, and no antifungal activity was seen in preliminary assays. Expression of the protein appears to be regulated at the gene level, with mRNA levels at their highest in the ripe fruit. PMID:8685266

In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

PubMed Central

Tridente, Giuseppe; Bason, Caterina; Sivori, Simona; Beri, Ruggero; Dolcino, Marzia; Valletta, Enrico; Corrocher, Roberto; Puccetti, Antonio

2006-01-01

Background Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. Methods and Findings In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. Conclusions Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are

Influence of temperature, pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis.

PubMed

Hu, Xia; Wang, Chunyan; Wang, Le; Zhang, Ranran; Chen, Hui

2014-04-01

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K(m) and V(max) determined for substrate guaiacol were 15.4 μM and 372.9 IU mg⁻¹, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg⁻¹. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.

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

PubMed Central

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

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

PubMed

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.

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

PubMed

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.

Cyclo(l-Leucyl-l-Prolyl) Produced by Achromobacter xylosoxidans Inhibits Aflatoxin Production by Aspergillus parasiticus

PubMed Central

Yan, Pei-Sheng; Song, Yuan; Sakuno, Emi; Nakajima, Hiromitsu; Nakagawa, Hiroyuki; Yabe, Kimiko

2004-01-01

Aflatoxins are potent carcinogenic and toxic substances that are produced primarily by Aspergillus flavus and Aspergillus parasiticus. We found that a bacterium remarkably inhibited production of norsolorinic acid, a precursor of aflatoxin, by A. parasiticus. This bacterium was identified as Achromobacter xylosoxidans based on its 16S ribosomal DNA sequence and was designated A. xylosoxidans NFRI-A1. A. xylosoxidans strains commonly showed similar inhibition. The inhibitory substance(s) was excreted into the medium and was stable after heat, acid, or alkaline treatment. Although the bacterium appeared to produce several inhibitory substances, we finally succeeded in purifying a major inhibitory substance from the culture medium using Diaion HP20 column chromatography, thin-layer chromatography, and high-performance liquid chromatography. The purified inhibitory substance was identified as cyclo(l-leucyl-l-prolyl) based on physicochemical methods. The 50% inhibitory concentration for aflatoxin production by A. parasiticus SYS-4 (= NRRL2999) was 0.20 mg ml−1, as determined by the tip culture method. High concentrations (more than 6.0 mg ml−1) of cyclo(l-leucyl-l-prolyl) further inhibited fungal growth. Similar inhibitory activities were observed with cyclo(d-leucyl-d-prolyl) and cyclo(l-valyl-l-prolyl), whereas cyclo(d-prolyl-l-leucyl) and cyclo(l-prolyl-d-leucyl) showed weaker activities. Reverse transcription-PCR analyses showed that cyclo(l-leucyl-l-prolyl) repressed transcription of the aflatoxin-related genes aflR, hexB, pksL1, and dmtA. This is the first report of a cyclodipeptide that affects aflatoxin production. PMID:15574949

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

PubMed

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.

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

PubMed

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.

Reduction of reactive red 241 by oxygen insensitive azoreductase purified from a novel strain Staphylococcus KU898286

PubMed Central

Nisar, Numrah; Aleem, Amber; Saleem, Faiza; Aslam, Fakhra; Shahid, Ammara; Chaudhry, Hina; Malik, Kausar; Albaser, Abdulhadi; Iqbal, Amjad; Yang, Yaodong

2017-01-01

An oxygen insensitive azoreductase was purified from a novel bacterial strain (Staphylococcus sp. KU898286) that was isolated from an abandoned site of the textile waste discharge unit. The isolated enzyme had efficiently cleaved the azo-bonds through reductive transformation under aerobic conditions. Initial phenotypic characterization and final construction of phylogenetic tree on the basis of 16s rDNA demonstrated 99% resemblance of the isolate to Staphylococcus aureus. The purified azoreductase was found to have a broad spectrum activity that reduced RR241 at a concentration of 50mg/L with pH between 6–8 and 30°C temperature). Besides, the reactive red 241 (RR241) was reduced at extracellular level as well as NADH dependent intracellular level. Complete reduction/ decolourization of RR241 were achieved after 18 hrs of exposure. The final degradation product observed to be 2-nephthol was purified by High Pressure Liquid Chromatography (HPLC) and the molecular mass was computed by Gas Chromatography-Mass spectroscopy (GC-MS). The study revealed a cost effective and eco-friendly approach to degrade the toxic dyes into less toxic products by Staphylococcus sp. KU898286. PMID:28467413

Process for purifying geothermal steam

DOEpatents

Li, Charles T.

1980-01-01

Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

Process for purifying geothermal steam

DOEpatents

Li, C.T.

Steam containing hydrogen sulfide is purified and sulfur recovered by passing the steam through a reactor packed with activated carbon in the presence of a stoichiometric amount of oxygen which oxidizes the hydrogen sulfide to elemental sulfur which is adsorbed on the bed. The carbon can be recycled after the sulfur has been recovered by vacuum distillation, inert gas entrainment or solvent extraction. The process is suitable for the purification of steam from geothermal sources which may also contain other noncondensable gases.

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

PubMed

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.

PubMed

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.

Purified silicon production system

DOEpatents

Wang, Tihu; Ciszek, Theodore F.

2004-03-30

Method and apparatus for producing purified bulk silicon from highly impure metallurgical-grade silicon source material at atmospheric pressure. Method involves: (1) initially reacting iodine and metallurgical-grade silicon to create silicon tetraiodide and impurity iodide byproducts in a cold-wall reactor chamber; (2) isolating silicon tetraiodide from the impurity iodide byproducts and purifying it by distillation in a distillation chamber; and (3) transferring the purified silicon tetraiodide back to the cold-wall reactor chamber, reacting it with additional iodine and metallurgical-grade silicon to produce silicon diiodide and depositing the silicon diiodide onto a substrate within the cold-wall reactor chamber. The two chambers are at atmospheric pressure and the system is open to allow the introduction of additional source material and to remove and replace finished substrates.

Cytokine-like Activity of Liver Type Fatty Acid Binding Protein (L-FABP) Inducing Inflammatory Cytokine Interleukin-6

PubMed Central

Kim, Hyunwoo; Gil, Gaae; Lee, Siyoung; Kwak, Areum; Jo, Seunghyun; Kim, Ensom; Nguyen, Tam T.; Kim, Sinae; Jhun, Hyunjhung; Kim, Somi; Kim, Miyeon; Lee, Youngmin

2016-01-01

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation. PMID:27799875

Air Purifiers Eliminate Pathogens, Preserve Food

NASA Technical Reports Server (NTRS)

2009-01-01

NASA-funded researchers produced an ethylene reduction device for a plant growth unit. KES Science & Technology Inc., a Kennesaw, Georgia-based company specializing in sustaining perishable foods, licensed the ethylene scrubbing technology. KES partnered with Akida Holdings, of Jacksonville, Florida, which now markets the NASA-developed technology as AiroCide. According to the company, it is the only air purifier that completely destroys airborne bacteria, mold, fungi, mycotoxins, viruses, volatile organic compounds (like ethylene), and odors. What?s more, the devices have no filters that need changing and produce no harmful byproducts, such as the ozone created by some filtration systems.

Natural Air Purifier

NASA Technical Reports Server (NTRS)

1993-01-01

NASA environmental research has led to a plant-based air filtering system. Dr. B.C. Wolverton, a former NASA engineer who developed a biological filtering system for space life support, served as a consultant to Terra Firma Environmental. The company is marketing the BioFilter, a natural air purifier that combines activated carbon and other filter media with living plants and microorganisms. The filter material traps and holds indoor pollutants; plant roots and microorganisms then convert the pollutants into food for the plant. Most non-flowering house plants will work. After pollutants have been removed, the cleansed air is returned to the room through slits in the planter. Terra Firma is currently developing a filter that will also disinfect the air.

Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism.

PubMed

Watanabe, Seiya; Kodaki, Tsutomu; Kodak, Tsutomu; Makino, Keisuke

2006-02-03

Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP+ to NAD+ as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.

[Preparation and activity detection of chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein].

PubMed

Yang, Jun; Zhang, Ming-juan; Qiang, Lei; Su, Bao-shan; Wang, Yi-li; Si, Lü-sheng

2008-03-01

To prepare highly specific chicken egg yolk IgY antibody against human papillomavirus 16 type L1 main capsid protein (HPV16L1) for detection of HPV16L1. Purified HPV16L1 protein was used to immunize the hens, from which the eggs were collected since one week after the first immunization. The egg yolk was separated and the IgY antibody purified by PEG-6000 method. The bioactivity of the antibody was tested using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was performed to detect the HPV16L1 in the CHO cells transfected with the recombinant pcDNA-EGFP-HPV16L1 plasmid (containing EGFP-HPV16L1 fusion gene) for assessing the specific affinity of IgY to HPV16L1. After 3 immunizations of the hens, the titer of the purified IgY antibody against HPV16L1 from the egg yolk reached 1:10240. The IgY bound specifically to the EGFP-HPV16L1 protein expressed in the transfected CHO cells. High titer IgY can be prepared by immunization of the hens with HPV16L1 protein, and the prepared IgY can be used for HPV16L1 detection at the cellular level.

Investigation of hydrazide derivatives inhibitory effect on peroxidase enzyme purified from turnip roots

NASA Astrophysics Data System (ADS)

Almaz, Züleyha; Öztekin, Aykut; Özdemir, Hasan

2017-04-01

Peroxidases (EC: 1.11.1.7) are haem proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group [1]. They are found in all cells and play a critical role in many biological processes, such as the host-defense mechanism [2]. Peroxidases (PODs) are widely used in clinical biochemistry, enzyme immunoassays, synthesis of various aromatic chemicals, treatment of waste water containing phenolic compounds [3, 4]. In this study, peroxidase enzyme was purified with Para amino benzohydrazide (PABH)-L-Tyrosine Sepharose 4B affinity chromatography to investigate the inhibitory effect of hydrazide derivatives on Turnip (Brassica rapa L.). 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 enzyme and inhibition type of these molecules were determined.

Could Ultracool Dwarfs Have Sun-Like Activity?

NASA Astrophysics Data System (ADS)

Kohler, Susanna

2016-11-01

Solar-like stars exhibit magnetic cycles; our Sun, for instance, displays an 11-year period in its activity, manifesting as cyclic changes in radiation levels, the number of sunspots and flares, and ejection of solar material. Over the span of two activity cycles, the Suns magnetic field flips polarity and then returns to its original state.An artists illustration comparing the Sun to TRAPPIST-1, an ultracool dwarf star known to host several planets. [ESO]But what about the magnetic behavior of objects near the cooler end of the stellar main sequence do they exhibit similar activity cycles?Effects of a Convecting InteriorDwarf stars have made headlines in recent years due to their potential to harbor exoplanets. Because these cooler stars have lower flux levels compared to the Sun, their habitable zones lie much closer to the stars. The magnetic behavior of these stars is therefore important to understand: could ultracool dwarfs exhibit solar-like activity cycles that would affect planets with close orbits?The differences in internal structure between different mass stars. Ultracool dwarfs have fully convective interiors. [www.sun.org]Theres a major difference between ultracool dwarfs (stars of spectral type higher than M7 and brown dwarfs) and Sun-like stars: their internal structures. Sun-like stars have a convective envelope that surrounds a radiative core. The interiors of cool, low-mass objects, on the other hand, are fully convective.Based on theoretical studies of how magnetism is generated in stars, its thought that the fully convective interiors of ultracool dwarfs cant support large-scale magnetic field formation. This should prevent these stars from exhibiting activity cycles like the Sun. But recent radio observations of dwarf stars have led scientist Matthew Route (ITaP Research Computing, Purdue University) to question these models.A Reversing Field?During observations of the brown dwarf star J1047+21 in 20102011, radio flares were detected with

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

PubMed

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).

Two systems developed for purifying inert atmospheres

NASA Technical Reports Server (NTRS)

Foster, M. S.; Johnson, C. E.; Kyle, M. L.

1969-01-01

Two systems, one for helium and one for argon, are used for purifying inert atmospheres. The helium system uses an activated charcoal bed at liquid nitrogen temperature to remove oxygen and nitrogen. The argon system uses heated titanium sponge to remove nitrogen and copper wool beds to remove oxygen. Both use molecular sieves to remove water vapor.

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

PubMed

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.

Protective Effect of Active Immunization with Purified Escherichia coli Heat-Labile Enterotoxin in Rats

PubMed Central

Klipstein, Frederick A.; Engert, Richard F.

1979-01-01

The protective effect of active immunization by different routes with a purified preparation of the polymyxin-release form of Escherichia coli heat-labile toxin was evaluated in rats. Immunized animals were challenged by placing toxin into ligated ileal loops at dosages which produced either 50% or the maximum secretory response in unimmunized rats. Immunization exclusively by the parenteral route yielded significant protection. Rats were also protected when parenteral priming was followed by boosting given either directly into the duodenum or perorally 2 h after intragastric cimetidine, but not when the peroral boosts were given with bicarbonate. Immunization administered entirely by the peroral route with cimetidine yielded protection but only when the immunizing dosage was fivefold greater than that found effective in the parenteral-peroral approach. Rats immunized exclusively by the parenteral route and those boosted perorally with cimetidine were also tested, and found to be protected, against challenge with viable organisms of strains that produce either heat-labile toxin alone or both heat-labile and heat-stable toxin, but they were not protected against a strain which produces just heat-stable toxin. Geometric mean serum antibody titers were increased by 16-fold or more over control values in those groups of rats in which protection was achieved, with the exception of those immunized exclusively by the peroral route. These observations demonstrate that (i) active immunization with purified E. coli heat-labile toxin results in significant protection against both this toxin as well as viable organisms which produce it, but not against viable strains which produce heat-stable toxin only, and (ii) concomitant ablation of gastric secretion by the use of cimetidine renders the peroral route of immunization effective. They suggest that prophylactic immunization against diarrheal disease caused by heat-labile toxin-producing strains of E. coli may be feasible in

HnRNP L and L-like cooperate in multiple-exon regulation of CD45 alternative splicing

PubMed Central

Preußner, Marco; Schreiner, Silke; Hung, Lee-Hsueh; Porstner, Martina; Jäck, Hans-Martin; Benes, Vladimir; Rätsch, Gunnar; Bindereif, Albrecht

2012-01-01

CD45 encodes a trans-membrane protein-tyrosine phosphatase expressed in diverse cells of the immune system. By combinatorial use of three variable exons 4–6, isoforms are generated that differ in their extracellular domain, thereby modulating phosphatase activity and immune response. Alternative splicing of these CD45 exons involves two heterogeneous ribonucleoproteins, hnRNP L and its cell-type specific paralog hnRNP L-like (LL). To address the complex combinatorial splicing of exons 4–6, we investigated hnRNP L/LL protein expression in human B-cells in relation to CD45 splicing patterns, applying RNA-Seq. In addition, mutational and RNA-binding analyses were carried out in HeLa cells. We conclude that hnRNP LL functions as the major CD45 splicing repressor, with two CA elements in exon 6 as its primary target. In exon 4, one element is targeted by both hnRNP L and LL. In contrast, exon 5 was never repressed on its own and only co-regulated with exons 4 and 6. Stable L/LL interaction requires CD45 RNA, specifically exons 4 and 6. We propose a novel model of combinatorial alternative splicing: HnRNP L and LL cooperate on the CD45 pre-mRNA, bridging exons 4 and 6 and looping out exon 5, thereby achieving full repression of the three variable exons. PMID:22402488

Structural features of immunostimulatory polysaccharide purified from pectinase hydrolysate of barley leaf.

PubMed

Kim, Hoon; Kwak, Bong-Shin; Hong, Hee-Do; Suh, Hyung-Joo; Shin, Kwang-Soon

2016-06-01

Four polysaccharide fractions were isolated from young barley leaves treated with or without pectinase followed by ethanol fractionation. Among the polysaccharide fractions, BLE-P isolated from pectinase digested with a high molecular weight had the most enhanced macrophage stimulatory activity, indicating that pectinase digestion of barley leaf is a useful method for enhancement of its activity. BLE-P was further purified by column chromatography to identify the chemical and structural properties. BLE-P-I eluted in void volume fraction showed potent macrophage stimulatory activity. Monosaccharide composition and linkage analysis indicated that at least three kinds of polysaccharide, that is, glucuronoarabinoxylan (GAX; 40-45%), rhamnogalacturonan-I (RG-I) with branching mainly involving a type II arabinogalactan (AG-II) side chain (30-35%), and linear glucan such as starch and cellulose (less than 10%) coexisted in BLE-P-I. Given the association with macrophage stimulatory activity, it is likely that the GAX and to the RG-I polysaccharide branched with an AG-II side chain may be important for expression of the activity in barley leaf. Copyright © 2016 Elsevier B.V. All rights reserved.

Co-operative actions and degradation analysis of purified xylan-degrading enzymes from Thermomonospora fusca BD25 on oat-spelt xylan.

PubMed

Tuncer, M; Ball, A S

2003-01-01

To determine and quantify the products from the degradation of xylan by a range of purified xylan-degrading enzymes, endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase produced extracellularly by Thermomonospora fusca BD25. The amounts of reducing sugars released from oat-spelt xylan by the actions of endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase were equal to 28.1, 4.6 and 7% hydrolysis (as xylose equivalents) of the substrate used, respectively. However, addition of beta-xylosidase and alpha-l-arabinofuranosidase preparation to endoxylanase significantly enhanced (70 and 20% respectively) the action of endoxylanase on the substrate. The combination of purified endoxylanase, beta-xylosidase and alpha-l-arabinofuranosidase preparations produced a greater sugar yield (58.6% hydrolysis) and enhanced the total reducing sugar yield by around 50%. The main xylooligosaccharide products released using the action of endoxylanase alone on oat-spelt xylan were identified as xylobiose and xylopentose. alpha-l-Arabinofuranosidase was able to release arabinose and xylobiose from oat-spelt xylan. In the presence of all three purified enzymes the hydrolysis products of oat-spelt xylan were mainly xylose, arabinose and substituted xylotetrose with lesser amount of substituted xylotriose. The addition of the beta-xylosidase and alpha-l-arabinofuranosidase enzymes to purified xylanases more than doubled the degradation of xylan from 28 to 58% of the total substrate with xylose and arabinose being the major sugars produced. The results highlight the role of xylan de-branching enzymes in the degradation of xylan and suggest that the use of enzyme cocktails may significantly improve the hydrolysis of xylan in industrial processes.

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

PubMed

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.

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

PubMed

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.

Production and Evaluation of a Purified Adenovirus Group-Specific (Hexon) Antigen for Use in the Diagnostic Complement Fixation Test

PubMed Central

Dowdle, W. R.; Lambriex, M.; Hierholzer, J. C.

1971-01-01

A simple procedure for the production of large volumes of purified adenovirus group-specific complement-fixing (CF) (hexon) antigen by selective adsorption to and elution from CaHPO4 is described. Results of immunodiffusion tests, electrophoresis, electron microscopy, and tests for hemagglutination and infectivity indicate that the purified antigen consisted of a single virus component (hexon). The purified product contained little host materials. Unlike the crude virus harvest usually employed for serodiagnostic CF tests, the purified antigen demonstrated no anticomplementary activity and did not develop such activity during storage. The purified antigen was equal to or slightly more sensitive than crude virus harvests for serodiagnosis of adenovirus infections. Images PMID:4325021

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women.

PubMed

Aranha, C; Natraj, U; Iyer, K S; Shahani, S

1998-03-01

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. Lymphocyte proliferation assay and 35S-methionine labeling were done to study de novo synthesis of proteins followed by autoradiography to confirm synthesis of proteins. The rosette inhibition assay was used for detection of the EPF-like molecule. Gel filtration on Sephadex G-100 and RPHPLC were used for purification of the EPF-like molecule. The rate of incorporation of 35S-methionine was significantly higher in the lymphocytes of pregnant women compared to those of the control, and autoradiography confirmed the synthesis of proteins during pregnancy. There is a total protein enhancement trend observed during the first trimester that declines toward term. The EPF-like molecule is observed to be synthesized during all the trimesters of pregnancy. This molecule, when purified, showed a single homogeneous biologically active peak. The results indicated that there is an enhancement of existing protein or synthesis of new proteins during pregnancy. The EPF-like molecule is one of the many proteins synthesized and secreted by lymphocytes during pregnancy that, when purified, is biologically active.

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

PubMed

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.

A New Ligand-Based Method for Purifying Active Human Plasma-Derived Ficolin-3 Complexes Supports the Phenomenon of Crosstalk between Pattern-Recognition Molecules and Immunoglobulins

PubMed Central

Man-Kupisinska, Aleksandra; Michalski, Mateusz; Maciejewska, Anna; Swierzko, Anna S.; Cedzynski, Maciej; Lugowski, Czeslaw; Lukasiewicz, Jolanta

2016-01-01

Despite recombinant protein technology development, proteins isolated from natural sources remain important for structure and activity determination. Ficolins represent a class of proteins that are difficult to isolate. To date, three methods for purifying ficolin-3 from plasma/serum have been proposed, defined by most critical step: (i) hydroxyapatite absorption chromatography (ii) N-acetylated human serum albumin affinity chromatography and (iii) anti-ficolin-3 monoclonal antibody-based affinity chromatography. We present a new protocol for purifying ficolin-3 complexes from human plasma that is based on an exclusive ligand: the O-specific polysaccharide of Hafnia alvei PCM 1200 LPS (O-PS 1200). The protocol includes (i) poly(ethylene glycol) precipitation; (ii) yeast and l-fucose incubation, for depletion of mannose-binding lectin; (iii) affinity chromatography using O-PS 1200-Sepharose; (iv) size-exclusion chromatography. Application of this protocol yielded average 2.2 mg of ficolin-3 preparation free of mannose-binding lectin (MBL), ficolin-1 and -2 from 500 ml of plasma. The protein was complexed with MBL-associated serine proteases (MASPs) and was able to activate the complement in vitro. In-process monitoring of MBL, ficolins, and total protein content revealed the presence of difficult-to-remove immunoglobulin G, M and A, in some extent in agreement with recent findings suggesting crosstalk between IgG and ficolin-3. We demonstrated that recombinant ficolin-3 interacts with IgG and IgM in a concentration-dependent manner. Although this association does not appear to influence ficolin-3-ligand interactions in vitro, it may have numerous consequences in vivo. Thus our purification procedure provides Ig-ficolin-3/MASP complexes that might be useful for gaining further insight into the crosstalk and biological activity of ficolin-3. PMID:27232184

Physalins B, F and G, seco-steroids purified from Physalis angulata L., inhibit lymphocyte function and allogeneic transplant rejection.

PubMed

Soares, M B P; Brustolim, D; Santos, L A; Bellintani, M C; Paiva, F P; Ribeiro, Y M; Tomassini, T C B; Ribeiro Dos Santos, R

2006-03-01

Physalis angulata is a solanaceae widely used in folk medicine in various tropical countries in the world. We have previously described that seco-steroids (physalins) purified from P. angulata are potent inhibitors of macrophage activation, blocking the production of pro-inflammatory cytokines and LPS-induced lethality. Herein we investigated the immunomodulatory activities of these substances in lymphocyte proliferation and cytokine production and in transplantation. The addition of physalins B, F or G to concanavalin A-activated splenocyte cultures induced a concentration-dependent inhibition of proliferation. Physalin B also inhibited IL-2 production by Con A-activated spleen cells. The addition of 2 mug/ml physalin B to mixed lymphocyte reaction (MLR) caused a 100% inhibition of proliferation. More importantly, treatment of mice with physalin B, F or G prevented the rejection of allogeneic heterotopic heart transplant. Our results demonstrate the suppressive activity of physalins B, F and G in lymphocyte function and indicate the potential use of physalins as immunosuppressive agents for treatments of pathologies in which inhibition of immune responses is desired.

