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Sample records for active soluble form

  1. Solubility Enhancement of a Poorly Water Soluble Drug by Forming Solid Dispersions using Mechanochemical Activation

    PubMed Central

    Rojas-Oviedo, I.; Retchkiman-Corona, B.; Quirino-Barreda, C. T.; Cárdenas, J.; Schabes-Retchkiman, P. S.

    2012-01-01

    Mechanochemical activation is a practical cogrinding operation used to obtain a solid dispersion of a poorly water soluble drug through changes in the solid state molecular aggregation of drug-carrier mixtures and the formation of noncovalent interactions (hydrogen bonds) between two crystalline solids such as a soluble carrier, lactose, and a poorly soluble drug, indomethacin, in order to improve its solubility and dissolution rate. Samples of indomethacin and a physical mixture with a weight ratio of 1:1 of indomethacin and lactose were ground using a high speed vibrating ball mill. Particle size was determined by electron microscopy, the reduction of crystallinity was determined by calorimetry and transmission electron microscopy, infrared spectroscopy was used to find evidence of any interactions between the drug and the carrier and the determination of apparent solubility allowed for the corroboration of changes in solubility. Before grinding, scanning electron microscopy showed the drug and lactose to have an average particle size of around 50 and 30 μm, respectively. After high speed grinding, indomethacin and the mixture had a reduced average particle size of around 5 and 2 μm, respectively, showing a morphological change. The ground mixture produced a solid dispersion that had a loss of crystallinity that reached 81% after 30 min of grinding while the drug solubility of indomethacin within the solid dispersion increased by 2.76 fold as compared to the pure drug. Drug activation due to hydrogen bonds between the carboxylic group of the drug and the hydroxyl group of lactose as well as the decrease in crystallinity of the solid dispersion and the reduction of the particle size led to a better water solubility of indomethacin. PMID:23798775

  2. Activation of human natural killer cells by the soluble form of cellular prion protein

    SciTech Connect

    Seong, Yeon-Jae; Sung, Pil Soo; Jang, Young-Soon; Choi, Young Joon; Park, Bum-Chan; Park, Su-Hyung; Park, Young Woo; Shin, Eui-Cheol

    2015-08-21

    Cellular prion protein (PrP{sup C}) is widely expressed in various cell types, including cells of the immune system. However, the specific roles of PrP{sup C} in the immune system have not been clearly elucidated. In the present study, we investigated the effects of a soluble form of recombinant PrP{sup C} protein on human natural killer (NK) cells. Recombinant soluble PrP{sup C} protein was generated by fusion of human PrP{sup C} with the Fc portion of human IgG{sub 1} (PrP{sup C}-Fc). PrP{sup C}-Fc binds to the surface of human NK cells, particularly to CD56{sup dim} NK cells. PrP{sup C}-Fc induced the production of cytokines and chemokines and the degranulation of granzyme B from NK cells. In addition, PrP{sup C}-Fc facilitated the IL-15-induced proliferation of NK cells. PrP{sup C}-Fc induced phosphorylation of ERK-1/2 and JNK in NK cells, and inhibitors of the ERK or the JNK pathways abrogated PrP{sup C}-Fc-induced cytokine production in NK cells. In conclusion, the soluble form of recombinant PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways. - Highlights: • Recombinant soluble PrP{sup C} (PrP{sup C}-Fc) was generated by fusion of human PrP{sup C} with IgG1 Fc portion. • PrP{sup C}-Fc protein induces the production of cytokines and degranulation from human NK cells. • PrP{sup C}-Fc protein enhances the IL-15-induced proliferation of human NK cells. • PrP{sup C}-Fc protein activates human NK cells via the ERK and JNK signaling pathways.

  3. Scube/You activity mediates release of dually lipid-modified Hedgehog signal in soluble form

    PubMed Central

    Creanga, Adrian; Glenn, Thomas D.; Mann, Randall K.; Saunders, Adam M.; Talbot, William S.; Beachy, Philip A.

    2012-01-01

    Owing to their covalent modification by cholesterol and palmitate, Hedgehog (Hh) signaling proteins are localized predominantly to the plasma membrane of expressing cells. Yet Hh proteins are also capable of mobilizing to and eliciting direct responses from distant cells. The zebrafish you gene, identified genetically >15 years ago, was more recently shown to encode a secreted glycoprotein that acts cell-nonautonomously in the Hh signaling pathway by an unknown mechanism. We investigated the function of the protein encoded by murine Scube2, an ortholog of you, and found that it mediates release in soluble form of the mature, cholesterol- and palmitate-modified Sonic hedgehog protein signal (ShhNp) when added to cultured cells or purified detergent-resistant membrane microdomains containing ShhNp. The efficiency of Scube2-mediated release of ShhNp is enhanced by the palmitate adduct of ShhNp and by coexpression in ShhNp-producing cells of mDispatchedA (mDispA), a transporter-like protein with a previously defined role in the release of lipid-modified Hh signals. The structural determinants of Scube2 required for its activity in cultured cell assays match those required for rescue of you mutant zebrafish embryos, and we thus conclude that the role of Scube/You proteins in Hh signaling in vivo is to facilitate the release and mobilization of Hh proteins for distant action. PMID:22677548

  4. Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

    PubMed

    Rudenko, Natalia N; Ignatova, Lyudmila K; Ivanov, Boris N

    2007-01-01

    Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids. PMID:17347907

  5. Characterization of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of detergent or phospholipid for optimal activity.

    PubMed

    Tschantz, W R; Paetzel, M; Cao, G; Suciu, D; Inouye, M; Dalbey, R E

    1995-03-28

    Leader peptidase is a novel serine protease in Escherichia coli, which functions to cleave leader sequences from exported proteins. Its catalytic domain extends into the periplasmic space and is anchored to the membrane by two transmembrane segments located at the N-terminal end of the protein. At present, there is no information on the structure of the catalytic domain. Here, we report on the properties of a soluble form of leader peptidase (delta 2-75), and we compare its properties to those of the wild-type enzyme. We find that the truncated leader peptidase has a kcat of 3.0 S-1 and a Km of 32 microM with a pro-OmpA nuclease A substrate. In contrast to the wild-type enzyme (pI of 6.8), delta 2-75 is water-soluble and has an acidic isoelectric point of 5.6. We also show with delta 2-75 that the replacement of serine 90 and lysine 145 with alanine residues results in a 500-fold reduction in activity, providing further evidence that leader peptidase employs a catalytic serine/lysine dyad. Finally, we find that the catalysis of delta 2-75 is accelerated by the presence of the detergent Triton X-100, regardless if the substrate is pro-OmpA nuclease A or a peptide substrate. Triton X-100 is required for optimal activity of delta 2-75 at a level far below the critical micelle concentration. Moreover, we find that E. coli phospholipids stimulate the activity of delta 2-75, suggesting that phospholipids may play an important physiological role in the catalytic mechanism of leader peptidase. PMID:7696258

  6. Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.

    PubMed Central

    Ni, L.; Zhou, J.; Hurley, T. D.; Weiner, H.

    1999-01-01

    Human liver cytosolic and mitochondrial isozymes of aldehyde dehydrogenase share 70% sequence identity. However, the first 21 residues are not conserved between the human isozymes (15% identity). The three-dimensional structures of the beef mitochondrial and sheep cytosolic forms have virtually identical three-dimensional structures. Here, we solved the structure of the human mitochondrial enzyme and found it to be identical to the beef enzyme. The first 21 residues are found on the surface of the enzyme and make no contact with other subunits in the tetramer. A pair of chimeric enzymes between the human isozymes was made. Each chimera had the first 21 residues from one isozyme and the remaining 479 from the other. When the first 21 residues were from the mitochondrial isozyme, an enzyme with cytosolic-like properties was produced. The other was expressed but was insoluble. It was possible to restore solubility and activity to the chimera that had the first 21 cytosolic residues fused to the mitochondrial ones by making point mutations to residues at the N-terminal end. When residue 19 was changed from tyrosine to a cysteine, the residue found in the mitochondrial form, an active enzyme could be made though the Km for NAD+ was 35 times higher than the native mitochondrial isozyme and the specific activity was reduced by 75%. This residue interacts with residue 203, a nonconserved, nonactive site residue. A mutation of residue 18, which also interacts with 203, restored solubility, but not activity. Mutation to residue 15, which interacts with 104, also restored solubility but not activity. It appears that to have a soluble or active enzyme a favorable interaction must occur between a residue in a surface loop and a residue elsewhere in the molecule even though neither make contact with the active site region of the enzyme. PMID:10631996

  7. Isolation of a biologically active soluble form of the hemagglutinin-neuraminidase protein of Sendai virus.

    PubMed Central

    Thompson, S D; Laver, W G; Murti, K G; Portner, A

    1988-01-01

    As a first step in establishing the three-dimensional structure of the Sendai virus hemagglutinin-neuraminidase (HN), we have isolated and characterized a potentially crystallizable form of the molecule. The sequence of HN, a surface glycoprotein, predicts a protein with an uncharged hydrophobic region near the amino terminus which is responsible for anchorage in the viral envelope. To avoid rosette formation (aggregation), which would preclude crystallization, this hydrophobic tail was removed from a membrane-free form of HN by proteolytic digestion. This digestion resulted in a single product with a molecular weight of about 10,000 less than native HN. N-terminal amino acid sequence analysis of cleaved HN (C-HN) indicated a single cleavage site at amino acid residue 131, resulting in a product consisting of the carboxyl-terminal 444 amino acids of HN. Functional analyses revealed that C-HN retained full neuraminidase activity and was able to bind erythrocytes, indicating that the N-terminal 131 residues were not necessary for these biological activities. Furthermore, this cleavage product retained the antigenic structure of intact HN, since monoclonal antibodies still bound to C-HN in enzyme-linked immunosorbent assay and Western (immuno-) blot analysis. Viewed by electron microscopy, the dimeric and tetrameric forms of intact HN form rosettes while C-HN maintains the oligomeric structure but no longer aggregates. Furthermore, the electron micrographs revealed a C-HN tetramer strikingly similar to the influenza virus neuraminidase in both size and gross structural features. Images PMID:2846877

  8. Production and characterization of a soluble, active form of Tva, the subgroup A avian sarcoma and leukosis virus receptor.

    PubMed

    Balliet, J W; Berson, J; D'Cruz, C M; Huang, J; Crane, J; Gilbert, J M; Bates, P

    1999-04-01

    The receptor for the subgroup A avian sarcoma and leukosis viruses [ASLV(A)] is the cellular glycoprotein Tva. A soluble form of Tva, sTva, was produced and purified with a baculovirus expression system. Using this system, 7 to 10 mg of purified sTva per liter of cultured Sf9 cells was obtained. Characterization of the carbohydrate modification of sTva revealed that the three N glycosylation sites in sTva were differentially utilized; however, the O glycosylation common to Tva produced in mammalian and avian cells was not observed. Purified sTva demonstrates significant biological activity, specifically blocking infection of avian cells by ASLV(A) with a 90% inhibitory concentration of approximately 25 pM. A quantitative enzyme-linked immunosorbent assay, developed to assess the binding of sTva to ASLV envelope glycoprotein, demonstrates that sTva has a high affinity for EnvA, with an apparent dissociation constant of approximately 0.3 nM. Once they are bound, a very stable complex is formed between EnvA and sTva, with an estimated complex half-life of 6 h. The soluble receptor protein described here represents a valuable tool for analysis of the receptor-envelope glycoprotein interaction and for structural analysis of Tva. PMID:10074155

  9. Active and water-soluble form of lipidated Wnt protein is maintained by a serum glycoprotein afamin/α-albumin.

    PubMed

    Mihara, Emiko; Hirai, Hidenori; Yamamoto, Hideki; Tamura-Kawakami, Keiko; Matano, Mami; Kikuchi, Akira; Sato, Toshiro; Takagi, Junichi

    2016-01-01

    Wnt plays important role during development and in various diseases. Because Wnts are lipidated and highly hydrophobic, they can only be purified in the presence of detergents, limiting their use in various in vitro and in vivo assays. We purified N-terminally tagged recombinant Wnt3a secreted from cells and accidentally discovered that Wnt3a co-purified with a glycoprotein afamin derived from the bovine serum included in the media. Wnt3a forms a 1:1 complex with afamin, which remains soluble in aqueous buffer after isolation, and can induce signaling in various cellular systems including the intestical stem cell growth assay. By co-expressing with afamin, biologically active afamin-Wnt complex can be easily obtained in large quantity. As afamin can also solubilize Wnt5a, Wnt3, and many more Wnt subtypes, afamin complexation will open a way to put various Wnt ligands and their signaling mechanisms under a thorough biochemical scrutiny that had been difficult for years. PMID:26902720

  10. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOEpatents

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  11. How Soluble GARP Enhances TGFβ Activation

    PubMed Central

    Fridrich, Sven; Hahn, Susanne A.; Linzmaier, Marion; Felten, Matthias; Zwarg, Jenny; Lennerz, Volker; Tuettenberg, Andrea; Stöcker, Walter

    2016-01-01

    GARP (glycoprotein A repetitions predominant) is a cell surface receptor on regulatory T-lymphocytes, platelets, hepatic stellate cells and certain cancer cells. Its described function is the binding and accommodation of latent TGFβ (transforming growth factor), before the activation and release of the mature cytokine. For regulatory T cells it was shown that a knockdown of GARP or a treatment with blocking antibodies dramatically decreases their immune suppressive capacity. This confirms a fundamental role of GARP in the basic function of regulatory T cells. Prerequisites postulated for physiological GARP function include membrane anchorage of GARP, disulfide bridges between the propeptide of TGFβ and GARP and connection of this propeptide to αvβ6 or αvβ8 integrins of target cells during mechanical TGFβ release. Other studies indicate the existence of soluble GARP complexes and a functionality of soluble GARP alone. In order to clarify the underlying molecular mechanism, we expressed and purified recombinant TGFβ and a soluble variant of GARP. Surprisingly, soluble GARP and TGFβ formed stable non-covalent complexes in addition to disulfide-coupled complexes, depending on the redox conditions of the microenvironment. We also show that soluble GARP alone and the two variants of complexes mediate different levels of TGFβ activity. TGFβ activation is enhanced by the non-covalent GARP-TGFβ complex already at low (nanomolar) concentrations, at which GARP alone does not show any effect. This supports the idea of soluble GARP acting as immune modulator in vivo. PMID:27054568

  12. Production of aggregation prone human interferon gamma and its mutant in highly soluble and biologically active form by SUMO fusion technology.

    PubMed

    Tileva, M; Krachmarova, E; Ivanov, I; Maskos, K; Nacheva, G

    2016-01-01

    The Escherichia coli expression system is a preferable choice for production of recombinant proteins. A disadvantage of this system is the target protein aggregation in "inclusion bodies" (IBs) that further requires solubilisation and refolding, which is crucial for the properties and the yield of the final product. In order to prevent aggregation, SUMO fusion tag technology has been successfully applied for expression of eukaryotic proteins, including human interferon gamma (hIFNγ) that was reported, however, with no satisfactory biological activity. We modified this methodology for expression and purification of both the wild type hIFNγ and an extremely prone to aggregation mutant hIFNγ-K88Q, whose recovery from IBs showed to be ineffective upon numerous conditions. By expression of the N-terminal His-SUMO fusion proteins in the E. coli strain BL21(DE3)pG-KJE8, co-expressing two chaperone systems, at 24 °C a significant increase in solubility of both target proteins (1.5-fold for hIFNγ and 8-fold for K88Q) was achieved. Two-step chromatography (affinity and ion-exchange) with on-dialysis His-SUMO-tag cleavage was applied for protein purification that yielded 6.0-7.0mg/g wet biomass for both proteins with >95% purity and native N-termini. The optimised protocol led to increased yields from 5.5 times for hIFNγ up to 100 times for K88Q in comparison to their isolation from IBs. Purified hIFNγ showed preserved thermal stability and antiproliferative activity corresponding to that of the native reference sample (3 × 10(7)IU/mg). The developed methodology represents an optimised procedure that can be successfully applied for large scale expression and purification of aggregation-prone proteins in soluble native form. PMID:26407523

  13. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility

    PubMed Central

    Smith, Madison A.; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N.; Johnston, Kathryn A.; Lopez, Karlo M.

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM−1 cm−1. No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  14. Overexpression of Soluble Recombinant Human Lysyl Oxidase by Using Solubility Tags: Effects on Activity and Solubility.

    PubMed

    Smith, Madison A; Gonzalez, Jesica; Hussain, Anjum; Oldfield, Rachel N; Johnston, Kathryn A; Lopez, Karlo M

    2016-01-01

    Lysyl oxidase is an important extracellular matrix enzyme that has not been fully characterized due to its low solubility. In order to circumvent the low solubility of this enzyme, three solubility tags (Nus-A, Thioredoxin (Trx), and Glutathione-S-Transferase (GST)) were engineered on the N-terminus of mature lysyl oxidase. Total enzyme yields were determined to be 1.5 mg for the Nus-A tagged enzyme (0.75 mg/L of media), 7.84 mg for the Trx tagged enzyme (3.92 mg/L of media), and 9.33 mg for the GST tagged enzyme (4.67 mg/L of media). Enzymatic activity was calculated to be 0.11 U/mg for the Nus-A tagged enzyme and 0.032 U/mg for the Trx tagged enzyme, and no enzymatic activity was detected for the GST tagged enzyme. All three solubility-tagged forms of the enzyme incorporated copper; however, the GST tagged enzyme appears to bind adventitious copper with greater affinity than the other two forms. The catalytic cofactor, lysyl tyrosyl quinone (LTQ), was determined to be 92% for the Nus-A and Trx tagged lysyl oxidase using the previously reported extinction coefficient of 15.4 mM(-1 )cm(-1). No LTQ was detected for the GST tagged lysyl oxidase. Given these data, it appears that Nus-A is the most suitable tag for obtaining soluble and active recombinant lysyl oxidase from E. coli culture. PMID:26942005

  15. α1 Soluble Guanylyl Cyclase (sGC) Splice Forms as Potential Regulators of Human sGC Activity*S⃞

    PubMed Central

    Sharina, Iraida G.; Jelen, Filip; Bogatenkova, Elena P.; Thomas, Anthony; Martin, Emil; Murad, Ferid

    2008-01-01

    Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one α- and one β-subunit. The α1/β1 sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new α1 sGC protein variants generated by alternative splicing. The 363 residue N1-α1 sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-α1 sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-α1 sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-α1, N2-α1 sGC mRNA levels together with RT-PCR analysis for C-α1 sGC demonstrated that the expression of the α1 sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-α1 sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the α1/β1 heterodimer. The C-α1 sGC variant heterodimerizes with the β1 subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-α1 band did not change in response to ODQ treatments, while the level of 83 kDa α1 band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues. PMID:18381288

  16. Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

    PubMed

    Schneider, P; Holler, N; Bodmer, J L; Hahne, M; Frei, K; Fontana, A; Tschopp, J

    1998-04-20

    Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high. PMID:9547332

  17. High-performance CaMKI: A highly active and stable form of CaMKIδ produced by high-level soluble expression in Escherichia coli.