Purification and antibacterial activity of recombinant warnericin RK expressed in Escherichia coli.

PubMed

Verdon, Julien; Girardin, Nicolas; Marchand, Adrienne; Héchard, Yann; Berjeaud, Jean-Marc

2013-06-01

Warnericin RK is a small cationic peptide produced by Staphylococcus warneri RK. This peptide has an antimicrobial spectrum of activity almost restricted to the Legionella genus. It is a membrane-active peptide with a proposed detergent-like mechanism of action at high concentration. Moreover, the fatty acids content of Legionella was shown to modulate the peptide activity. In order to decipher the mode of action in details using solid-state NMR spectroscopy, large amount of an isotopic labeled peptide is required. Since it is less expensive to obtain such a peptide biologically, we report here methods to express warnericin RK in Escherichia coli with or without a fusion partner and to purify resulting recombinant peptides. The cDNA fragment encoding warnericin RK was synthesized and ligated into three expression vectors. Two fusion peptides, carrying polyhistidine tag in N- or C-terminal and a native peptide, without tag, were expressed in E. coli cells. Fusion peptides were purified, with a yield of 3 mg/l, by affinity chromatography and reverse-phase HPLC. The recombinant native peptide was purified using a two-step purification method consisting of a hydrophobic chromatography followed by a reverse-phase HPLC step with a yield of 1.4 mg/l. However, the anti-Legionella activity was lower for both tagged peptide probably because of structural modifications. So, the native recombinant peptide was preferentially chosen for (15)N-labeling experiments. Our results suggest that the developed production and purification procedures will be useful in obtaining a large quantity of recombinant isotope-labeled warnericin RK for further studies.

A new TRAF-like protein from B. oleracea ssp. botrytis with lectin activity and its effect on macrophages.

PubMed

Duarte, Christiane E M; Abranches, Monise V; Silva, Patrick F; de Paula, Sérgio O; Cardoso, Silvia A; Oliveira, Leandro L

2017-01-01

Lectins are involved in a wide range of biological mechanisms, like immunomodulatory agent able to activate the innate immunity. In this study, we purified and characterized a new lectin from cauliflower (Brassica oleracea ssp. botrytis - BOL) by three sequential chromatographic steps and confirmed the purity by SDS-PAGE. Additionally, we evaluated the role of the lectin in innate immunity by a phagocytosis assay, production of H 2 O 2 and NO. BOL was characterized like a non-glycosylated protein that showed a molecular mass of ∼34kDa in SDS-PAGE. Its N-terminal sequence (ETRAFREERPSSKIVTIAG) did not reveal any similarity to the other lectins; nevertheless, it showed 100% homology to a putative TRAF-like protein from Brassica rapa and Brassica napus. This is a first report of the TRAF-protein with lectinic activity. The BOL retained its complete hemagglutination activity from 4°C up to 60°C, with stability being more apparent between pH 7.0 and 8.0. Moreover, the lectin was able to stimulate phagocytosis and induce the production of H 2 O 2 and NO. Therefore, BOL can be explored as an immunomodulatory agent by being able to activate the innate immunity and favor antigen removal. Copyright © 2016 Elsevier B.V. All rights reserved.

Guanine nucleotide regulatory protein co-purifies with the D/sub 2/-dopamine receptor

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

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

1986-05-01

The D/sub 2/-dopamine receptor from bovine anterior pituitary was purified approx.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 /sup 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/sub 2/ receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 ..mu..M NPA. /sup 35/S-GTP..gamma..Smore » 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/sub 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/sub 2/-dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes.« less

A trypsin inhibitor from Sapindus saponaria L. seeds: purification, characterization, and activity towards pest insect digestive enzyme.

PubMed

Macedo, Maria Lígia R; Diz Filho, Eduardo B S; Freire, Mariadas Graças M; Oliva, Maria Luiza V; Sumikawa, Joana T; Toyama, Marcos H; Marangoni, Sérgio

2011-01-01

The present paper describes the purification, characterization and determination of the partial primary structure of the first trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity. SDS-PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15 and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 10⁻⁹ M for trypsin. The partial NH₂- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis.

Lysophosphatidylinositol-induced activation of the cation channel TRPV2 triggers glucagon-like peptide-1 secretion in enteroendocrine L cells.

PubMed

Harada, Kazuki; Kitaguchi, Tetsuya; Kamiya, Taichi; Aung, Kyaw Htet; Nakamura, Kazuaki; Ohta, Kunihiro; Tsuboi, Takashi

2017-06-30

The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca 2+ concentrations ([Ca 2+ ] i ) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca 2+ ] i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of G q and G 12/13 signaling pathways in the LPI-induced increased [Ca 2+ ] i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca 2+ ] i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca 2+ ] i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by G q and G 12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Methods for Purifying Enzymes for Mycoremediation

NASA Technical Reports Server (NTRS)

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.

Biochemical characterization of a novel L-Asparaginase with low glutaminase activity from Rhizomucor miehei and its application in food safety and leukemia treatment.

PubMed

Huang, Linhua; Liu, Yu; Sun, Yan; Yan, Qiaojuan; Jiang, Zhengqiang

2014-03-01

A novel fungal gene encoding the Rhizomucor miehei l-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported l-asparaginases. The purified l-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this l-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia.

Optimization, purification, and characterization of L-asparaginase from Actinomycetales bacterium BkSoiiA.

PubMed

Dash, Chitrangada; Mohapatra, Sukanti Bala; Maiti, Prasanta Kumar

2016-01-01

Actinobacteria are promising source of a wide range of important enzymes, some of which are produced in industrial scale, with others yet to be harnessed. L-Asparaginase is used as an antineoplastic agent. The present work deals with the production and optimization of L-asparaginase from Actinomycetales bacterium BkSoiiA using submerged fermentation in M9 medium. Production optimization resulted in a modified M9 medium with yeast extract and fructose as carbon and nitrogen sources, respectively, at pH 8.0, incubated for 120 hr at 30 ± 2 °C. The crude enzyme was purified to near homogeneity by ammonium sulfate precipitation following dialysis, ion-exchange column chromatography, and finally gel filtration. The sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed an apparent molecular weight of 57 kD. The enzyme was purified 95.06-fold and showed a final specific activity of 204.37 U/mg with 3.49% yield. The purified enzyme showed maximum activity at a pH 10.0 and was stable at pH 7.0 to 9.0. The enzyme was activated by Mn(2+) and strongly inhibited by Ba(2+). All these preliminary characterization suggests that the L-asparaginase from the source may be a tool useful to pharmaceutical industries after further research.

Purification and Characterization of a Novel Thermostable α-l-Arabinofuranosidase from a Color-Variant Strain of Aureobasidium pullulans

PubMed Central

Saha, Badal C.; Bothast, Rodney J.

1998-01-01

A color-variant strain of Aureobasidium pullulans (NRRL Y-12974) produced α-l-arabinofuranosidase (α-l-AFase) when grown in liquid culture on oat spelt xylan. An extracellular α-l-AFase was purified 215-fold to homogeneity from the culture supernatant by ammonium sulfate treatment, DEAE Bio-Gel A agarose column chromatography, gel filtration on a Bio-Gel A-0.5m column, arabinan-Sepharose 6B affinity chromatography, and SP-Sephadex C-50 column chromatography. The purified enzyme had a native molecular weight of 210,000 and was composed of two equal subunits. It had a half-life of 8 h at 75°C, displayed optimal activity at 75°C and pH 4.0 to 4.5, and had a specific activity of 21.48 μmol · min−1 · mg−1 of protein against p-nitrophenyl-α-l-arabinofuranoside (pNPαAF). The purified α-l-AFase readily hydrolyzed arabinan and debranched arabinan and released arabinose from arabinoxylans but was inactive against arabinogalactan. The Km values of the enzyme for the hydrolysis of pNPαAF, arabinan, and debranched arabinan at 75°C and pH 4.5 were 0.26 mM, 2.14 mg/ml, and 3.25 mg/ml, respectively. The α-l-AFase activity was not inhibited at all by l-arabinose (1.2 M). The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). PMID:9435077

Potency of microfiltration membrane process in purifying broccoli (Brassica oleracea L.) fermented by lactic acid bacteria (LAB) as functional food

NASA Astrophysics Data System (ADS)

Susilowati, Agustine; Aspiyanto, Maryati, Yati; Melanie, Hakiki; Lotulung, Puspa D.

2017-01-01

Purifying broccoli (Brassica oleracea L.) fermented by Lactic Acid Bacteria (LAB) using mixture of L. bulgaricus, S. thermopillus, L. acidophillusand Bifidobacteriumbifidum and fructooligosaccharides (FOS) as carbon source have been performed to recover biomass concentrate for probiotic and antioxidant. Purification of fermented broccoli was conducted through microfiltration (MF) membrane of 0.15 µm at stirrer rotation speed 400 rpm, room temperature and pressure 40 psia for 30 minutes. Fermented broccoli produced via fermentation process with fermentation time 0 (initial) and 48 hours, and LAB concentration 10% and 20% (v/v) represented as biomass of A, B, C and D. The experimental result showed that based on selectivity of total organic acids, separating optimization was achieved at biomass D (fermentation time 48 hours and mixed LAB culture concentration 20%). Concentrate composition produced in this condition were total acids 6.04%, total solids 24.31%, total polyphenol 0.0252%, reducing sugar 68.25 mg/mL, total sugars 30.89 mg/mL, and dissolved protein 28.54 mg/mL with pH 3.94. In this condition, recovery of biomass concentrate of D for total acids 5.64 folds, total solids 1.82 folds, total polyphenol 3.03 folds, reducing sugar 1.16 folds, total sugars 1.19 folds, and dissolved protein 0.67 folds compared with feed (initial process). Identification of monomer of biomass concentrate D as polyphenol derivatives at T2,01 and T3.01 gave monomer with molecular weight (MW) 192.78 Dalton (Da.), and monomer with MW 191.08, 191.49 and 192.07 Da., while lactic acid derivatives showed MW 251.13, 251.6 and 252.14, and monomer with MW 250.63, 252.14 and 254.22 Da.

Thermostable purified endoglucanase from thermophilic bacterium acidothermus cellulolyticus

DOEpatents

Tucker, Melvin P.; Grohmann, Karel; Himmel, Michael E.; Mohagheghi, Ali

1992-01-01

A substantially purified high molecular weight cellulase enzyme having a molecular weight of between about 156,000 to about 203,400 daltons isolated from the bacterium Acidothermus cellulolyticus (ATCC 43068) and a method of producing it are disclosed. The enzyme is water soluble, possesses both C.sub.1 and C.sub.x types of enzymatic activity, has a high degree of stability toward heat and exhibits both a high optimum temperature activity and high inactivation characteristics.

Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

DOEpatents

Adney, William S.; Thomas, Steven R.; Nieves, Rafael A.; Himmel, Michael E.

1994-01-01

A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C.sub.1, and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81.degree. C. at pH's from about 2 to about 9, and at a inactivation temperature of about 100.degree. C. at pH's from about 2 to about 9.

Thermostable purified endoglucanase II from Acidothermus cellulolyticus ATCC

DOEpatents

Adney, W.S.; Thomas, S.R.; Nieves, R.A.; Himmel, M.E.

1994-11-22

A purified low molecular weight endoglucanase II from Acidothermus cellulolyticus (ATCC 43068) is disclosed. The endoglucanase is water soluble, possesses both C[sub 1], and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 81 C at pH's from about 2 to about 9, and at a inactivation temperature of about 100 C at pH's from about 2 to about 9. 9 figs.

Heparin-like activity in uterine fluid.

PubMed Central

Foley, M E; Griffin, B D; Zuzel, M; Aparicio, S R; Bradbury, K; Bird, C C; Clayton, J K; Jenkins, D M; Scott, J S; Rajah, S M; McNichol, G P

1978-01-01

Uterine fluid was collected from a group of normal patients and a group of patients with menorrhagia. Heparin-like activity was detected in 34 out of 38 samples using an anti-Xa heparin assay. The heparin-like activity in uterine fluid was inhibited by adding the heparin antagonist hexadimethrine bromide to the assay. Concentrations of fibrinogen-fibrin degradation products (FDPs) were measured in five samples of uterine fluid. FDPs in the concentration detected had no effect on the anti-Xa assay. Heparin-like activity was higher in the group with menorrhagia, although the differences were not significant. Heparin-like activity increased throughout the menstrual cycle and decreased during menstruation, suggesting a possible cyclical variation in activity. There was no correlation between mast cell numbers in the endometrium and myometrium and heparin-like activity in uterine fluid and no correlation between the numbers and the stage in the menstrual cycle. In a few patients with intrauterine contraceptive devices (IUCDs) heparin-like activity was increased. PMID:687899

17α-ethinyl estradiol attenuates depressive-like behavior through GABAA receptor activation/nitrergic pathway blockade in ovariectomized mice.

PubMed

Saeedi Saravi, Seyed Soheil; Arefidoust, Alireza; Yaftian, Rahele; Saeedi Saravi, Seyed Sobhan; Dehpour, Ahmad Reza

2016-04-01

This study was performed to investigate the antidepressant-like effect of 17α-ethinyl estradiol (EE2) in ovariectomized (OVX) mice and the possible role of nitrergic and gamma aminobutyric acid (GABA)ergic pathways in this paradigm. Bilateral ovariectomy was performed in female mice, and different doses of EE2 were intraperitoneally injected either alone or combined with GABAA agonist, diazepam, GABAA antagonist, flumazenil, non-specific nitric oxide synthase (NOS) inhibitor, N(ω)-nitro-L-arginine methyl ester (L-NAME), specific nNOS inhibitor, 7-nitroindazole (7-NI), a nitric oxide (NO) precursor, L-arginine, and selective PDE5I, sildenafil. After locomotion assessment, immobility times were recorded in the forced swimming test (FST) and tail suspension test (TST). Moreover, hippocampal nitrite concentrations were measured in the examined groups. Ten days after ovariectomy, a significant prolonged immobility times were observed. EE2 (0.3 and 1μg/kg and 0.03, 0.1, and 1mg/kg) caused antidepressant-like activity in OVX mice in FST and TST. Diazepam (1 and 5mg/kg), L-NAME (30mg/kg), and 7-NI (100mg/kg) significantly reduced the immobility times. Co-administration of minimal and sub-effective doses of EE2 and diazepam (0.3μg/kg and 0.5mg/kg, respectively) exerted a significant antidepressant-like effect. The same effect was observed in combination of minimal and sub-effective doses of EE2 and either L-NAME or 7-NI. Moreover, combination of minimal and sub-effective doses of EE2, diazepam either L-NAME, or 7-NI emphasized the significant robust antidepressant-like activity. The study has demonstrated that lowest dose of EE2 exerts a significant antidepressant-like behavior. It is suggested that suppression of NO system, as well as GABAA activation, may be responsible for antidepressant-like activity of EE2 in OVX mice. Moreover, GABAA activation may inhibit nitrergic pathway.

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

PubMed Central

2012-01-01

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. PMID:22958433

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

PubMed

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.

Construction and immunological characterization of CD40L or GM-CSF incorporated Hantaan virus like particle

PubMed Central

Zhang, Xiaoxiao; Truax, Agnieszka D.; Ma, Ruixue; Liu, Ziyu; Lei, Yingfeng; Zhang, Liang; Ye, Wei; Zhang, Fanglin; Xu, Zhikai; Shang, Lei; Liu, Rongrong; Wang, Fang; Wu, Xingan

2016-01-01

Infection of Hantaan virus (HTNV) usually causes hemorrhagic fever with renal syndrome (HFRS). China has the worst epidemic incidence of HFRS as well as high fatality. Inactivated whole virus has been used for HFRS vaccination, however there are still problems such as safety concerns. CD40 ligand (CD40L) and granulocyte macrophage colony-stimulating factor (GM-CSF) are well-known immune stimulating molecules that can enhance antigen presenting, lymphocytes activation and maturation, incorporation of CD40L and GM-CSF to the surface of virus like particles (VLPs) can greatly improve the vaccination effect. We constructed eukaryotic vectors expressing HTNV M segment and S segment, as well as vectors expressing HTNV M segment with CD40L or GM-CSF, our results showed successful production of CD40L or GM-CSF incorporated HTNV VLPs. In vitro stimulation with CD40L or GM-CSF anchored HTNV VLP showed enhanced activation of macrophages and DCs. CD40L/GM-CSF incorporated VLP can induce higher level of HTNV specific antibody and neutralizing antibody in mice. Immunized mice splenocytes showed higher ability of secreting IFN-γ and IL-2, as well as enhancing CTL activity. These results suggest CD40L/GM-CSF incorporated VLP can serve as prospective vaccine candidate. PMID:27542281

A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection.

PubMed

Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Blee, Elizabeth

2015-09-01

Caleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein in Aspergillus flavus that is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway, aflR and aflD, were downregulated in the strains in which A. flavus PXG (AfPXG) was silenced, leading to reduced aflatoxin B1 production in vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in which AfPXG was silenced. PXG-deficient A. flavus strains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Comparative Analysis of the Flax Immune Receptors L6 and L7 Suggests an Equilibrium-Based Switch Activation Model

PubMed Central

Chen, Chunhong; Newell, Kim; Lawrence, Gregory J.; Ellis, Jeffrey G.; Anderson, Peter A.; Dodds, Peter N.

2016-01-01

NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling. PMID:26744216

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

PubMed

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.

PubMed

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.

Isolation of purified oocyst walls and sporocysts from Toxoplasma gondii.

PubMed

Everson, William V; Ware, Michael W; Dubey, J P; Lindquist, H D Alan

2002-01-01

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.

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

PubMed

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.

PubMed

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.

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

PubMed

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

DOEpatents

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.

General Toxicity and Antifungal Activity of a New Dental Gel with Essential Oil from Abies Sibirica L

PubMed Central

Noreikaitė, Aurelija; Ayupova, Rizvangul; Satbayeva, Elmira; Seitaliyeva, Aida; Amirkulova, Marzhan; Pichkhadze, Guram; Datkhayev, Ubaidilla; Stankeviandccaron;ius, Edgaras

2017-01-01

Background The aim of this study was to analyze the antifungal activity and the general toxicity of a new dental gel containing essential oil from the tree Abies sibirica L., which grows in the Republic of Kazakhstan. Material/Methods The essential oil from Abies sibirica L. was obtained by microwave heating method using the STARTE Microwave Extraction System. Adjutants used to prepare the oil were carbomer 974P, glycerin, polysorbate 80, xylitol, triethanolamine, and purified water, all allowed for medical usage. The antifungal activity of the essential oil was assessed by monitoring the optical density of Candida albicans in a microplate reader. The safety was determined by analyzing the acute and subacute toxicity. Results The essential oil obtained by the microwave heating method revealed a higher antifungal activity in comparison with the essential oil obtained by the steam distillation method. No obvious changes were detected in guinea pigs following cutaneous application of the gel. Enteral administration of the essential oil caused minimal functional and histological changes in mice after 4 weeks. The new harmless dental gel containing pine oil from Abies sibirica L. was provided for the purposes of this particular clinical research. Conclusions The high antifungal activity of the gel is the basis for more in-depth studies on its safety and pharmacological activity. PMID:28132065

General Toxicity and Antifungal Activity of a New Dental Gel with Essential Oil from Abies Sibirica L.

PubMed

Noreikaitė, Aurelija; Ayupova, Rizvangul; Satbayeva, Elmira; Seitaliyeva, Aida; Amirkulova, Marzhan; Pichkhadze, Guram; Datkhayev, Ubaidilla; Stankevičius, Edgaras

2017-01-29

BACKGROUND The aim of this study was to analyze the antifungal activity and the general toxicity of a new dental gel containing essential oil from the tree Abies sibirica L., which grows in the Republic of Kazakhstan. MATERIAL AND METHODS The essential oil from Abies sibirica L. was obtained by microwave heating method using the STARTE Microwave Extraction System. Adjutants used to prepare the oil were carbomer 974P, glycerin, polysorbate 80, xylitol, triethanolamine, and purified water, all allowed for medical usage. The antifungal activity of the essential oil was assessed by monitoring the optical density of Candida albicans in a microplate reader. The safety was determined by analyzing the acute and subacute toxicity. RESULTS The essential oil obtained by the microwave heating method revealed a higher antifungal activity in comparison with the essential oil obtained by the steam distillation method. No obvious changes were detected in guinea pigs following cutaneous application of the gel. Enteral administration of the essential oil caused minimal functional and histological changes in mice after 4 weeks. The new harmless dental gel containing pine oil from Abies sibirica L. was provided for the purposes of this particular clinical research. CONCLUSIONS The high antifungal activity of the gel is the basis for more in-depth studies on its safety and pharmacological activity.

Associations between fatigue, physical activity, and QoL in patients with myeloproliferative neoplasms.

PubMed

Tolstrup Larsen, Rasmus; Tang, Lars H; Brochmann, Nana; Meulengracht Flachs, Esben; Illemann Christensen, Anne; Hasselbalch, Hans C; Zwisler, Ann-Dorthe

2018-06-01

Patients with Philadelphia chromosome-negative Myeloproliferative Neoplasms (MPNs) report fatigue as the most common symptom and contributing significantly to reduction in their quality of life (QoL). Targeted non-pharmacological intervention to increase levels of physical activity is suggested as a fatigue-reducing and QoL-enhancing intervention in MPN patients. The interrelationship between physical activity, fatigue, and QoL has, to our knowledge, never been reported. We analyzed data from 1807 MPN patients. The primary analysis included a multiple regression model allowing fatigue to mediate the relationship between physical activity and QoL. We herein report the first and the largest study of patients with MPNs, in whom we have investigated the interrelationship between fatigue, physical activity, and QoL. Sedentary patients were more likely to report fatigue compared to highly active patients. There was a negative association between fatigue and QoL, and there was a positive association between physical activity and QoL. There was no interaction between fatigue and physical activity in the association with QoL. We found positive associations between level of physical activity and QoL, independently of fatigue being present. More research is needed before physical activity can be introduced as a targeted intervention to reduce fatigue and increase QoL in the management of patients. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

PubMed Central

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-01-01

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. PMID:28264934

Antifungal activity and cytotoxicity of extracts and triterpenoid saponins obtained from the aerial parts of Anagallis arvensis L.

PubMed

Soberón, José R; Sgariglia, Melina A; Pastoriza, Ana C; Soruco, Estela M; Jäger, Sebastián N; Labadie, Guillermo R; Sampietro, Diego A; Vattuone, Marta A

2017-05-05

Anagallis arvensis L. (Primulaceae) is used in argentinean northwestern traditional medicine to treat fungal infections. We are reporting the isolation and identification of compounds with antifungal activity against human pathogenic yeast Candida albicans, and toxicity evaluation. to study the antifungal activity of extracts and purified compounds obtained form A. arvensis aerial parts, alone and in combinations with fluconazole (FLU), and to study the toxicity of the active compounds. Disk diffusion assays were used to perform an activity-guided isolation of antifungal compounds from the aerial parts of A. arvensis. Broth dilution checkerboard and viable cell count assays were employed to determine the effects of samples and combinations of FLU + samples against Candida albicans. The chemical structures of active compounds were elucidated by spectroscopic analysis. Genotoxic and haemolytic effects of the isolated compounds were determined. Four triterpenoid saponins (1-4) were identified. Anagallisin C (AnC), exerted the highest inhibitory activity among the assayed compounds against C. albicans reference strain (ATCC 10231), with MIC-0 =1µg/mL. The Fractional Inhibitory Concentration Index (FICI=0.129) indicated a synergistic effect between AnC (0.125µg/mL) and FLU (0.031µg/mL) against C. albicans ATCC 10231. AnC inhibited C. albicans 12-99 FLU resistant strain (MIC-0 =1µg/mL), and the FICI=0.188 indicated a synergistic effect between AnC (0.125µg/mL) and fluconazole (16µg/mL). The combination AnC+ FLU exerted fungicidal activity against both C. albicans strains. AnC exerted inhibitory activity against C. albicans ATCC 10231 sessile cells (MIC 5 0=0.5µg/mL and MIC 80 =1µg/mL) and against C. albicans 12-99 sessile cells (MIC 5 0=0.75µg/mL and MIC 80 =1.25µg/mL). AnC exerted haemolytic effect against human red blood cells at 15µg/mL and did not exerted genotoxic effect on Bacillus subtilis rec strains. The antifungal activity and lack of genotoxic

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

PubMed

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.

PubMed

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.

PubMed

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.

Extraction, antioxidant and antibacterial activities of Broussonetia papyrifera fruits polysaccharides.

PubMed

Han, Qiaohong; Wu, Zili; Huang, Bo; Sun, Liangqi; Ding, Chunbang; Yuan, Shu; Zhang, Zhongwei; Chen, Yanger; Hu, Chao; Zhou, Lijun; Liu, Jing; Huang, Yan; Liao, Jinqiu; Yuan, Ming

2016-11-01

Polysaccharides were extracted from Broussonetia papyrifera ((L.) L'Herit. ex Vent.) fruits (BPP), and response surface methodology was used to maximize extraction yield. The optimum extraction conditions were: ratio of water to solid, 30mL/g; extraction duration, 50min; extraction power, 180W; and extraction temperature, 60°C. Under these conditions, the yield of BPP was 8.61%. Then, BPP was purified, and three purified fractions (designated BPP-1, BPP-2 and BPP-3) were obtained for further physicochemical properties, antioxidant activity and antibacterial activity analysis. These fractions were mainly composed of glucose, mannose and arabinose residue, meanwhile, BPP-3 had a significantly higher rhamnose and uronic acid content than BPP-1 and BPP-2. And BPP-3 showed the best hydroxyl radial scavenging activity, ferric reducing activity power (FRAP), antihemolytic activity and antibacterial activity. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PubMed

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 < 0.01). The addition of earthworms into IVFCW increased enzyme activity and nitrification potentials in December, which resulted in improving purifying capability.

Adenylate cyclase-stimulating, bone-resorbing and B TGF-like activities in canine apocrine cell adenocarcinoma of the anal sac.

PubMed

Weir, E C; Centrella, M; Matus, R E; Brooks, M L; Wu, T; Insogna, K L

1988-12-01

Canine apocrine cell adenocarcinoma of the anal sac (APO-AS) is a spontaneously occurring tumor that causes humorally mediated hypercalcemia in 90% of cases. To further define the nature of the responsible mediator in APO-AS, we examined tumor extracts from five APO-AS and four control tumors for adenylate cyclase-stimulating activity (ACSA). All extracts from APO-AS contained potent ACSA, whereas the four control tumors did not. The ACSA extracted from one tumor demonstrated a dose response curve parallel to that of synthetic bovinePTH-(1-34) and was 80% inhibited by Nle8,18,Tyr34 bPTH-(3-34)amide at a concentration of 10(-5) M. Extracts from three APO-AS and three control tumors were also examined for in vitro bone-resorbing activity (BRA). All APO-AS contained significant BRA, stimulating resorption 1.47 to 2.13-fold over basal, whereas none of the control tumors stimulated resorption. Purification of one extract using C18 reverse-phase high pressure liquid chromatography (RP-HPLC) resulted in a single sharp peak of ACSA which was 400-fold purified compared with the initial extract. This pool also contained significant bone-resorbing activity, whereas none of the adjacent pools did. Purification of a second extract using sequential CN and C18 RP-HPLC followed by size exclusion HPLC resulted in material that was at least 10,000-fold purified, and showed co-purification of ACSA and B TGF-like activity.

Safety of purified decolorized (low anthraquinone) whole leaf Aloe vera (L) Burm. f. juice in a 3-month drinking water toxicity study in F344 rats.

PubMed

Shao, A; Broadmeadow, A; Goddard, G; Bejar, E; Frankos, V

2013-07-01

Decolorized (purified, low anthraquinone) whole leaf Aloe vera (L.) Burm. f. juice was administered at concentrations of 0%, 0.5%, 1% and 2% in the drinking water of F344Du rats for 3 months without any adverse effect. The no-observed-adverse-effect level (NOAEL) in this study was considered to be >2%w/v (>1845 mg/kg bodyweight/day for males and >2920 mg/kg bodyweight for females). The test material contained total anthraquinones at <0.1 parts per million. In the present study there was a complete absence of any histological alteration in samples from the cecum, colon (proximal, mid and distal regions). Similar concentrations of non-decolorized (unpurified, high anthraquinone) Aloe vera extracts tested in other studies have resulted in an increased incidence and severity of diarrhea and colon adenomas and carcinomas. The results of this study supports the assertion that the high levels of anthraquinone present in orally administered, non-purified whole leaf Aloe vera extract may be responsible for the adverse effects observed on the colon. Copyright © 2013 Elsevier Ltd. All rights reserved.

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2010 CFR

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...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 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...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 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...

21 CFR 880.6710 - Medical ultraviolet water purifier.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 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...

Imprinting regulator DNMT3L is a transcriptional repressor associated with histone deacetylase activity.

PubMed

Aapola, Ulla; Liiv, Ingrid; Peterson, Pärt

2002-08-15

DNMT3L is a regulator of imprint establishment of normally methylated maternal genomic sequences. DNMT3L shows high similarity to the de novo DNA methyltransferases, DNMT3A and DNMT3B, however, the amino acid residues needed for DNA cytosine methyltransferase activity have been lost from the DNMT3L protein sequence. Apart from methyltransferase activity, Dnmt3a and Dnmt3b serve as transcriptional repressors associating with histone deacetylase (HDAC) activity. Here we show that DNMT3L can also repress transcription by binding directly to HDAC1 protein. We have identified the PHD-like zinc finger of the ATRX domain as a main repression motif of DNMT3L, through which DNMT3L recruits the HDAC activity needed for transcriptional silencing. Furthermore, we show that DNMT3L protein contains an active nuclear localisation signal at amino acids 156-159. These results describe DNMT3L as a co-repressor protein and suggest that a transcriptionally repressed chromatin organisation through HDAC activity is needed for establishment of genomic imprints.

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

NASA Astrophysics Data System (ADS)

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.

PubMed

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.

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

21 CFR 880.6500 - Medical ultraviolet air purifier.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Medical ultraviolet air purifier. 880.6500 Section... Miscellaneous Devices § 880.6500 Medical ultraviolet air purifier. (a) Identification. A medical ultraviolet air purifier is a device intended for medical purposes that is used to destroy bacteria in the air by exposure...

Antioxidant and immunobiological activity of water-soluble polysaccharide fractions purified from Acanthopanax senticosu.

PubMed

Chen, Ruizhan; Liu, Zhiqiang; Zhao, Jimin; Chen, Ruiping; Meng, Fanlei; Zhang, Min; Ge, Wencheng

2011-07-15

A water-soluble polysaccharide obtained from Acanthopanax senticosus leaves (ASL), was fractionated by DEAE-Sepharose fast-flow column chromatography, and purified by Sephadex G-75 gel-permeation column chromatography. The characteristics of ASP-2-1 were determined by chemical analysis, high-performance capillary electrophoresis (HPCE), high-performance gel-permeation chromatography (HPGPC). The results show that ASP-2-1 contained 89.47% carbohydrate, 7.45% uronic acid, 2.16% protein and seven kinds of monosaccharides including rhamnose, xylose, glucose, mannose, arabinose, galactose and glucuronic acid in a molar ratio of 7.45:18.63:25.15:0.93:8.35:2.79:5.69, with an average molecular weight of about 14,573Da. Furthermore, the immunobiological and antioxidant activities, in vitro, of ASP-2-1 were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and ferric-reducing antioxidant power assay (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH()), superoxide radical (()O(2)(-)) and hydroxyl radical (()OH) free radical-scavenging assay, respectively. The results showed that ASP-2-1 exhibited significantly higher immunomodulatory activities against the lymphocyte proliferation in vitro, pronounced reductive power (FRAP value: 785.1μM at 0.2mg/ml), strong hydroxyl radical (89.56% at 1mg/ml) scavenging activity, moderate superoxide radicals (65.32% at 1mg/ml) and DPPH radicals (68.9% at 1mg/ml) scavenging activities. ASP-2-1 should be explored as a novel and potential natural antioxidant and immunostimulating agent for use in functional foods or medicine. Copyright © 2011 Elsevier Ltd. All rights reserved.

Purification, characterization and kinetic properties of extracellular L-asparaginase produced by Cladosporium sp.

PubMed

Mohan Kumar, N S; Manonmani, H K

2013-04-01

L-asparaginase from Cladosporium sp. grown on wheat bran by SSF was purified. Enzyme appeared to be a trimer with homodimer of 37 kDa and another 47 kDa amounting to total mass of 121 kDa as estimated by SDS-PAGE and 120 kDa on gel filtration column. The optimum temperature and pH of the enzyme were 30 °C and 6.3, respectively with Vmax of 4.44 μmol/mL/min and Km of 0.1 M. Substrate specificity studies indicated that, L-asparaginase has greater affinity towards L-asparagine with substrate hydrolysis efficiency (Vmax/Km ratio) eightfold higher than that of L-glutamine. L-asparaginase activity in presence of thiols studied showed decrease in Vmax and increase in Km, indicating nonessential mode of inactivation. Among the thiols tested, β-mercaptomethanol, exerted inhibitory effect, suggesting a critical role of disulphide linkages in maintaining a suitable conformation of the enzyme. Metal ions such as Ca(2+), Co(2+), Cu(2+), Mg(2+), Na(+), K(+) and Zn(2+) significantly affected enzyme activity whereas presence of Fe(3+), Pb(2+) and KI stimulated the activity. Detergents studied also enhanced L-asparaginase activity. In-vitro half-life of purified L-asparaginase in mammalian blood serum was 93.69 h. The enzyme inhibited acrylamide formation in potato chips by 96 % making it a potential candidate for food industry to reduce acrylamide content in starchy fried food commodities.

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

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

Takahashi, Hitoshi; Akazawa, Daisuke; Toray Industries, Inc., Kanagawa

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.more » 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.« less

Purification and characterization of a papaya (Carica papaya L.) pectin methylesterase isolated from a commercial papain preparation

USDA-ARS?s Scientific Manuscript database

We purified a single stable pectin methylesterase (CpL-PME; EC 3.1.1.11) from a commercial papain preparation, which is isolated from Carica papaya (L.) fruit latex. This CpL-PME was separated from the abundant cysteine endopeptidases activities using sequential hydrophobic interaction and cation-ex...

Characterization of the co-purified invertase and β-glucosidase of a multifunctional extract from Aspergillus terreus.

PubMed

Giraldo, Marielle Aleixo; Gonçalves, Heloísa Bressan; Furriel, Rosa Dos Prazeres Melo; Jorge, João Atílio; Guimarães, Luis Henrique Souza

2014-05-01

The filamentous fungus Aspergillus terreus secretes both invertase and β-glucosidase when grown under submerged fermentation containing rye flour as the carbon source. The aim of this study was to characterize the co-purified fraction, especially the invertase activity. An invertase and a β-glucosidase were co-purified by two chromatographic steps, and the isolated enzymatic fraction was 139-fold enriched in invertase activity. SDS-PAGE analysis of the co-purified enzymes suggests that the protein fraction with invertase activity was heterodimeric, with subunits of 47 and 27 kDa. Maximal invertase activity, which was determined by response surface methodology, occurred in pH and temperature ranges of 4.0-6.0 and 55-65 °C, respectively. The invertase in co-purified enzymes was stable for 1 h at pH 3.0-10.0 and maintained full activity for up to 1 h at 55 °C when diluted in water. Invertase activity was stimulated by 1 mM concentrations of Mn²⁺ (161 %), Co²⁺ (68 %) and Mg²⁺ (61 %) and was inhibited by Al³⁺, Ag⁺, Fe²⁺ and Fe³⁺. In addition to sucrose, the co-purified enzymes hydrolyzed cellobiose, inulin and raffinose, and the apparent affinities for sucrose and cellobiose were quite similar (K(M) = 22 mM). However, in the presence of Mn²⁺, the apparent affinity and V(max) for sucrose hydrolysis increased approximately 2- and 2.9-fold, respectively, while for cellobiose, a 2.6-fold increase in V(max) was observed, but the apparent affinity decreased 5.5-fold. Thus, it is possible to propose an application of this multifunctional extract containing both invertase and β-glucosidase to degrade plant biomass, thus increasing the concentration of monosaccharides obtained from sucrose and cellobiose.

Thermostable purified endoglucanas from acidothermus cellulolyticus ATCC 43068

DOEpatents

Himmel, Michael E.; Adney, William S.; Tucker, Melvin P.; Grohmann, Karel

1994-01-01

A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068). The cellulase is water soluble, possesses both C.sub.1 and C.sub.x types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83.degree. C. at pH's from about 2 to about 9, and in inactivation temperature of about 110.degree. C. at pH's from about 2 to about 9.

Thermostable purified endoglucanase from Acidothermus cellulolyticus ATCC 43068

DOEpatents

Himmel, M.E.; Adney, W.S.; Tucker, M.P.; Grohmann, K.

1994-01-04

A purified low molecular weight cellulase endoglucanase I having a molecular weight of between about 57,420 to about 74,580 daltons from Acidothermus cellulolyticus (ATCC 43068) is presented. The cellulase is water soluble, possesses both C[sub 1] and C[sub x] types of enzyme activity, a high degree of stability toward heat, and exhibits optimum temperature activity at about 83 C at pH's from about 2 to about 9, and in inactivation temperature of about 110 C at pH's from about 2 to about 9. 7 figures.

Biochemical Characterization of a Novel l-Asparaginase with Low Glutaminase Activity from Rhizomucor miehei and Its Application in Food Safety and Leukemia Treatment

PubMed Central

Huang, Linhua; Liu, Yu; Sun, Yan

2014-01-01

A novel fungal gene encoding the Rhizomucor miehei l-asparaginase (RmAsnase) was cloned and expressed in Escherichia coli. Its deduced amino acid sequence shared only 57% identity with the amino acid sequences of other reported l-asparaginases. The purified l-asparaginase homodimer had a molecular mass of 133.7 kDa, a high specific activity of 1,985 U/mg, and very low glutaminase activity. RmAsnase was optimally active at pH 7.0 and 45°C and was stable at this temperature for 30 min. The final level of acrylamide in biscuits and bread was decreased by about 81.6% and 94.2%, respectively, upon treatment with 10 U RmAsnase per mg flour. Moreover, this l-asparaginase was found to potentiate a lectin's induction of leukemic K562 cell apoptosis, allowing lowering of the drug dosage and shortening of the incubation time. Overall, our findings suggest that RmAsnase possesses a remarkable potential for the food industry and in chemotherapeutics for leukemia. PMID:24362429

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

PubMed

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.

Water Purifiers

NASA Technical Reports Server (NTRS)

1992-01-01

Technology developed to purify the water aboard manned spacecraft has led to a number of spinoff applications. One of them is the Ambassador line of bacteriostatic water treatment systems, which employ high grade, high absorption media to inhibit bacteria growth and remove the medicinal taste and odor of chlorine. Company President, Ray Ward, originally became interested in the technology because of the "rusty" taste of his water supply.

α-l-rhamnosidase selective for rutin to isoquercitrin transformation from Penicillium griseoroseum MTCC-9224.

PubMed

Yadav, Sarita; Yadava, Sudha; Yadav, Kapil D S

2017-02-01

An α-l-rhamnosidase secreting fungal strain has been isolated from the decaying goose berry (Emblica officinalis) fruit peel. The fungal strain has been identified as Penicillium greoroseum MTCC-9224. The α-l-rhamnosidase of this fungal strain has been purified to homogeneity using a simple procedure involving concentration by ultra filtration and an anion exchange chromatography on DEAE-cellulose. The purified enzyme gave a single protein band corresponding to molecular mass of 97kDa in SDS-PAGE analysis. The native-PAGE analysis also gave a single protein band confirming the purity of the enzyme. Using p-nitrophenyl-α-l-rhamnopyranoside as the substrate, K m and k cat values of the enzyme were 0.65mM and 43.65s -1 , respectively. The pH and temperature optima of the enzyme were 6.5 and 57°C, respectively. The activation energy for the thermal denaturation of the enzyme was 27.9kJ/mol. The purified α-l-rhamnosidase hydrolyzed rutin to isoquercitrin and l-rhamnose but has no effect on naringin and hesperidin. Copyright © 2017 Elsevier Inc. All rights reserved.

Physical, chemical and biochemical studies with isolated chloroplasts and purified enzymes

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

El-Badry, A. M.

1971-01-01

Fructose-1,6-diphosphatase has been isolated, purified, and crystallized from previously isolated spinach chloroplasts. The effects of various anions, cations, and sulfhydryl reagent were tested, and activation by Mg ++, glycine, HCO 3 -, and sulfhydryl reagent is described.

Determination of dioxin-like polychlorinated biphenyls in 1 mL whole blood using programmable temperature vaporization large volume injection coupled to gas chromatogram and high-resolution mass spectrometry.

PubMed

Shen, Haitao; Guan, Rongfa; Li, Jingguang; Zhang, Lei; Ren, Yiping; Xu, Xiaomin; Song, Yang; Zhao, Yunfeng; Han, Jianlong; Wu, Yongning

2013-03-12

A sensitive method based on programmable temperature vaporization large volume injection coupled to gas chromatogram and high-resolution mass spectrometry (PTV-GC-HRMS) has been developed for the determination of ultra trace levels of dioxin-like polychlorinated biphenyls (DL PCBs) in small amounts of human blood. Blood samples (1mL) were first extracted by column extraction and then purified with column chromatorgraphies. Final extracts (20μL) were introduced to the PTV injector under the solvent vent mode and detected by GC-HRMS (SIM mode). PTV parameters were observed by changing one factor at a time (practical conditions: vent flow: 50mLmin(-1), vent pressure: 0kPa and vent time: 0.1min), recoveries of most PCB congeners ranged from 55.1% to 108%, and method detection limits were in the range of 0.11-1.63pgg(-1). Copyright © 2013 Elsevier B.V. All rights reserved.

A highly specific l-galactose-1-phosphate phosphatase on the path to ascorbate biosynthesis

PubMed Central

Laing, William A.; Bulley, Sean; Wright, Michele; Cooney, Janine; Jensen, Dwayne; Barraclough, Di; MacRae, Elspeth

2004-01-01

Ascorbate is a critical compound in plants and animals. Humans are unable to synthesize ascorbate, and their main source of this essential vitamin are plants. However, the pathway of synthesis in plants is yet to be established, and several unknown enzymes are only postulated to exist. We describe a specific l-galactose-1-phosphate (l-gal-1-P) phosphatase that we partially purified from young kiwifruit (Actinidia deliciosa) berries. The enzyme had a native molecular mass of ≈65 kDa, was completely dependent on Mg2+ for activity and was very specific in its ability to hydrolyze l-gal-1-P. The activity had a pH optimum of 7.0, a KM(l-gal-1-P) of 20–40 μM and a Ka(Mg2+) of 0.2 mM. The activity was inhibited by Mg2+ at concentrations >2 mM. The enzyme from Arabidopsis thaliana shoots showed similar properties to the kiwifruit enzyme. The Arabidopsis thaliana enzyme preparation was digested with trypsin, and proteins present were identified by using liquid chromatography–MS. One of 24 proteins present in our preparation was an Arabidopsis thaliana protein, At3g02870, annotated myo-inositol-1-phosphate phosphatase in GenBank, that matched the characteristics of the purified l-gal-1-phosphate phosphatase. We then expressed a kiwifruit homologue of this gene in Escherichia coli and found that it showed 14-fold higher maximum velocity for l-gal-1-P than myo-inositol-1-P. The expressed enzyme showed very similar properties to the enzyme purified from kiwifruit and Arabidopsis, except that its KM(l-gal-1-P) and Ka(Mg2+) were higher in the expressed enzyme. The data are discussed in terms of the pathway to ascorbate biosynthesis in plants PMID:15550539

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

PubMed

Schmid, Stefan; Seiler, Cornelia; Gerecke, Andreas C; Hächler, Herbert; Hilbi, Hubert; Frey, Joachim; Weidmann, Simon; Meier, Lukas; Berchtold, Christian; Zenobi, Renato

2013-07-15

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 3.0m s(-1) (3200Lmin(-1)), much higher than other plasma-based reactors described in the literature, which generally are limited to air flow rates below 10Lmin(-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 10Hz or 50Hz, 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. Copyright © 2013 Elsevier B.V. All rights reserved.

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

PubMed Central

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

2014-01-01

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. PMID:24449906

Partial purification and anti-leukemic activity of L-asparaginase enzyme of the actinomycete strain LA-29 isolated from the estuarine fish, Mugil cephalus (Linn.).

PubMed

Sahu, Maloy Kumar; Poorani, E; Sivakumar, K; Thangaradjou, T; Kannan, L

2007-07-01

The actinomycete strain LA-29 isolated from the gut contents of the fish, Mugil cephalus of the Vellar estuary showed excellent L-asparaginase activity The enzyme was purified 18-fold and the final recovery of protein was 1.9%, which exhibited an activity of 13.57 IU/mg protein. The partially purified L-asparaginase inhibited the growth of leukemia cells in male wistar rats. Average survival period of the rats was more in an optimum enzyme dose of 100 units and the survival period was less when the dosages were increased and at the same time the enzyme became less effective when the dosages were decreased. Higher survival of 17.2 days was recorded when 100 units of the enzyme was given in three intermittent doses (50/25/25 units) at the interval of 24 hr. Analysis of cell components of the strain LA-29 has revealed the wall type-I which is the characteristic of the genus Streptomyces. Further the morphological, physiological and biochemical features along with the micromorphological results obtained for the strain LA-29 were compared with that of the Streptomyces species found in Bergey's Manual of Determinative Bacteriology and the strain LA-29 has been tentatively identified as Streptomyces canus.

Low cost thermoformed solar still water purifier for D&E countries

NASA Astrophysics Data System (ADS)

Flendrig, L. M.; Shah, B.; Subrahmaniam, N.; Ramakrishnan, V.

IntroductionSolar distillation mimics nature’s hydrologic water cycle by purifying water through evaporation (using solar energy) and condensation (rain). It is one of the most basic purification systems available today to obtain high quality drinking water and can remove non-volatile contamination from almost any water source. This low-tech technology should therefore be ideally suited for developing and emerging countries where sun shines in abundance. In the past century numerous designs have been realised with footprints ranging from 0.5 m 2 to thousands of square meters. Despite all efforts, this intriguing technology has not been applied widely yet. Among the challenges that remain are: (1) its low yield, (2) obtaining local commitment to operate/maintain large scale systems properly, and (3) relatively high initial investment costs. The objective of this study has been to address challenges 1 and 3 by using standard plastic thermoforming technology to realize a small scale single slope solar still for personal use (2-4 l per day) with adequate efficiency and at low production costs. Materials and methodsThe solar still consists of two parts: a basin that holds the dirty water and a transparent tilted cover onto which the clean water vapour can condense. The basin has a footprint of 1.34 m 2 and is made of a 3 mm thick sheet of black high-density polyethylene (HDPE) which is thermoformed using standard equipment for making fish-ponds. This allows for the incorporation of detailed features, like reinforcements and a clean-water collection gutter, at no extra cost. The transparent cover is made of UV stabilised low-density PE-foil which is under a slope of 10° to transport condensed water droplets to the lower located collection gutter. Throughput and purification performance were evaluated in duplicate at our Bangalore R&D facilities in India, over a short term (5 day) period. Solar radiation was measured using a Pyranometer. The system was loaded with 40 l

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

PubMed Central

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

Characterization of an RNA aptamer against HPV-16 L1 virus-like particles.