    PubMed

    Senga, Yukako; Akizuki, Kazutoshi; Katayama, Syouichi; Shigeri, Yasushi; Kameshita, Isamu; Ishida, Atsuhiko; Sueyoshi, Noriyuki

    2016-07-01

    We describe here the expression and characterization of a constitutively active fragment of zebrafish Ca(2+)/calmodulin-dependent protein kinase (CaMK) Iδ designated zCaMKIδ(1-299) that lacks an autoinhibitory domain. We used a simple one-step purification method to isolate the recombinant enzyme at high yield (220 mg/l of the culture medium) from the soluble fraction of lysates prepared from Escherichia coli. Unlike the corresponding fragment of CaMKIα (CaMKΙα(1-294)), the kinase activity of zCaMKIδ(1-299), without activation procedures, was comparable to that of wild-type zCaMKIδ activated by CaMK kinase. zCaMKIδ(1-299) exhibited broad substrate specificity highly similar to that of wild-type zCaMKIδ, and complementary to that of the cAMP-dependent protein kinase catalytic subunit (PKAc). The protein kinase activity of zCaMKIδ(1-299) was higher compared with that of PKAc as well as CX-30K-CaMKII that comprises a constitutively active fragment of CaMKII fused to the N-terminal region of Xenopus CaMKI. Furthermore, kinase activity was highly stable against thermal inactivation and repeated freezing-thawing. Thus, zCaMKIδ(1-299) represents a readily available alternative that can be used as a "High-performance phosphorylating reagent" alone or in combination with PKAc in diverse experiments on protein phosphorylation and dephosphorylation. PMID:27207832

  18. Multiple forms of soluble monophenol, dihydroxyphenylalanine: oxygen oxidoreductase (EC 1.14.18.1) from potato tubers (Solanum tuberosum). III. Influence of pH on the molecular weight distribution of enzyme activity in potato juice.

    PubMed

    Matheis, G; Belitz, H D

    1979-09-01

    Gel chromatography on Sepharose and on Sephadex was used to separate the soluble phenol oxidase in various potato juices into multiple molecular forms ranging from 36,000 to 800,000 daltons. Adjustment of potato juice from physiological pH (ca. 6) to pH 4.5 or to pH 7.8 resulted in the predominance of low-mol.-wt. (less than 150,000 daltons) or high-mol.-wt. (greater than 150,000 daltons) enzyme forms, respectively. This suggests association phenomena of subunits. In potato juice of physiological pH and in potato juice adjusted to pH 4.5, all enzyme forms exhibited both monophenol and o-diphenol oxidase activities (assayed at pH 6.0). In potato juice adjusted to pH 7.8 considerable loss of monophenol oxidase activity (assayed at pH 6.0) occurred. This suggests that o-diphenol oxidase is more alkali-stable than monophenol oxidase. The significance of these findings for enzyme purifications and for the in vivo action of the enzyme is discussed. PMID:41378

  19. Structure, Solubility and Stability of Orbifloxacin Crystal Forms: Hemihydrate versus Anhydrate.

    PubMed

    Santos, Olimpia Maria Martins; Freitas, Jennifer Tavares Jacon; Cazedey, Edith Cristina Laignier; de Araújo, Magali Benjamim; Doriguetto, Antonio Carlos

    2016-01-01

    Orbifloxacin (ORBI) is a widely used antimicrobial drug of the fluoroquinolone class. In the official pharmaceutical compendia the existence of polymorphism in this active pharmaceutical ingredient (API) is reported. No crystal structure has been reported for this API and as described in the literature, its solubility is very controversial. Considering that different solid forms of the same API may have different physicochemical properties, these different solubilities may have resulted from analyses inadvertently carried out on different polymorphs. The solubility is the most critical property because it can affect the bioavailability and may compromise the quality of a drug product. The crystalline structure of ORBI determined by SCXRD is reported here for the first time. The structural analysis reveals that the ORBI molecule is zwitterionic and hemihydrated. ORBI hemihydrated form was characterized by the following techniques: TG/DTA, FTIR-ATR, and PXRD. A second crystalline ORBI form is also reported: the ORBI anhydrous form was obtained by heating the hemihydrate. These ORBI solid forms were isomorphous, since no significant change in unit cell and space group symmetry were observed. The solid-state phase transformation between these forms is discussed and the equilibrium solubility data were examined in order to check the impact of the differences observed in their crystalline structures. PMID:27005603

  20. Expression of a soluble form of iodotyrosine deiodinase for active site characterization by engineering the native membrane protein from Mus musculus

    SciTech Connect

    Buss, Jennifer M.; McTamney, Patrick M.; Rokita, Steven E.

    2012-06-27

    Reductive deiodination is critical for thyroid function and represents an unusual exception to the more common oxidative and hydrolytic mechanisms of dehalogenation in mammals. Studies on the reductive processes have been limited by a lack of convenient methods for heterologous expression of the appropriate proteins in large scale. The enzyme responsible for iodide salvage in the thyroid, iodotyrosine deodinase, is now readily generated after engineering its gene from Mus musculus. High expression of a truncated derivative lacking the membrane domain at its N-terminal was observed in Sf9 cells, whereas expression in Pichia pastoris remained low despite codon optimization. Ultimately, the desired expression in Escherichia coli was achieved after replacing the two conserved Cys residues of the deiodinase with Ala and fusing the resulting protein to thioredoxin. This final construct provided abundant enzyme for crystallography and mutagenesis. Utility of the E. coli system was demonstrated by examining a set of active site residues critical for binding to the zwitterionic portion of substrate.

  1. Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

    PubMed Central

    Quade-Lyssy, Patricia; Kanarek, Anna Maria; Baiersdörfer, Markus; Postina, Rolf; Kojro, Elzbieta

    2013-01-01

    The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis. PMID:23966666

  2. Functional expression of soluble forms of human CD38 in Escherichia coli and Pichia pastoris.

    PubMed

    Fryxell, K B; O'Donoghue, K; Graeff, R M; Lee, H C; Branton, W D

    1995-06-01

    Cyclic adenosine diphosphate (ADP)-ribose (cADPR), a metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes calcium from intracellular stores in many cells. The synthesis of cADPR from NAD+ and its subsequent hydrolysis to ADPR is catalyzed by an ADP-ribosyl cyclase and a cADPR hydrolase, respectively. The ADP-ribosyl cyclase cloned from the ovotestis of the marine invertebrate Aplysia californica has amino acid sequence homology to the human lymphocyte surface antigen CD38. CD38 has been shown to catalyze both the formation and the hydrolysis of cADPR. In this study, we produced soluble, enzymatically active CD38 using recombinant expression techniques in bacteria and yeast. We engineered a gene coding for a soluble form of CD38 by excision of the region of the gene coding for the N-terminal amino acids representing the putative membrane spanning sequence and short putative intracellular sequence. For expression in bacteria (Escherichia coli), this construct was cloned into the pFlag-1 plasmid which allows induced, periplasmic expression and relatively simple purification of the soluble CD38. For expression in yeast (Pichia pastoris) the CD38 sequence was further modified to eliminate four putative N-linked glycosylation sites and the resulting construct was expressed as a secreted protein. Both systems produce soluble enzymes of approximately 30 kDa and both recombinant enzymes display similar cyclase and hydrolase activities. PMID:7663169

  3. The soluble form of LR11 protein is a regulator of hypoxia-induced, urokinase-type plasminogen activator receptor (uPAR)-mediated adhesion of immature hematological cells.

    PubMed

    Nishii, Keigo; Nakaseko, Chiaki; Jiang, Meizi; Shimizu, Naomi; Takeuchi, Masahiro; Schneider, Wolfgang J; Bujo, Hideaki

    2013-04-26

    A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit(+) Lin(-) cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit(+) Lin(-) cells of lr11(-/-) mice was reduced by hypoxia much more than of lr11(+/+) animals. sLR11 induced the adhesion of U937 and c-Kit(+) Lin(-) cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1α, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1α-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1α binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche. PMID:23486467

  4. Egg Activation at Fertilization by a Soluble Sperm Protein.

    PubMed

    Swann, Karl; Lai, F Anthony

    2016-01-01

    The most fundamental unresolved issue of fertilization is to define how the sperm activates the egg to begin embryo development. Egg activation at fertilization in all species thus far examined is caused by some form of transient increase in the cytoplasmic free Ca(2+) concentration. What has not been clear, however, is precisely how the sperm triggers the large changes in Ca(2+) observed within the egg cytoplasm. Here, we review the studies indicating that the fertilizing sperm stimulates a cytosolic Ca(2+) increase in the egg specifically by delivering a soluble factor that diffuses into the cytosolic space of the egg upon gamete membrane fusion. Evidence is primarily considered in species of eggs where the sperm has been shown to elicit a cytosolic Ca(2+) increase by initiating Ca(2+) release from intracellular Ca(2+) stores. We suggest that our best understanding of these signaling events is in mammals, where the sperm triggers a prolonged series of intracellular Ca(2+) oscillations. The strongest empirical studies to date suggest that mammalian sperm-triggered Ca(2+) oscillations are caused by the introduction of a sperm-specific protein, called phospholipase C-zeta (PLCζ) that generates inositol trisphosphate within the egg. We will discuss the role and mechanism of action of PLCζ in detail at a molecular and cellular level. We will also consider some of the evidence that a soluble sperm protein might be involved in egg activation in nonmammalian species. PMID:26631595

  5. Soluble Proteins Form Film by the Treatment of Low Temperature Plasma

    NASA Astrophysics Data System (ADS)

    Ikehara, Sanae; Sakakita, Hajime; Ishikawa, Kenji; Akimoto, Yoshihiro; Nakanishi, Hayao; Shimizu, Nobuyuki; Hori, Masaru; Ikehara, Yuzuru

    2015-09-01

    It has been pointed out that low temperature plasma in atmosphere was feasible to use for hemostasis without heat injury. Indeed, earlier studies demonstrated that low temperature plasma played an important role to stimulate platelets to aggregate and turned on the proteolytic activities of coagulation factors, resulting in the acceleration of the natural blood coagulation process. On the other hands, our developed equips could immediately form clots upon the contact with plasma flair, while the histological appearance was different from natural coagulation. Based on these findings in formed clots, we sought to determine if plasma flair supplied by our devices was capable of forming film using a series of soluble proteins Following plasma treatment, films were formed from bovine serum albumin, and the other plasma proteins at physiological concentration. Analysis of trans-electron microscope demonstrated that plasma treatment generated small protein particles and made them fuse to be larger aggregations The combined results demonstrated that plasma are capable of aggregating soluble proteins and that platelets and coagulation factors are not necessary for plasma induced blood coagulation. Supported in part by Grants-in-Aid for Scientific Research on Priority Area (21590454, 24590498, and 24108006 to Y. I.).

  6. CCN activation of fumed silica aerosols mixed with soluble pollutants

    NASA Astrophysics Data System (ADS)

    Dalirian, M.; Keskinen, H.; Ahlm, L.; Ylisirniö, A.; Romakkaniemi, S.; Laaksonen, A.; Virtanen, A.; Riipinen, I.

    2014-09-01

    Particle-water interactions of completely soluble or insoluble particles are fairly well understood but less is known of aerosols consisting of mixtures of soluble and insoluble components. In this study, laboratory measurements were performed to investigate cloud condensation nuclei (CCN) activity of silica particles coated with ammonium sulphate (a salt), sucrose (a sugar) and bovine serum albumin known as BSA (a protein). In addition, the agglomerated structure of the silica particles was investigated by estimating the surface equivalent diameter based on measurements with a Differential Mobility Analyzer (DMA) and an Aerosol Particle Mass Analyzer (APM). By using the surface equivalent diameter the non-sphericity of the particles containing silica was accounted for when estimating CCN activation. Furthermore, characterizing critical supersaturations of particles consisting of pure soluble on insoluble compounds using existing frameworks showed that the CCN activation of single component particles was in good agreement with Köhler and adsorption theory based models when the agglomerated structure was accounted for. For mixed particles the CCN activation was governed by the soluble components, and the soluble fraction varied considerably with particle size for our wet-generated aerosols. Our results confirm the hypothesis that knowing the soluble fraction is the key parameter needed for describing the CCN activation of mixed aerosols, and highlight the importance of controlled coating techniques for acquiring a detailed understanding of the CCN activation of atmospheric insoluble particles mixed with soluble pollutants.

  7. CCN activation of fumed silica aerosols mixed with soluble pollutants

    NASA Astrophysics Data System (ADS)

    Dalirian, M.; Keskinen, H.; Ahlm, L.; Ylisirniö, A.; Romakkaniemi, S.; Laaksonen, A.; Virtanen, A.; Riipinen, I.

    2015-04-01

    Particle-water interactions of completely soluble or insoluble particles are fairly well understood but less is known of aerosols consisting of mixtures of soluble and insoluble components. In this study, laboratory measurements were performed to investigate cloud condensation nuclei (CCN) activity of silica particles mixed with ammonium sulfate (a salt), sucrose (a sugar) and bovine serum albumin known as BSA (a protein). The agglomerated structure of the silica particles was investigated using measurements with a differential mobility analyser (DMA) and an aerosol particle mass analyser (APM). Based on these data, the particles were assumed to be compact agglomerates when studying their CCN activation capabilities. Furthermore, the critical supersaturations of particles consisting of pure and mixed soluble and insoluble compounds were explored using existing theoretical frameworks. These results showed that the CCN activation of single-component particles was in good agreement with Köhler- and adsorption theory based models when the agglomerated structure was accounted for. For mixed particles the CCN activation was governed by the soluble components, and the soluble fraction varied considerably with particle size for our wet-generated aerosols. Our results confirm the hypothesis that knowing the soluble fraction is the key parameter needed for describing the CCN activation of mixed aerosols, and highlight the importance of controlled coating techniques for acquiring a detailed understanding of the CCN activation of atmospheric insoluble particles mixed with soluble pollutants.

  8. [Novel biomarker for pathological immature cells--soluble form of LR11].

    PubMed

    Bujo, Hideaki

    2012-05-01

    LR11 (also called SorLA or SORL1), a member of the LDL receptor family, was originally discovered in 1996 from genes specifically expressed in the intimal smooth muscle cells of atherosclerotic plaques. The soluble form of LR11 (sLR11) as well as the membrane-bound form plays a key role in the phenotype conversion of medial smooth muscle cells into intimal smooth muscle cells through the activation of urokinase receptor/integrin-mediated intracellular pathways. The levels of sLR11 in serum or CSF are increased in patients with atherosclerotic diseases, Alzheimer's disease or malignant diseases including acute leukemias. The recently developed ELISA system using two specific antibodies against LR11 made it possible to measure sLR11 quantitatively and stably for many samples. Thus, a novel clinical examination is expected to detect the pathological immature cells important for the pathophysiology of the above diseases. The soluble receptor-based clinical approach, together with basic studies about the structure-function relationship, may shed light on the development of novel target therapy against pathological immature cells in the science fields of so far independently categorized diseases. PMID:22774576

  9. Polymerization process for carboxyl containing polymers utilizing oil soluble ionic surface active agents

    SciTech Connect

    Uebele, C.E.; Ball, L.E.; Jorkasky, R.J. II; Wardlow, E. Jr.

    1987-09-08

    This patent describes a method for polymerizing olefinically unsaturated carboxylic acid monomers containing at least one activated carbon to carbon olefinic double bond and at least one carboxyl group. The monomers are polymerized in an organic media consisting essentially of organic liquids, in the presence of free radical forming catalysts and at least one oil soluble ionic surface active agent selected from the group consisting of: (a) anionic surface active agents; (b) cationic surface active agents; and (c) amphoteric surface active agents.

  10. Soluble complement complex C5b-9 promotes microglia activation.

    PubMed

    Yang, Chao; Yang, Li; Liu, Yong

    2014-02-15

    Soluble C5b-9 has been described as a pro-inflammatory mediator that triggers cell activation rather than inducing cell death. Microglia is the most important immune cell involved in inflammatory response in the CNS. Although microglia activation induced by various stimuli has been well characterized, the role of C5b-9 in microglia has not been well studied. In the current experiment, we utilized assembled functional C5b-9 to treat microglia and analyzed the function. We found that soluble C5b-9 could promote microglia activation by up-regulation of costimulatory molecules and increase cytokine secretion. Our results suggested that soluble C5b-9 possessed immunoregulatory potential on microglia. PMID:24434076

  11. A recipe for designing water-soluble, beta-sheet-forming peptides.

    PubMed Central

    Mayo, K. H.; Ilyina, E.; Park, H.

    1996-01-01

    Based on observations of solubility and folding properties of peptide 33-mers derived from the beta-sheet domains of platelet factor-4 (PF4), interleukin-8 (IL-8), and growth related protein (Gro-alpha), as well as other beta-sheet-forming peptides, general guidelines have been developed to aid in the design of water soluble, self-association-induced beta-sheet-forming peptides. CD, 1H-NMR, and pulsed field gradient NMR self-diffusion measurements have been used to assess the degree of folding and state of aggregation. PF4 peptide forms native-like beta-sheet tetramers and is sparingly soluble above pH 6. IL-8 peptide is insoluble between pH 4.5 and pH 7.5, yet forms stable, native-like beta-sheet dimers at higher pH. Gro-alpha peptide is soluble at all pH values, yet displays no discernable beta-sheet structure even when diffusion data indicate dimer-tetramer aggregation. A recipe used in the de novo design of water-soluble beta-sheet-forming peptides calls for the peptide to contain 40-50% hydrophobic residues, usually aliphatic ones (I, L, V, A, M) (appropriately paired and mostly but not always alternating with polar residues in the sheet sequence), a positively charged (K, R) to negatively charged (E, D) residue ratio between 4/2 and 6/2, and a noncharged polar residue (N, Q, T, S) composition of about 20% or less. Results on four de novo designed, 33-residue peptides are presented supporting this approach. Under near physiologic conditions, all four peptides are soluble, form beta-sheet structures to varying degrees, and self-associate. One peptide folds as a stable, compact beta-sheet tetramer, whereas the others are transient beta-sheet-containing aggregates. PMID:8819163

  12. Cryptic clues as to how water-soluble protein toxins form pores in membranes.

    PubMed

    Parker, Michael W

    2003-07-01

    Pore-forming protein toxins possess the remarkable property that they can exist either in a stable water-soluble state or as an integral membrane pore. In order to convert from the water-soluble to the membrane state, the toxin must undergo large conformational changes. Recent work on a class of pore-forming toxins that are rich in beta-sheet content suggests a common mechanism of membrane insertion may exist despite these toxins possessing very different primary, tertiary and quaternary structures. PMID:12893054

  13. Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form

    PubMed Central

    Pagani, Alessia; Vieillevoye, Maud; Nai, Antonella; Rausa, Marco; Ladli, Meriem; Lacombe, Catherine; Mayeux, Patrick; Verdier, Frédérique; Camaschella, Clara; Silvestri, Laura

    2015-01-01

    Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency. PMID:25637053

  14. Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form.