PubMed

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores; Alvarez-Salas, Luis Marat

2014-10-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3'end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection.

Characterization of an RNA Aptamer Against HPV-16 L1 Virus-Like Particles

PubMed Central

Leija-Montoya, Ana Gabriela; Benítez-Hess, María Luisa; Toscano-Garibay, Julia Dolores

2014-01-01

The human papillomavirus (HPV) capsid is mainly composed of the L1 protein that can self-assemble into virus-like particles (VLPs) that are structurally and immunologically similar to the infectious virions. We report here the characterization of RNA aptamers that recognize baculovirus-produced HPV-16 L1 VLPs. Interaction and slot-blot binding assays showed that all isolated aptamers efficiently bound HPV-16 VLPs, although the Sc5-c3 aptamer showed the highest specificity and affinity (Kd=0.05 pM). Sc5-c3 secondary structure consisted of a hairpin with a symmetric bubble and an unstructured 3′end. Biochemical and genetic analyses showed that the Sc5-c3 main loop is directly involved on VLPs binding. In particular, binding specificity appeared mediated by five non-consecutive nucleotide positions. Experiments using bacterial-produced HPV-16 L1 resulted in low Sc5-c3 binding, suggesting that recognition of HPV-16 L1 VLPs relies on quaternary structure features not present in bacteria-produced L1 protein. Sc5-c3 produced specific and stable binding to HPV-16 L1 VLPs even in biofluid protein mixes and thus it may provide a potential diagnostic tool for active HPV infection. PMID:25111024

Cloning and expression of phosphoenolpyruvate carboxykinase from a cestode parasite and its solubilization from inclusion bodies using l-arginine.

PubMed

Dutta, Asim K; Ramnath; Dkhar, Barilin; Tandon, Veena; Das, Bidyadhar

2016-09-01

Phosphoenolpyruvate carboxykinase is an essential regulatory enzyme of glycolysis in the cestode parasite, Raillietina echinobothrida, and is considered a potential target for anthelmintic action because of its differential activity from that of its avian host. However, due to the unavailability of its structure, the mechanism of regulation of PEPCK from R. echinobothrida (rePEPCK) and its interaction with possible modulators remain unclear. Hence, in this study, the rePEPCK gene was cloned into pGEX-4T-3 and overexpressed for its characterization. On being induced by IPTG, the recombinant rePEPCK was expressed as inclusion bodies (IBs); hence, various agents, like different inducer concentrations, temperature, time, host cell types, culture media, pH, and additives, were used to bring the protein to soluble form. Finally, a significant amount (∼46%) of rePEPCK was solubilized from IBs by adding 2M l-arginine. Near-UV circular dichroism spectra analysis indicated that l-arginine (2M) had no effect on the conformation of the protein. In this study, we have reported a yield of ∼73mg of purified rePEPCK per 1L of culture. The purified rePEPCK retained its biological activity, and Km of the enzyme for its substrate was determined and discussed. The availability of recombinant rePEPCK may help in biochemical- and biophysical-studies to explore its molecular mechanisms and regulations. Copyright © 2015 Elsevier Inc. All rights reserved.

Respirators: Air Purifying, Self-Study, Course 40723

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

Chochoms, Michael

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.

Domains involved in calcineurin phosphatase inhibition and nuclear localisation in the African swine fever virus A238L protein

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

Abrams, Charles C.; Chapman, Dave A.G.; Silk, Rhiannon

2008-05-10

The African swine fever virus A238L protein inhibits calcineurin phosphatase activity and activation of NF-{kappa}B and p300 co-activator. An 82 amino acid domain containing residues 157 to 238 at the C-terminus of A238L was expressed in E. coli and purified. This purified A238L fragment acted as a potent inhibitor of calcineurin phosphatase in vitro with an IC{sub 50} of approximately 70 nM. Two putative nuclear localisation signals were identified between residues 80 to 86 (NLS-1) and between residues 203 to 207 overlapping with the N-terminus of the calcineurin docking motif (NLS-2). Mutation of these motifs independently did not reduce nuclearmore » localisation compared to the wild type A238L protein, whereas mutation of both motifs significantly reduced nuclear localisation of A238L. Mutation of the calcineurin docking motif resulted in a dramatic increase in the nuclear localisation of A238L provided an intact NLS was present. We propose that binding of calcineurin to A238L masks NLS-2 contributing to the cytoplasmic retention of A238L.« less

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

PubMed

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.

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

PubMed

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.

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

PubMed

Chen, Jialin; De Raeymaecker, Joren; Hovgaard, Jannik Brøndsted; Smaardijk, Susanne; Vandecaetsbeek, Ilse; Wuytack, Frank; Møller, Jesper Vuust; Eggermont, Jan; De Maeyer, Marc; Christensen, Søren Brøgger; Vangheluwe, Peter

2017-04-28

The Golgi/secretory pathway Ca 2+ /Mn 2+ -transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca 2+ -dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca 2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca 2+ -ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di- tert -butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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

PubMed

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 < 0.05). Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis, the hydrolysate with 40% DH had a MW lower than 6.5 kDa. The purified protease from 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.

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

PubMed

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.

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

PubMed

Scaglia, Barbara; Nunes, Ramom Rachide; Rezende, Maria Olímpia Oliveira; Tambone, Fulvia; Adani, Fabrizio

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 100-6000mgcarbonL(-1). (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 (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(2)=-0.85; p<0.01, n=6). Copyright © 2016 Elsevier B.V. All rights reserved.

METHOD FOR PURIFYING URANIUM

DOEpatents

Knighton, J.B.; Feder, H.M.

1960-04-26

A process is given for purifying a uranium-base nuclear material. The nuclear material is dissolved in zinc or a zinc-magnesium alloy and the concentration of magnesium is increased until uranium precipitates.

Antimicrobial activity in the common seawhip, Leptogorgia virgulata (Cnidaria: Gorgonaceae).

PubMed

Shapo, Jacqueline L; Moeller, Peter D; Galloway, Sylvia B

2007-09-01

Antimicrobial activity was examined in the gorgonian Leptogorgia virgulata (common seawhip) from South Carolina waters. Extraction and assay protocols were developed to identify antimicrobial activity in crude extracts of L. virgulata. Detection was determined by liquid growth inhibition assays using Escherichia coli BL21, Vibrio harveyii, Micrococcus luteus, and a Bacillus sp. isolate. This represents the first report of antimicrobial activity in L. virgulata, a temperate/sub-tropical coral of the western Atlantic Ocean. Results from growth inhibition assays guided a fractionation scheme to identify active compounds. Reverse-phase HPLC, HPLC-mass spectrometry, and 1H and 13C NMR spectroscopy were used to isolate, purify, and characterize metabolites in antimicrobial fractions of L. virgulata. Corroborative HPLC-MS/NMR evidence validated the presence of homarine and a homarine analog, well-known emetic metabolites previously isolated from L. virgulata, in coral extracts. In subsequent assays, partially-purified L. virgulata fractions collected from HPLC-MS fractionation were shown to contain antimicrobial activity using M. luteus and V. harveyii. This study provides evidence that homarine is an active constituent of the innate immune system in L. virgulata. We speculate it may act synergistically with cofactors and/or congeners in this octocoral to mount a response to microbial invasion and disease.

Acaricidal activity of essential oils from Lippia alba genotypes and its major components carvone, limonene, and citral against Rhipicephalus microplus.

PubMed

Peixoto, Magna Galvão; Costa-Júnior, Livio Martins; Blank, Arie Fitzgerald; Lima, Aldilene da Silva; Menezes, Thays Saynara Alves; Santos, Darlisson de Alexandria; Alves, Péricles Barreto; Cavalcanti, Sócrates Cabral de Holanda; Bacci, Leandro; Arrigoni-Blank, Maria de Fátima

2015-05-30

The goal of the present study was to evaluate the acaricidal potential of Lippia alba essential oil, citral chemotypes (LA-10 and LA-44 genotypes) and carvone chemotypes (LA-13 and LA-57 genotypes), as well as purified citral and enantiomers of carvone and limonene. Efficacy against Rhipicephalus microplus was assessed by the larval packet and the engorged female immersion tests. Citral chemotypes had greater larvicidal activity than carvone chemotypes, and this was further supported by larvicidal and adulticidal activity of purified citral with LC50 values of 7.0 and 29.8 mg/mL, respectively. While purified enantiomers of carvone exhibited greater larvicidal activity than those of limonene, enantioselectivity of limonene was observed with R-(+) displaying significantly higher efficacy (LC50 of 31.2mg/mL) than S-(-) (LC50 of 54.5mg/mL). The essential oils and purified compounds were much less toxic toward engorged adult females, with the exception of citral, and this may be due to limited cuticular penetration. Published by Elsevier B.V.

Purified human breast milk MUC1 and MUC4 inhibit human immunodeficiency virus.

PubMed

Mthembu, Yolanda; Lotz, Zoe; Tyler, Marilyn; de Beer, Corena; Rodrigues, Jeronimo; Schoeman, Leann; Mall, Anwar Suleman

2014-01-01

The HIV-AIDS pandemic is prevalent in sub-Saharan Africa. Breastfeeding is a risk factor, with transmission from mother to child being as high as 40%. To determine the antiviral activity of crude breast milk and its purified mucins MUC1 and MUC4 against HIV-1 in patients who were HIV positive compared to those who were not. Twenty-one human milk samples were taken from both groups. Breast milk mucins were purified by density-gradient ultracentrifugation in caesium chloride and analyzed by SDS-PAGE, Western blotting and amino acid content. The inhibition of the virus by crude milk and purified mucin was assayed by an in vitro HIV-1 p24 assay. SDS-PAGE for purified mucin showed several high-molecular-weight bands for the HIV-negative group and prominently stained single bands on the stacking gel with faintly periodic acid Schiff-positive glycoprotein bands observed in some cases in the running gel for the HIV-positive mucins. Western blot analysis identified the mucins in both groups to be MUC1 and MUC4. Both mucins showed more intensity on Western blotting for the HIV-positive group. There was no difference in the content of serine, threonine and proline of purified mucins for both groups. HIV-1 was not inhibited by crude breast milk from normal (13/14 samples) and infected individuals (19/19 samples). Fifteen of 20 and 16/18 samples of purified mucin from the uninfected and HIV-positive groups, respectively, inhibited the virus. Crude breast milk does not inhibit HIV-1, whilst purified mucins do in an in vitro assay. © 2014 S. Karger AG, Basel.

In vitro antioxidant, anti-inflammatory and anticancer activities of ethyl acetate soluble proanthocyanidins of the inflorescence of Cocos nucifera L.

PubMed

Padumadasa, Chayanika; Dharmadana, Durga; Abeysekera, Ajit; Thammitiyagodage, Mayuri

2016-09-05

Proanthocyanidins belong to a class of polyphenolic compounds called flavonoids and have been reported to exhibit important biological activities. The immature inflorescence of Cocos nucifera L. is used by Ayurvedic and traditional medical practitioners for the treatment of menorrhagia in Sri Lanka. Our studies have shown that the inflorescence of Cocos nucifera L. predominantly contains proanthocyanidins. To determine the antioxidant, anti-inflammatory and anticancer activities of ethyl acetate soluble proanthocyanidins (EASPA) of immature inflorescence of Cocos nucifera L. EASPA fraction of an acetone/water (7:3) extract of Cocos nucifera L. inflorescence was purified on Sephadex LH-20 and was used for the study. Antioxidant activity of EASPA was determined using DPPH and SOR scavenging assays. Anti-inflammatory activity of EASPA was determined by oxidative burst assay using chemiluminescence technique. MTT colorimetric assay was used to evaluate the cytotoxicity of EASPA to both PC3 and HeLa cells. EASPA showed radical scavenging activity against both DPPH and superoxide radicals with IC50 values of 11.02 ± 0.60 μg/mL and 26.11 ± 0.72 μg/mL. In both assays, EASPA showed less antioxidant activity than the standards used. It exhibited similar anti-inflammatory activity (IC50 = 10.31 ± 1.11 μg/mL) to ibuprofen (IC50 = 11.20 ± 1.90 μg/mL) (P ≥ 0.05). EASPA also showed stronger cytotoxic activity towards Hela cells (IC50 = 18.78 ± 0.90 μg/mL) than tamoxifen (IC50 = 28.80 ± 1.94 μg/mL) (P ≤ 0.05), while low cytotoxicity was observed against PC3 cells (IC50 = 44.21 ± 0.73 μg/mL) compared to doxorubicin (IC50 = 1.38 ± 0.16 μg/mL). EASPA showed antioxidant, anti-inflammatory and anticancer activities.

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

PubMed

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.

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

PubMed Central

2014-01-01

Background 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. Methods 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. Results 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. Conclusions 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. PMID:24678592

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

PubMed

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.

PubMed

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.

PubMed

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).

PubMed

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.

PubMed

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.

Glucagon-like peptide-1 receptor expression on human eosinophils and its regulation of eosinophil activation.

PubMed

Mitchell, P D; Salter, B M; Oliveria, J P; El-Gammal, A; Tworek, D; Smith, S G; Sehmi, R; Gauvreau, G M; Butler, M; O'Byrne, P M

2017-03-01

Glucagon-like peptide-1 (GLP-1) and its receptor are part of the incretin family of hormones that regulate glucose metabolism. GLP-1 also has immune modulatory roles. To measure the expression of the GLP-1 receptor (GLP-1R) on eosinophils and neutrophils in normal and asthmatic subjects and evaluate effects of a GLP-1 analog on eosinophil function. Peripheral blood samples were taken from 10 normal and 10 allergic asthmatic subjects. GLP-1R expression was measured on eosinophils and neutrophils. Subsequently, the asthmatic subjects underwent allergen and diluent inhalation challenges, and GLP-1R expression was measured. Purified eosinophils, collected from mild asthmatic subjects, were stimulated with lipopolysaccharide (LPS) and a GLP-1 analog to evaluate eosinophil cell activation markers CD11b and CD69 and cytokine (IL-4, IL-5, IL-8 and IL-13) production. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. Eosinophil, but not neutrophil, expression of GLP-1R is significantly higher in normal controls compared to allergic asthmatics. The expression of GLP-1R did not change on either eosinophils or neutrophils following allergen challenge. A GLP-1 analog significantly decreased the expression of eosinophil-surface activation markers following LPS stimulation and decreased eosinophil production of IL-4, IL-8 and IL-13, but not IL-5. Glucagon-like peptide-1 receptor is expressed on human eosinophils and neutrophils. A GLP-1 analog attenuates LPS-stimulated eosinophil activation. GLP-1 agonists may have additional adjunctive indications in treating persons with concomitant type 2 diabetes mellitus and asthma. © 2016 John Wiley & Sons Ltd.

Purification and characterization of substance P endopeptidase activities in the rat spinal cord.

PubMed

Karlsson, K; Eriksson, U; Andrén, P; Nyberg, F

1997-02-01

Two enzymes with substance P degrading activity were purified from the membrane bound fraction of the rat spinal cord. The purified enzymes were characterized with regard to biochemical and kinetic properties. One of the enzymes exhibited close similarity to neutral endopeptidase 24.11 (NEP, EC 3.4.24.11), while the other resembled a substance P converting endopeptidase (SPE), which has previously been identified and purified from human cerebrospinal fluid (CSF). Detergent treated spinal cord homogenates from male Sprague Dawley rats were purified by anion-exchange chromatography (DEAE-sepharose CL-6B), hydrophobic-interaction chromatography (phenyl-sepharose CL-4B) and molecular sieving (Sephadex G-50). Two fractions with enzymes differing in size were recovered and allowed for further purification to apparent homogeneity by ion-exchange chromatography and molecular sieving on a micro-purification system (SMART). The enzyme activities were monitored by following the conversion of synthetic substance P using a radioimmunoassay specific for the heptapeptide product, substance P (1-7). By SDS-polyacrylamide gel electrophoresis of the purified enzymes molecular weights of 43 and 70 kDa were estimated for the SPE-like and NEP-like activity, respectively. A K(m) of 5 microM was determined for the conversion of substance P to its (1-7) fragment by the SPE-like activity. Reversed-phase HPLC together with mass spectrometry permitted identification of all fragments released from substance P by the peptidases. The released fragments were for both enzymes identified as substance P (1-7), substance P (8-11), substance P (1-8), substance P (9-11). The NEP-like enzyme preparation also gave substance P (1-6) as a major product.

Purified Dendritic Cell-Tumor Fusion Hybrids Supplemented with Non-Adherent Dendritic Cells Fraction Are Superior Activators of Antitumor Immunity

PubMed Central

Wang, Yucai; Liu, Yunyan; Zheng, Lianhe

2014-01-01

Background Strong evidence supports the DC-tumor fusion hybrid vaccination strategy, but the best fusion product components to use remains controversial. Fusion products contain DC-tumor fusion hybrids, unfused DCs and unfused tumor cells. Various fractions have been used in previous studies, including purified hybrids, the adherent cell fraction or the whole fusion mixture. The extent to which the hybrids themselves or other components are responsible for antitumor immunity or which components should be used to maximize the antitumor immunity remains unknown. Methods Patient-derived breast tumor cells and DCs were electro-fused and purified. The antitumor immune responses induced by the purified hybrids and the other components were compared. Results Except for DC-tumor hybrids, the non-adherent cell fraction containing mainly unfused DCs also contributed a lot in antitumor immunity. Purified hybrids supplemented with the non-adherent cell population elicited the most powerful antitumor immune response. After irradiation and electro-fusion, tumor cells underwent necrosis, and the unfused DCs phagocytosed the necrotic tumor cells or tumor debris, which resulted in significant DC maturation. This may be the immunogenicity mechanism of the non-adherent unfused DCs fraction. Conclusions The non-adherent cell fraction (containing mainly unfused DCs) from total DC/tumor fusion products had enhanced immunogenicity that resulted from apoptotic/necrotic tumor cell phagocytosis and increased DC maturation. Purified fusion hybrids supplemented with the non-adherent cell population enhanced the antitumor immune responses, avoiding unnecessary use of the tumor cell fraction, which has many drawbacks. Purified hybrids supplemented with the non-adherent cell fraction may represent a better approach to the DC-tumor fusion hybrid vaccination strategy. PMID:24466232

Production, isolation, and purification of L-asparaginase from Pseudomonas aeruginosa 50071 using solid-state fermentation.

PubMed

El-Bessoumy, Ashraf A; Sarhan, Mohamed; Mansour, Jehan

2004-07-31

The L-asparaginase (E. C. 3. 5. 1. 1) enzyme was purified to homogeneity from Pseudomonas aeruginosa 50071 cells that were grown on solid-state fermentation. Different purification steps (including ammonium sulfate fractionation followed by separation on Sephadex G-100 gel filtration and CM-Sephadex C50) were applied to the crude culture filtrate to obtain a pure enzyme preparation. The enzyme was purified 106-fold and showed a final specific activity of 1900 IU/mg with a 43% yield. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme revealed it was one peptide chain with M(r) of 160 kDa. A Lineweaver-Burk analysis showed a K(m) value of 0.147 mM and V(max) of 35.7 IU. The enzyme showed maximum activity at pH 9 when incubated at 37 degrees C for 30 min. The amino acid composition of the purified enzyme was also determined.

Polygalacturonase from Rhizopus stolonifer, an Elicitor of Casbene Synthetase Activity in Castor Bean (Ricinus communis L.) Seedlings 1

PubMed Central

Lee, Sung-Chul; West, Charles A.

1981-01-01

Apparently homogeneous polygalacturonase-elicitor purified from the filtrates of Rhizopus stolonifer cultures stimulates germinating castor bean seedlings to produce greatly increased levels of casbene synthetase activity. The purification procedure involved gel-filtration chromatography on Sephadex G-25 and G-75 columns followed by cation-exchange chromatography on a Sephadex CM C-50 column. Homogeneity of the purified preparation was indicated by the results of cationic polyacrylamide disc gel electrophoresis and isoelectric focusing (pI = 8.0). The identity of the casbene elicitor activity and polygalacturonase were indicated by the coincidence of the two activities at all stages of purification, the coincidence of both activities with the single protein-staining band detected on a cationic polyacrylamide disc gel and an isoelectric focusing gel, and the identical behavior of both activities on an agarose gel affinity column. The purified polygalacturonase-elicitor is a glycoprotein with approximately 20% carbohydrate content and an estimated molecular weight of 32,000 by polyacrylamide disc gel electrophoresis. PMID:16661728

Methods for purifying carbon materials

DOEpatents

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.

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

PubMed

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.

Anti-oxidative and Anti-microbial Activities of Purified MPN-1-1 from Persicaria nepalensis (Meisn.) Miyabe.

PubMed

Yang, Woong-Suk; Yang, Seung-Hoon; Lee, Jae-Yong; Jang, Seong-Ho; Kim, Cheorl-Ho; Hwnag, Cher-Won

2017-01-01

Persicaria is a genus of flowering plants generally used for traditional medicine and nutritional supplements in tropical and subtropical East Asian countries. Previous studies have shown that Persicaria extracts alleviate lipid peroxidation, hypertension, and inflammation. We investigated the anti-oxidative and anti-microbial effects of ethanol extracts of Persicaria nepalensis (Meisn.) Miyabe, and isolated and identified an active compound, MPN-1-1 from the ethanol extracts. Anti-oxidative values, as indicated by the Oxygen Radical Absorbance Capacity (ORAC) assay, were enhanced by treatment with Persicaria nepalensis (Meisn.) Miyabe ethanol extracts, and bacterial growth was inhibited. The active compound (MPN-1-1), which was further isolated and purified from a Persicaria nepalensis (Meisn.) Miyabe ethanol extract by medium pressure liquid chromatography (MPLC), also had strong anti-oxidative and anti-microbial activity. 1H-NMR spectroscopy identified MPN-1-1 as a 1-ethenyl-4,8-dimethoxy-9H-pyrido(3,4-β) indole compound, which is an alkaloid. Our results provide evidence that Persicaria nepalensis (Meisn.) Miyabe extract has strong physiological activity without any toxic effects, and furthermore, MPN-1-1 can be potentially utilized as a natural dietary supplement as well as an anti-oxidant. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Phantom Word Activation in L2

ERIC Educational Resources Information Center

Broersma, Mirjam; Cutler, Anne

2008-01-01

L2 listening can involve the phantom activation of words which are not actually in the input. All spoken-word recognition involves multiple concurrent activation of word candidates, with selection of the correct words achieved by a process of competition between them. L2 listening involves more such activation than L1 listening, and we report two…

Sequence, overproduction and purification of Vibrio proteolyticus ribosomal protein L18 for in vitro and in vivo studies

NASA Technical Reports Server (NTRS)

Setterquist, R. A.; Smith, G. K.; Oakley, T. H.; Lee, Y. H.; Fox, G. E.