    PubMed

    Pagani, Alessia; Vieillevoye, Maud; Nai, Antonella; Rausa, Marco; Ladli, Meriem; Lacombe, Catherine; Mayeux, Patrick; Verdier, Frédérique; Camaschella, Clara; Silvestri, Laura

    2015-04-01

    Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency. PMID:25637053

  15. 300-Fold Increase in Production of the Zn2+-Dependent Dechlorinase TrzN in Soluble Form via Apoenzyme Stabilization

    PubMed Central

    Coppin, Christopher W.; Carr, Paul D.; Aleksandrov, Alexey; Wilding, Matthew; Sugrue, Elena; Ubels, Joanna; Paks, Michael; Newman, Janet; Peat, Thomas S.; Russell, Robyn J.; Field, Martin; Weik, Martin; Oakeshott, John G.

    2014-01-01

    Microbial metalloenzymes constitute a large library of biocatalysts, a number of which have already been shown to catalyze the breakdown of toxic chemicals or industrially relevant chemical transformations. However, while there is considerable interest in harnessing these catalysts for biotechnology, for many of the enzymes, their large-scale production in active, soluble form in recombinant systems is a significant barrier to their use. In this work, we demonstrate that as few as three mutations can result in a 300-fold increase in the expression of soluble TrzN, an enzyme from Arthrobacter aurescens with environmental applications that catalyzes the hydrolysis of triazine herbicides, in Escherichia coli. Using a combination of X-ray crystallography, kinetic analysis, and computational simulation, we show that the majority of the improvement in expression is due to stabilization of the apoenzyme rather than the metal ion-bound holoenzyme. This provides a structural and mechanistic explanation for the observation that many compensatory mutations can increase levels of soluble-protein production without increasing the stability of the final, active form of the enzyme. This study provides a molecular understanding of the importance of the stability of metal ion free states to the accumulation of soluble protein and shows that differences between apoenzyme and holoenzyme structures can result in mutations affecting the stability of either state differently. PMID:24771025

  16. A minichaperone-based fusion system for producing insoluble proteins in soluble stable forms.

    PubMed

    Sharapova, Olga A; Yurkova, Maria S; Fedorov, Alexey N

    2016-02-01

    We have developed a fusion system for reliable production of insoluble hydrophobic proteins in soluble stable forms. A carrier is thermophilic minichaperone, GroEL apical domain (GrAD), a 15 kDa monomer able to bind diverse protein substrates. The Met-less variant of GrAD has been made for further convenient use of Met-specific CNBr chemical cleavage, if desired. The Met-less GrAD retained stability and solubility of the original protein. Target polypeptides can be fused to either C-terminus or N-terminus of GrAD. The system has been tested with two unrelated insoluble proteins fused to the C-terminus of GrAD. One of the proteins was also fused to GrAD N-terminus. The fusions formed inclusion bodies at 25°C and above and were partly soluble only at lower expression temperatures. Most importantly, however, after denaturation in urea, all fusions without exception were completely renatured in soluble stable forms that safely survived freezing-thawing as well as lyophilization. All fusions for both tested target proteins retained solubility at high concentrations for days. Functional analysis revealed that a target protein may retain functionality in the fusion. Convenience features include potential thermostability of GrAD fusions, capacity for chemical and enzymatic cleavage of a target and His6 tag for purification. PMID:26612097

  17. Highly active water-soluble olefin metathesis catalyst.

    PubMed

    Hong, Soon Hyeok; Grubbs, Robert H

    2006-03-22

    A novel water-soluble ruthenium olefin metathesis catalyst supported by a poly(ethylene glycol) conjugated saturated 1,3-dimesityl-4,5-dihydroimidazol-2-ylidene ligand is reported. The catalyst displays improved activity in ring-opening metathesis polymerization, ring-closing metathesis, and cross-metathesis reactions in aqueous media. PMID:16536510

  18. [New conjugates of antitumor antibiotic doxorubicin with water-soluble galactomannan: synthesis and biological activity].

    PubMed

    Teviashova, A N; Olsuf'eva, E N; Preobrazhenskaia, M N; Klesov, A A; Zomer, E; Platt, D

    2007-01-01

    New water-soluble conjugates in the form of Schiff bases (DGM-1 and DGM-2) were prepared by the interaction of water-soluble periodate-oxidized galactomannan with doxorubicin or N-(L-lysyl)doxorubicin, respectively. The water-soluble galactomannan (DAVANAT a commercial product of Pro-Pharmaceuticals company) was obtained by partial acidic hydrolysis of high-molecular-mass galactomannan from Cyamopsis tetragonoloba (guar gum) seeds. The conjugate stability was studied in aqueous solutions. The DGM-1 antiproliferative activity was comparable with that of doxorubicin on three models: cell lines of murine melanoma B 16-F1, human breast cancer MCF-7 (HTB-22), and human colon cancer HT-29 (HTB-38). DGM-2 was poorly active in all the three tests. DGM- 1 can thus be regarded as a high-molecular-mass depot form of doxorubicin. PMID:17375669

  19. Bacterial activities driving arsenic speciation and solubility in marine sediments

    NASA Astrophysics Data System (ADS)

    Battaglia-Brunet, F.; Seby, F.; Crouzet, C.; Joulian, C.; Mamindy-Pajany, Y.; Guezennec, A. G.; Hurel, C.; Marmier, N.; Bataillard, P.

    2012-04-01

    Harbour and marina sediments represent particular environments, with high concentrations in organic carbon and pollutants. Over 50 million m3 of marine sediments are dredged every year in French maritime and commercial ports, to maintain the water depth suitable for navigation, and the most part of them is discharged in deeper sea zones. The present study aimed to elucidate, using a range of complementary approaches, the influence of bacterial activity on arsenic speciation and mobility in marina sediments. Two sites were considered: L'Estaque, impacted by metallurgical activities and by the commercial port of Marseille, and St-Mandrier, less polluted, affected by classical chemical pollutants associated to professional and recreational boating. Arsenic concentration was noticeably higher in l'Estaque sediment (200-350 mg/kg) than in St-Mandrier sediment (15-50 mg/kg). In the solid phases, As(III) was the dominant species in L'Estaque sediment, whereas As(V) was the main form in St Mandrier sediment. At both sites, arsenic was the major trace element detected in interstitial water. Free sulfide and thio-arsenic complexes were detected in the interstitial water of l'Estaque sediment, suggesting a role of sulfate-reduction bacterial activity on arsenic solubility. Anaerobic microcosm experiments confirmed this hypothesis, as stimulation of sulfate-reduction induced a dramatic increase of arsenic concentration in the liquid phase, linked to the formation of soluble thio-arsenic complexes. Nevertheless, microcosms performed in aerobic conditions showed that bacterial activity globally decreased the transfer of arsenic from the sediment toward the overlying water. A red-brown fine layer developed at the sediment-water interface. Altogether, these results suggest that the sediment-water interface zone and the close transition area between aerobic and anaerobic conditions host intense biogeochemical reactions involving As, Fe and S species. These reactions most probably

  20. Teratogenicity and metabolism of water-soluble forms of vitamin A in the pregnant rat

    SciTech Connect

    Gunning, D.B.; Barua, A.B.; Olson, J.A. )

    1990-02-26

    Retinoyl {beta}-glucuronide, unlike retinoic acid, has been shown to be non-teratogenic when administered orally, even in large doses, to pregnant rats. The degree to which water-solubility is associated with low teratogenicity is not known. Other water-soluble forms of vitamin A have now been synthesized in our laboratory and are being evaluated for teratogenicity. New water-soluble forms of vitamin A were administered orally to pregnant Sprague-Dawley rats in a single dose of 0.35 mmole/kg bw on day 8 of gestation. On day 19, the dams were sacrificed and the litters were examined. Control animals received either vehicle only or an equivalent dose of all-trans retinoic acid. Maternal and fetal tissues were taken and analyzed by HPLC for vitamin A metabolites. In another experiment, a large single oral dose of the radiolabelled water-soluble compound was administered on day 10. At either 30 minutes or 1 hour after the dose, dams were sacrificed and the embryos analyzed both for radioactivity and for specific metabolites. In contrast to retinoyl {beta}-glucuronide, retinoyl {beta}-glucose is highly teratogenic under identical conditions. Thus, water-solubility does not seem to be the determining factor in the teratogenicity of retinoic acid conjugates.

  1. Multiple forms of soluble monophenol, dihydroxyphenylalanine: oxygen-oxidoreductase (EC 1.14.18.1) from potato tubers (Solanum tuberosum).

    PubMed

    Matheis, G; Belitz, H D

    1975-04-01

    Upon polyacrylamide gel electrophoresis, a soluble phenoloxidase from potatoes (var. Maritta) revealed 17 multiple forms with activity towards dopa and almost all other o-diphenols tested, but only 5 of the forms reacted with monophenols. Isoelectric focusing of the crude enzyme resulted in 2 main peaks with activity towards dopa, having isoelectric points at pH ranges 4.0-4.7 and 5.1-5.4: smaller amounts of the enzyme at higher pI values were also detected. When activity peaks were controlled by polyacrylamide gel electrophoresis, all bands previously detected by electrophoresis of the crude enzyme were recovered, but all peaks were electrophoretically heterogeneous. Gel chromatography of the crude enzyme showed different molecular forms. Their molecular weights indicated monomer, dimer, tetramer, octamer and polymer (at least hexadecamer) forms with a monomer molecular weight of about 36000. PMID:820122

  2. Characterization of the conglomerate form of acetyl-DL-leucine by thermal analysis and solubility measurements

    NASA Astrophysics Data System (ADS)

    Estime, N.; Pena, R.; Teychené, S.; Autret, J. M.; Biscans, B.

    2012-03-01

    Starting from a mixture of enantiomers in solution, crystallization can generate different types of crystals. In order to determine which type of crystal is obtained in the case of acetyl leucine, an active pharmaceutical ingredient (API), analytical methods have been used to partially elucidate the binary and ternary phase diagrams of the system composed of the two enantiomers and water. The melting temperature phase diagram of this compound has been obtained by using differential scanning calorimetry (DSC) analyzes. The results show that it is characteristic of a conglomerate. This mode of crystallization has also been confirmed by X-ray powder diffraction analysis. Solubility measurements of enantiomerical mixtures in water enabled the determination of the ternary diagram of solubility. The empiric Meyerhoffer double solubility rule has been modified, due to the characterization of interactions between enantiomers.

  3. Solubility Prediction of Active Pharmaceutical Compounds with the UNIFAC Model

    NASA Astrophysics Data System (ADS)

    Nouar, Abderrahim; Benmessaoud, Ibtissem; Koutchoukali, Ouahiba; Koutchoukali, Mohamed Salah

    2016-03-01

    The crystallization from solution of an active pharmaceutical ingredient requires the knowledge of the solubility in the entire temperature range investigated during the process. However, during the development of a new active ingredient, these data are missing. Its experimental determination is possible, but tedious. UNIFAC Group contribution method Fredenslund et al. (Vapor-liquid equilibria using UNIFAC: a group contribution method, 1977; AIChE J 21:1086, 1975) can be used to predict this physical property. Several modifications on this model have been proposed since its development in 1977, modified UNIFAC of Dortmund Weidlich et al. (Ind Eng Chem Res 26:1372, 1987), Gmehling et al. (Ind Eng Chem Res 32:178, 1993), Pharma-modified UNIFAC Diedrichs et al. (Evaluation und Erweiterung thermodynamischer Modelle zur Vorhersage von Wirkstofflöslichkeiten, PhD Thesis, 2010), KT-UNIFAC Kang et al. (Ind Eng Chem Res 41:3260, 2002), ldots In this study, we used UNIFAC model by considering the linear temperature dependence of interaction parameters as in Pharma-modified UNIFAC and structural groups as defined by KT-UNIFAC first-order model. More than 100 binary datasets were involved in the estimation of interaction parameters. These new parameters were then used to calculate activity coefficient and solubility of some molecules in various solvents at different temperatures. The model gives better results than those from the original UNIFAC and shows good agreement between the experimental solubility and the calculated one.

  4. Chemical Characterization of Soluble Phosphorus Forms along a Hydrologic Flowpath of a Forested Stream Ecosystem

    SciTech Connect

    Segars, J.E.

    1999-01-01

    The concentration and distribution of soluble phosphorus (P) forms were determined in compartments of a hydrologic pathway in a forested watershed (Walker Branch, Tennessee). Rainfall, throughfall, soil water, groundwater, stream water, and water from two sites in Melton Hill reservoir downstream of Walker Branch were examined for soluble reactive and total soluble phosphorus (SRP and TSP). Soluble unreactive P (SUP) was determined from their difference. An increase of TSP from rainfall to throughfall indicated leaching or wash off of P from the canopy. SRP and SUP decreased markedly as water percolated through the soil, suggesting biological uptake and/or geochemical adsorption of phosphate groups on soil particles. Changes in soluble P. concentrations within the stream channel supported previous evidence for biological control of P dynamics in Walker Branch. Overall, SUP (an estimate of soluble organic P) constituted a significant fraction of the total soluble P present in each compartment of the flowpath. An analytical technique using high-performance liquid chromatography (HPLC) to separate the inositol phosphates (IP's) was developed and used in characterizing organic P fractions of natural systems. Commercial orthophosphate, inositol monophosphate (IMP), and inositol hexaphosphate (IHP) were adequately separated from each other on Aminex A-27 resin using a sodium chloride/tetrasodium EDTA gradient elution. The technique was used to separate an enzyme hydrolysate mixture of IP's into five components. IHP was separated from PO{sub 4} and IMP in a wheat bran extract using the HPLC method. Alkaline bromination was used to extract IP's from a Walker Branch soil sample and HPLC was used to examine the extract; at least three IP peaks were recognized. Using the HPLC technique, an attempt was made to detect the presence of IP's in a Walker Branch groundwater sample concentration by ultrafiltration. The concentration process was unsuccessful possibly due to filtration

  5. Organic soluble and uniform film forming oligoethylene glycol substituted BODIPY small molecules with improved hole mobility.

    PubMed

    Singh, Saumya; Venugopalan, Vijay; Krishnamoorthy, Kothandam

    2014-07-14

    Judiciously chosen side chains of conjugated molecules have a positive impact on charge transport properties when used as the active material in organic electronic devices. Amongst the side chains, oligoethylene glycols (OEGs) have been relatively unexplored due to their hydrophilic nature. OEGs also affect the smooth film formation of conjugated molecules, which preclude device fabrication. However, X-ray diffraction studies have shown that OEGs facilitate intermolecular contact, which is a desirable property for the fabrication of organic electronic devices. Thus the challenge is to design and synthesize organic solvent soluble and uniform film forming conjugated molecules with OEG side chains. We have designed and synthesized conjugated small molecules (CSMs) comprising BODIPY as acceptor and triphenylamine as donor with an OEG side chain. This molecule forms smooth films when processed from organic solvents. In order to understand the impact of the OEG side chain, we have also synthesized alkyl chain analogs. All the molecules exhibit exactly the same HOMO and LUMO energy levels, but the packing in the solid state is different. CSM with methyl side chains exhibit an inter planar distance of 4.15 Å. Contrary to this, the OEG side chain containing CSM showed an inter planar spacing of 4.30 Å, which is 0.2 Å less than the alkyl side chain comprising CSMs. Please note that the length of the hydrophobic and hydrophilic side chains is the same. Interestingly, the OEG side chain comprising CSM showed two orders of higher hole carrier mobilities compared to all the other derivatives. The same molecule also showed an extremely low threshold voltage of -0.27 V indicating the OEG side chains' favourable interaction between substrate as well as between molecules. PMID:24874914

  6. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli

    PubMed Central

    Suryanarayana, Nagendra; Vanlalhmuaka; Mankere, Bharti; Verma, Monika; Thavachelvam, Kulanthaivel; Tuteja, Urmil

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L−1) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. PMID:26966576

  7. Soluble Expression and Characterization of Biologically Active Bacillus anthracis Protective Antigen in Escherichia coli.

    PubMed

    Suryanarayana, Nagendra; Vanlalhmuaka; Mankere, Bharti; Verma, Monika; Thavachelvam, Kulanthaivel; Tuteja, Urmil

    2016-01-01

    Bacillus anthracis secretory protein protective antigen (PA) is primary candidate for subunit vaccine against anthrax. Attempts to obtain large quantity of PA from Escherichia coli expression system often result in the formation of insoluble inclusion bodies. Therefore, it is always better to produce recombinant proteins in a soluble form. In the present study, we have obtained biologically active recombinant PA in small scale E. coli shake culture system using three different expression constructs. The PA gene was cloned in expression vectors bearing trc, T5, and T7 promoters and transformed into their respective E. coli hosts. The growth conditions were optimized to obtain maximum expression of PA in soluble form. The expression construct PA-pET32c in DE3-pLysS E. coli host resulted in a maximum production of soluble PA (15 mg L(-1)) compared to other combinations. Purified PA was subjected to trypsin digestion and binding assay with lethal factor to confirm the protein's functionality. Biological activity was confirmed by cytotoxicity assay on J774.1 cells. Balb/c mice were immunized with PA and the immunogenicity was tested by ELISA and toxin neutralization assay. This study highlights the expression of soluble and biologically active recombinant PA in larger quantity using simpler E. coli production platform. PMID:26966576

  8. Biological activities of water-soluble fullerene derivatives

    NASA Astrophysics Data System (ADS)

    Nakamura, S.; Mashino, T.

    2009-04-01

    Three types of water-soluble fullerene derivatives were synthesized and their biological activities were investigated. C60-dimalonic acid, an anionic fullerene derivative, showed antioxidant activity such as quenching of superoxide and relief from growth inhibition of E. coli by paraquat. C60-bis(7V,7V-dimethylpyrrolidinium iodide), a cationic fullerene derivative, has antibacterial activity and antiproliferative effect on cancer cell lines. The mechanism is suggested to be respiratory chain inhibition by reactive oxygen species produced by the cationic fullerene derivative. Proline-type fullerene derivatives showed strong inhibition activities on HIV-reverse transcriptase. The IC50 values were remarkably lower than nevirapine, a clinically used anti-HIV drug. Fullerene derivatives have a big potential for a new type of lead compound to be used as medicine.