1996-01-01

A strategy suggested by comparative genomic studies was used to amplify the entire Vibrio proteolyticus (Vp) gene for ribosomal protein L18. Vp L18 and its flanking regions were sequenced and compared with the deduced amino acid (aa) sequences of other known L18 proteins. A 26-aa residue segment at the carboxy terminus contains many strongly conserved residues and may be critical for the L18 interaction with 5S rRNA. This approach should allow rapid characterization of L18 from large numbers of bacteria. Both Vp L18 and Escherichia coli (Ec) L18 were overproduced and purified using a T7 expression vector which fuses an N-terminal peptide segment (His-tag) containing 6 histidine residues to the recombinant protein. The purified fusion proteins, Vp His::L18 and Ec His::L18, were both found to bind to either the Vp 5S or Ec 5S rRNAs in vitro. Vp His::L18 protein was also shown to incorporate into Ec ribosomes in vivo. This His-tag strategy likely will have general applicability for the study of ribosomal proteins in vitro and in vivo.

Purification and Properties of a New L-Sorbose Dehydrogenase Accelerative Protein from Bacillus megaterium Bred by Ion-Beam Implantation

NASA Astrophysics Data System (ADS)

Zhao, Shiguang; Yao, Liming; Su, Caixin; Wang, Tao; Wang, Jun; Tang, Mingli; Yu, Zengliang

2008-06-01

Bacillus megaterium BM302 bred by ion-beam implantation produces L-sorbose dehydrogenase accelerative protein (SAP) to accelerate the activity of L-sorbose dehydrogenase (SDH) of Gluconobacter oxydans in the 2-keto-L-gulonic acid (2KLG) fermentation from L-sorbose by the mixed culture of B. megaterium BM302 and G. oxydans. The SAP purified by three chromatographic steps gave 35-fold purification with a yield of 13% and a specific activity of 5.21 units/mg protein. The molecular weight of the purified SAP was about 58 kDa. The SDH accelerative activity of SAP at pH 7 and 50°C was the highest. Additionally, it retained 60% activity at a pH range of 6.5 ~ 10 and was stable at 20°C ~ 60°C. After 0.32-unit SAP was added to the single cultured G. oxydans strains, the SDH activity was apparently accelerated and the 2KLG yield of GO29, GO112, G0 and GI13 was enhanced 2.1, 3.3, 3.5 and 2.9 folds respectively over that of the strains without the addition of SAP.

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

PubMed

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.

Antiulcer and in vitro antioxidant activities of Jasminum grandiflorum L.

PubMed

Umamaheswari, M; Asokkumar, K; Rathidevi, R; Sivashanmugam, A T; Subhadradevi, V; Ravi, T K

2007-04-04

The study was aimed at evaluating the antiulcer and antioxidant activities of 70% ethanolic axtract of leaves of Jasminum grandiflorum L. (JGLE). The leaves of Jasminum grandiflorum L. (Family: Oleaceae) is used in folk medicine for treating ulcerative stomatitis, skin diseases, ulcers, wounds, corns - a hard or soft hyperkeratosis of the sole of the human foot secondary to friction and pressure (Stedman's Medical Dictionary, 28th ed. Lippincott Williams & Wilkins, Philadelphia. p. 443), etc., Antiulcerogenic activity of JGLE (100 and 200 mg/kg, b.w., orally) was evaluated employing aspirin + pylorus ligation (APL) and alcohol (AL) induced acute gastric ulcer models and ulcer-healing activity using acetic acid-induced (AC) chronic ulcer model in rats. Both the antisecretory and cytoprotection hypothesis were evaluated. The antioxidant activity of JGLE has been assayed by using in vitro methods like 2,2-diphenyl-1-picrylhydrazylhydrate (DPPH) assay, reductive ability, superoxide anion scavenging activity, nitric oxide scavenging activity and total phenolic content, in order to explain the role of antioxidant principles in the antiulcerogenic activity of the extract. There was a significant (P<0.01) dose-dependent decrease in the ulcerative lesion index produced by all the three models in rats as compared to the standard drug famotidine (20 mg/kg, b.w. orally). The reduction in gastric fluid volume, total acidity and an increase in the pH of the gastric fluid in APL rats proved the antisecretory activity of JGLE. Additionally, JGLE completely healed the ulcer within 20 days of treatment in AC model as evidenced by histopathological studies. Like antiulcer activity, the free radical scavenging activities of JGLE depends on concentration and increased with increasing amount of the extract. These results suggest that leaves of Jasminum grandiflorum possess potential antiulcer activity, which may be attributed to its antioxidant mechanism of action.

Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

PubMed

Palomino-Vizcaino, Giovanni; Valencia Reséndiz, Diana Gabriela; Benítez-Hess, María Luisa; Martínez-Acuña, Natalia; Tapia-Vieyra, Juana Virginia; Bahena, Daniel; Díaz-Sánchez, Mauricio; García-González, Octavio Patricio; Alvarez-Sandoval, Brenda Arizaí; Alvarez-Salas, Luis Marat

2018-02-15

Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

Preparation and photocatalytic activities of 3D flower-like CuO nanostructures

NASA Astrophysics Data System (ADS)

Qingfei, Fan; Qi, Lan; Meili, Zhang; Ximei, Fan; Zuowan, Zhou; Chaoliang, Zhang

2016-08-01

Hierarchical 3D flower-like CuO nanostructures on the Cu substrates were synthesized by a wet chemical method and subsequent heat treatment. The synthesis, structure and morphologies of obtained samples under different concentrations of Na2S2O3 were investigated in detail and the possible growth mechanisms of the 3D flower-like CuO nanostructures were discussed. Na2S2O3 plays a key role in the generation of the 3D flower-like CuO nanostructures. When the concentration of Na2S2O3 is more than 0.4 mol/L, the 3D flower-like CuO nanostructures can be prepared on the Cu foils. The photocatalytic performances were studied by analyzing the degradation of methyl orange (MO) in aqueous solution in the presence of hydroxide water (H2O2). The 3D flower-like CuO nanostructures exhibit higher photocatalytic activity (96.2% degradation rate) than commercial CuO particles (36.3% degradation rate). The origin of the higher photocatalytic activity of the 3D flower-like CuO nanostructures was also discussed. Project supported by the High-Tech Research and Development Program of China (No. 2009AA03Z427).

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

PubMed

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®

Effects of cosmetics containing purified honeybee (Apis mellifera L.) venom on acne vulgaris.

PubMed

Han, Sang Mi; Lee, Kwang Gill; Pak, Sok Cheon

2013-09-01

Acne vulgaris is a chronic dermatologic problem with multiple factors involved in its pathogenesis. Alternative solutions to acne treatment were instigated by antibiotic resistance despite of its extensive use. Purified bee venom (PBV) has been proposed as a promising candidate for that purpose. The present study was designed to confirm the antibacterial effect of PBV and access the efficacy of cosmetics containing PBV in subjects with acne vulgaris. The skin bacterium Propionibacterium acnes was incubated with PBV at various concentrations and bacterial growth was evaluated using the colony forming unit (CFU) assay. The mechanism of PBV employed in killing P. acnes was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, a total of 12 subjects were randomized in a double-blind, controlled trial to receive either cosmetics containing PBV or cosmetics without PBV for two weeks. Evaluations included lesion counts and skin microorganism. PBV exhibited antimicrobial activity in a concentration-dependent manner, reducing the number of P. acnes CFU by approximately 6 logs at a concentration of 0.5 mg. When PBV concentration was higher than 1.0 mg, no P. acnes colonies were spotted on an agar. TEM and SEM of untreated P. acnes illustrated the normal pleomorphic structure, whereas the PBV-treated bacterium lost the integrity of surface architecture. Significant difference (P=0.027) in the grading levels based on numbers of lesion counts for inflammatory and noninflammatory was observed in favour of the PBV group compared with the control group. In terms of average decrement of skin microorganism, subjects receiving cosmetics containing PBV experienced a significant 57.5% decrease of adenosine triphosphate levels, whereas participants receiving cosmetics without PBV experienced a nonsignificant decrease of 4.7%. These results show that the in vitro actions of antimicrobial activity of PBV were translated in vivo. Cosmetics

Reading Aloud Activity in L2 and Cerebral Activation

ERIC Educational Resources Information Center

Takeuchi, Osamu; Ikeda, Maiko; Mizumoto, Atsushi

2012-01-01

This article explores the cerebral mechanism of reading aloud activities in L2 learners. These activities have been widely used in L2 learning and teaching, and its effect has been reported in various Asian L2 learning contexts. However, the reasons for its effectiveness have not been examined. In order to fill in this gap, two studies using a…

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

PubMed Central

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

PubMed Central

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

Membrane-acting bacteriocin purified from a soil isolate Pediococcus pentosaceus LB44 shows broad host-range.

PubMed

Kaur, Ramanjeet; Tiwari, Santosh Kumar

2018-04-15

Bacteriocin LB44 was purified from cell-free supernatant (CFS) of Pediococcus pentosaceus LB44 using activity-guided chromatography techniques. It was stable up to 121 °C, pH 2.0-6.0, sensitive to proteinase K, papain and trypsin, and retained complete activity in the presence of organic solvents tested. The molecular weight of bacteriocin was ∼6 kDa and initial ten amino acid residues (GECGMCXECG) suggested a new compound. The loss in viable cell count and K + ion efflux of target cells of Micrococcus luteus suggested bactericidal activity. The cell membrane of bacteriocin-treated cells was found to be ruptured which was further confirmed by Fourier Transform Infrared (FTIR) analysis suggesting interaction of bacteriocin with phospholipids in cell membrane. It showed broad host-range and inhibited the growth of Lactobacillus delbrueckii NRRL B-4525, L. plantarum NRRL B-4496, L. acidophilus NRRL B-4495, Enterococcus hirae LD3, Weissella confusa LM85, Staphylococcus aureus, Salmonella typhi ATCC 13311, Serratia marcescens ATCC 27137, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 29905, Haloferax larsenii HA1, HA3, HA8, HA9 and HA10. These properties suggested a new bacteriocin from soil isolate P. pentosaceus LB44 which may offers possible applications in food-safety and therapeutics. Copyright © 2018 Elsevier Inc. All rights reserved.

A DKP Cyclo(L-Phe-L-Phe) Found in Chicken Essence Is a Dual Inhibitor of the Serotonin Transporter and Acetylcholinesterase

PubMed Central

Tsuruoka, Nobuo; Beppu, Yoshinori; Koda, Hirofumi; Doe, Nobutaka; Watanabe, Hiroshi; Abe, Keiichi

2012-01-01

Diketopiperazines (DKPs) are naturally-occurring cyclic dipeptides with a small structure and are found in many organisms and in large amounts in some foods and beverages. We found that a chicken essence beverage, which is popular among Southeast Asians as a traditional remedy and a rich source of DKPs, inhibited the serotonin transporter (SERT) and suppressed serotonin uptake from rat brain synaptosomes, which prompted us to isolate and identify the active substance(s). We purified a SERT inhibitor from the chicken essence beverage and identified it as the DKP cyclo(L-Phe-L-Phe). Interestingly, it was a naturally occurring dual inhibitor that inhibited both SERT and acetylcholinesterase (AChE) in vitro. The DKP increased extracellular levels of the cerebral monoamines serotonin, norepinephrine, and dopamine in the medial prefrontal cortex and acetylcholine in the ventral hippocampus of freely moving rats when administered orally. Moreover, cyclo(L-Phe-L-Phe) significantly shortened escape latency in the water maze test in depressed mice previously subjected to a repeated open-space swimming task, which induces a depression-like state. Cyclo(L-Phe-L-Phe) also significantly improved accuracy rates in a radial maze test in rats and increased step-through latencies in a passive avoidance test in mice with scopolamine-induced amnesia. These animal test results suggest that cyclo(L-Phe-L-Phe), which is present abundantly in some foods such as chicken essence, may abrogate the onset of depression and, thus, contribute to preventing the development of Alzheimer’s disease and other dementia, because senile depression is a risk factor for dementia. PMID:23209830

Phytochemical Analysis and Biological Activities of Cola nitida Bark

PubMed Central

Dah-Nouvlessounon, Durand; Adoukonou-Sagbadja, Hubert; Diarrassouba, Nafan; Sina, Haziz; Adjanohoun, Adolphe; Inoussa, Mariam; Akakpo, Donald; Gbenou, Joachim D.; Kotchoni, Simeon O.; Dicko, Mamoudou H.; Baba-Moussa, Lamine

2015-01-01

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. PMID:25767723

Antifungal Properties of Crude Extracts, Fractions, and Purified Compounds from Bark of Curatella americana L. (Dilleniaceae) against Candida Species

PubMed Central

Mendes de Toledo, Cleyton Eduardo; Santos, Patrícia Regina; Palazzo de Mello, João Carlos; Dias Filho, Benedito Prado; Ueda-Nakamura, Tânia

2015-01-01

The ethnomedicinal plant Curatella americana L. (Dilleniaceae) is a common shrub in the Brazilian cerrado, in which crude extract showed antifungal activity in a preliminary study. In this work, the antifungal and cytotoxic properties of the crude extract, fractions, and isolated compounds from C. americana were evaluated against the standard yeast strains Candida albicans, C. tropicalis, and C. parapsilosis, clinical isolates, and fluconazole-resistant strains. The combinatory effects between subfractions and isolated compounds and effects on cell morphology, virulence factors, and exogenous ergosterol were also evaluated. The MIC obtained against the Candida species including fluconazole-resistant strain ranged from 15.3 to 31.3 µg/mL for crude extract, 3.9 to 15.6 µg/mL for ethyl acetate fraction, and 7.8 to 31.3 µg/mL for subfractions. The isolated compounds identified as 4′-O-methyl-catechin, epicatechin-3-O-gallate, and 4′-O-methyl-catechin-3-O-gallate showed lower antifungal activity than the crude extract and fractions (MIC ranging from 31.3 to 125.0 µg/mL). The addition of exogenous ergosterol to yeast culture did not interfere in the antifungal activity of the extract and its fractions. Synergistic antifungal activity was observed between subfractions and isolated compounds. The effects on virulence factors and the different mechanisms of action compared to fluconazole and nystatin suggest that this ethnomedicinal plant may be an effective alternative treatment for candidiasis. PMID:26347790

Effect of Sildenafil on Pre-Eclampsia-Like Mouse Model Induced By L-Name.

PubMed

Motta, C; Grosso, C; Zanuzzi, C; Molinero, D; Picco, N; Bellingeri, R; Alustiza, F; Barbeito, C; Vivas, A; Romanini, M C

2015-08-01

N(omega)-nitro-L-arginine methyl ester (L-NAME) decreases the vasodilator effect of nitric oxide (NO) and induces pre-eclampsia in mouse. Sildenafil inhibits the degradation of nitric oxide and increases vasodilation. This study aimed to determine the effects of sildenafil citrate on angiogenesis and oxidative stress at the maternal foetal interface on pre-eclampsia-like mouse model induced by L-NAME. Twenty pregnant mice were divided into four groups: (i) vehicle control; (ii) L-NAME; (iii) sildenafil; (4) L-NAME+sildenafil. L-NAME was administered from day 7 of pregnancy and sildenafil from day 8 until day 16; animals were euthanized on day 17. Placental and foetal sizes and weights were measured; lipid peroxide levels and catalase activity in placental homogenates were determined, and placental vascular endothelia were identified by lectin-histochemistry using BSA-I lectin. Western blot analysis was used to determine VEGF expression in placental homogenates. No changes were seen in placental and foetal development in mice with normal pregnancies treated with sildenafil. Treatments with L-NAME reduced significantly the placental weight and average height and decreased the percentage of the endothelial surface. These alterations may be mediated by the reduction of NO levels in trophoblastic cells, due to the inhibitory effect of L-NAME on nitric oxide synthase (NOS) synthesis. This effect was offset by the treatment with sildenafil, with an increase in the percentage of the endothelial surface. In conclusion, our results indicate that treatment with sildenafil on pre-eclampsia mouse model can be used without adverse effects on the concept and its use in the treatment of pre-eclampsia is promising. © 2015 Blackwell Verlag GmbH.

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

PubMed

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.

Anxiolytic-like effect of Sonchus oleraceus L. in mice.

PubMed

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 (P<0.05) and time spent in the open-arm portions of the maze (P<0.05). The extracts induce an anti-thigmotactic effect, evidenced by increased locomotor activity into the central part of the open field set-up (P<0.05). The extracts administered at 30-300 mg/kg, p.o. had a similar anxiolytic effect to clonazepam (0.5 mg/kg, p.o.). These data indicate that Sonchus oleraceus extract exerts an anxiolytic-like effect on mice.

A purified truncated form of yeast Gal4 expressed in Escherichia coli and used to functionalize poly(lactic acid) nanoparticle surface is transcriptionally active in cellulo.

PubMed

Legaz, Sophie; Exposito, Jean-Yves; Borel, Agnès; Candusso, Marie-Pierre; Megy, Simon; Montserret, Roland; Lahaye, Vincent; Terzian, Christophe; Verrier, Bernard

2015-09-01

Gal4/UAS system is a powerful tool for the analysis of numerous biological processes. Gal4 is a large yeast transcription factor that activates genes including UAS sequences in their promoter. Here, we have synthesized a minimal form of Gal4 DNA sequence coding for the binding and dimerization regions, but also part of the transcriptional activation domain. This truncated Gal4 protein was expressed as inclusion bodies in Escherichia coli. A structured and active form of this recombinant protein was purified and used to cover poly(lactic acid) (PLA) nanoparticles. In cellulo, these Gal4-vehicles were able to activate the expression of a Green Fluorescent Protein (GFP) gene under the control of UAS sequences, demonstrating that the decorated Gal4 variant can be delivery into cells where it still retains its transcription factor capacities. Thus, we have produced in E. coli and purified a short active form of Gal4 that retains its functions at the surface of PLA-nanoparticles in cellular assay. These decorated Gal4-nanoparticles will be useful to decipher their tissue distribution and their potential after ingestion or injection in UAS-GFP recombinant animal models. Copyright © 2015 Elsevier Inc. All rights reserved.

Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from Punica granatum L.

PubMed

Reddy, Muntha K; Gupta, Sashi K; Jacob, Melissa R; Khan, Shabana I; Ferreira, Daneel

2007-05-01

The Punica granatum L. (pomegranate) by-product POMx was partitioned between water, EtOAc and n-BuOH, and the EtOAc and n-BuOH extracts were purified by XAD-16 and Sephadex LH-20 column chromatography to afford ellagic acid (1), gallagic acid (2), punicalins (3), and punicalagins (4). Compounds 1 - 4 and the mixture of tannin fractions (XAD-16 eluates) were evaluated for antioxidant, antiplasmodial, and antimicrobial activities in cell-based assays. The mixture of tannins (TPT), XAD-EtOAc, XAD-H2O, XAD-PJ and XAD-BuOH, exhibited IC50 values against reactive oxygen species (ROS) generation at 0.8 - 19 microg/mL. Compounds 1 - 4 showed IC50 values of 1.1, 3.2, 2.3 and 1.4 microM, respectively, against ROS generation and no toxicity up to 31.25 microg/mL against HL-60 cells. Gallagic acid (2) and punicalagins (4) exhibited antiplasmodial activity against Plasmodium falciparum D6 and W2 clones with IC50 values of 10.9, 10.6, 7.5 and 8.8 microM, respectively. Fractions XAD-EtOAc, XAD-BuOH, XAD-H2O and XAD-PJ compounds 1 - 4 revealed antimicrobial activity when assayed against Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Cryptococcus neoformans, methicillin-resistant Staphylococcus aureus (MRSA), Aspergillus fumigatus and Mycobacterium intracellulare. Compounds 2 and 4 showed activity against P. aeruginosa, C. neoformans, and MRSA. This is the first report on the antioxidant, antiplasmodial and antimicrobial activities of POMx isolates, including structure-activity relationships (SAR) of the free radical inhibition activity of compounds 1 - 4. Our results suggest a beneficial effect from the daily intake of POMx and pomegranate juice (PJ) as dietary supplements to augment the human immune system's antioxidant, antimalarial and antimicrobial capacities.

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

PubMed

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

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

PubMed Central

Duan, Yi; Sperber, Anthony M.

2016-01-01

ABSTRACT 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. IMPORTANCE 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

Artificial ribonucleases inactivate a wide range of viruses using their ribonuclease, membranolytic, and chaotropic-like activities.

PubMed

Fedorova, Antonina A; Goncharova, Elena P; Koroleva, Lyudmila S; Burakova, Ekatherina A; Ryabchikova, Elena I; Bichenkova, Elena V; Silnikov, Vladimir N; Vlassov, Valentin V; Zenkova, Marina A

2016-09-01

Artificial ribonucleases (aRNases) are small compounds catalysing RNA cleavage. Recently we demonstrated that aRNases readily inactivate various viruses in vitro. Here, for three series of aRNases (1,4-diazabicyclo [2.2.2]octane-based and peptide-like compounds) we show that apart from ribonuclease activity the aRNases display chaotropic-like and membranolytic activities. The levels of membranolytic and chaotropic-like activities correlate well with the efficiency of various viruses inactivation (enveloped, non-enveloped, RNA-, DNA-containing). We evaluated the impact of these activities on the efficiency of virus inactivation and found: i) the synergism between membranolytic and chaotropic-like activities is sufficient for the inactivation of enveloped viruses (influenza A, encephalitis, vaccinia viruses) for 1,4-diazabicyclo [2.2.2]octane based aRNases, ii) the inactivation of non-enveloped viruses (encephalomyocarditis, acute bee paralysis viruses) is totally dependent on the synergism of chaotropic-like and ribonuclease activities, iii) ribonuclease activity plays a leading role in the inactivation of RNA viruses by aRNases Dp12F6, Dtr12 and K-D-1, iv) peptide-like aRNases (L2-3, K-2) being effective virus killers have a more specific mode of action. Obtained results clearly demonstrate that aRNases represent a new class of broad-spectrum virus-inactivating agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Purifying Water by Imbibition

NASA Technical Reports Server (NTRS)

Lawton, E. A.

1986-01-01

Concept for purifying water uses absorbent material to remove organic substances. Entire bulk of material employed, not just surface. Proposed purification process uses inexpensive equipment and low energy. Material is methyl acrylate polymer. Material cheap and regenerated by rinsing with methanol or by allowing absorbed compounds to evaporate from it.

Method of purifying isosaccharinate

DOEpatents

Rai, Dhanpat; Moore, Robert C.; Tucker, Mark D.

2010-09-07

A method of purifying isosaccharinate by mixing sodium carbonate, potassium carbonate, sodium hydroxide or potassium hydroxide with calcium isosaccharinate, removing the precipitated calcium carbonate and adjusting the pH to between approximately 4.5 to 5.0 thereby removing excess carbonate and hydroxide to provide an acidic solution containing isosaccharinate.

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates.

PubMed

Seidegård, J; DePierre, J W; Guenthner, T M; Oesch, F

1986-09-01

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were

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

PubMed

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.

PubMed

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.

Development of a Novel Human scFv Against EGFR L2 Domain by Phage Display Technology.