  9. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    PubMed

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

  10. A Soluble Adenylyl Cyclase Form Targets to Axonemes and Rescues Beat Regulation in Soluble Adenylyl Cyclase Knockout Mice

    PubMed Central

    Chen, Xi; Baumlin, Nathalie; Buck, Jochen; Levin, Lonny R.; Fregien, Nevis

    2014-01-01

    Ciliary beating is important for effective mucociliary clearance. Soluble adenylyl cyclase (sAC) regulates ciliary beating, and a roughly 50-kD sAC variant is expressed in axonemes. Normal human bronchial epithelial (NHBE) cells express multiple sAC splice variants: full-length sAC; variants with catalytic domain 1 (C1) deletions; and variants with partial C1. One variant, sACex5v2-ex12v2, contains two alternative splices creating new exons 5 (ex5v2) and 12 (ex12v2), encoding a roughly 45-kD protein. It is therefore similar in size to ciliary sAC. The variant increases in expression upon ciliogenesis during differentiation at the air–liquid interface. When expressed in NHBE cells, this variant was targeted to cilia. Exons 5v2–7 were important for ciliary targeting, whereas exons 2–4 prevented it. In vitro, cytoplasmic sACex2-ex12v2 (containing C1 and C2) was the only variant producing cAMP. Ciliary sACex5v2-ex12v2 was not catalytically active. Airway epithelial cells isolated from wild-type mice revealed sAC-dependent ciliary beat frequency (CBF) regulation, analogous to NHBE cells: CBF rescue from HCO3−/CO2–mediated intracellular acidification was sensitive to the sAC inhibitor, KH7. Compared with wild type, sAC C2 knockout (KO) mice revealed lower CBF baseline, and the HCO3−/CO2–mediated CBF decrease was not inhibited by KH7, confirming lack of functional sAC. Human sACex5v2-ex12v2 was targeted to cilia and sACex2-ex12v2 to the cytoplasm in these KO mice. Introduction of the ciliary sACex5v2-ex12v2 variant, but not the cytoplasmic sACex2-ex12v2, restored functional sAC activity in C2 KO mice. Thus, we show, for the first time, a mammalian axonemal targeting sequence that localizes a sAC variant to cilia to regulate CBF. PMID:24874272

  11. Determination of water-soluble forms of oxalic and formic acids in soils by ion chromatography

    NASA Astrophysics Data System (ADS)

    Karicheva, E.; Guseva, N.; Kambalina, M.

    2016-03-01

    Carboxylic acids (CA) play an important role in the chemical composition origin of soils and migration of elements. The content of these acids and their salts is one of the important characteristics for agrochemical, ecological, ameliorative and hygienic assessment of soils. The aim of the article is to determine water-soluble forms of same carboxylic acids — (oxalic and formic acids) in soils by ion chromatography with gradient elution. For the separation and determination of water-soluble carboxylic acids we used reagent-free gradient elution ion-exchange chromatography ICS-2000 (Dionex, USA), the model solutions of oxalate and formate ions, and leachates from soils of the Kola Peninsula. The optimal gradient program was established for separation and detection of oxalate and formate ions in water solutions by ion chromatography. A stability indicating method was developed for the simultaneous determination of water-soluble organic acids in soils. The method has shown high detection limits such as 0.03 mg/L for oxalate ion and 0.02 mg/L for formate ion. High signal reproducibility was achieved in wide range of intensities which correspond to the following ion concentrations: from 0.04 mg/g to 10 mg/L (formate), from 0.1 mg/g to 25 mg/L (oxalate). The concentration of formate and oxalate ions in soil samples is from 0.04 to 0.9 mg/L and 0.45 to 17 mg/L respectively.

  12. Expression and crystallization of a soluble form of Drosophila fasciclin III.

    PubMed

    Strong, R K; Vaughn, D E; Bjorkman, P J; Snow, P M

    1994-08-19

    A truncated form of Drosophila fasciclin III has been engineered by site-directed mutagenesis. Secreted fasciclin III is expressed at 35 to 40 mg/l in insect cells with baculovirus carrying the recombinant gene. Single crystals of purified soluble fasciclin III have been grown by vapor diffusion versus polyethylene glycol 8000/sodium citrate at low pH. The space group is P6(1)22 or its enantiomorph P6(5)22, with unit cell dimensions a = b = 140 A, c = 260 A. Cryo-preserved crystals diffract to reciprocal lattice spacings beyond 3.0 A. PMID:8064861

  13. Soluble Epoxide Hydrolase Dimerization Is Required for Hydrolase Activity*

    PubMed Central

    Nelson, Jonathan W.; Subrahmanyan, Rishi M.; Summers, Sol A.; Xiao, Xiangshu; Alkayed, Nabil J.

    2013-01-01

    Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity. PMID:23362272

  14. Soluble epoxide hydrolase dimerization is required for hydrolase activity.

    PubMed

    Nelson, Jonathan W; Subrahmanyan, Rishi M; Summers, Sol A; Xiao, Xiangshu; Alkayed, Nabil J

    2013-03-15

    Soluble epoxide hydrolase (sEH) plays a key role in the metabolic conversion of the protective eicosanoid 14,15-epoxyeicosatrienoic acid to 14,15-dihydroxyeicosatrienoic acid. Accordingly, inhibition of sEH hydrolase activity has been shown to be beneficial in multiple models of cardiovascular diseases, thus identifying sEH as a valuable therapeutic target. Recently, a common human polymorphism (R287Q) was identified that reduces sEH hydrolase activity and is localized to the dimerization interface of the protein, suggesting a relationship between sEH dimerization and activity. To directly test the hypothesis that dimerization is essential for the proper function of sEH, we generated mutations within the sEH protein that would either disrupt or stabilize dimerization. We quantified the dimerization state of each mutant using a split firefly luciferase protein fragment-assisted complementation system. The hydrolase activity of each mutant was determined using a fluorescence-based substrate conversion assay. We found that mutations that disrupted dimerization also eliminated hydrolase enzymatic activity. In contrast, a mutation that stabilized dimerization restored hydrolase activity. Finally, we investigated the kinetics of sEH dimerization and found that the human R287Q polymorphism was metastable and capable of swapping dimer partners faster than the WT enzyme. These results indicate that dimerization is required for sEH hydrolase activity. Disrupting sEH dimerization may therefore serve as a novel therapeutic strategy for reducing sEH hydrolase activity. PMID:23362272

  15. "JCE" Classroom Activity #105. A Sticky Situation: Chewing Gum and Solubility

    ERIC Educational Resources Information Center

    Montes-Gonzalez, Ingrid; Cintron-Maldonado, Jose A.; Perez-Medina, Ilia E.; Montes-Berrios, Veronica; Roman-Lopez, Saurie N.

    2010-01-01

    In this Activity, students perform several solubility tests using common food items such as chocolate, chewing gum, water, sugar, and oil. From their observations during the Activity, students will initially classify the substances tested as soluble or insoluble. They will then use their understanding of the chemistry of solubility to classify the…

  16. Water-soluble ruthenium complexes bearing activity against protozoan parasites.

    PubMed

    Sarniguet, Cynthia; Toloza, Jeannette; Cipriani, Micaella; Lapier, Michel; Vieites, Marisol; Toledano-Magaña, Yanis; García-Ramos, Juan Carlos; Ruiz-Azuara, Lena; Moreno, Virtudes; Maya, Juan Diego; Azar, Claudio Olea; Gambino, Dinorah; Otero, Lucía

    2014-06-01

    Parasitic illnesses are major causes of human disease and misery worldwide. Among them, both amebiasis and Chagas disease, caused by the protozoan parasites, Entamoeba histolytica and Trypanosoma cruzi, are responsible for thousands of annual deaths. The lack of safe and effective chemotherapy and/or the appearance of current drug resistance make the development of novel pharmacological tools for their treatment relevant. In this sense, within the framework of the medicinal inorganic chemistry, metal-based drugs appear to be a good alternative to find a pharmacological answer to parasitic diseases. In this work, novel ruthenium complexes [RuCl2(HL)(HPTA)2]Cl2 with HL=bioactive 5-nitrofuryl containing thiosemicarbazones and PTA=1,3,5-triaza-7-phosphaadamantane have been synthesized and fully characterized. PTA was included as co-ligand in order to modulate complexes aqueous solubility. In fact, obtained complexes were water soluble. Their activity against T. cruzi and E. histolytica was evaluated in vitro. [RuCl2(HL4)(HPTA)2]Cl2 complex, with HL4=N-phenyl-5-nitrofuryl-thiosemicarbazone, was the most active compound against both parasites. In particular, it showed an excellent activity against E. histolytica (half maximal inhibitory concentration (IC50)=5.2 μM), even higher than that of the reference drug metronidazole. In addition, this complex turns out to be selective for E. histolytica (selectivity index (SI)>38). The potential mechanism of antiparasitic action of the obtained ruthenium complexes could involve oxidative stress for both parasites. Additionally, complexes could interact with DNA as second potential target by an intercalative-like mode. Obtained results could be considered a contribution in the search for metal compounds that could be active against multiple parasites. PMID:24740394

  17. Nutritive activity of soluble rice brain arabinoxylans in broiler diets.

    PubMed

    Annison, G; Moughan, P J; Thomas, D V

    1995-07-01

    1. A soluble material (703 g/kg non-starch polysaccharide, 141 g/kg starch and 166 g/kg protein) of low viscosity (termed RB-NSP), was isolated in large quantities from defatted Australian rice bran using a mild alkaline extraction and ethanol precipitation. 2. The soluble non-starch polysaccharide fraction of RB-NSP comprised arabinose (0.40 mol%), xylose (0.32 mol%) galactose (0.17 mol%), glucose (0.08 mol%) and mannose (0.03 mol%). 3. RB-NSP was included at graded concentrations (0, 20, 40 and 60 g/kg) in a sorghum/casein basal diet and the diet fed to male broilers in a classical balance trial to determine apparent metabolisable energy (AME). The AME values recorded were 13.26, 13.85, 14.26 and 14.00 MJ/kg DM with a significant correlation (r = 0.65, P < 0.001) between dietary RB-NSP inclusion rate and dietary AME. 4. Feeding RB-NSP had no effect on growth, food conversion ratio or the digestibilities of starch and protein which were both high (0.98-0.99 and 0.88-0.89, respectively). 5. It was concluded that the RB-NSP may have been a substrate for hindgut fermentation in the broiler but that it possessed no anti-nutritive activity. PMID:7583378

  18. Solubility and changes of mercury binding forms in contaminated soils after immobilization treatment

    SciTech Connect

    Biester, H.; Zimmer, H.

    1998-09-15

    Mobility at different pH and binding forms of mercury (Hg) have been investigated in three Hg-contaminated soils after immobilization treatment with alkali-polysulfide (APS) and trimercapto-s-triazine trisodium salt solution (TMT). Changes of solid-phase Hg binding forms after immobilization were determined by Hg pyrolysis. Hg concentrations in the water extracts of all samples increased after treatments due to the formation of soluble mercury sulfides (APS treatment), and the mobilization of humic acid bound Hg at the high pH of the reagents. In contrast, Hg concentrations decreased sharply at low pH due to decomposition of soluble mercury sulfides and precipitation of humic acid-bound Hg. Inorganic Hg compounds such as Hg{sup 0} or HgCl{sub 2} are effectively transformed to mercury sulfides by APS treatment, whereas TMT could transform HgCl{sub 2} but not Hg{sup 0}. Both reagents were found to affect humic acid bound Hg by way of increasing Hg desorption temperatures, although APS was found not to desorb Hg completely from humic acids and TMT-Hg complexes are actually incorporated into humic acids.

  19. Submit and disulfide structure of monomeric and dimeric forms of detergent-soluble HLA antigens.

    PubMed

    Springer, T A; Robb, R J; Terhorst, C; Strominger, J L

    1977-07-10

    The structure of monomeric and disulfide-bonded dimeric forms of HLA antigens has been studied. Detergent-soluble HLA antigen heavy chains contain one or two easily reduced sulfhydryl groups not found in papain-solubilized HLA antigens, as demonstrated by amino acid analysis (Springer, T. A., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2481-2485, and Terhorst, C., Parham, P., Mann, D.L., and Strominger, J.L. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 910-914) and by labeling with iodo[3H]acetate. Dimer formation occurred during purification, since it was prevented by pretreatment of membranes containing HLA antigen with iodoacetamide. Cross-linking studies showed that the non-disulfide-bonded form of HLA antigens contains one subunit each of the Mr = 44,000 heavy chain and the Mr = 12,000 light chain (beta2-microglobulin). PMID:873911

  20. High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

    PubMed

    Gerli, R; Muscat, C; Bistoni, O; Falini, B; Tomassini, C; Agea, E; Tognellini, R; Biagini, P; Bertotto, A

    1995-12-01

    The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA. PMID:8536371

  1. 77 FR 20987 - Oral Dosage Form New Animal Drugs; Change of Sponsor; Lincomycin Hydrochloride Soluble Powder...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-04-09

    ... Sponsor; Lincomycin Hydrochloride Soluble Powder; Penicillin G Potassium in Drinking Water; Tetracycline Powder AGENCY: Food and Drug Administration, HHS. ACTION: Final rule; technical amendment. SUMMARY: The...; penicillin G potassium, USP; and tetracycline hydrochloride soluble powders administered in drinking...

  2. Antioxidant Activity of Water-soluble Polysaccharides from Brasenia schreberi

    PubMed Central

    Xiao, Huiwen; Cai, Xueru; Fan, Yijun; Luo, Aoxue

    2016-01-01

    Objective: In order to investigate the antioxidant activities of polysaccharides (BPL-1 and BPL-2), one of the most important functional constituents in Brasenia schreberi was isolated from the external mucilage of B. schreberi (BPL-1) and the plant in vivo (BPL-2). This paper examines the relationship between the content of sulfuric radicals and uronic acid in BPL and the antioxidant activity of BPL. Materials and Methods: The free radicals, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS+) and 1,1-diphnyl-2-picrylhydrazyl (DPPH-), were used to determine the antioxidant activity of BPL. The Fourier-transform infrared spectroscopy of BPL-1 and BPL-2 revealed typical characteristics of polysaccharides. Results: The two sample types had different contents. This was proved by their different adsorption peak intensities. The IC50 values of BPL-1 (31.189 mg/ml) and BPL-2 (1.863 mg/ml) showed significant DPPH radical scavenging activity. Based on the quantification of ABTS radical scavenging, the IC50 value of BPL-1 (5.460 mg/ml) was higher than that of BPL-2 (0.239 mg/ml). Therefore, in terms of the reducing power, the IC50 value of BPL-1 was too high to determine, and the IC50 value of BPL-2 was found to be 50.557 mg/ml. Hence, the antioxidant activity and total reducing power were high, and they were greater in BPL-2 than in BPL-1. In addition, BPL-2 was found to have more sulfuric radicals and uronic acid than BPL-1. Conclusion: The contents of sulfuric radicals and uronic acid are significantly correlated to the antioxidant activity and reducing power of BPL; the more sulfuric radicals and uronic acid, the more antioxidant activity and reducing power BPL has. SUMMARY The water-soluble crude polysaccharides obtained from the external mucilage and the Brasenia schreberi plant in vivo were confirmed to have high contents of sulfuric radicals and uronic acidBoth BPL-1 and BPL-2 exhibited antioxidative activity and reducing power, and their antioxidative

  3. Calcium-dependent Dimerization of Human Soluble Calcium Activated Nucleotidase: Characterization of the Dimer Interface

    SciTech Connect

    Yang,M.; Horii, K.; Herr, A.; Kirley, T.

    2006-01-01

    Mammals express a protein homologous to soluble nucleotidases used by blood-sucking insects to inhibit host blood clotting. These vertebrate nucleotidases may play a role in protein glycosylation. The activity of this enzyme family is strictly dependent on calcium, which induces a conformational change in the secreted, soluble human nucleotidase. The crystal structure of this human enzyme was recently solved; however, the mechanism of calcium activation and the basis for the calcium-induced changes remain unclear. In this study, using analytical ultracentrifugation and chemical cross-linking, we show that calcium or strontium induce noncovalent dimerization of the soluble human enzyme. The location and nature of the dimer interface was elucidated using a combination of site-directed mutagenesis and chemical cross-linking, coupled with crystallographic analyses. Replacement of Ile{sup 170}, Ser{sup 172}, and Ser{sup 226} with cysteine residues resulted in calcium-dependent, sulfhydryl-specific intermolecular cross-linking, which was not observed after cysteine introduction at other surface locations. Analysis of a super-active mutant, E130Y, revealed that this mutant dimerized more readily than the wild-type enzyme. The crystal structure of the E130Y mutant revealed that the mutated residue is found in the dimer interface. In addition, expression of the full-length nucleotidase revealed that this membrane-bound form can also dimerize and that these dimers are stabilized by spontaneous oxidative cross-linking of Cys{sup 30}, located between the single transmembrane helix and the start of the soluble sequence. Thus, calcium-mediated dimerization may also represent a mechanism for regulation of the activity of this nucleotidase in the physiological setting of the endoplasmic reticulum or Golgi.

  4. A soluble form of GAS1 inhibits tumor growth and angiogenesis in a triple negative breast cancer model.

    PubMed

    Jiménez, Adriana; López-Ornelas, Adolfo; Estudillo, Enrique; González-Mariscal, Lorenza; González, Rosa O; Segovia, José

    2014-10-01

    We previously demonstrated the capacity of GAS1 (Growth Arrest Specific 1) to inhibit the growth of gliomas by blocking the GDNF-RET signaling pathway. Here, we show that a soluble form of GAS1 (tGAS1), decreases the number of viable MDA MB 231 human breast cancer cells, acting in both autocrine and paracrine manners when secreted from producing cells. Moreover, tGAS1 inhibits the growth of tumors implanted in female nu/nu mice through a RET-independent mechanism which involves interfering with the Artemin (ARTN)-GFRα3-(GDNF Family Receptor alpha 3) mediated intracellular signaling and the activation of ERK. In addition, we observed that the presence of tGAS1 reduces the vascularization of implanted tumors, by preventing the migration of endothelial cells. The present results support a potential adjuvant role for tGAS1 in the treatment of breast cancer, by detaining tumor growth and inhibiting angiogenesis. PMID:24992044

  5. Soluble forms of polyQ-expanded huntingtin rather than large aggregates cause endoplasmic reticulum stress

    NASA Astrophysics Data System (ADS)

    Leitman, Julia; Ulrich Hartl, F.; Lederkremer, Gerardo Z.

    2013-11-01

    In Huntington’s disease, as in other neurodegenerative diseases, it was initially thought that insoluble protein aggregates are the toxic species. However, growing evidence implicates soluble oligomeric polyglutamine-expanded huntingtin in cytotoxicity. Here we show that pathogenic huntingtin inhibits endoplasmic reticulum (ER)-associated degradation and induces ER stress before its aggregation into visible inclusions. All three branches of the unfolded protein response are activated. ER stress can be compensated by overexpression of p97/VCP, suggesting its sequestration by pathogenic huntingtin as a main cause. Stress correlates with the presence of huntingtin oligomers and is independent of continual huntingtin synthesis. Stress levels, measured in striatal neurons, are stabilized but only slowly subside on huntingtin aggregation into inclusions. Our results can be explained by the constant conversion of huntingtin monomers to toxic oligomers; large aggregates sequester the former, precluding further conversion, whereas pre-existing toxic oligomers are only gradually depleted.

  6. Efficient soluble expression of active recombinant human cyclin A2 mediated by E. coli molecular chaperones.

    PubMed

    Grigoroudis, Asterios I; McInnes, Campbell; Premnath, Padmavathy Nandha; Kontopidis, George

    2015-09-01

    Bacterial expression of human proteins continues to present a critical challenge in protein crystallography and drug design. While human cyclin A constructs have been extensively characterized in complex with cyclin dependent kinase 2 (CDK2), efforts to express the monomeric human cyclin A2 in Escherichia coli in a stable form, without the kinase subunit, have been laden with technical difficulties, including solubility, yield and purity. Here, optimized conditions are described with the aim of generating for first time, sufficient quantities of human recombinant cyclin A2 in a soluble and active form for crystallization and ligand characterization purposes. The studies involve implementation of a His-tagged heterologous expression system under conditions of auto-induction and mediated by molecular chaperone-expressing plasmids. A high yield of human cyclin A2 was obtained in natively folded and soluble form, through co-expression with groups of molecular chaperones from E. coli in various combinations. A one-step affinity chromatography method was utilized to purify the fusion protein products to homogeneity, and the biological activity confirmed through ligand-binding affinity to inhibitory peptides, representing alternatives for the key determinants of the CDK2 substrate recruitment site on the cyclin regulatory subunit. As a whole, obtaining the active cyclin A without the CDK partner (referred to as monomeric in this work) in a straightforward and facile manner will obviate protein--production issues with the CDK2/cyclin A complex and enable drug discovery efforts for non-ATP competitive CDK inhibition through the cyclin groove. PMID:25956535

  7. Cryomilling-induced solid dispersion of poor glass forming/poorly water-soluble mefenamic acid with polyvinylpyrrolidone K12.

    PubMed

    Kang, Naewon; Lee, Jangmi; Choi, Ji Na; Mao, Chen; Lee, Eun Hee

    2015-06-01

    The effect of mechanical impact on the polymorphic transformation of mefenamic acid (MFA) and the formation of a solid dispersion of mefenamic acid, a poor glass forming/poorly-water soluble compound, with polyvinylpyrrolidone (PVP) K12 was investigated. The implication of solid dispersion formation on solubility enhancement of MFA, prepared by cryomilling, was investigated. Solid state characterization was conducted using powder X-ray diffraction (PXRD) and Fourier-transform infrared (FTIR) spectroscopy combined with crystal structure analysis. Apparent solubility of the mixtures in pH 7.4 buffer was measured. A calculation to compare the powder patterns and FTIR spectra of solid dispersions with the corresponding physical mixtures was conducted. Solid state characterization showed that (1) MFA I transformed to MFA II when pure MFA I was cryogenically milled (CM); and (2) MFA forms a solid dispersion when MFA was cryogenically milled with PVP K12. FTIR spectral analysis showed that hydrogen bonding facilitated by mechanical impact played a major role in forming solid dispersions. The apparent solubility of MFA was significantly improved by making a solid dispersion with PVP K12 via cryomilling. This study highlights the importance of cryomilling with a good hydrogen bond forming excipient as a technique to prepare solid dispersion, especially when a compound shows a poor glass forming ability and therefore, is not easy to form amorphous forms by conventional method. PMID:24849785

  8. Pharmacokinetics of salicylic acid following administration of aspirin tablets and three different forms of soluble aspirin in normal subjects.

    PubMed

    Gatti, G; Barzaghi, N; Attardo Parrinello, G; Vitiello, B; Perucca, E

    1989-01-01

    The pharmacokinetic profile of an innovative formulation of soluble aspirin (l-ornithine acetylsalicylate, ldB 1003) was compared with that of conventional tablets and two other soluble dosage forms (d, l-lysine acetylsalicylate and a buffered effervescent formulation of acetylsalicylic acid) after administration of single oral doses in six normal volunteers. All soluble forms showed a rapid absorption profile, peak plasma salicylic acid levels being attained after about 30 min on average and without statistically significant differences among the solutions tested. As compared to the soluble formulations, acetylsalicylic acid given as tablets resulted in slower absorption, with peak plasma salicylic acid levels being reached more than 1 h after dosing. Despite these differences in time course of plasma level profiles, the extent of absorption was similar for all formulations. Apart from the potential advantages in terms of improved gastric tolerability, the increased rate of absorption of aspirin solutions is therapeutically useful whenever a rapid onset of action is required. In this respect, the kinetic pattern of the innovative formulation compares favourably with that of other available soluble dosage forms. PMID:2517497

  9. Using Computational Fluid Dynamics to investigate the generation of soluble bedrock forms

    NASA Astrophysics Data System (ADS)

    Myre, J. M.; Covington, M. D.

    2014-12-01

    The roles and interactions of processes that generate bedforms in soluble bedrock settings are not yet fully understood. One outstanding puzzle is the morphogenesis of scallops. Current chemical theory does not allow dissolution rate to spatially vary under many of the conditions at which scallops are thought to form. This lack of variation in dissolution rate disagrees with the creation and existence of scallops for a large portion of the range of scallop sizes that are found in nature. Previous studies that have examined processes that generate bedforms using computational fluid dynamics (CFD) have made simplifying assumptions to ensure tractability. Consequently, tractability is sacrificed and some fine scale effects associated with the driving processes are not modeled. We incorporate Large-Eddy Simulation (LES) and adaptive meshing into a lattice-Boltzmann CFD method for fluid flow, chemical dissolution, and solute transport allowing relaxation of simplifying assumptions employed in previous models. These methods can model sub-grid scale turbulence and resolve the dissolution and precipitation processes occurring at the fluid-solid interface. We use this CFD model to simulate flow and dissolution over scallops and flutes to investigate the formational processes responsible for variations in dissolution rate that drive the spatio-temporal evolution of these features.

  10. Summary of Uranium Solubility Studies in Concrete Waste Forms and Vadose Zone Environments

    SciTech Connect

    Golovich, Elizabeth C.; Wellman, Dawn M.; Serne, R. Jeffrey; Bovaird, Chase C.

    2011-09-30

    One of the methods being considered for safely disposing of Category 3 low-level radioactive wastes is to encase the waste in concrete. Concrete encasement would contain and isolate the waste packages from the hydrologic environment and act as an intrusion barrier. The current plan for waste isolation consists of stacking low-level waste packages on a trench floor, surrounding the stacks with reinforced steel, and encasing these packages in concrete. These concrete-encased waste stacks are expected to vary in size with maximum dimensions of 6.4 m long, 2.7 m wide, and 4 m high. The waste stacks are expected to have a surrounding minimum thickness of 15 cm of concrete encasement. These concrete-encased waste packages are expected to withstand environmental exposure (solar radiation, temperature variations, and precipitation) until an interim soil cover or permanent closure cover is installed and to remain largely intact thereafter. Any failure of concrete encasement may result in water intrusion and consequent mobilization of radionuclides from the waste packages. This report presents the results of investigations elucidating the uranium mineral phases controlling the long-term fate of uranium within concrete waste forms and the solubility of these phases in concrete pore waters and alkaline, circum-neutral vadose zone environments.

  11. Soluble Oligomers of the Pore-forming Toxin Cytolysin A from Escherichia coli Are Off-pathway Products of Pore Assembly.

    PubMed

    Roderer, Daniel; Benke, Stephan; Schuler, Benjamin; Glockshuber, Rudi

    2016-03-11

    The α-pore-forming toxin Cytolysin A (ClyA) is responsible for the hemolytic activity of various Escherichia coli and Salmonella enterica strains. Soluble ClyA monomers spontaneously assemble into annular dodecameric pore complexes upon contact with membranes or detergent. At ClyA monomer concentrations above ∼100 nm, the rate-limiting step in detergent- or membrane- induced pore assembly is the unimolecular reaction from the monomer to the assembly-competent protomer, which then oligomerizes rapidly to active pore complexes. In the absence of detergent, ClyA slowly forms soluble oligomers. Here we show that soluble ClyA oligomers cannot form dodecameric pore complexes after the addition of detergent and are hemolytically inactive. In addition, we demonstrate that the natural cysteine pair Cys-87/Cys-285 of ClyA forms a disulfide bond under oxidizing conditions and that both the oxidized and reduced ClyA monomers assemble to active pores via the same pathway in the presence of detergent, in which an unstructured, monomeric intermediate is transiently populated. The results show that the oxidized ClyA monomer assembles to pore complexes about one order of magnitude faster than the reduced monomer because the unstructured intermediate of oxidized ClyA is less stable and dissolves more rapidly than the reduced intermediate. Moreover, we show that oxidized ClyA forms soluble, inactive oligomers in the absence of detergent much faster than the reduced monomer, providing an explanation for several contradictory reports in which oxidized ClyA had been described as inactive. PMID:26757820

  12. [Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast].

    PubMed

    Kasho, V N; Bakuleva, N P; Baĭkov, A A; Avaeva, S M

    1982-06-01

    Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH. The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form. The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center. The enzyme binding to PPi is strongly stabilized by fluoride. The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule. PMID:6126223

  13. The soluble form of BMPRIB is a novel therapeutic candidate for treating bone related disorders.

    PubMed

    Yamawaki, Kengo; Kondo, Yuichiro; Okada, Tsutomu; Oshima, Takeshi; Kakitani, Makoto; Tomizuka, Kazuma

    2016-01-01

    Bone morphogenetic proteins (BMPs) are multi-functional growth factors that belong to the TGF-beta superfamily. Recently, several soluble BMP receptors, such as ActRIIA-Fc, ActRIIB-Fc, and ALK1-Fc, are undergoing clinical trials. Both BMPRIA and BMPRIB are type I BMP receptors, and while BMPRIA-Fc has been reported to have bone-increasing properties, there have been no investigations concerning the biological functions of BMPRIB-Fc. Therefore, comparing the effects of BMPRIA-Fc and BMPRIB-Fc in vivo should be helpful in revealing the differences in biological function between BMPRIA and BMPRIB, and would also aid in the evaluation of BMPRIB-Fc as a therapeutic agent. Here, we produced Tg chimeras in which BMPRIA-Fc and BMPRIB-Fc proteins circulated at high concentrations (36.8-121.4 μg/mL). Both Tg chimeras showed a significant increase of bone volume and strength. Using histological analysis, adenoma of the glandular stomach was observed only in BMPRIA-Fc chimeras suggesting the tumorigenic activity of this protein. Administration of recombinant BMPRIB-Fc protein to normal mice also increased bone volumes. Finally, treatment with BMPRIB-Fc decreased the area of osteolytic regions in a mouse model of breast cancer metastasis. In conclusion, our data suggest that BMPRIB-Fc can be used for the treatment of bone-related disorders with a lower risk than BMPRIA-Fc. PMID:26732094

  14. Impairement of HT29 Cancer Cells Cohesion by the Soluble Form of Neurotensin Receptor-3

    PubMed Central

    Lacas-Gervais, Sandra; Béraud-Dufour, Sophie; Coppola, Thierry; Mazella, Jean

    2014-01-01

    The neurotensin (NT) receptor-3 (NTSR3), also called sortilin is a multifunctional protein localized at the intracellular and plasma membrane level. The extracellular domain of NTSR3 (sNTSR3) is released by shedding from several cell lines including colonic cancer cells. This soluble protein acts as an active ligand through its ability to bind, to be internalized in the human adenocarcinoma epithelial HT29 cells and to stimulate the PI3 kinase pathway. The aim of this study was to investigate cellular responses induced by sNTSR3 in HT29 cells. The cellular functions of sNTSR3 were monitored by immunofluocytochemistry, electron microscopy and quantitative PCR in order to characterize the cell shape and the expression of adhesion proteins. We evidenced that sNTSR3 significantly regulates the cellular morphology as well as the cell-cell and the cell-matrix adherens properties by decreasing the expession of several integrins and by modifying the structure of desmosomes. Altogether, these properties lead to an increase of cell detachment upon sNTSR3 treatment on HT29, HCT116 and SW620 cancer cells. Our results indicate that sNTSR3 may induce the first phase of a process which weaken HT29 epithelial properties including desmosome architecture, cell spreading, and initiation of cell separation, all events which could be responsible for cancer metastasis. PMID:25221642

  15. Solubilities of biologically active phenolic compounds: measurements and modeling.

    PubMed

    Queimada, António J; Mota, Fátima L; Pinho, Simão P; Macedo, Eugénia A

    2009-03-19

    Aqueous solubilities of natural phenolic compounds from different families (hydroxyphenyl, polyphenol, hydroxybenzoic, and phenylpropenoic) were experimentally obtained. Measurements were performed on tyrosol and ellagic, protocatechuic, syringic, and o-coumaric acids, at five different temperatures (from 288.2 to 323.2 K), using the standard shake-flask method, followed by compositional analysis using UV spectrophotometry. To verify the accuracy of the spectrophotometric method, some data points were measured by gravimetry, and in general, the values obtained with the two methods are in good agreement (deviations lower than 11%). To adequately understand the solubilization process, melting properties of the pure phenolics were obtained by differential scanning calorimetry (DSC), and apparent acid dissociation constants were measured by potentiometry titration. The aqueous solubilities followed the expected general exponential trend. The melting temperatures did not follow the same solubility tendency, and for tyrosol and ellagic acid, not only the size and extent of hydrogen bonding, but also the energy associated with their crystal structures, determine the solubility. For these binary systems, acid dissociation is not important. Approaches for modeling the measured data were evaluated. These included an excess Gibbs energy equation, the modified UNIQUAC model, and the cubic-plus-association (CPA) equation of state. Particularly for the CPA approach, a new methodology that explicitly takes into account the number and nature of the associating sites and the prediction of the pure-component parameters from molecular structure is proposed. The results indicate that these are appropriate tools for representing the water solubilities of these molecules. PMID:19243119

  16. Influence of glucosamine on oligochitosan solubility and antibacterial activity.

    PubMed

    Blagodatskikh, Inesa V; Kulikov, Sergey N; Vyshivannaya, Oxana V; Bezrodnykh, Evgeniya A; Yamskov, Igor A; Tikhonov, Vladimir E

    2013-11-15

    Light scattering studies indicate that oligochitosan (short-chain chitosan) solutions contain aggregates at pH values below the critical pH of phase separation, while at or above this point the gel phase coexists with the aggregate solution. This work demonstrates for the first time that the presence of D-glucosamine in an oligochitosan solution shifts the critical pH to a higher value and improves the oligochitosan antibacterial activity against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermis in neutral and slightly alkaline aqueous media. By comparing the results of light scattering studies and antimicrobial assays one can conclude that the antimicrobial activity of oligochitosan is dependent on its unimolecular form, not its supramolecular structures. The widening of the homogeneity region of an oligochitosan solution could lead to promising biomedical applications. PMID:24056011

  17. Biochemical, conformational, and immunogenic analysis of soluble trimeric forms of henipavirus fusion glycoproteins.

    PubMed

    Chan, Yee-Peng; Lu, Min; Dutta, Somnath; Yan, Lianying; Barr, Jennifer; Flora, Michael; Feng, Yan-Ru; Xu, Kai; Nikolov, Dimitar B; Wang, Lin-Fa; Skiniotis, Georgios; Broder, Christopher C

    2012-11-01

    The henipaviruses, Hendra virus (HeV) and Nipah virus (NiV), are paramyxoviruses discovered in the mid- to late 1990s that possess a broad host tropism and are known to cause severe and often fatal disease in both humans and animals. HeV and NiV infect cells by a pH-independent membrane fusion mechanism facilitated by their attachment (G) and fusion (F) glycoproteins. Here, several soluble forms of henipavirus F (sF) were engineered and characterized. Recombinant sF was produced by deleting the transmembrane (TM) and cytoplasmic tail (CT) domains and appending a glycosylphosphatidylinositol (GPI) anchor signal sequence followed by GPI-phospholipase D digestion, appending a trimeric coiled-coil (GCNt) domain (sF(GCNt)), or deleting the TM, CT, and fusion peptide domain. These sF glycoproteins were produced as F(0) precursors, and all were apparent stable trimers recognized by NiV-specific antisera. Surprisingly, however, only the GCNt-appended constructs (sF(GCNt)) could elicit cross-reactive henipavirus-neutralizing antibody in mice. In addition, sF(GCNt) constructs could be triggered in vitro by protease cleavage and heat to transition from an apparent prefusion to postfusion conformation, transitioning through an intermediate that could be captured by a peptide corresponding to the C-terminal heptad repeat domain of F. The pre- and postfusion structures of sF(GCNt) and non-GCNt-appended sF could be revealed by electron microscopy and were distinguishable by F-specific monoclonal antibodies. These data suggest that only certain sF constructs could serve as potential subunit vaccine immunogens against henipaviruses and also establish important tools for further structural, functional, and diagnostic studies on these important emerging viruses. PMID:22915804

  18. Combining inkjet printing and amorphous nanonization to prepare personalized dosage forms of poorly-soluble drugs.

    PubMed

    Cheow, Wean Sin; Kiew, Tie Yi; Hadinoto, Kunn

    2015-10-01

    Inkjet printing of drug nanosuspension on edible porous substrates was carried out for the first time with the objective of preparing personalized dosage forms of poorly soluble drugs. Amorphous drug-polysaccharide nanoparticle complex (or drug nanoplex in short) was used as the nanosuspension ink, instead of the conventional crystalline nanodrug. The amorphous drug nanoplex exhibited low propensity to Ostwald ripening growth, high colloidal stability, and supersaturation generation capability making it ideal for printing. Nanoplexes of ciprofloxacin - a BCS Class IV compound - prepared by complexation with dextran sulfate were used as the nanosuspension ink at two different sizes (i.e. ≈265nm and 188nm). Inkjet printing was performed on cellulose substrate at 0.25% (w/v) nanosuspension concentration and 5% (w/v) polyethylene glycol. For both nanoplex sizes, the results indicated that the printed dose could be increased by increasing the number of droplets dispensed. However, exact correlations between the achievable dose and the number of droplets dispensed were not evident, which was likely caused by the spatial non-homogeneity in the nanosuspension concentration. Compared to the larger nanoplex, printed nanodrugs of the smaller nanoplex consistently exhibited higher payload with better batch-to-batch reproducibility (<6%). The maximum achievable payload was equal to ≈2.5μg/cm(2), which was multifold higher than that achieved had inkjet printing of ciprofloxacin solution been performed. Nevertheless, print substrate with higher liquid uptake capacity is needed to increase the payload nearer to the therapeutic dose. Lastly, the drug release and non-cytotoxicity of the printed nanodrug were successfully established in vitro. PMID:26325060

  19. Lubrication of starch in ionic liquid-water mixtures: Soluble carbohydrate polymers form a boundary film on hydrophobic surfaces.

    PubMed

    Yakubov, Gleb E; Zhong, Lei; Li, Ming; Boehm, Michael W; Xie, Fengwei; Beattie, David A; Halley, Peter J; Stokes, Jason R

    2015-11-20

    Soluble starch polymers are shown to enhance the lubrication of ionic liquid-water solvent mixtures in low-pressure tribological contacts between hydrophobic substrates. A fraction of starch polymers become highly soluble in 1-ethyl-3-methylimidazolium acetate (EMIMAc)-water solvents with ionic liquid fraction ≥60wt%. In 65wt% EMIMAc, a small amount of soluble starch (0.33wt%) reduces the boundary friction coefficient by up to a third in comparison to that of the solvent. This low-friction is associated with a nanometre thick film (ca. 2nm) formed from the amylose fraction of the starch. In addition, under conditions where there is a mixture of insoluble starch particles and solubilised starch polymers, it is found that the presence of dissolved amylose enhances the lubrication of starch suspensions between roughened substrates. These findings open up the possibility of utilising starch biopolymers, as well as other hydrocolloids, for enhancing the performance of ionic liquid lubricants. PMID:26344308

  20. Soluble HLA class I antigen secretion by normal lymphocytes: relationship with cell activation and effect of interferon-gamma.