PubMed

Rahbarnia, Leila; Farajnia, Safar; Babaei, Hossein; Majidi, Jafar; Veisi, Kamal; Khosroshahi, Shiva Ahdi; Tanomand, Asghar

2017-01-01

Epidermal growth factor receptor (EGFR) as a transmembrane tyrosine kinase receptor frequently overexpresses in tumors with epithelial origin. The L2 domain from extracellular part of EGFR is involved in ligand binding and the blockage of this domain prevents activation of related signaling pathways. This study was aimed to develop a novel human scFv against EGFR L2 domain as a promising target for cancer therapy. The L2 recombinant protein was purified and used for panning a human scFv phage library (Tomlinson I). In this study, a novel screening strategy was applied to select clones with high binding and enrichment of rare specific phage clones of the L2 protein. After five biopanning rounds several specific clones were isolated which among them one phage clone with high binding was purified for further analysis. The specific interaction of selected clone against target antigen was confirmed by ELISA and western blotting. Immunofluorescence staining showed that purified scFv binds to A431 cells surface, displaying EGFR surface receptor. In the present study, we isolated for the first time a novel human scFv against EGFR L2 domain. This study can be the groundwork for developing more effective diagnostic and therapeutic agents against EGFR overexpressing cancers using this novel human anti-L2 ScFv. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Characterization of purified and xerogel immobilized novel lignin peroxidase produced from Trametes versicolor IBL-04 using solid state medium of corncobs.

PubMed

Asgher, Muhammad; Iqbal, Hafiz Muhammad Nasir; Irshad, Muhammad

2012-08-03

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. 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 K(m) and V(max) 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 Cu(2+), Mn(2+) and Fe(2+) whereas, Cystein, EDTA and Ag(+) showed inhibitory effects. The indigenously isolated white rot fungal strain

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

PubMed Central

2012-01-01

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. Conclusions The indigenously

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.

PubMed

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.

A colorimetric aptasensor for sulfadimethoxine detection based on peroxidase-like activity of graphene/nickel@palladium hybrids.

PubMed

Wang, Aicheng; Zhao, Huimin; Chen, Xiaochi; Tan, Bing; Zhang, Yaobin; Quan, Xie

2017-05-15

A sensitive, rapid and label-free colorimetric aptasensor for sulfadimethoxine (SDM) detection was developed based on the tunable peroxidase-like activity of graphene/nickel@palladium nanoparticle (Gr/Ni@Pd) hybrids. The addition of the SDM aptamer could inhibit the peroxidase-like catalytic activity of the hybrids. However, the target SDM and aptamer could be triggered tightly and recover the catalytic activity of the Gr/Ni@Pd hybrids. Due to the peroxidase-like catalytic activity, Gr/Ni@Pd could catalyze the decomposition of H 2 O 2 with releasing hydroxyl radicals which further oxidized reagent 3, 3', 5, 5'-Tetramethylbenzidine (TMB) to oxTMB accompanied with a colorless-to-blue color change. The original color change could be applied to obtain quantitative detection of SDM, due to the relationship between the concentration of the target and the color difference. As a result, this approach performed a linear response for SDM from 1 to 500 ng/mL with a limit detection of 0.7 ng/mL (S/N = 3) under the optimized conditions and realized the detection of SDM in spiked lake water samples. Therefore, this colorimetric aptasensor was an alternative assay for SDM detection in real water. Moreover, with its design principle, this work might be applied to detecting other small molecule by employing appropriate aptamer. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification and structural characterization of a new pro-apoptotic cyclic octapeptide cyclosaplin from somatic seedlings of Santalum album L.

PubMed

Mishra, Abheepsa; Gauri, Samiran S; Mukhopadhyay, Sourav K; Chatterjee, Soumya; Das, Shibendu S; Mandal, Santi M; Dey, Satyahari

2014-04-01

Small cyclic peptides exhibiting potent biological activity have great potential for anticancer therapy. An antiproliferative cyclic octapeptide, cyclosaplin was purified from somatic seedlings of Santalum album L. (sandalwood) using gel filtration and RP-HPLC separation process. The molecular mass of purified peptide was found to be 858 Da and the sequence was determined by MALDI-ToF-PSD-MS as 'RLGDGCTR' (cyclic). The cytotoxic activity of the peptide was tested against human breast cancer (MDA-MB-231) cell line in a dose and time-dependent manner. The purified peptide exhibited significant antiproliferative activity with an IC50 2.06 μg/mL. In a mechanistic approach, apoptosis was observed in differential microscopic studies for peptide treated MDA-MB-231 cells, which was further confirmed by mitochondrial membrane potential, DNA fragmentation assay, cell cycle analysis and caspase 3 activities. The modeling and docking experiments revealed strong affinity (kcal/mol) of peptide toward EGFR and procaspase 3. The co-localization studies revealed that the peptide sensitizes MDA-MB-231 cells by possibly binding to EGFR and induces apoptosis. This unique cyclic octapeptide revealed to be a favorable candidate for development of anticancer agents. Copyright © 2014 Elsevier Inc. All rights reserved.

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

PubMed

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.

A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L

PubMed Central

Thapa, Hem R.; Naik, Mandar T.; Okada, Shigeru; Takada, Kentaro; Molnár, István; Xu, Yuquan; Devarenne, Timothy P.

2016-01-01

The green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C30 squalene. Confirming this hypothesis, the current study identifies C20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encoding a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C15 and C20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. This discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii. PMID:27050299

A squalene synthase-like enzyme initiates production of tetraterpenoid hydrocarbons in Botryococcus braunii Race L

DOE PAGES

Thapa, Hem R.; Naik, Mandar T.; Okada, Shigeru; ...

2016-04-06

Here, the green microalga Botryococcus braunii is considered a promising biofuel feedstock producer due to its prodigious accumulation of hydrocarbon oils that can be converted into fuels. B. braunii Race L produces the C 40 tetraterpenoid hydrocarbon lycopadiene via an uncharacterized biosynthetic pathway. Structural similarities suggest this pathway follows a biosynthetic mechanism analogous to that of C 30 squalene. Confirming this hypothesis, the current study identifies C 20 geranylgeranyl diphosphate (GGPP) as a precursor for lycopaoctaene biosynthesis, the first committed intermediate in the production of lycopadiene. Two squalene synthase (SS)-like complementary DNAs are identified in race L with one encodingmore » a true SS and the other encoding an enzyme with lycopaoctaene synthase (LOS) activity. Interestingly, LOS uses alternative C 15 and C 20 prenyl diphosphate substrates to produce combinatorial hybrid hydrocarbons, but almost exclusively uses GGPP in vivo. In conclusion, this discovery highlights how SS enzyme diversification results in the production of specialized tetraterpenoid oils in race L of B. braunii.« less

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells.

PubMed

Wan, Aini; Xu, Dongsheng; Liu, Kedong; Peng, Lin; Cai, Yanfei; Chen, Yun; He, Yang; Yang, Jianfeng; Jin, Jian; Li, Huazhong

2017-08-09

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500 µg/L) and the half-life of IGF-1 in blood circulation is only 4.5 min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA-IGF-1 reached 100 mg/L. The fusion protein HSA-IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA-IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA-IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA-IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.

Crystal Structure of a Josephin-Ubiquitin Complex: Evolutionary Restraints on Ataxin-3 Deubiquitinating Activity

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

S Weeks; K Grasty; L Hernandez-Cuebas

2011-12-31

The Josephin domain is a conserved cysteine protease domain found in four human deubiquitinating enzymes: ataxin-3, the ataxin-3-like protein (ATXN3L), Josephin-1, and Josephin-2. Josephin domains from these four proteins were purified and assayed for their ability to cleave ubiquitin substrates. Reaction rates differed markedly both among the different proteins and for different substrates with a given protein. The ATXN3L Josephin domain is a significantly more efficient enzyme than the ataxin-3 domain despite their sharing 85% sequence identity. To understand the structural basis of this difference, the 2.6 {angstrom} x-ray crystal structure of the ATXN3L Josephin domain in complex with ubiquitinmore » was determined. Although ataxin-3 and ATXN3L adopt similar folds, they bind ubiquitin in different, overlapping sites. Mutations were made in ataxin-3 at selected positions, introducing the corresponding ATXN3L residue. Only three such mutations are sufficient to increase the catalytic activity of the ataxin-3 domain to levels comparable with that of ATXN3L, suggesting that ataxin-3 has been subject to evolutionary restraints that keep its deubiquitinating activity in check.« less

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

PubMed

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

NASA Astrophysics Data System (ADS)

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).

Characterization and isolation of highly purified porcine satellite cells

PubMed Central

Ding, Shijie; Wang, Fei; Liu, Yan; Li, Sheng; Zhou, Guanghong; Hu, Ping

2017-01-01

Pig is an important food source and an excellent system to model human diseases. Careful characterization of the swine skeletal muscle stem cells (satellite cells) will shed lights on generation of swine skeletal muscle disease model and efficient production of porcine meat for the food industry. Paired box protein 7 (Pax7) is a highly conserved transcription factor shared by satellite cells from various species. However, the sequence of Pax7 has not been characterized in pig. The lack of method to isolate highly purified satellite cells hinders the thorough characterization of the swine satellite cells. Here we found molecular markers for swine satellite cells and revealed that the porcine satellite cells were heterogeneous in various pieces of skeletal muscle. We further developed a method to isolate highly purified satellite cells directly from porcine muscles using fluorescence-activated cell sorting. We next characterized the proliferation and differentiation abilities of isolated satellite cells in vitro; and found that long-term culturing of satellite cells in vitro led to stemness loss. PMID:28417015

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

PubMed

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.

PubMed

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.

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

PubMed Central

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

2015-01-01

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. PMID:26473845

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase.

PubMed

Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; Oliveira, Úrsula Castro de; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

2016-11-24

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

Insights into the Hypertensive Effects of Tityus serrulatus Scorpion Venom: Purification of an Angiotensin-Converting Enzyme-Like Peptidase

PubMed Central

Cajado-Carvalho, Daniela; Kuniyoshi, Alexandre Kazuo; Duzzi, Bruno; Iwai, Leo Kei; de Oliveira, Úrsula Castro; Junqueira de Azevedo, Inácio de Loiola Meirelles; Kodama, Roberto Tadashi; Portaro, Fernanda Vieira

2016-01-01

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension. PMID:27886129

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

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

Lorenzato, Annalisa; Biolatti, Marta; Institute for Cancer Research at Candiolo, Candiolo, Torino

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 cancermore » 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.« less

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

PubMed

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.

Purified isolation of vacuoles from Sedum alfredii leaf-derived protoplasts.

PubMed

Gao, Xiao-Yu; Liao, Xing-Cheng; Wu, Ruo-Lai; Liu, Ting; Wang, Hai-Xing; Lu, Ling-Li

This study aims to develop a method for isolating and purifying protoplasts/vacuoles from fresh leaves of the Cd hyperaccumulator plant species, Sedum alfredii. The results revealed that preheating cellulase and macerozyme at 50 °C for 5 min significantly accelerated the cell wall degradation. For the most optimal conditions for mesophyll protoplast isolation, the mixture of fresh leaves and cell lysates was followed by a 2-h-long vibration. The protoplast lysate for vacuole isolation was diluted, and 0.675 mmol/L was identified as the most appropriate 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS) level, in which S. alfredii large vacuoles are characterized by a high metal and malic acid content. For the best vacuole purification results, we established that 0.8 mol/L was the most optimal mannitol level in the vacuole buffer in terms of vacuole protection during centrifugation, whereas a Ficoll concentration of 0.10 g/ml was adopted in the density-gradient centrifugation.

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

PubMed

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.

Antimycobacterial physalins from Physalis angulata L. (Solanaceae).

PubMed

Januário, A H; Filho, E Rodrigues; Pietro, R C L R; Kashima, S; Sato, D N; França, S C

2002-08-01

Crude extracts and fractions from aerial parts of Physalis angulata have been bioassayed for antimycobacterial activity. Fraction A1-29-12 containing physalins B, F and D exhibited a minimum inhibitory concentration value (MIC) against Mycobacterium tuberculosis H(37)Rv strain of 32 microg/mL. Purified physalin B and physalin D were also tested showing MIC values against Mycobacterium tuberculosis H(37)Rv strain of > 128 microg/mL and 32 microg/mL respectively, suggesting that physalin D plays a relevant role in the antimycobacterial activity displayed. Structural elucidation of both physalins D and B was based on detailed (13)C and (1)H NMR spectral analysis with the aid of 2D-correlation spectroscopy ((1)H-(1)H, COSY, HSQC and HMBC). The assignment of the (13)C chemical shift for physalin D is reported here for the first time. Copyright 2002 John Wiley & Sons, Ltd.

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

PubMed Central

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 < 0.01). Further investigations showed that relative to that of model control, the respective blood lactate (BL) concentrations of low- and medium-dose were 28.52% and 28.81% lower (P < 0.05), whereas the levels of blood urea nitrogen (BUN) were 36.83% and 40.54% lower (P < 0.05), and that of liver glycogen (LG) levels were 12.81% and 17.22% lower (P < 0.05). The results indicated that GABA has an advantage over taurine of anti-fatigue effect. These findings were indicative of the anti-fatigue activity of GABA. PMID:26743028

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

PubMed

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

2016-01-08

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 < 0.01). Further investigations showed that relative to that of model control, the respective blood lactate (BL) concentrations of low- and medium-dose were 28.52% and 28.81% lower (P < 0.05), whereas the levels of blood urea nitrogen (BUN) were 36.83% and 40.54% lower (P < 0.05), and that of liver glycogen (LG) levels were 12.81% and 17.22% lower (P < 0.05). The results indicated that GABA has an advantage over taurine of anti-fatigue effect. These findings were indicative of the anti-fatigue activity of GABA.

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

NASA Astrophysics Data System (ADS)

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 < 0.01). Further investigations showed that relative to that of model control, the respective blood lactate (BL) concentrations of low- and medium-dose were 28.52% and 28.81% lower (P < 0.05), whereas the levels of blood urea nitrogen (BUN) were 36.83% and 40.54% lower (P < 0.05), and that of liver glycogen (LG) levels were 12.81% and 17.22% lower (P < 0.05). The results indicated that GABA has an advantage over taurine of anti-fatigue effect. These findings were indicative of the anti-fatigue activity of GABA.

Immunoreactivity of Biochemically Purified Amandin from Thermally Processed Almonds (Prunus dulcis L.).

PubMed

Zaffran, Valerie D; Sathe, Shridhar K

2018-06-15

Almond seeds were subjected to select thermal processing and amandin was purified from processed and unprocessed (control) seeds using cryoprecipitation. Amandin immunoreactivity was assessed using two murine monoclonal antibodies (mAbs)-4C10 and 4F10 detecting human IgE-relevant conformational and linear epitopes, respectively. Overall amandin immunoreactivity following thermal treatment ranged from 64.9% to 277.8% (4C10) and 81.3% to 270.3% (4F10). Except for autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min), the tested processing conditions resulted in increased immunoreactivity as determined by mAbs 4C10 and 4F10-based enzyme-linked immunosorbent assays (ELISAs). A significant, yet not complete, reduction in immunoreactivity was caused by autoclaving (121 °C, 15 psi, 30 min) and roasting (160 °C, 30 min). Western- and dot-blot immunoassays corroborated the ELISA results, confirming amandin thermal stability. The tested immunoassays indicated amandin to be stable, regardless of the targeted epitope and the processing method that whole almond seeds were subjected to. © 2018 Institute of Food Technologists®.

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

PubMed

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.

Physicochemical and Biological Characterization of Fucoidan from Fucus vesiculosus Purified by Dye Affinity Chromatography

PubMed Central

Zayed, Ahmed; Muffler, Kai; Hahn, Thomas; Rupp, Steffen; Finkelmeier, Doris; Burger-Kentischer, Anke; Ulber, Roland

2016-01-01

A comparative study concerning the physicochemical, monomeric composition and biological characters among different fucoidan fractions is presented. Common purification techniques for fucoidan usually involve many steps. During these steps, the important structural features might be affected and consequently alter its biological activities. Three purified fractions were derived from Fucus vesiculosus water extract which, afterwards, were purified by a recently-developed dye affinity chromatography protocol. This protocol is based on dye-sulfated polysaccharide interactions. The first two fractions were obtained from crude precipitated fucoidan at different pH values of the adsorption phase: pH 1 and 6. This procedure resulted in fucoidan_1 and 6 fractions. The other, third, fraction: fucoidan_M, however, was obtained from a buffered crude extract at pH 1, eliminating the ethanol precipitation step. All of the three fractions were then further evaluated. Results revealed that fucoidan_M showed the highest sulfur content (S%), 12.11%, with the lowest average molecular weight, 48 kDa. Fucose, galactose, and uronic acid/glucose dimers were detected in all fractions, although, xylose was only detected in fucoidan_1 and 6. In a concentration of 10 µg·mL−1, Fucoidan_6 showed the highest heparin-like anticoagulant activity and could prolong the APTT and TT significantly to 66.03 ± 2.93 and 75.36 ± 1.37 s, respectively. In addition, fucoidan_M demonstrated the highest potency against HSV-1 with an IC50 of 2.41 µg·mL−1. The technique proved to be a candidate for fucoidan purifaction from its crude extract removing the precipitation step from common purification protocols and produced different fucoidan qualities resulted from the different incubation conditions with the immobilized thiazine toluidine blue O dye. PMID:27092514

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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.

Phase change liquid purifier and pump

DOEpatents

Steinhour, Leif Alexi

2017-05-23

Systems, methods, and apparatus are provided for purifying and pumping liquids, and more particularly, for purifying and pumping water. The apparatus includes a chamber including a top portion and a bottom portion. A surface configured to be heated is proximate the bottom portion of the chamber. A baffle is disposed within the chamber and above the surface. The baffle is disposed at an angle relative to a vertical direction. The chamber further includes an inlet and a first outlet. The surface heats a liquid in the chamber, causing the liquid to boil. In operation, bubbles rise from the surface and are forced in a horizontal direction by the baffle disposed in the chamber.

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

PubMed

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.

Anti-Plasmodial Polyvalent Interactions in Artemisia annua L. Aqueous Extract – Possible Synergistic and Resistance Mechanisms

PubMed Central

Suberu, John O.; Gorka, Alexander P.; Jacobs, Lauren; Roepe, Paul D.; Sullivan, Neil

2013-01-01

Artemisia annua hot water infusion (tea) has been used in in vitro experiments against P. falciparum malaria parasites to test potency relative to equivalent pure artemisinin. High performance liquid chromatography (HPLC) and mass spectrometric analyses were employed to determine the metabolite profile of tea including the concentrations of artemisinin (47.5±0.8 mg L-1), dihydroartemisinic acid (70.0±0.3 mg L-1), arteannuin B (1.3±0.0 mg L-1), isovitexin (105.0±7.2 mg L-1) and a range of polyphenolic acids. The tea extract, purified compounds from the extract, and the combination of artemisinin with the purified compounds were tested against chloroquine sensitive and chloroquine resistant strains of P. falciparum using the DNA-intercalative SYBR Green I assay. The results of these in vitro tests and of isobologram analyses of combination effects showed mild to strong antagonistic interactions between artemisinin and the compounds (9-epi-artemisinin and artemisitene) extracted from A. annua with significant (IC50 <1 μM) anti-plasmodial activities for the combination range evaluated. Mono-caffeoylquinic acids, tri-caffeoylquinic acid, artemisinic acid and arteannuin B showed additive interaction while rosmarinic acid showed synergistic interaction with artemisinin in the chloroquine sensitive strain at a combination ratio of 1:3 (artemisinin to purified compound). In the chloroquine resistant parasite, using the same ratio, these compounds strongly antagonised artemisinin anti-plasmodial activity with the exception of arteannuin B, which was synergistic. This result would suggest a mechanism targeting parasite resistance defenses for arteannuin B’s potentiation of artemisinin. PMID:24244716

Antioxidant activity and diffusion of catechin and epicatechin from antioxidant active films made of poly(L-lactic acid).

PubMed

Iñiguez-Franco, Fabiola; Soto-Valdez, Herlinda; Peralta, Elizabeth; Ayala-Zavala, J Fernando; Auras, Rafael; Gámez-Meza, Nohemí

2012-07-04

Active membranes and food packaging containing antioxidants like catechin and epicatechin, combined with the use of materials made of biopolymers obtained from renewable sources, could create a novel alternative to reduce oxidation in food, pharmaceutical, and cosmetic products. Poly(94% L-lactic acid) films containing 1.28% catechin and 1.50% epicatechin were extruded in a pilot plant-scale extrusion machine. The diffusion kinetics of catechin and epicatechin into 95% ethanol at 20, 30, 40, and 50 °C and 50% ethanol at 40 °C displayed Fickian release behavior and diffusion coefficients between 0.5 and 50 × 10(-11) cm(2)/s. According to the Arrhenius equation, the energy of activation for the diffusion of catechin and epicatechin in the films was 110.43 and 98.92 kJ/mol, respectively. The antioxidant activity of the films was measured in methanol extracts containing 46.42 μg/mL of catechin and 57.52 μg/mL of epicatechin as 32.90 and 36.68% of scavenging the 2,2-diphenyl-1-picrylhydrazyl radical, respectively.

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

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

Öztekin, Aykut, E-mail: aoztekin@agri.edu.tr; Agri Ibrahim Cecen University Faculty of Arts and Sciences, Department of Chemistry, 04100-Agri; Almaz, Züleyha, E-mail: zturkoglu-2344@hotmail.com

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. (Thismore » research was supported by Ataturk University. Project Number: BAP-2015/98).« less

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

PubMed Central

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

Discovery of small molecule inhibitors of ubiquitin-like poxvirus proteinase I7L using homology modeling and covalent docking approaches

NASA Astrophysics Data System (ADS)

Katritch, Vsevolod; Byrd, Chelsea M.; Tseitin, Vladimir; Dai, Dongcheng; Raush, Eugene; Totrov, Maxim; Abagyan, Ruben; Jordan, Robert; Hruby, Dennis E.

2007-10-01

Essential for viral replication and highly conserved among poxviridae, the vaccinia virus I7L ubiquitin-like proteinase (ULP) is an attractive target for development of smallpox antiviral drugs. At the same time, the I7L proteinase exemplifies several interesting challenges from the rational drug design perspective. In the absence of a published I7L X-ray structure, we have built a detailed 3D model of the I7L ligand binding site (S2-S2' pocket) based on exceptionally high structural conservation of this site in proteases of the ULP family. The accuracy and limitations of this model were assessed through comparative analysis of available X-ray structures of ULPs, as well as energy based conformational modeling. The 3D model of the I7L ligand binding site was used to perform covalent docking and VLS of a comprehensive library of about 230,000 available ketone and aldehyde compounds. Out of 456 predicted ligands, 97 inhibitors of I7L proteinase activity were confirmed in biochemical assays (˜20% overall hit rate). These experimental results both validate our I7L ligand binding model and provide initial leads for rational optimization of poxvirus I7L proteinase inhibitors. Thus, fragments predicted to bind in the prime portion of the active site can be combined with fragments on non-prime side to yield compounds with improved activity and specificity.

Inhibition of purified enolases from oral bacteria by fluoride.

PubMed

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.

PubMed

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.