    PubMed Central

    Brieva, J A; Villar, L M; Leoro, G; Alvarez-Cermeño, J C; Roldán, E; Gonzalez-Porqué, P

    1990-01-01

    HLA class I antigens are thought to be integral membrane proteins. However, soluble forms of these molecules have been detected. Our laboratory has recently shown that the predominant form of these soluble proteins present in human serum, spleen tissue and culture supernatant of activated lymphocytes exhibits molecular weight and structure similar to classical HLA class I antigens, but lacks HLA A or B polymorphic determinants. In the present study, the secretion of such soluble proteins by lymphocytes has been further explored. Phytohaemagglutinin-stimulated normal lymphocytes secrete considerable quantities of soluble HLA (sHLA) class I proteins. This secretion seems to be a general property of lymphocytes, since activation of T as well as B cells by appropriate mitogens equally induce sHLA I secretion. Lymphocytes require RNA and protein synthesis, but not DNA synthesis, for the secretion to occur. Kinetic studies reveal that maximal sHLA I secretion precedes the peak of DNA synthesis by 24 h. In vitro stimulation with antigens or alloantigens also provokes sHLA I secretion. Moreover, this phenomenon has also been detected for in vivo-activated lymphocytes, as enhanced spontaneous sHLA I secretion was observed in cultures of low-density blastic B and T cells, and of blood lymphocytes obtained from normal subjects who had received a booster immunization 5 days earlier. Interferon-gamma (IFN-gamma) increases the expression of membrane-bound class I antigens but does not induce any sHLA I secretion, suggesting that both molecules are under different regulatory mechanisms. Our results indicate that human lymphocytes, upon stimulation, actively secrete considerable amounts of a soluble form of these biologically relevant proteins. PMID:2122936

  1. Synthesis of water-soluble dinuclear mn-porphyrin with multiple antioxidative activities.

    PubMed

    Kubota, Riku; Imamura, Shinya; Shimizu, Takahiko; Asayama, Shoichiro; Kawakami, Hiroyoshi

    2014-06-12

    Superoxide dismutase (SOD) and catalase activities of a drug are of great importance for its effective protection against reactive oxygen species (ROS)-induced injury. Achievement of catalase activity of a synthetic compound remains a challenge. Water-soluble Mn-porphyrins have high SOD and peroxynitrite (ONOO(-)) reducing activities, but not catalase-like activity. Herein, we are able to retain the fair SOD-like activity of a mononuclear Mn-5-(N-methylpyridinium-4-yl)-10,15,20-triphenyl porphyrin (MnM4PyP3P), while gaining in catalase-like activity with its dinuclear complex, 1,3-di[5-(N-methylene-pyridinium-4-yl)-10,15,20-triphenyl porphynato manganese] benzene tetrachloride (MnPD). Mechanistic study indicates that catalase-like activity of MnPD is due to synergism of two Mn active sites, where hydroxo-Mn(IV) complex is formed as an intermediate. The in vivo experiments demonstrate that MnPD significantly restores the treadmill-running ability of SOD-deficient mouse and thus indicates the therapeutic potential of MnPD. Furthermore, MnPD may serve as a mechanistic tool and indicate the new directions in the synthesis of catalase-like mimics. PMID:24944735

  2. Influence of honey-roasting on the main pharmacological activities and the water-soluble active glycosides of licorice.

    PubMed

    Wang, Mengyue; Zhang, Min; Tang, Qiyu; Li, Xiaobo

    2012-01-01

    In traditional Chinese medicine (TCM), licorice is usually processed with honey and traditionally used in decoction form. However, the influence of honey-roasting on the main pharmacological activities and the water-soluble active constituents of licorice has not been reported. The aim of the present study is to determine whether honey-roasting can modify the main pharmacological activities and the active constituents of licorice. According to licorice clinical application and processing method, the mainly related pharmacological activities of crude licorice, processed licorice and refined honey, such as enhancing immune function, relieving cough, eliminating phlegm and detoxication, were compared. The results showed that honey-roasting obviously reinforced the licorice activity of enhancing Pi-deficiency mice's immune function, and significantly weaken the licorice activity of relieving cough, removing phlegm and detoxication. However, honey didn't show the significant activity of relieving cough, removing phlegm and detoxication. The influence of honey-roasting on the chemical compositions in licorice slice and licorice decoction was investigated by using HPLC. The results showed that the content and the decocting quantity of mainly 5 active glycosides in licorice, i.e. liquiritin apioside, liquiritin, licuraside, isoliquiritin and glycyrrhizin, obviously changed after processing; glycyrrhizin and liquiritin obviously decomposed during honey-roasting. In conclusion, honey-roasting obviously modified the main pharmacological activities and the water-soluble compositions of licorice. The modification was not cause by honey only. This finding may shed some light on understanding the differences in the therapeutic values of crude and processed licorice. PMID:23983334

  3. Simultaneous quantification of 21 water soluble vitamin circulating forms in human plasma by liquid chromatography-mass spectrometry.

    PubMed

    Meisser Redeuil, Karine; Longet, Karin; Bénet, Sylvie; Munari, Caroline; Campos-Giménez, Esther

    2015-11-27

    This manuscript reports a validated analytical approach for the quantification of 21 water soluble vitamins and their main circulating forms in human plasma. Isotope dilution-based sample preparation consisted of protein precipitation using acidic methanol enriched with stable isotope labelled internal standards. Separation was achieved by reversed-phase liquid chromatography and detection performed by tandem mass spectrometry in positive electrospray ionization mode. Instrumental lower limits of detection and quantification reached <0.1-10nM and 0.2-25nM, respectively. Commercially available pooled human plasma was used to build matrix-matched calibration curves ranging 2-500, 5-1250, 20-5000 or 150-37500nM depending on the analyte. The overall performance of the method was considered adequate, with 2.8-20.9% and 5.2-20.0% intra and inter-day precision, respectively and averaged accuracy reaching 91-108%. Recovery experiments were also performed and reached in average 82%. This analytical approach was then applied for the quantification of circulating water soluble vitamins in human plasma single donor samples. The present report provides a sensitive and reliable approach for the quantification of water soluble vitamins and main circulating forms in human plasma. In the future, the application of this analytical approach will give more confidence to provide a comprehensive assessment of water soluble vitamins nutritional status and bioavailability studies in humans. PMID:26522745

  4. Soluble adhesion molecules correlate with surface expression in an in vitro model of endothelial activation.

    PubMed

    Kjaergaard, Anders G; Dige, Anders; Krog, Jan; Tønnesen, Else; Wogensen, Lise

    2013-10-01

    Endothelial activation is a pivotal event in the development and progression of inflammation. Central to endothelial activation is the up-regulation of cellular adhesion molecules (CAMs) including E-selectin (CD62E), ICAM-1 (CD54), VCAM-1 (CD106) and PECAM-1 (CD31). These CAMs are also found in soluble forms (sCAMs). In this in vitro study of endothelial activation, we examined whether the levels of sCAMs correlate with the endothelial surface expression of CAMs in a dose-dependent and time-dependent manner. Such a correlation would support the use of sCAMs as surrogate markers for endothelial activation in inflammatory conditions. Human umbilical vein endothelial cells (HUVEC) were cultured with various concentrations of TNF-α for 8 hr and at a fixed concentration of TNF-α for various durations. The levels of soluble and surface-bound E-selectin, ICAM-1, VCAM-1 and PECAM-1 were quantified by flow cytometry. TNF-α stimulation increased CAM and sCAM expression in a dose-dependent and time-dependent manner. There was a significant positive correlation between the levels of ICAM-1 and sICAM-1 and between the levels of VCAM and sVCAM-1 in both the dose-response and time-response experiments. A positive correlation between the levels of E-selectin and sE-selectin was observed in the time-response experiment. This study supports the use of sCAMs as potential biomarkers of endothelial activation. In particular, the use of sICAM-1, sVCAM-1 and sE-selectin seems promising. PMID:23724832

  5. Multiple forms of soluble monophenol, dihydroxyphenylalamine: oxygen-oxidoreductase (EC 1.14.18.1) from potato tubers (Solanum tuberosum). II. Partial characterization of the enzyme forms with different molecular weights.

    PubMed

    Matheis, G; Belitz, H D

    1977-04-28

    Gel chromatography on Sephadex G-200 was used to separate a soluble phenoloxidase from potatoes (var. Maritta) into at least six active fractions with dopa (dihydroxyphenylalanine) as substrate. Only high-molecular-weight-enzyme forms exhibited monophenoloxidase activity. Re-chromatography of the highest-molecular-weight form gave the same molecular weight distribution as with the crude enzyme. The molecular weights indicate association phenomena of subunits with a molecular weight of about 36000 daltons. According to polyacrylamide-gel electrophoresis, several monomeric forms with differnt isoelectric points seem to be present. This suggests that the large number of multiple forms of the enzyme arises from various combinations of identical and/or different subunits. SDS polyacrylamide gel electrophoresis failed to show the monomeric forms; the dimer and higher oligomers were obtained. PMID:404778

  6. Active curcumin nanoparticles formed from a volatile microemulsion template.

    PubMed

    Margulis, K; Srinivasan, S; Ware, M J; Summers, H D; Godin, B; Magdassi, S

    2014-01-01

    We report on biological performance of organic nanoparticles formed by a simple method based on rapid solvent removal from a volatile microemulsion. The particular focus of the study was on testing the suitability of the method for substances soluble in partially water-miscible organic solvents as well as on evaluating the therapeutic activity of the resultant nanoparticles. Curcumin was employed as a model for hydrophobic drug, and, as it is soluble in water-miscible organic solvents, it was successfully incorporated into a new cyclopentanone-water microemulsion system. During rapid solvent removal by spray-drying, the nanometric droplets of the microemulsion were converted into nanoparticles containing amorphous curcumin with the average size of 20.2±3.4 nm, having ζ potential of -36.2 ±1.8 mV. These nanoparticles were dispersible in water and retained the high loading of the active substance. The therapeutic activity of the resulting nanoparticles was demonstrated in a pancreatic cancer cell line Panc-1. The effective concentration for reducing the metabolic activity was found to be 11.5 μM for nanoparticles compared with 19.5 μM for free curcumin. PMID:25485110

  7. Solubility and some crystallization properties of conglomerate forming chiral drug guaifenesin in water.

    PubMed

    Fayzullin, Robert R; Lorenz, Heike; Bredikhina, Zemfira A; Bredikhin, Alexander A; Seidel-Morgenstern, Andreas

    2014-10-01

    The solubility of 3-(2-methoxyphenoxy)-propane-1,2-diol, the well-known chiral drug guaifenesin 1, in water has been investigated by means of polythermal and isothermal approaches. It was found that the solubilities of racemic and enantiomeric diols rac- and (R)-1 depend strongly on temperature. The ternary phase diagram of the guaifenesin enantiomers in water in the temperature range between 10°C and 40°C was constructed. Clear evidence was obtained that rac-1 crystallizes as a stable conglomerate. The Meyerhoffer coefficient for the guaifenesin-water system is more than two and strongly depends on temperature. Neither crystalline hydrates nor polymorphs were detected within the range of conditions covered. Metastable zone width data with regard to primary nucleation were also collected for rac-1 and (R)-1. On the basis of the knowledge acquired, the resolution of racemic guaifenesin by preferential crystallization from solution could be realized successfully. PMID:25091705

  8. Different forms of soluble cytoplasmic mRNA binding proteins and particles in Xenopus laevis oocytes and embryos

    SciTech Connect

    Murray, M.T.; Krohne, G.; Franke, W.W. )

    1991-01-01

    To gain insight into the mechanisms involved in the formation of maternally stored mRNPs during Xenopus laevis development, we searched for soluble cytoplasmic proteins of the oocyte that are able to selectively bind mRNAs, using as substrate radiolabeled mRNA. In vitro mRNP assembly in solution was followed by UV-cross-linking and RNase digestion, resulting in covalent tagging of polypeptides by nucleotide transfer. Five polypeptides of approximately 54, 56 60, 70, and 100 kD (p54, p56, p60, p70, and p100) have been found to selectively bind mRNA and assemble into mRNPs. These polypeptides, which correspond to previously described native mRNP components, occur in three different particle classes of approximately 4.5S, approximately 6S, and approximately 15S, as also determined by their reactions with antibodies against p54 and p56. Whereas the approximately 4.5S class contains p42, p60, and p70, probably each in the form of individual molecules or small complexes, the approximately 6S particles appears to consist only of p54 and p56, which occur in a near-stoichiometric ratio suggestive of a heterodimer complex. The approximately 15S particles contain, in addition to p54 and p56, p60 and p100 and this is the single occurring form of RNA-binding p100. We have also observed changes in the in vitro mRNA binding properties of these polypeptides during oogenesis and early embryonic development, in relation to their phosphorylation state and to the activity of an approximately 15S particle-associated protein kinase, suggesting that these proteins are involved in the developmental translational regulation of maternal mRNAs.

  9. Enzymatic activity of soluble and membrane tethered peptide pro-hormone convertase 1.

    PubMed

    Bruzzaniti, Angela; Mains, Richard E

    2002-05-01

    Pro-hormone convertases PC1 and PC2 perform endoproteolytic cleavages of precursors in peptide-containing secretory granules. PC1 and PC2 are soluble, secreted with bioactive peptides. Evolutionarily related PCs have membrane tethers, not secreted. We tethered PC1 to the transmembrane-cytoplasmic domains (CD) of a granule enzyme (peptidylglycine-alpha-amidating monooxygenase; PAM) and Golgi-localized PC8. The tethered PC1 is far more stable to elevated temperature and denaturants than soluble PC1, and more active. Both tethers allow PC1 to visit the cell surface transiently, cleaving soluble molecules outside the cell. Both membrane-bound PC1 chimeras cleave membrane PAM into soluble active fragments when PAM is expressed on adjacent cells. PMID:12084516

  10. WATER ACTIVITY DATA ASSESSMENT TO BE USED IN HANFORD WASTE SOLUBILITY CALCULATIONS

    SciTech Connect

    DISSELKAMP RS

    2011-01-06

    The purpose of this report is to present and assess water activity versus ionic strength for six solutes:sodium nitrate, sodium nitrite, sodium chloride, sodium carbonate, sodium sulfate, and potassium nitrate. Water activity is given versus molality (e.g., ionic strength) and temperature. Water activity is used to estimate Hanford crystal hydrate solubility present in the waste.

  11. Increased serum levels of soluble l-selectin (CD62L) in patients with active systemic lupus erythematosus (SLE)

    PubMed Central

    Font, J; Pizcueta, P; Ramos-Casals, M; Cervera, R; García-Carrasco, M; Navarro, M; Ingelmo, M; Engel, P

    2000-01-01

    The adhesion molecule l-selectin (CD62L) mediates lymphocyte recirculation and leucocyte rolling on vascular endothelium at sites of inflammation. Serum levels of soluble l-selectin (sl-selectin) were measured in patients with SLE in order to relate these levels to clinical activity and immunological parameters. An ELISA was used to detect the soluble form of human l-selectin (CD62L) in 42 patients with SLE and in 33 healthy individuals. The mean ± s.e.m. values of sl-selectin were 1285 ± 121 ng/ml for patients with SLE and 986 ± 180 ng/ml for healthy blood donors, but there was no significant difference. When patients with active SLE were analysed, higher levels of circulating sl-selectin were found when compared with patients without activity (1497 ± 167 ng/ml versus 941 ± 150 ng/ml; P = 0.028). We found a significant correlation between the levels of sl-selectin and of dsDNA antibodies (r = 0.36, P = 0.044) and between levels of sl-selectin and SLE Disease Activity Index (SLEDAI) score (r = 0.42, P = 0.003). Patients with active SLE studied cross-sectionally showed significant elevations of sl-selectin (CD62L) compared with controls. Thus, the levels of this soluble adhesion molecule correlated with active disease and levels of anti-dsDNA antibodies. PMID:10606979

  12. Effects of hydroxyl radical scavengers KCN and CO on ultraviolet light-induced activation of crude soluble guanylate cyclase

    SciTech Connect

    Karlsson, J.O.; Axelsson, K.L.; Andersson, R.G.

    1985-01-01

    The crude soluble guanylate cyclase (GC) from bovine mesenteric artery was stimulated by ultraviolet (UV) light (366 nm). Addition of free radical scavengers, dimethylsulfoxide or superoxide dismutase and/or catalase to the GC assay did not abolish the stimulatory effect of UV light. On the contrary, the UV light-induced activation was enhanced in the presence of these scavengers. KCN (1 mM) did not affect the UV light-induced activation, while 0.1 mM of CO potentiated the activation. These results may indicate that UV light is operating through a direct interaction with the ferrous form of the GC-heme.

  13. Dermal nanocrystals from medium soluble actives - physical stability and stability affecting parameters.

    PubMed

    Zhai, Xuezhen; Lademann, Jürgen; Keck, Cornelia M; Müller, Rainer H

    2014-09-01

    Nanocrystals are meanwhile applied to increase the dermal penetration of drugs, but were applied by now only to poorly soluble drugs (e.g. 1-10 μg/ml). As a new concept nanocrystals from medium soluble actives were produced, using caffeine as model compound (solubility 16 mg/ml at 20 °C). Penetration should be increased by (a) further increase in solubility and (b) mainly by increased hair follicle targeting of nanocrystals compared to pure solution. Caffeine nanocrystal production in water lead to pronounced crystal growth. Therefore the stability of nanocrystals in water-ethanol (1:9) and ethanol-propylene glycol (3:7) mixtures with lower dielectric constant D was investigated, using various stabilizers. Both mixtures in combination with Carbopol 981 (non-neutralized) yielded stable nanosuspensions over 2 months at 4 °C and room temperature. Storage at 40 °C lead to crystal growth, attributed to too strong solubility increase, supersaturation and Ostwald ripening effects. Stability of caffeine nanocrystals at lower temperatures could not only be attributed to lower solubility, because the solubilities of caffeine in mixtures and in water are not that much different. Other effects such as quantified by reduced dielectric constant D, and specific interactions between dispersion medium and crystal surface seem to play a role. With the 2 mixtures and Carbopol 981, a basic formulation composition for this type of nanocrystals has been established, to be used in the in vivo proof of principle of the new concept. PMID:25016978

  14. Lipid peroxides as endogenous oxidants forming 8-oxo-guanosine and lipid-soluble antioxidants as suppressing agents

    PubMed Central

    Kanazawa, Kazuki; Sakamoto, Miku; Kanazawa, Ko; Ishigaki, Yoriko; Aihara, Yoshiko; Hashimoto, Takashi; Mizuno, Masashi

    2016-01-01

    The oxidation of guanosine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is closely associated with induction of various diseases, but the endogenous oxidant species involved remains unclear. Hydrogen peroxides (H2O2) have been considered to be the oxidant, while lipid peroxides are another possible oxidant because generated easily in bio-membranes surrounding DNA. The oxidant potency was compared between lipid peroxides and H2O2. Linoleic acid hydroperoxides (LOOH) formed 8-oxo-dG at a higher level than H2O2 in guanosine or double-stranded DNA. In the presence of a physiological concentration of Fe2+ to produce hydroxyl radicals, LOOH was also a stronger oxidant. In a lipid micelle, LOOH markedly produced 8-oxo-dG at a concentration one-tenth of that of H2O2. Upon adding to rat hepatic mitochondria, phosphatidylcholine hydroperoxides produced 8-oxo-dG abundantly. Employing HepG2 cells after pretreated with glutathione peroxidase inhibitor, LOOH formed 8-oxo-dG more abundantly than H2O2. Then, antioxidants to suppress the 8-oxo-dG formation were examined, when the nuclei of pre-incubated HepG2 with antioxidants were exposed to LOOH. Water-soluble ascorbic acid, trolox, and N-acetyl cysteine showed no or weak antioxidant potency, while lipid-soluble 2,6-dipalmitoyl ascorbic acid, α-tocopherol, and lipid-soluble phytochemicals exhibited stronger potency. The present study shows preferential formation of 8-oxo-dG upon LOOH and the inhibition by lipid-soluble antioxidants. PMID:27499574

  15. Lipid peroxides as endogenous oxidants forming 8-oxo-guanosine and lipid-soluble antioxidants as suppressing agents.

    PubMed

    Kanazawa, Kazuki; Sakamoto, Miku; Kanazawa, Ko; Ishigaki, Yoriko; Aihara, Yoshiko; Hashimoto, Takashi; Mizuno, Masashi

    2016-07-01

    The oxidation of guanosine to 8-oxo-2'-deoxyguanosine (8-oxo-dG) in DNA is closely associated with induction of various diseases, but the endogenous oxidant species involved remains unclear. Hydrogen peroxides (H2O2) have been considered to be the oxidant, while lipid peroxides are another possible oxidant because generated easily in bio-membranes surrounding DNA. The oxidant potency was compared between lipid peroxides and H2O2. Linoleic acid hydroperoxides (LOOH) formed 8-oxo-dG at a higher level than H2O2 in guanosine or double-stranded DNA. In the presence of a physiological concentration of Fe(2+) to produce hydroxyl radicals, LOOH was also a stronger oxidant. In a lipid micelle, LOOH markedly produced 8-oxo-dG at a concentration one-tenth of that of H2O2. Upon adding to rat hepatic mitochondria, phosphatidylcholine hydroperoxides produced 8-oxo-dG abundantly. Employing HepG2 cells after pretreated with glutathione peroxidase inhibitor, LOOH formed 8-oxo-dG more abundantly than H2O2. Then, antioxidants to suppress the 8-oxo-dG formation were examined, when the nuclei of pre-incubated HepG2 with antioxidants were exposed to LOOH. Water-soluble ascorbic acid, trolox, and N-acetyl cysteine showed no or weak antioxidant potency, while lipid-soluble 2,6-dipalmitoyl ascorbic acid, α-tocopherol, and lipid-soluble phytochemicals exhibited stronger potency. The present study shows preferential formation of 8-oxo-dG upon LOOH and the inhibition by lipid-soluble antioxidants. PMID:27499574

  16. Analysis of organic gas phase compounds formed by hydrothermal liquefaction of Dried Distillers Grains with Solubles.

    PubMed

    Madsen, René B; Christensen, Per S; Houlberg, Kasper; Lappa, Elpiniki; Mørup, Anders J; Klemmer, Maika; Olsen, Eva M; Jensen, Mads M; Becker, Jacob; Iversen, Bo B; Glasius, Marianne

    2015-09-01

    This work provides a comprehensive characterization of the gas phase from hydrothermal liquefaction of Dried Distillers Grains with Solubles (DDGS) collected during a 24-h continuous experiment. The gas consisted mainly of CO2, CO, H2, CH4 and C2H6 accounting for 96 v/v% while further analysis by gas chromatography coupled to mass spectrometry (GC-MS) showed additionally 62 compounds of which 54 were tentatively identified. These products included methanethiol, dimethyl sulfide, various olefins and several aromatic compounds. The composition provided clear indication of the steady state of the system. Apart from CO2, olefins were the most abundant compound class and could provide a source of revenue. PMID:26051525

  17. Activity-Based Approach for Teaching Aqueous Solubility, Energy, and Entropy

    ERIC Educational Resources Information Center

    Eisen, Laura; Marano, Nadia; Glazier, Samantha

    2014-01-01

    We describe an activity-based approach for teaching aqueous solubility to introductory chemistry students that provides a more balanced presentation of the roles of energy and entropy in dissolution than is found in most general chemistry textbooks. In the first few activities, students observe that polar substances dissolve in water, whereas…

  18. A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice

    SciTech Connect

    Tomioka, Yukiko; Morimatsu, Masami; Nishijima, Ken-ichi; Usui, Tatsufumi; Yamamoto, Sayo; Suyama, Haruka; Ozaki, Kinuyo; Ito, Toshihiro; and others

    2014-07-18

    Highlights: • Tumor-associated antigen MUC1 binds to Siglec-9. • Soluble Siglec-9 reduced proliferation of MUC1-positive tumor in transgenic mice. • Soluble Siglec-9 and MUC1 on tumor cells were colocalized in transgenic mice. • MUC1 expression on tumor cells were reduced in soluble Siglec-9 transgenic mice. - Abstract: Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

  19. The solubility of the tetragonal form of hen egg white lysozyme from pH 4.0 to 5.4

    NASA Technical Reports Server (NTRS)

    Cacioppo, Elizabeth; Pusey, Marc L.

    1991-01-01

    Hen egg white lysozyme solubilities in the presence of the tetragonal crystal form have been determined. Conditions investigated cover the pH range 4.0 to 5.4, varying from 2.0 to 7.0 percent NaCl concentrations and from 4 to 25 C. In all instances, the solubilities were found to increase with temperature and decrease with increasing salt concentration. The effects of pH were more complex, showing a decreasing solubility with increasing pH at low salt concentration and an increasing solubility with increasing pH at high salt concentration.

  20. Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana

    PubMed Central

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Steffen; Reinbothe, Christiane

    2015-01-01

    Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (RESPONSIVE TO DESICCATION 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles’ heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP. PMID:26016527

  1. Water-soluble chlorophyll protein is involved in herbivore resistance activation during greening of Arabidopsis thaliana.

    PubMed

    Boex-Fontvieille, Edouard; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Steffen; Reinbothe, Christiane

    2015-06-01

    Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (responsive to desiccation 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles' heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP. PMID:26016527

  2. Redox-Dependent Solubility of Technetium in Low Activity Waste Glass

    SciTech Connect

    Soderquist, Chuck Z.; Schweiger, Michael J.; Kim, Dong-Sang; Lukens, Wayne W.; Mccloy, John S.

    2014-03-01

    The solubility of technetium was measured in a Hanford low activity waste glass simulant. The simulant glass was melted, quenched and pulverized to make a stock of powdered glass. A series of glass samples were prepared using the powdered glass and varying amounts of solid potassium pertechnetate. Samples were melted at 1000°C in sealed fused quartz ampoules. After cooling, the bulk glass and the salt phase above the glass (when present) were sampled for physical and chemical characterization. Technetium was found in the bulk glass up to 2000 ppm (using the glass as prepared) and 3000 ppm (using slightly reducing conditions). The chemical form of technetium obtained by x-ray absorption near edge spectroscopy can be mainly assigned to isolated Tc(IV), with a minority of Tc(VII) in some glasses and TcO2 in two glasses. The concentration and speciation of technetium depends on glass redox and amount of technetium added. Solid crystals of pertechnetate salts were found in the salt cake layer that formed at the top of some glasses during the melt.

  3. Soluble interleukin 2 receptors are released from activated human lymphoid cells in vitro

    SciTech Connect

    Rubin, L.A.; Kurman, C.C.; Fritz, M.E.; Biddison, W.E.; Boutin, B.; Yarchoan, R.; Nelson, D.L.

    1985-11-01

    With the use of an enzyme-linked immunoabsorbent assay to measure soluble human interleukin 2 receptors (IL 2R), certain human T cell leukemia virus I (HTLV I)-positive T cell lines were found to spontaneously release large quantities of IL 2R into culture supernatants. This was not found with HTLV I-negative and IL 2 independent T cell lines, and only one of seven B cell-derived lines examined produced small amounts of IL 2R. In addition to this constitutive production of soluble IL 2R by certain cell lines, normal human peripheral blood mononuclear cells (PBMC) could be induced to release soluble IL 2R by plant lectins, the murine monoclonal antibody OKT3, tetanus toxoid, and allogeneic cells. Such activated cells also expressed cellular IL 2R measurable in detergent solubilized cell extracts. The generation of cellular and supernatant IL 2R was: dependent on cellular activation, rapid, radioresistant (3000 rad), and inhibited by cycloheximide treatment. NaDodSO4-polyacrylamide gel electrophoresis analysis of soluble IL 2R demonstrated molecules of apparent Mr = 35,000 to 40,000, and 45,000 to 50,000, respectively, somewhat smaller than the mature surface receptor on these cells. The release of soluble IL 2R appears to be a characteristic marker of T lymphocyte activation and might serve an immunoregulatory function during both normal and abnormal cell growth and differentiation.

  4. Experimental Solubility Approach to Determine PDMS-Water Partition Constants and PDMS Activity Coefficients.

    PubMed

    Grant, Sharon; Schacht, Veronika J; Escher, Beate I; Hawker, Darryl W; Gaus, Caroline

    2016-03-15

    Freely dissolved aqueous concentration and chemical activity are important determinants of contaminant transport, fate, and toxic potential. Both parameters are commonly quantified using Solid Phase Micro-Extraction (SPME) based on a sorptive polymer such as polydimethylsiloxane (PDMS). This method requires the PDMS-water partition constants, KPDMSw, or activity coefficient to be known. For superhydrophobic contaminants (log KOW >6), application of existing methods to measure these parameters is challenging, and independent measures to validate KPDMSw values would be beneficial. We developed a simple, rapid method to directly measure PDMS solubilities of solid contaminants, SPDMS(S), which together with literature thermodynamic properties was then used to estimate KPDMSw and activity coefficients in PDMS. PDMS solubility for the test compounds (log KOW 7.2-8.3) ranged over 3 orders of magnitude (4.1-5700 μM), and was dependent on compound class. For polychlorinated biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins (PCDDs), solubility-derived KPDMSw increased linearly with hydrophobicity, consistent with trends previously reported for less chlorinated congeners. In contrast, subcooled liquid PDMS solubilities, SPDMS(L), were approximately constant within a compound class. SPDMS(S) and KPDMSw can therefore be predicted for a compound class with reasonable robustness based solely on the class-specific SPDMS(L) and a particular congener's entropy of fusion, melting point, and aqueous solubility. PMID:26881312

  5. Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione.

    PubMed

    Schrammel, A; Pfeiffer, S; Schmidt, K; Koesling, D; Mayer, B

    1998-07-01

    Soluble guanylyl cyclase (sGC) is the major physiological target of sydnonimine-based vasodilators such as molsidomine. Decomposition of sydnonimines results in the stoichiometric formation of nitric oxide (NO) and superoxide (O2-), which rapidly react to form peroxynitrite. Inasmuch as sGC is activated by NO but not by peroxynitrite, we investigated the mechanisms underlying sGC activation by 3-morpholinosydnonimine (SIN-1). Stimulation of purified bovine lung sGC by SIN-1 was found to be strongly dependent on glutathione (GSH). By contrast, GSH did not affect sGC activation by NO released from 2,2-diethyl-1-nitroso-oxyhydrazine, indicating that NO/O2- released from SIN-1 converted GSH to an activator of sGC. High performance liquid chromatography identified this product as the thionitrite S-nitrosoglutathione. Further, the reaction product decomposed to release NO upon addition of Cu(NO3)2 in the presence of GSH. Activation of sGC was antagonized by the Cu(I)-specific chelator neocuproine, whereas the Cu(II)-selective drug cuprizone was less potent. Carbon dioxide (delivered as NaHCO3) antagonized S-nitrosation by peroxynitrite but not by SIN-1. Thus, NO/O2- released from SIN-1 mediates a CO2-insensitive conversion of GSH to S-nitrosoglutathione, a thionitrite that activates sGC via trace metal-catalyzed release of NO. These results may provide novel insights into the molecular mechanism underlying the nitrovasodilator action of SIN-1. PMID:9658207

  6. Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

    PubMed

    Jeong, Jipseol; Kim, Woo H; Fernandez, Cherry P; Kim, Suk; Kim, Yong-Hwan; Jang, Hyung-Kwan; Lillehoj, Hyun S; Woo, Hee-Jong; Min, Wongi

    2015-01-01

    The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens. PMID:25173813

  7. A Novel Soluble Form of Tim-3 Associated with Severe Graft-versus-Host Disease

    PubMed Central

    Hansen, John A.; Hanash, Samir M.; Tabellini, Laura; Baik, Chris; Lawler, Richard L.; Grogan, Bryan M.; Storer, Barry; Chin, Alice; Johnson, Melissa; Wong, Chee-Hong; Zhang, Qing; Martin, Paul J.; McDonald, George B.

    2014-01-01

    The T cell Ig and mucin domain 3 (Tim-3) receptor has been implicated as a negative regulator of adaptive immune responses. We have utilized a proteomic strategy to identify novel proteins associated with graft versus host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT). Mass spectrometry analysis of plasma from subjects with mid-gut and upper-gut GVHD compared with those without GVHD identified increased levels of a protein identified with high confidence as Tim-3. A follow-up validation study using an immunoassay to measure Tim-3 levels in individual plasma samples from 127 patients demonstrated significantly higher plasma Tim-3 concentrations in patients with the more severe mid-gut GVHD, compared with those with upper-gut GVHD (P = .005), patients without GVHD (P = .002), and normal controls (P < .0001). Surface expression of Tim-3 was increased on CD8+ T cells from patients with grade 2 to 4 acute GVHD (P = .01). Mass spectrometry–based profiling of plasma from multiple subjects diagnosed with common diseases provided evidence for restricted release of soluble Tim-3 in the context of GVHD. These findings have mechanistic implications for the development of novel strategies for targeting the Tim-3 immune regulatory pathway as an approach to improving control of GVHD. PMID:23791624

  8. Tunable functional hydrogels formed from a versatile water-soluble chitosan.

    PubMed

    Xiao, Congming; You, Rongrui; Fan, Ying; Zhang, Yue

    2016-04-01

    A versatile water-soluble chitosan (WSC) was applied to construct two kinds of controllable functional hydrogels. Magnetic beads were prepared by physical cross-linking WSC with sodium alginate, soaking particles with ferrous chloride and being subjected to self-oxidation. Magnetic character of the beads was tunable by simply changing the initial concentration of ferrous ions. The beads could bind compounds that contained different charges. Their adsorption capacities for coomassie brilliant blue, rhodamine and hemoglobin were 1, 0.5 and 2.3mg/g respectively. Another kind of functional hydrogel was prepared through radical cross-linking reaction between WSC and a macromonomer (PVAM) derived from well-defined polyvinyl alcohol. The dynamic mechanical thermal analysis and thermogravimetric analysis results revealed that the mechanical strength and thermal stability of this hydrogel depended on the structure of PVAM. The capability to bind heavy metal ions of the hydrogel also relied on the structure of PVAM. The adsorption capacities of the hydrogels for Cu(2+) and Pb(2+) could reach 20.3 and 60.1mg/g respectively. PMID:26772916

  9. DISTRIBUTION AND SOLUBILITY OF RADIONUCLIDES AND NEUTRON ABSORBERS IN WASTE FORMS FOR DISPOSITION OF PLUTONIUM ASH AND SCRAPS, EXCESS PLUTONIUM, AND MISCELLANEOUS SPENT NUCLEAR FUELS

    EPA Science Inventory

    The objective of this multi-institutional, multi-national research effort is to understand the distributions, solubilities, and releases of radionuclides and neutron absorbers in waste forms. The results will provide the underpinning knowledge for developing, evaluating, selectin...

  10. Active superconducting devices formed of thin films

    DOEpatents

    Martens, Jon S.; Beyer, James B.; Nordman, James E.; Hohenwarter, Gert K. G.

    1991-05-28

    Active superconducting devices are formed of thin films of superconductor which include a main conduction channel which has an active weak link region. The weak link region is composed of an array of links of thin film superconductor spaced from one another by voids and selected in size and thickness such that magnetic flux can propagate across the weak link region when it is superconducting. Magnetic flux applied to the weak link region will propagate across the array of links causing localized loss of superconductivity in the links and changing the effective resistance across the links. The magnetic flux can be applied from a control line formed of a superconducting film deposited coplanar with the main conduction channel and weak link region on a substrate. The devices can be formed of any type to superconductor but are particularly well suited to the high temperature superconductors since the devices can be entirely formed from coplanar films with no overlying regions. The devices can be utilized for a variety of electrical components, including switching circuits, amplifiers, oscillators and modulators, and are well suited to microwave frequency applications.

  11. Thin Films Formed from Conjugated Polymers with Ionic, Water-Soluble Backbones.

    PubMed

    Voortman, Thomas P; Chiechi, Ryan C

    2015-12-30

    This paper compares the morphologies of films of conjugated polymers in which the backbone (main chain) and pendant groups are varied between ionic/hydrophilic and aliphatic/hydrophobic. We observe that conjugated polymers in which the pendant groups and backbone are matched, either ionic-ionic or hydrophobic-hydrophobic, form smooth, structured, homogeneous films from water (ionic) or tetrahydrofuran (hydrophobic). Mismatched conjugated polymers, by contrast, form inhomogeneous films with rough topologies. The polymers with ionic backbone chains are conjugated polyions (conjugated polymers with closed-shell charges in the backbone), which are semiconducting materials with tunable bad-gaps, not unlike uncharged conjugated polymers. PMID:25723354

  12. Phenolic acids and antioxidant activity of distillers dried grains with solubles (DDGS) as compared with corn

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Sample sets of ground corn and the corresponding distillers dried grains with solubles (DDGS) were collected from three commercial plants in Iowa. Phenolic acids were analyzed by high performance liquid chromatography coupled with diode array and/or mass spectrometry. The antioxidant activity was ...

  13. Soluble urokinase plasminogen activation receptor - An emerging new biomarker of cardiovascular disease and critical illness

    PubMed Central

    Cyrille, Nicole B.; Villablanca, Pedro A.; Ramakrishna, Harish

    2016-01-01

    Soluble urokinase plasminogen activation receptor (suPAR) is an emerging new biomarker, which has been shown to not only correlate with traditional biomarkers but also outperform CRP at prognosticating CVD. More clinical trials on suPAR is in the future research agenda. PMID:27052059

  14. FATE OF WATER SOLUBLE AZO DYES IN THE ACTIVATED SLUDGE PROCESS

    EPA Science Inventory

    The objective of this study was to determine the partitioning of water soluble azo dyes in the activated sludge process (ASP). Azo dyes are of concern because some of the dyes, dye precursors , and/or their degradation products such as aromatic amines (which are also dye precurso...