Murine Coronavirus Ubiquitin-Like Domain Is Important for Papain-Like Protease Stability and Viral Pathogenesis

PubMed Central

Mielech, Anna M.; Deng, Xufang; Chen, Yafang; Kindler, Eveline; Wheeler, Dorthea L.; Mesecar, Andrew D.; Thiel, Volker; Perlman, Stanley

2015-01-01

ABSTRACT Ubiquitin-like domains (Ubls) now are recognized as common elements adjacent to viral and cellular proteases; however, their function is unclear. Structural studies of the papain-like protease (PLP) domains of coronaviruses (CoVs) revealed an adjacent Ubl domain in severe acute respiratory syndrome CoV, Middle East respiratory syndrome CoV, and the murine CoV, mouse hepatitis virus (MHV). Here, we tested the effect of altering the Ubl adjacent to PLP2 of MHV on enzyme activity, viral replication, and pathogenesis. Using deletion and substitution approaches, we identified sites within the Ubl domain, residues 785 to 787 of nonstructural protein 3, which negatively affect protease activity, and valine residues 785 and 787, which negatively affect deubiquitinating activity. Using reverse genetics, we engineered Ubl mutant viruses and found that AM2 (V787S) and AM3 (V785S) viruses replicate efficiently at 37°C but generate smaller plaques than wild-type (WT) virus, and AM2 is defective for replication at higher temperatures. To evaluate the effect of the mutation on protease activity, we purified WT and Ubl mutant PLP2 and found that the proteases exhibit similar specific activities at 25°C. However, the thermal stability of the Ubl mutant PLP2 was significantly reduced at 30°C, thereby reducing the total enzymatic activity. To determine if the destabilizing mutation affects viral pathogenesis, we infected C57BL/6 mice with WT or AM2 virus and found that the mutant virus is highly attenuated, yet it replicates sufficiently to elicit protective immunity. These studies revealed that modulating the Ubl domain adjacent to the PLP reduces protease stability and viral pathogenesis, revealing a novel approach to coronavirus attenuation. IMPORTANCE Introducing mutations into a protein or virus can have either direct or indirect effects on function. We asked if changes in the Ubl domain, a conserved domain adjacent to the coronavirus papain-like protease, altered

Kinase domain-targeted isolation of defense-related receptor-like kinases (RLK/Pelle) in Platanus×acerifolia: phylogenetic and structural analysis.

PubMed

Pilotti, Massimo; Brunetti, Angela; Uva, Paolo; Lumia, Valentina; Tizzani, Lorenza; Gervasi, Fabio; Iacono, Michele; Pindo, Massimo

2014-12-08

Plant receptor-like kinase (RLK/Pelle) family regulates growth and developmental processes and interaction with pathogens and symbionts.Platanaceae is one of the earliest branches of Eudicots temporally located before the split which gave rise to Rosids and Asterids. Thus investigations into the RLK family in Platanus can provide information on the evolution of this gene family in the land plants.Moreover RLKs are good candidates for finding genes that are able to confer resistance to Platanus pathogens. Degenerate oligonucleotide primers targeting the kinase domain of stress-related RLKs were used to isolate for the first time 111 RLK gene fragments in Platanus×acerifolia. Sequences were classified as candidates of the following subfamilies: CrRLK1L, LRR XII, WAK-like, and LRR X-BRI1 group. All the structural features typical of the RLK kinase domain were identified, including the non-RD motif which marks potential pathogen recognition receptors (PRRs). The LRR XII candidates, whose counterpart in Arabidopsis and rice comprises non-RD PRRs, were mostly non-RD kinases, suggesting a group of PRRs. Region-specific signatures of a relaxed purifying selection in the LRR XII candidates were also found, which is novel for plant RLK kinase domain and further supports the role of LRR XII candidates as PRRs. As we obtained CrRLK1L candidates using primers designed on Pto of tomato, we analysed the phylogenetic relationship between CrRLK1L and Pto-like of plant species. We thus classified all non-solanaceous Pto-like genes as CrRLK1L and highlighted for the first time the close phylogenetic vicinity between CrRLK1L and Pto group. The origins of Pto from CrRLK1L is proposed as an evolutionary mechanism. The signatures of relaxed purifying selection highlight that a group of RLKs might have been involved in the expression of phenotypic plasticity and is thus a good candidate for investigations into pathogen resistance.Search of Pto-like genes in Platanus highlighted the close

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

PubMed

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

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

Murata, Tomonori; Yamauchi, Kiyoshi

2008-02-01

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-[{sup 125}I]triiodo-L-thyronine ([{sup 125}I]T{sub 3}) binding to the plasma protein transthyretin (TTR assay) and thyroid hormone receptor (TR assay) and T{sub 3}-dependent luciferase assay (Luc assay). Subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) of the dichloromethane/methanolmore » (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{sub 3}-like activity of {approx} 10{sup -10} M T{sub 3}-equivalent concentration (T{sub 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.« less

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

PubMed

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

The Effects of Different Purifying Methods on the Chemical Properties, in Vitro Anti-Tumor and Immunomodulatory Activities of Abrus cantoniensis Polysaccharide Fractions

PubMed Central

Wu, Shaowei; Fu, Xiong; Brennan, Margaret A.; Brennan, Charles S.; Chun, Chen

2016-01-01

Abrus cantoniensis (Hance) is a popular Chinese vegetable consumed as a beverage, soup or folk medicine. To fully exploit the potential of the polysaccharide in Abrus cantoniensis, nine polysaccharide fractions of Abrus cantoniensis were isolated and purified (AP-AOH30-1, AP-AOH30-2, AP-AOH80-1, AP-AOH80-2, AP-ACl-1, AP-ACl-2, AP-ACl-3, AP-H and AP-L). Fourier-transform infrared spectroscopy (FT-IR) and gas chromatography (GC) were used to characterize these Abrus polysaccharides fractions (APF). In vitro anti-tumor and immunomodulatory activities were also investigated and compared using the rank-sum ratio (RSR) method. Results demonstrated significant differences in the structure and bioactivities among APF, which were associated to the process used for their purification. Among the APF, AP-ACl-3 yield was 613.5 mg/kg of product and consisted of rhamnose (9.8%), arabinose (8.9%), fructose (3.0%), galactose (9.9%), glucose (4.3%), galacturonic acid (3.0%) and glucuronic acid (61.1%) with a molecular weight of 4.4 × 104 Da. Furthermore, AP-ACl-3 exhibited considerable bioactivities significantly preventing the migration of MCF-7 cells and stimulating lymphocyte proliferation along with nitric oxide (NO) production of peritoneal macrophages. AP-ACl-3 could be explored as a novel potential anti-tumor and immunomodulatory agent. PMID:27058538

L1 English/L2 Spanish: Orthography-Phonology Activation without Contrasts

ERIC Educational Resources Information Center

Shea, Christine

2017-01-01

We consider how orthography activates sounds that are in a noncontrastive relationship in the second language (L2) and for which only one variant exists in the first language (L1). Participants were L1 English / L2 Spanish and native Spanish listeners. Intervocalically, Spanish graphemes "b d g" correspond phonetically to stops and…

Tea Derived Galloylated Polyphenols Cross-Link Purified Gastrointestinal Mucins

PubMed Central

Georgiades, Pantelis; Pudney, Paul D. A.; Rogers, Sarah; Thornton, David J.; Waigh, Thomas A.

2014-01-01

Polyphenols derived from tea are thought to be important for human health. We show using a combination of particle tracking microrheology and small-angle neutron scattering that polyphenols acts as cross-linkers for purified gastrointestinal mucin, derived from the stomach and the duodenum. Both naturally derived purified polyphenols, and green and black tea extracts are shown to act as cross-linkers. The main active cross-linking component is found to be the galloylated forms of catechins. The viscosity, elasticity and relaxation time of the mucin solutions experience an order of magnitude change in value upon addition of the polyphenol cross-linkers. Similarly small-angle neutron scattering experiments demonstrate a sol-gel transition with the addition of polyphenols, with a large increase in the scattering at low angles, which is attributed to the formation of large scale (>10 nm) heterogeneities during gelation. Cross-linking of mucins by polyphenols is thus expected to have an impact on the physicochemical environment of both the stomach and duodenum; polyphenols are expected to modulate the barrier properties of mucus, nutrient absorption through mucus and the viscoelastic microenvironments of intestinal bacteria. PMID:25162539

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

PubMed Central

Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.; Rawat, Swati; Solano, Carlos; Kumar, Abhay; Grøtli, Morten; Stemmler, Timothy L.; Rosen, Barry P.

2015-01-01

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeast Saccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)] in vitro and in vivo and that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8 is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation. PMID:26711267

Arsenic Directly Binds to and Activates the Yeast AP-1-Like Transcription Factor Yap8

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

Kumar, Nallani Vijay; Yang, Jianbo; Pillai, Jitesh K.

The AP-1-like transcription factor Yap8 is critical for arsenic tolerance in the yeastSaccharomyces cerevisiae. However, the mechanism by which Yap8 senses the presence of arsenic and activates transcription of detoxification genes is unknown. Here we demonstrate that Yap8 directly binds to trivalent arsenite [As(III)]in vitroandin vivoand that approximately one As(III) molecule is bound per molecule of Yap8. As(III) is coordinated by three sulfur atoms in purified Yap8, and our genetic and biochemical data identify the cysteine residues that form the binding site as Cys132, Cys137, and Cys274. As(III) binding by Yap8 does not require an additional yeast protein, and Yap8more » is regulated neither at the level of localization nor at the level of DNA binding. Instead, our data are consistent with a model in which a DNA-bound form of Yap8 acts directly as an As(III) sensor. Binding of As(III) to Yap8 triggers a conformational change that in turn brings about a transcriptional response. Thus, As(III) binding to Yap8 acts as a molecular switch that converts inactive Yap8 into an active transcriptional regulator. This is the first report to demonstrate how a eukaryotic protein couples arsenic sensing to transcriptional activation.« less

Isolation and purification of an early pregnancy factor-like molecule from culture supernatants obtained from lymphocytes of pregnant women: II. Identification of the molecule as a Fc-receptor-like molecule: a preliminary report.

PubMed

Aranha, C; Bordekar, A; Shahani, S

1998-11-01

Early pregnancy factor (EPF)-like activity from culture supernatants obtained from stimulated lymphocytes of pregnant women was characterized and identified. The enzyme-linked immunosorbent assay depending on the presence of "Fc" receptors on bovine spermatozoa was used to identify the EPF-like molecule purified by gel filtration and reverse-phase high-performance liquid chromatography. The results indicated that the crude lymphocyte culture supernatant, the EPF-positive G IV fraction obtained on gel filtration, and the EPF-positive reverse-phase high-performance liquid chromatography protein readily bound with the different concentrations of aggregated human gamma-globulin in a manner similar to that in which the standard control of aggregated human gamma-globulin binds to the bovine spermatozoa. EPF-like activity synthesized and secreted by lymphocytes during pregnancy may be a Fc-receptor-like molecule.

Structure characteristics of a water-soluble polysaccharide purified from dragon fruit (Hylocereus undatus) pulp.

PubMed

Xu, Lishan; Zhang, Yaojie; Wang, Lizhi

2016-08-01

Dragon fruit is a tropical fruit with good taste. It can bring health benefits to human body. As one of the major bioactive components in this fruit, the polysaccharides might contribute to the health benefits. However, the precise structure information remains unknown. A leading polysaccharide of dragon fruit pulp, DFPP, was purified and identified by NMR and GC-MS. →4-β-d-GlcpA-1→, →6-β-d-Galp-1→ and →4-α-l-Rhap-1→ constituted the backbone and α-l-Araf-1→5-α-l-Araf-1→ formed the branch chain. The precise structure was putatively identified as below. The molecular weight was 2.2×10(3)kDa. The structure information of polysaccharides will be helpful to understand this fruit. Copyright © 2016 Elsevier Ltd. All rights reserved.

Purified frequency modulation of a quantum cascade laser with an all-optical approach.

PubMed

Peng, Chen; Zhou, Haijun; Zhu, Liguo; Chen, Tao; Liu, Qiao; Wang, Detian; Li, Jiang; Peng, Qixian; Chen, Gang; Li, Zeren

2017-11-01

Purified frequency modulation (FM) is demonstrated in a standard middle-infrared quantum cascade laser by illuminating its front facet with two near-infrared (NIR) lasers. A 2 mW laser at 1550 nm is utilized to modulate the amplitude and frequency of a quantum cascade laser, and the associated amplitude modulation (AM) is suppressed by a 1.85 mW laser at 850 nm. Due to the hot carrier effect and the increment of electron temperature, the AM has been decreased. In addition, the free carrier concentration increases in the active region due to the two NIR illuminations, which enhance the FM. Purified FM is beneficial in improving the signal fidelity for free-space optical communication and high-speed FM spectroscopy.

Molecular and immunological characterization of subtilisin like serine protease, a major allergen of Curvularia lunata.

PubMed

Tripathi, Prabhanshu; Nair, Smitha; Singh, B P; Arora, Naveen

2011-03-01

Serine protease from numerous sources have been identified and characterized as major allergens. The present study aimed to clone, express and characterize a serine protease from Curvularia lunata. cDNA library screening identified partial protease clones. A clone showed significant homology to subtilisin like serine proteases from Aspergillus and Penicillium species. Full length sequence was generated by RACE PCR, subcloned in pET vector, protein expressed in Escherichia coli and purified from inclusion bodies yielding 0.5 mg/L of culture. Bioinformatic analysis identified serine protease motifs of subtilase family, catalytic triad and N-glycosylation sites on the primary sequence. The protein resolved at 54-kDa on SDS-PAGE and was recognized as a major allergen on immunoblot with 13/16 C. lunata sensitive patients' sera in ELISA and immunoblot. Recombinant protein reacted with rabbit polyclonal antibodies against alkaline serine proteases from C. lunata. Recombinant protein required 50-56 ng of same protein for 50% inhibition of IgE binding in competitive ELISA. In addition, 13 of 16 patients' samples showed significant basophil histamine release upon stimulation with purified recombinant protein. In conclusion, a 54 kDa major allergen of C. lunata was cloned, expressed, characterized and showed biological activity. It has potential to be used in molecule based approach for allergy diagnosis and therapy. Copyright © 2010 Elsevier GmbH. All rights reserved.

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover.

PubMed

Xu, Zhenshang; He, Huiying; Zhang, Susu; Guo, Tingting; Kong, Jian

2017-01-01

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains ( L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli . The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility.

Characterization of Feruloyl Esterases Produced by the Four Lactobacillus Species: L. amylovorus, L. acidophilus, L. farciminis and L. fermentum, Isolated from Ensiled Corn Stover

PubMed Central

Xu, Zhenshang; He, Huiying; Zhang, Susu; Guo, Tingting; Kong, Jian

2017-01-01

Lactic acid bacteria (LAB) play important roles in silage fermentation, which depends on the production of sufficient organic acids to inhibit the growth of undesirable microorganisms. However, LAB are not able to degrade cellulose and hemicellulose. Bacteria and fibrolytic enzymes are usually used as inoculants to improve the silage quality and digestibility. In the present study, we isolated four Lactobacillus strains (L. amylovorus CGMCC 11056, L. acidophilus CCTCC AB2010208, L. farciminis CCTCC AB2016237 and L. fermentum CCTCC AB2010204) with feruloyl esterase (FAE) activities from ensiled corn stover (CS) by a plate screening assay. The genes encoding FAEs were cloned and hetero-expressed in Escherichia coli. The optimal temperature and pH of these purified enzymes ranged from 45 to 50°C and from 7.0 to 8.0, respectively. They could hydrolyze hydroxycinnamoyl esters in a substrate-specific manner when methyl ferulate, methyl caffeate, methyl ρ-coumarate and methyl sinapinate were used as substrates. Moreover, these four FAEs were able to hydrolyze CS to release hydroxycinnamic acids. Furthermore, these strains could degrade hydroxycinnamic esters, and L. amylovorus CGMCC 11056 was the most efficient strain among these four isolates. These results provided a new target for the development of inoculants to improve silage quality and digestibility. PMID:28626449

Recombinant expression and characterization of L-asparaginase II from a moderately thermotolerant bacterial isolate.

PubMed

Vidya, Jalaja; Pandey, Ashok

2012-07-01

A moderately thermotolerant bacterium belonging to Enterobacteriaceae, which can grow at 44.5 °C, was isolated from cow dung; L-asparaginase II gene was isolated by PCR, cloned, and expressed in pET 20b with pelB leader sequence and 6× Histidine tag at the C-terminal end. The active protein from the soluble sonicated fraction was purified through nickel affinity chromatography. After characterization, the purified protein showed optimum activities at a temperature of 37 °C and in a buffer system of pH 6 to 7. The enzyme exhibited thermostability at 50 °C with a 33% and 28% of activity retention after 45 and 60 min. The kinetic parameters for the enzyme were calculated from Lineweaver-Burk plot, and K(m) and V(max) were 0.89 mM and 0.18 U/mg, respectively.

A Tyrosine Residue Along with a Glutamic Acid of the Omega-Like Loop Governs the Beta-Lactamase Activity of MSMEG_4455 in Mycobacterium smegmatis.

PubMed

Bansal, Ankita; Kar, Debasish; Pandey, Satya Deo; Matcha, Ashok; Kumar, N Ganesh; Nathan, Soshina; Ghosh, Anindya S

2017-06-01

Mycobacterial beta-lactamases are involved in exerting beta-lactam resistance, though many of these proteins remain uncharacterized. Here, we have characterized MSMEG_4455 of Mycobacterium smegmatis as a beta-lactamase using molecular, biochemical and mutational techniques. To elucidate its nature in vivo and in vitro, and to predict its structure-function relationship in silico analysis is done. The MSMEG_4455 is cloned and expressed ectopically in a beta-lactamase deficient Escherichia coli mutant to establish the in vivo beta-lactamase like nature via minimum inhibitory concentration (MIC) determination. Likewise the in vivo results, purified soluble form of MSMEG_4455 showed beta-lactam hydrolysis pattern similar to group 2a penicillinase. In silico analyses of MSMEG_4455 reveal glutamic acid (E)193 and tyrosine (Y)194 of omega-like loop might have importance in strengthening hydrogen bond network around the active-site, though involvement of tyrosine is rare for beta-lactamase activity. Accordingly, these residues are mutated to alanine (A) and phenylalanine (F), respectively. The mutated proteins have partially lost their ability to exert beta-lactamase activity both in vivo and in vitro. The Y194F mutation had more prominent effect on the enzymatic activity. Therefore, we infer that Y194 is the key for beta-lactamase activity of MSMEG_4455.

Evolution of CCL11: genetic characterization in lagomorphs and evidence of positive and purifying selection in mammals.

PubMed

Neves, Fabiana; Abrantes, Joana; Esteves, Pedro J

2016-07-01

The interactions between chemokines and their receptors are crucial for differentiation and activation of inflammatory cells. CC chemokine ligand 11 (CCL11) binds to CCR3 and to CCR5 that in leporids underwent gene conversion with CCR2. Here, we genetically characterized CCL11 in lagomorphs (leporids and pikas). All lagomorphs have a potentially functional CCL11, and the Pygmy rabbit has a mutation in the stop codon that leads to a longer protein. Other mammals also have mutations at the stop codon that result in proteins with different lengths. By employing maximum likelihood methods, we observed that, in mammals, CCL11 exhibits both signatures of purifying and positive selection. Signatures of purifying selection were detected in sites important for receptor binding and activation. Of the three sites detected as under positive selection, two were located close to the stop codon. Our results suggest that CCL11 is functional in all lagomorphs, and that the signatures of purifying and positive selection in mammalian CCL11 probably reflect the protein's biological roles. © The Author(s) 2016.

Assessment of the opsonic activity of purified bovine sIgA following intramammary immunization of cows with Staphylococcus aureus.

PubMed

Barrio, M B; Rainard, P; Gilbert, F B; Poutrel, B

2003-09-01

The phagocytosis of Staphylococcus aureus by bovine polymorphonuclear neutrophils (PMN) requires the presence of antibodies. Among the major isotypes of bovine antibodies, IgG2 and IgM are considered opsonic for bovine PMN. However, the role of purified bovine secretory IgA (sIgA) as an opsonin has not been assessed. In the present study, IgG2 were obtained from serum and sIgA, IgG1, and IgM were purified from the colostrums of three cows intramammarily immunized with heat-killed Staphylococcus aureus. The Ig preparations were assayed for specific antibodies, and the opsonic capacity of every isotype was investigated. Despite the presence of antibodies, we observed no distinct chemiluminescence response of PMN stimulated with sIgA- or IgG1-opsonized S. aureus, whereas IgM or IgG2 bound to bacteria induced a marked chemiluminescence response. Moreover, the counting of internalized bacteria per PMN after phagocytosis revealed a low uptake of S. aureus opsonized with sIgA or IgG1, in contrast to IgM or IgG2, which triggered efficient ingestion of bacteria. Priming of neutrophils by TNF-alpha, IFN-gamma, or C5adesArg did not promote an oxidative burst or uptake of sIgA-opsonized S. aureus to a greater extent than with IgG1-opsonized bacteria. Furthermore, analysis of uningested bacteria by flow cytometry after incubation with PMN showed a preferential uptake of IgM-opsonized S. aureus by PMN and only few sIgA-positive stained bacteria were PMN-associated. These experiments indicate that sIgA, like IgG1 and unlike IgM or IgG2, could not be considered as a major opsonin for phagocytosis of S. aureus by bovine blood PMN.

Purified horse milk exosomes contain an unpredictable small number of major proteins.

PubMed

Sedykh, Sergey E; Purvinish, Lada V; Monogarov, Artem S; Burkova, Evgeniya E; Grigor'eva, Alina E; Bulgakov, Dmitrii V; Dmitrenok, Pavel S; Vlassov, Valentin V; Ryabchikova, Elena I; Nevinsky, Georgy A

2017-06-01

Exosomes are 40-100 nm nanovesicles containing RNA and different proteins. Exosomes containing proteins, lipids, mRNAs, and microRNAs are important in intracellular communication and immune function. Exosomes from different sources are usually obtained by combination of centrifugation and ultracentrifugation and according to published data can contain from a few dozens to thousands of different proteins. Crude exosome preparations from milk of eighteen horses were obtained for the first time using several standard centrifugations. Exosome preparations were additionally purified by FPLC gel filtration. Individual preparations demonstrated different profiles of gel filtration showing well or bad separation of exosome peaks and one or two peaks of co-isolating proteins and their complexes. According to the electron microscopy, well purified exosomes displayed a typical exosome-like size (30-100 nm) and morphology. It was shown that exosomes may have several different biological functions, but detection of their biological functions may vary significantly depending on the presence of exosome contaminating proteins and proteins directly into exosomes. Exosome proteins were identified before and after gel filtration by MALDI MS and MS/MS spectrometry of protein tryptic hydrolyzates derived by SDS PAGE and 2D electrophoresis. The results of protein identification were unexpected: one or two peaks co-isolating proteins after gel-filtration mainly contained kappa-, beta-, alpha-S1-caseins and its precursors, but these proteins were not found in well-purified exosomes. Well-purified exosomes contained from five to eight different major proteins: CD81, CD63 receptors, beta-lactoglobulin and lactadherin were common to all preparations, while actin, butyrophilin, lactoferrin, and xanthine dehydrogenase were found only in some of them. The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of

Deacylation of Purified Lipopolysaccharides by Cellular and Extracellular Components of a Sterile Rabbit Peritoneal Inflammatory Exudate

PubMed Central

Weinrauch, Yvette; Katz, Seth S.; Munford, Robert S.; Elsbach, Peter; Weiss, Jerrold

1999-01-01

The extent to which the mammalian host is capable of enzymatic degradation and detoxification of bacterial lipopolysaccharides (LPS) is still unknown. Partial deacylation of LPS by the enzyme acyloxyacyl hydrolase (AOAH) provides such a mechanism, but its participation in the disposal of LPS under physiological conditions has not been established. In this study, deacylation of isolated radiolabeled LPS by both cellular and extracellular components of a sterile inflammatory peritoneal exudate elicited in rabbits was examined ex vivo. AOAH-like activity, tested under artificial conditions (pH 5.4, 0.1% Triton X-100), was evident in all components of the exudate (mononuclear cells [MNC] > polymorphonuclear leukocytes [PMN] > inflammatory [ascitic] fluid [AF]). Under more physiological conditions, in a defined medium containing purified LPS-binding protein, the LPS-deacylating activity of MNC greatly exceeded that of PMN. In AF, MNC (but not PMN) also produced rapid and extensive CD14-dependent LPS deacylation. Under these conditions, almost all MNC-associated LPS underwent deacylation within 1 h, a rate greatly exceeding that previously found in any cell type. The remaining extracellular LPS was more slowly subject to CD14-independent deacylation in AF. Quantitative analysis showed a comparable release of laurate and myristate but no release of 3-hydroxymyristate, consistent with an AOAH-like activity. These findings suggest a major role for CD14+ MNC and a secondary role for AF in the deacylation of cell-free LPS at extravascular inflammatory sites. PMID:10377115

Analysis of immune responses in genital tracts of mice immunised with purified ribosomal fractions of Neisseria gonorrhoeae.

PubMed Central

Kita, E; Kashiba, S

1984-01-01

Immunisation of ddY mice with the purified ribosomal fraction of Neisseria gonorrhoeae was found to protect against intravaginal challenge with homologous organisms. This protection correlated with the presence of bactericidal antibody to purified ribosomal fraction in serum as well as in vaginal secretions. Analysis of the vaginal fluids from control mice and those immunised with purified ribosomal fraction showed that the enhanced elimination of gonococci in immune mice might be because of an early response of leucocytes generated by the reaction mediated by antibody and complement. Absorption studies showed that there was at least one major protective antigen in purified ribosomal fraction, other than cell surface substances such as lipopolysaccharide, outer membrane proteins, and pili. Bactericidal assays mediated by antibody and complement showed that matched samples of serum and vaginal fluid from immune mice had comparable gonococcidal activity, which was augmented by the effect of progesterone. Although delayed hypersensitivity was produced in immune mice that were resistant to N gonorrhoeae, the exact role of cellular immunity could not be clarified in this study. These results suggest that antibody to purified ribosomal fraction plays a major part in protection against gonococcal infection in the genital tract, and that such protection may entail both cellular immunity and hormonal changes. PMID:6430462

Microheterogeneity in Purified Broad Bean Polyphenol Oxidase

PubMed Central

Ganesa, Chandrashekar; Fox, Mary T.; Flurkey, William H.

1992-01-01

Polyphenoloxidase was purified from chloroplasts of broad bean leaves (Vicia faba L.) to apparent homogeneity. The enzyme was composed of two proteins with an apparent mass of 65 and 68 kilodaltons after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated enzyme contained covalently attached carbohydrates and bound concanavalin A, Phaseolus vulgaris erythroagglutinin, and Ricinus communis agglutinin lectins. Under native isoelectric focusing, several charged isoforms were present in the pH range of 4 to 6. Many, if not all, of the isoforms separated by isoelectric focusing were glycosylated and bound concanavalin A. All these isoforms shared a 65 kilodalton protein in common, and some of the isoforms were associated with both a 65 and 68 kilodalton protein. Isoforms separated by isoelectric focusing in the presence of 9 molar urea followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a similar pattern of proteins within a slightly higher pH range from 5 to 6.5. ImagesFigure 1Figure 2Figure 3Figure 4Figure 5Figure 6 PMID:16668664

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

PubMed

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.

Isolation, characterization, and biological properties of an endotoxin-like material from the gram-positive organism Listeria monocytogenes.

PubMed

Wexler, H; Oppenheim, J D

1979-03-01

The bacterial component responsible for the induction of transient cold agglutinin syndrome in rabbits after intravenous injection of heat-killed Listeria monocytogenes type 4B has been purified and biologically and chemically characterized. A purified immunoglobulin M cold agglutinin was prepared from high-titer sera resulting from the immunization of rabbits with heat-killed L. monocytogenes type 4B and was subsequently used to monitor the purification of the bacterial component responsible for its induction. The bacterial component was isolated from a hot phenol-water extract of lyophilized L. monocytogenes type 4B by multiple molecular sieve chromatography. Upon chemical analysis the purified material was found to be strikingly similar in chemical composition to gram-negative lipopolysaccharide endotoxins. The material contained 15% total fatty acid (of which 50% was beta-hydroxymyristic acid), 40 to 45% neutral sugar (glucose, galactose, and rhamnose), 11.5% amino sugar, 12% uronic acid, 2.5% 2-keto-3-deoxyoctonic acid, 2% heptose, 0.87% phosphorus, and 1.6% amino acid, thereby accounting for 85 to 90% of the weight of the component. Electron micrographs of the purified material were similar to those of lipopolysaccharide preparations from gram-negative organisms. The purified material exist in aqueous solutions as large aggregates, but can be dissociated into a single smaller subunit (3.1S) by dialysis against sodium dodecyl sulfate buffer. The listerial component was toxic and pyrogenic to rabbits, producing symptoms typical of gram-negative endotoxins. Activity in the limulus lysate gelation assay and in the carbocyanine dye assay provides a further link of this material with classical gram-negative endotoxins.

Role of an expansin-like molecule in Dictyostelium morphogenesis and regulation of its gene expression by the signal transducer and activator of transcription protein Dd-STATa.

PubMed

Ogasawara, Shun; Shimada, Nao; Kawata, Takefumi

2009-02-01

Expansins are proteins involved in plant morphogenesis, exerting their effects on cellulose to extend cell walls. Dictyostelium is an organism that possesses expansin-like molecules, but their functions are not known. In this study, we analyzed the expL7 (expansin-like 7) gene, which has been identified as a putative target of Dd-STATa, a Dictyostelium homolog of the metazoan signal transducer and activator of transcription (STAT) proteins. Promoter fragments of the expL7 were fused to a lacZ reporter and the expression patterns determined. As expected from the behavior of the endogenous expL7 gene, the expL7/lacZ fusion gene was downregulated in Dd-STATa null slugs. In the parental strain, the expL7 promoter was activated in the anterior tip region. Mutational analysis of the promoter identified a sequence that was necessary for expression in tip cells. In addition, an activator sequence for pstAB cells was identified. These sequences act in combination with the repressor region to prevent ectopic expL7 expression in the prespore and prestalk regions of the slug and culminant. Although the expL7 null mutant showed no phenotypic change, the expL7 overexpressor showed aberrant stalk formation. These results indicate that the expansin-like molecule is important for morphogenesis in Dictyostelium.

Phytochemical screening and in-vitro antioxidant activity isolated bioactive compounds from Tridax procumbens Linn.

PubMed

Saxena, Manjusha; Mir, Abrar Hussain; Sharma, Manik; Malla, Mohd Yousu; Qureshi, Sumeerah; Mir, Mohd Iqbal; Chaturvedi, Yogesh

2013-12-15

Tridax procumbens L., Asteraceae, has been extensively used for various ailments in the Ayurvedic system of medicine. Previous studies have revealed remarkable phytoconstituents from Tridax procumbens L. with significant antioxidant activity. The aim of the present study is to measure the anti-DPPH activity of the purified isolated compounds from n butanol soluble part and ethyl acetate soluble part of successive methanolic extract of Tridax procumbens L. We thus quantified the total phenolic and total flavonoids in different purified isolated compounds, the whole of the tests were evaluated with a sample cone. of 100 microg mL(-1) and were determined spectrophotometrically using Folin-ciocaltue and AlCl3 reagents, respectively. DPPH (1,1-diphenyl, 2-picryl hydrazyl) assay was used to determine the in vitro antioxidant activity of different isolated compounds. Isolated compounds, one from ethyl acetate soluble part (EF-I) and one from n butanol soluble part (BF-II) were reported to possess a significant anti DPPH activity with lowest IC50 values 67.26 and 80.90 microg mL(-1), respectively while comparable to standard ascorbic acid with IC50 value of 59.62 microg mL(-1), due to the high concentration of phenols 146.4 microg mL(-1) from EF-I and 142.2 microg mL(-1) from BF-II and flavonoids 48 and 42.5 microg mL(-1) found in EF-I and BF-II isolated compounds, respectively.

Unique gene alterations are induced in FACS-purified Fos-positive neurons activated during cue-induced relapse to heroin seeking.

PubMed

Fanous, Sanya; Guez-Barber, Danielle H; Goldart, Evan M; Schrama, Regina; Theberge, Florence R M; Shaham, Yavin; Hope, Bruce T

2013-01-01

Cue-induced heroin seeking after prolonged withdrawal is associated with neuronal activation and altered gene expression in prefrontal cortex (PFC). However, these previous studies assessed gene expression in all neurons regardless of their activity state during heroin seeking. Using Fos as a marker of neural activity, we describe distinct molecular alterations induced in activated versus non-activated neurons during cue-induced heroin seeking after prolonged withdrawal. We trained rats to self-administer heroin for 10 days (6 h/day) and assessed cue-induced heroin seeking in extinction tests after 14 or 30 days. We used fluorescent-activated cell sorting (FACS) to purify Fos-positive and Fos-negative neurons from PFC 90 min after extinction testing. Flow cytometry showed that Fos-immunoreactivity was increased in less than 10% of sparsely distributed PFC neurons. mRNA levels of the immediate early genes fosB, arc, egr1, and egr2, as well as npy and map2k6, were increased in Fos-positive, but not Fos-negative, neurons. In support of these findings, double-label immunohistochemistry indicated substantial coexpression of neuropeptide Y (NPY)- and Arc-immunoreactivity in Fos-positive neurons. Our data indicate that cue-induced relapse to heroin seeking after prolonged withdrawal induces unique molecular alterations within activated PFC neurons that are distinct from those observed in the surrounding majority of non-activated neurons. Published 2012. This article is a US Government work and is in the public domain in the USA.

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

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

Valtieri, M.; Venturelli, D.; Care, A.

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,more » 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.« less

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

PubMed Central

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

2011-01-01

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. PMID:22247887

Antiviral activities of extracts and selected pure constituents of Ocimum basilicum.

PubMed

Chiang, Lien-Chai; Ng, Lean-Teik; Cheng, Pei-Win; Chiang, Win; Lin, Chun-Ching

2005-10-01

1. Ocimum basilicum (OB), also known as sweet basil, is a well known medicinal herb in traditional Chinese medicine preparations. In the present study, extracts and purified components of OB were used to identify possible antiviral activities against DNA viruses (herpes viruses (HSV), adenoviruses (ADV) and hepatitis B virus) and RNA viruses (coxsackievirus B1 (CVB1) and enterovirus 71 (EV71)). 2. The results show that crude aqueous and ethanolic extracts of OB and selected purified components, namely apigenin, linalool and ursolic acid, exhibit a broad spectrum of antiviral activity. Of these compounds, ursolic acid showed the strongest activity against HSV-1 (EC50 = 6.6 mg/L; selectivity index (SI) = 15.2), ADV-8 (EC50 = 4.2 mg/L; SI = 23.8), CVB1 (EC50 = 0.4 mg/L; SI = 251.3) and EV71 (EC50 = 0.5 mg/L; SI = 201), whereas apigenin showed the highest activity against HSV-2 (EC50 = 9.7 mg/L; SI = 6.2), ADV-3 (EC50 = 11.1 mg/L; SI = 5.4), hepatitis B surface antigen (EC50 = 7.1 mg/L; SI = 2.3) and hepatitis B e antigen (EC50 = 12.8 mg/L; SI = 1.3) and linalool showed strongest activity against AVD-II (EC50 = 16.9 mg/L; SI = 10.5). 3. No activity was noted for carvone, cineole, beta-caryophyllene, farnesol, fenchone, geraniol, beta-myrcene and alpha-thujone. 4. The action of ursolic acid against CVB1 and EV71 was found to occur during the infection process and the replication phase. 5. With SI values greater than 200, the potential use of ursolic acid for treating infection with CVB1 and EV71 merits further investigation.

Microfluidic device coupled with a microfabricated oxygen electrode for the measurement of bactericidal activity of neutrophil-like cells.

PubMed

Yamagishi, Anna; Tanabe, Koji; Yokokawa, Masatoshi; Morimoto, Yuji; Kinoshita, Manabu; Suzuki, Hiroaki

2017-09-08

A microfluidic device coupled with a microfabricated Clark-type oxygen electrode was used to measure the bactericidal activity of neutrophil-like cells differentiated from HL-60 cells. The neutrophil-like cells and Escherichia coli (E. coli) cells were cultured in the same medium, which was introduced into the flow channel of the device. Changes in the respiratory activity of E. coli were measured as changes in the consumption of dissolved oxygen. As the activity of the neutrophil-like cells increased, the rate of elimination of E. coli increased. The accompanying decrease in the number of E. coli reduced the consumption of dissolved oxygen. The changes were actually observed as changes in generated current. A distinct difference in changes in dissolved oxygen concentrations was observed between E. coli cells co-incubated with IFN-γ-activated or non-activated neutrophil-like cells. The required sample volume was less than 10 μL, and results could be obtained within 1-2 h. The device may be useful for the assessment of psychological stresses that affect the activity of neutrophils. Copyright © 2017 Elsevier B.V. All rights reserved.

Structural and functional analysis of virus factories purified from Rabbit vesivirus-infected Vero cells.

PubMed

Casais, Rosa; Molleda, Lorenzo González; Machín, Angeles; del Barrio, Gloria; Manso, Alberto García; Dalton, Kevin P; Coto, Ana; Alonso, José Manuel Martín; Prieto, Miguel; Parra, Francisco

2008-10-01

Rabbit vesivirus infection induces membrane modifications and accumulation of vesicular structures in the cytoplasm of infected Vero cells. Crude RaV replication complexes (RCs) have been purified and their structural and functional properties have been characterized. We show that calnexin, an ER-resident protein, RaV non-structural proteins 2AB-, 2C-, 3A-, 3B- and 3CD-like as well as viral RNAs co-localize within membranous structures which are able to replicate the endogenous RNA templates. The purified virus factories protected their viral RNA contents from microccocal nuclease degradation and were inaccessible to exogenously added synthetic transcripts. In addition, we have shown that RCs can be used to investigate uridylylation of native endogenous VPg. In contrast to the observation that the virus factories were inaccessible to RNAs, RCs were accessible to added recombinant VPg which was subsequently nucleotidylylated. Nevertheless no elongation of an RNA chain attached to native or recombinant VPg could be demonstrated.

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

NASA Astrophysics Data System (ADS)

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.

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

PubMed

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

NASA Astrophysics Data System (ADS)

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.

Method for purifying bidentate organophosphorus compounds

DOEpatents

Schulz, Wallace 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.

Regulation of Drosophila transient receptor potential-like (TrpL) channels by phospholipase C-dependent mechanisms.

PubMed

Estacion, M; Sinkins, W G; Schilling, W P

2001-01-01

Patch clamp and fura-2 fluorescence were employed to characterize receptor-mediated activation of recombinant Drosophila TrpL channels expressed in Sf9 insect cells. TrpL was activated by receptor stimulation and by exogenous application of diacylglycerol (DAG) or poly-unsaturated fatty acids (PUFAs). Activation of TrpL was blocked more than 70% by U73122, suggesting that the effect of these agents was dependent upon phospholipase C (PLC). In fura-2 assays, extracellular application of bacterial phosphatidylinositol (PI)-PLC or phosphatidylcholine (PC)-PLC caused a transient increase in TrpL channel activity, the magnitude of which was significantly less than that observed following receptor stimulation. TrpL channels were also activated in excised inside-out patches by cytoplasmic application of mammalian PLC-b2, bacterial PI-PLC and PC-PLC, but not by phospholipase D (PLD). The phospholipases had little or no effect when examined in either whole-cell or cell-attached configurations.TrpL activity was inhibited by addition of phosphatidylinositol-4,5-bisphosphate (PIP2) to excised inside-out membrane patches exhibiting spontaneous channel activity or to patches pre-activated by treatment with PLC. The effect was reversible, specific for PIP2, and was not observed with phosphatidylethanolamine (PE), PI, PC or phosphatidylserine (PS). However, antibodies against PIP2 consistently failed to activate TrpL in inside-out patches. It is concluded that both the hydrolysis of PIP2 and the generation of DAG are required to rapidly activate TrpL following receptor stimulation, or that some other PLC-dependent mechanism plays a crucial role in the activation process.

Purifying Aluminum by Vacuum Distillation

NASA Technical Reports Server (NTRS)

Du Fresne, E. R.

1985-01-01

Proposed method for purifying aluminum employs one-step vacuum distillation. Raw material for process impure aluminum produced in electrolysis of aluminum ore. Impure metal melted in vacuum. Since aluminum has much higher vapor pressure than other constituents, boils off and condenses on nearby cold surfaces in proportions much greater than those of other constituents.

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells

PubMed Central

Alabi, Ola D.; Gunnink, Stephen M.; Kuiper, Benjamin D.; Kerk, Samuel A.; Braun, Emily; Louters, Larry L.

2016-01-01

Aims Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. Main Methods We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Key findings Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 µM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. Significance The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. PMID:24657891

Osthole activates glucose uptake but blocks full activation in L929 fibroblast cells, and inhibits uptake in HCLE cells.

PubMed

Alabi, Ola D; Gunnink, Stephen M; Kuiper, Benjamin D; Kerk, Samuel A; Braun, Emily; Louters, Larry L

2014-05-02

Osthole, a coumarin derivative, has been used in Chinese medicine and studies have suggested a potential use in treatment of diabetes and cancers. Therefore, we investigated the effects of osthole and other coumarins on GLUT1 activity in two cell lines that exclusively express GLUT1. We measured the magnitude and time frame of the effects of osthole and related coumarins on glucose uptake in two cells lines; L929 fibroblast cells which have low GLUT1 expression levels and low basal glucose uptake and HCLE cells which have high GLUT1 concentrations and high basal uptake. We also explored the effects of these coumarins in combination with other GLUT1 activators. Osthole activates glucose uptake in L929 cells with a modest maximum 1.7-fold activation achieved by 50 μM with both activation and recovery occurring within minutes. However, osthole blocks full acute activation of glucose uptake by other, more robust activators. This behavior mimics the effects of other thiol reactive compounds and suggests that osthole is interacting with cysteine residues, possibly within GLUT1 itself. Coumarin, 7-hydroxycoumarin, and 7-methoxycoumarin, do not affect glucose uptake, which is consistent with the notion that the isoprenoid structure in osthole may be important to gain membrane access to GLUT1. In contrast to its effects in L929 cells, osthole inhibits basal glucose uptake in the more active HCLE cells. The differential effects of osthole in L929 and HCLE cells indicated that regulation of GLUT1 varies, likely depending on its membrane concentration. Copyright © 2014 Elsevier Inc. All rights reserved.

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

ERIC Educational Resources Information Center

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…

Antifungal activity of the lipopeptides produced by Bacillus amyloliquefaciens anti-CA against Candida albicans isolated from clinic.

PubMed

Song, Bo; Rong, Yan-Jun; Zhao, Ming-Xin; Chi, Zhen-Ming

2013-08-01

The bacterium Bacillus amyloliquefaciens anti-CA isolated from mangrove system was found to be able to actively kill Candida albicans isolated from clinic. The bacterial strain anti-CA could produce high level of bioactive substance, amylase and protease in the cheap medium containing 2.0 % soybean meal, 2.0 % wheat flour, pH 6.5 within 26 h. After purification, the main bioactive substance was confirmed to be a cyclic lipopeptide containing a heptapeptide, L-Asp→L-Leu→L-Leu→L-Val→L-Val→L-Glu→L-Leu and a 3-OH fatty acid (15 carbons). In addition to C. albicans, the purified lipopeptide can also kill many yeast strains including Metschnikowia bicuspidata, Candida tropicalis, Yarrowia lipolytica and Saccharomyces cerevisiae. After treated by the purified lipopeptide, both the whole cells and protoplasts of C. albicans were destroyed.

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

PubMed

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.

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

PubMed Central

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. PMID:28609478

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

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

Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared

2006-03-15

The Nipah virus fusion (F) protein is proteolytically processed to F{sub 1} + F{sub 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 cathepsinsmore » can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.« less

An efficient process for obtaining prebiotic oligosaccharides derived from lactulose using isomerized and purified whey permeate.

PubMed

Sabater, Carlos; Olano, Agustín; Prodanov, Marin; Montilla, Antonia; Corzo, Nieves

2017-12-01

One of the most promising uses of whey permeate (WP) is the synthesis of prebiotic oligosaccharides. Herein, commercial WP was submitted to chemical isomerization catalysed by sodium borate at an alkaline pH and subsequent purification using anion-exchange resins to remove boron. Subsequently, purified mixtures were used to synthesize prebiotic oligosaccharides using β-galactosidase from Bacillus circulans. Isomerization of concentrated WP (200 g L -1 lactose) gave rise to levels of lactulose up to 155.5 g L -1 after 30 min of reaction (molar ratio of boron/lactose, 1/1; pH 12; 70 °C). Boron was removed from the isomerized WP (IWP) using the combination of a strong acid (IR-120, H + ) and a weak base (IRA-743) anion-exchange resins, reducing its level to <1 ppm, without loss of lactulose. During the transglycosylation reaction of purified IWP (lactose/lactulose ratio, 1/2.4) maximum content of prebiotic compounds was achieved, i.e. 690 g kg -1 WP after 3 h of reaction. This study shows that combined chemical-enzymatic reactions together with the purification of IWP results in an efficient synthesis of prebiotic oligosaccharides. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

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

PubMed

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.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified Aromatic-Turmerone (AR)

PubMed Central

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

2012-01-01

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. PMID:23095866

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

PubMed

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.

The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor*

PubMed Central

2016-01-01

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli. PMID:27875296

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

PubMed

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.

In vitro and in vivo analyses of the Bacillus anthracis spore cortex lytic protein SleL

PubMed Central

Lambert, Emily A.; Sherry, Nora

2012-01-01

The bacterial endospore is the most resilient biological structure known. Multiple protective integument layers shield the spore core and promote spore dehydration and dormancy. Dormancy is broken when a spore germinates and becomes a metabolically active vegetative cell. Germination requires the breakdown of a modified layer of peptidoglycan (PG) known as the spore cortex. This study reports in vitro and in vivo analyses of the Bacillus anthracis SleL protein. SleL is a spore cortex lytic enzyme composed of three conserved domains: two N-terminal LysM domains and a C-terminal glycosyl hydrolase family 18 domain. Derivatives of SleL containing both, one or no LysM domains were purified and characterized. SleL is incapable of digesting intact cortical PG of either decoated spores or purified spore sacculi. However, SleL derivatives can hydrolyse fragmented PG substrates containing muramic-δ-lactam recognition determinants. The muropeptides that result from SleL hydrolysis are the products of N-acetylglucosaminidase activity. These muropeptide products are small and readily released from the cortex matrix. Loss of the LysM domain(s) decreases both PG binding and hydrolysis activity but these domains do not appear to determine specificity for muramic-δ-lactam. When the SleL derivatives are expressed in vivo, those proteins lacking one or both LysM domains do not associate with the spore. Instead, these proteins remain in the mother cell and are apparently degraded. SleL with both LysM domains localizes to the coat or cortex of the endospore. The information revealed by elucidating the role of SleL and its domains in B. anthracis sporulation and germination is important in designing new spore decontamination methods. By exploiting germination-specific lytic enzymes, eradication techniques may be greatly simplified. PMID:22343356

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

PubMed

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.

Functional Reconstitution into Liposomes of Purified Human RhCG Ammonia Channel

PubMed Central

Mouro-Chanteloup, Isabelle; Cochet, Sylvie; Chami, Mohamed; Genetet, Sandrine; Zidi-Yahiaoui, Nedjma; Engel, Andreas; Colin, Yves; Bertrand, Olivier; Ripoche, Pierre

2010-01-01

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 NH3, 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 C12E8 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 NH3 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 PunitNH3 (around 1×10−3 µm3.s−1) is close to the permeability measured in HEK293E cells expressing a recombinant human RhCG (1.60×10−3 µm3.s−1), and in human red

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

PubMed

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.