  15. Highly Potent, Water Soluble Benzimidazole Antagonist for Activated (alpha)4(beta)1 Integrin

    SciTech Connect

    Carpenter, R D; Andrei, M; Lau, E Y; Lightstone, F C; Liu, R; Lam, K S; Kurth, M J

    2007-08-29

    The cell surface receptor {alpha}{sub 4}{beta}{sub 1} integrin, activated constitutively in lymphoma, can be targeted with the bisaryl urea peptidomimetic antagonist 1 (LLP2A). However, concerns on its preliminary pharmacokinetic (PK) profile provided an impetus to change the pharmacophore from a bisaryl urea to a 2-arylaminobenzimidazole moiety resulting in improved solubility while maintaining picomolar potency [5 (KLCA4); IC{sub 50} = 305 pM]. With exceptional solubility, this finding has potential for improving PK to help diagnose and treat lymphomas.

  16. Chemokines derived from soluble fusion proteins expressed in Escherichia coli are biologically active

    SciTech Connect

    Magistrelli, Giovanni; Gueneau, Franck; Muslmani, Machadiya; Ravn, Ulla; Kosco-Vilbois, Marie; Fischer, Nicolas . E-mail: nfischer@novimmune.com

    2005-08-26

    Chemokines are a class of low molecular weight proteins that are involved in leukocytes trafficking. Due to their involvement in recruiting immune cells to sites of inflammation, chemokines, and chemokine receptors have become an attractive class of therapeutic targets. However, when expressed in Escherichia coli chemokines are poorly soluble and accumulate in inclusion bodies. Several purification methods have been described but involve time-consuming refolding, buffer exchange, and purification steps that complicate expression of these proteins. Here, we describe a simple and reliable method to express chemokines as fusions to the protein NusA. The fusion proteins were largely found in the soluble fraction and could be readily purified in a single step. Proteolytic cleavage was used to obtain soluble recombinant chemokines that were found to be very active in a novel in vitro chemotaxis assays. This method could be applied to several {alpha} and {beta} human chemokines, suggesting that it is generally applicable to this class of proteins.

  17. Formulation and particle size reduction improve bioavailability of poorly water-soluble compounds with antimalarial activity.

    PubMed

    Wang, Hongxing; Li, Qigui; Reyes, Sean; Zhang, Jing; Xie, Lisa; Melendez, Victor; Hickman, Mark; Kozar, Michael P

    2013-01-01

    Decoquinate (DQ) is highly effective at killing malaria parasites in vitro; however, it is extremely insoluble in water. In this study, solid dispersion method was used for DQ formulation which created a suitable physical form of DQ in aqueous phase for particle manipulation. Among many polymers and surfactants tested, polyvinylpyrrolidone 10, a polymer, and L- α -phosphatidylcholine or polysorbate, two surfactants, were chosen as DQ formulation components. The formulation particles were reduced to a mean size between 200 to 400 nm, which was stable in aqueous medium for at least three weeks. Pharmacokinetic (PK) studies showed that compared to DQ microparticle suspension, a nanoparticle formulation orally dosed to mice showed a 14.47-fold increase in area under the curve (AUC) of DQ plasma concentration and a 4.53-fold increase in AUC of DQ liver distribution. WR 299666, a poorly water-soluble compound with antimalarial activity, was also tested and successfully made into nanoparticle formulation without undergoing solid dispersion procedure. We concluded that nanoparticles generated by using appropriate formulation components and sufficient particle size reduction significantly increased the bioavailability of DQ and could potentially turn this antimalarial agent to a therapeutic drug. PMID:23766925

  18. Determination of soluble CD21 as a parameter of B cell activation.

    PubMed

    Huemer, H P; Larcher, C; Prodinger, W M; Petzer, A L; Mitterer, M; Falser, N

    1993-08-01

    In this study we established a novel solid-phase immunoassay for CD21 using the time-resolved fluorescence of lanthanide chelates. The capture assay was able to detect concentrations of as low as 100 pg of CD21 antigen per millilitre of sample and was used for quantitative determination of CD21 in lysates of different cell lines as well as in patient serum specimens. CD21 was measured in lysates of tonsils and cell lines of B, T cell and myelomonocyte lineage, and appeared to consist of monomeric antigen under the detergent conditions used. Elevated levels of soluble CD21 were observed in serum of patients with Epstein-Barr virus (EBV) infection, a disease known to be associated with polyclonal B cell activation, and in infection with the lymphotropic rubella virus. Significantly increased levels were also found in malignancies which are associated with EBV. In patients with nasopharyngeal carcinoma (NPC), a correlation with the titre of EBV-specific IgA was observed, thus supporting a possible role of soluble CD21 as a marker for disease activity in certain malignancies. Our data suggest that measurement of soluble CD21 could serve as a marker for activation of the immune system and diseases involving the B cell lymphoid system. Possible mechanisms and functions of soluble CD21 are discussed. PMID:8348744

  19. Nanocrystals of medium soluble actives--novel concept for improved dermal delivery and production strategy.

    PubMed

    Zhai, Xuezhen; Lademann, Jürgen; Keck, Cornelia M; Müller, Rainer H

    2014-08-15

    After use in oral pharmaceutical products, nanocrystals are meanwhile applied to improve the dermal penetration of cosmetic actives (e.g. rutin, hesperidin) and of drugs. By now, nanocrystals are only dermally applied made from poorly soluble actives. The novel concept is to formulate nanocrystals also from medium soluble actives, and to apply a dermal formulation containing additionally nanocrystals. The nanocrystals should act as fast dissolving depot, increase saturation solubility and especially accumulate in the hair follicles, to further increase skin penetration. Caffeine was used as model compound with relevance to market products, and a particular process was developed for the production of caffeine nanocrystals to overcome the supersaturation related effect of crystal growth and fiber formation - typical with medium soluble compounds. It is based on low energy milling (pearl milling) in combination with low dielectric constant dispersion media (water-ethanol or ethanol-propylene glycol mixtures) and optimal stabilizers. Most successful was Carbopol(®) 981 (e.g. 20% caffeine in ethanol-propylene glycol 3:7 with 2% Carbopol, w/w). Nanocrystals with varied sizes can now be produced in a controlled process e.g. 660 nm (optimal for hair follicle accumulation) to 250 nm (optimal for fast dissolution). The short term test proved stability over 2 months of the present formulation being sufficient to perform in vivo testing of the novel concept. PMID:24813782

  20. Serum albumin forms a lactoferrin-like soluble iron-binding complex in presence of hydrogen carbonate ions.

    PubMed

    Ueno, Hiroshi M; Urazono, Hiroshi; Kobayashi, Toshiya

    2014-02-15

    The iron-lactoferrin complex is a common food ingredient because of its iron-solubilizing capability in the presence of hydrogen carbonate ions. However, it is unclear whether the formation of a stable iron-binding complex is limited to lactoferrin. In this study, we investigated the effects of bovine serum albumin (BSA) on iron solubility and iron-catalyzed lipid oxidation in the presence of hydrogen carbonate ions. BSA could solubilize >100-fold molar equivalents of iron at neutral pH, exceeding the specific metal-binding property of BSA. This iron-solubilizing capability of BSA was impaired by thermally denaturing BSA at ≥ 70 °C for 10 min at pH 8.5. The resulting iron-BSA complex inhibited iron-catalyzed oxidation of soybean oil in a water-in-oil emulsion measured using the Rancimat test. Our study is the first to show that BSA, like lactoferrin, forms a soluble iron-binding complex in the presence of hydrogen carbonate ions. PMID:24128453

  1. Development of a new generation of waste form for entrapment and immobilization of highly volatile and soluble radionuclides.

    SciTech Connect

    Rodriguez, Mark Andrew; Bencoe, Denise Nora; Brinker, C. Jeffrey; Murphy, Andrew Wilson; Holt, Kathleen Caroline; Turnham, Rigney; Kruichak, Jessica Nicole; Tellez, Hernesto; Miller, Andy; Xiong, Yongliang; Pohl, Phillip Isabio; Ockwig, Nathan W.; Wang, Yifeng; Gao, Huizhen

    2010-09-01

    The United States is now re-assessing its nuclear waste disposal policy and re-evaluating the option of moving away from the current once-through open fuel cycle to a closed fuel cycle. In a closed fuel cycle, used fuels will be reprocessed and useful components such as uranium or transuranics will be recovered for reuse. During this process, a variety of waste streams will be generated. Immobilizing these waste streams into appropriate waste forms for either interim storage or long-term disposal is technically challenging. Highly volatile or soluble radionuclides such as iodine ({sup 129}I) and technetium ({sup 99}Tc) are particularly problematic, because both have long half-lives and can exist as gaseous or anionic species that are highly soluble and poorly sorbed by natural materials. Under the support of Sandia National Laboratories (SNL) Laboratory-Directed Research & Development (LDRD), we have developed a suite of inorganic nanocomposite materials (SNL-NCP) that can effectively entrap various radionuclides, especially for {sup 129}I and {sup 99}Tc. In particular, these materials have high sorption capabilities for iodine gas. After the sorption of radionuclides, these materials can be directly converted into nanostructured waste forms. This new generation of waste forms incorporates radionuclides as nano-scale inclusions in a host matrix and thus effectively relaxes the constraint of crystal structure on waste loadings. Therefore, the new waste forms have an unprecedented flexibility to accommodate a wide range of radionuclides with high waste loadings and low leaching rates. Specifically, we have developed a general route for synthesizing nanoporous metal oxides from inexpensive inorganic precursors. More than 300 materials have been synthesized and characterized with x-ray diffraction (XRD), BET surface area measurements, and transmission electron microscope (TEM). The sorption capabilities of the synthesized materials have been quantified by using stable

  2. Zinc oxide nanoparticles and monocytes: Impact of size, charge and solubility on activation status

    SciTech Connect

    Prach, Morag; Stone, Vicki; Proudfoot, Lorna

    2013-01-01

    Zinc oxide (ZnO) particle induced cytotoxicity was dependent on size, charge and solubility, factors which at sublethal concentrations may influence the activation of the human monocytic cell line THP1. ZnO nanoparticles (NP; average diameter 70 nm) were more toxic than the bulk form (< 44 μm mesh) and a positive charge enhanced cytotoxicity of the NP despite their relatively high dissolution. A positive charge of the particles has been shown in other studies to have an influence on cell viability. Centrifugal filtration using a cut off of 5 kDa and Zn element analysis by atomic absorption spectroscopy confirmed that exposure of the ZnO particles and NP to 10% foetal bovine serum resulted in a strong association of the Zn{sup 2+} ion with protein. This association with protein may influence interaction of the ZnO particles and NP with THP1 cells. After 24 h exposure to the ZnO particles and NP at sublethal concentrations there was little effect on immunological markers of inflammation such as HLA DR and CD14, although they may induce a modest increase in the adhesion molecule CD11b. The cytokine TNFα is normally associated with proinflammatory immune responses but was not induced by the ZnO particles and NP. There was also no effect on LPS stimulated TNFα production. These results suggest that ZnO particles and NP do not have a classical proinflammatory effect on THP1 cells. -- Highlights: ► ZnO is cytotoxic to THP-1 monocytes. ► ZnO nanoparticles are more toxic than the bulk form. ► Positive charge enhances ZnO nanoparticle cytotoxicity. ► Sublethal doses of ZnO particles do not induce classical proinflammatory markers.

  3. Soluble Immune Mediators and Vaginal Bacteria Impact Innate Genital Mucosal Antimicrobial Activity in Young Women

    PubMed Central

    Madan, Rebecca Pellett; Dezzutti, Charlene S.; Rabe, Lorna; Hillier, Sharon L.; Marrazzo, Jeanne; McGowan, Ian; Richardson, Barbra A.; Herold, Betsy C.

    2015-01-01

    Introduction Innate activity against Escherichia coli in female genital secretions may represent contributions from vaginal bacteria and host soluble immune mediators. We analyzed the relationship between E. coli inhibitory activity, soluble immune mediators, and vaginal bacteria in participants in MTN-004, a placebo-controlled trial of VivaGel®, a candidate product for topical HIV pre-exposure prophylaxis. Methods Escherichia coli inhibitory activity was quantified by colony reduction assay. Endocervical concentrations of interleukin (IL)-1β, IL-6, IL-12p40, macrophage inflammatory protein (MIP)-1α, granulocyte– macrophage colony-stimulating factor (GM-CSF), lactoferrin, and secretory leukocyte protease inhibitor (SLPI) were quantified to generate a cumulative mediator score. Vaginal bacteria were characterized by quantitative cultures. Results In the two placebo arms, higher soluble immune mediator score was associated with greater E. coli inhibitory activity (β = 17.49, 95% CI [12.77, 22.21] and β = 13.28, 95% CI [4.76, 21.80]). However, in the VivaGel arm, higher concentrations of E. coli (β = −3.80, 95% CI [−6.36, −1.25]) and group B Streptococcus (β = −3.91, 95% CI [−6.21, −1.60]) were associated with reduced E. coli inhibitory activity. Conclusions Both host mediators and vaginal bacteria impact E. coli inhibition in genital secretions. The relative contributions of host mediators and bacteria varied between women who used VivaGel vs placebos. PMID:26118476

  4. Thermodynamic assessment of solubility and activity of iron, chromium, and nickel in lead bismuth eutectic

    NASA Astrophysics Data System (ADS)

    Gossé, Stéphane

    2014-06-01

    Lead-Bismuth Eutectic (LBE) is a heavy liquid alloy used as a coolant for the Lead-Cooled Fast Reactors and spallation target for Accelerator Driven Systems. LBE is also considered in sodium fast reactor designs as coolant in secondary circuit to avoid any occurrence of the reaction between sodium and water in steam generators. Even if this coolant presents many advantages due to its thermophysical properties, corrosion towards structural materials remains one of the major issues of LBE. Because corrosion in LBE is partly driven by dissolution processes, the solubility and chemical activity of the main elements of the alloy are the key parameters to model the related corrosion processes. Using the Calphad method and the Thermo-Calc software, a thermodynamic database was developed to assess the interaction between Cr-Ni-Fe alloys and LBE. The current thermodynamic data on the Cr-Fe-Ni + Bi-Pb quinary system was reviewed and the Bi-Cr and Cr-Pb binary phase diagrams were assessed. Fe, Cr and Ni solubilities (in at. fraction, T in K) at LBE composition were calculated: Fe solubility at LBE composition: log10 (SFe)=0.5719-4398.6T (399-1173 K) Cr solubility at LBE composition: log10 (SCr)=-0.2757-3056.1T (399-1173 K) Ni solubility at LBE composition: log10 (SNi)=2.8717-2932.9T (528-742 K) log10 (SNi)=0.2871-1006.3T (742-1173 K) Then, the thermodynamic assessment performed in this study was used to predict more accurately the Fe, Cr and Ni activities and solubilities in the case of four austenitic model alloys also studied in the framework of corrosion tests [1]. The calculated activities and solubilities provide thermodynamic data to better understand dissolution or precipitation phenomena observed during LBE corrosion processes.

  5. Critical evaluation of changes in the ratio of insoluble bound to soluble phenolics on antioxidant activity of lentils during germination.

    PubMed

    Yeo, JuDong; Shahidi, Fereidoon

    2015-01-21

    A new indicator, the ratio of insoluble bound phenolics (IBPs) to soluble phenolics (SPs), is suggested as an effective means to monitor changes in the antioxidant activity of lentils during germination. This indicator may be used to monitor other process-induced changes in antioxidant potential of food phenolics in other foods. The antioxidant activity of SPs, IBPs, and total value, the sum of both free and esterified phenolics, of germinated CDC Richlea lentil variety was evaluated for 4 days. Total phenolic content (TPC), total flavonoid content (TFC), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical cation scavenging ability were employed to record antioxidant activities. An incremental increase in IBPs was found in TPC, TFC, DPPH, and ABTS radical cation scavenging ability, whereas SPs showed a declining trend in TFC, DPPH, and ABTS, except TPC during 4 days of germination. The ratio of IBPs to SPs increased using most methods, and this may be possibly due to the changes of phenolic compound formation from soluble into insoluble bound form during germination process. The ratio can be used as a novel method for monitoring process-induced changes in the antioxidant activity of foods. PMID:25560637

  6. Enhanced water-solubility, antibacterial activity and biocompatibility upon introducing sulfobetaine and quaternary ammonium to chitosan.

    PubMed

    Chen, Yuxiang; Li, Jianna; Li, Qingqing; Shen, Yuanyuan; Ge, Zaochuan; Zhang, Wenwen; Chen, Shiguo

    2016-06-01

    Chitosan (CS) has attracted much attention due to its good antibacterial activity and biocompatibility. However, CS is insoluble in neutral and alkaline aqueous solution, limiting its biomedical application to some extent. To circumvent this drawback, we have synthesized a novel N-quaternary ammonium-O-sulfobetaine-chitosan (Q3BCS) by introducing quaternary ammonium compound (QAC) and sulfobetaine, and its water-solubility, antibacterial activity and biocompatibility were evaluated compare to N-quaternary ammonium chitosan and native CS. The results showed that by introducing QAC, antibacterial activities and water-solubilities increase with degrees of substitution. The largest diameter zone of inhibition (DIZ) was improved from 0 (CS) to 15mm (N-Q3CS). And the water solution became completely transparent from pH 6.5 to pH 11; the maximal waters-solubility was improved from almost 0% (CS) to 113% at pH 7 (N-Q3CS). More importantly, by further introducing sulfobetaine, cell survival rate of Q3BCS increased from 30% (N-Q3CS) to 85% at 2000μg/ml, which is even greater than that of native CS. Furthermore, hemolysis of Q3BCS was dropped sharply from 4.07% (N-Q3CS) to 0.06%, while the water-solution and antibacterial activity were further improved significantly. This work proposes an efficient strategy to prepare CS derivatives with enhanced antibacterial activity, biocompatibility and water-solubility. Additionally, these properties can be finely tailored by changing the feed ratio of CS, glycidyl trimethylammonium chloride and NCO-sulfobetaine. PMID:27083366

  7. Kex2 protease converts the endoplasmic reticulum α1,2-mannosidase of Candida albicans into a soluble cytosolic form

    PubMed Central

    Mora-Montes, Héctor M.; Bader, Oliver; López-Romero, Everardo; Zinker, Samuel; Ponce-Noyola, Patricia; Hube, Bernhard; Gow, Neil A. R.; Flores-Carreón, Arturo

    2008-01-01

    Cytosolic α-mannosidases are glycosyl hydrolases that participate in the catabolism of cytosolic free N-oligosaccharides. Two soluble α-mannosidases (E-I and E-II) belonging to glycosyl hydrolases family 47 have been described in Candida albicans. We demonstrate that addition of pepstatin A during the preparation of cell homogenates enriched α-mannosidase E-I at the expense of E-II, indicating that the latter is generated by proteolysis during cell disruption. E-I corresponded to a polypeptide of 52 kDa that was associated with mannosidase activity and was recognized by an anti-α1,2-mannosidase antibody. The N-mannan core trimming properties of the purified enzyme E-I were consistent with its classification as a family 47 α1,2-mannosidase. Differential density-gradient centrifugation of homogenates revealed that α1,2-mannosidase E-I was localized to the cytosolic fraction and Golgi-derived vesicles, and that a 65 kDa membrane-bound α1,2-mannosidase was present in endoplasmic reticulum and Golgi-derived vesicles. Distribution of α-mannosidase activity in a kex2Δ null mutant or in wild-type protoplasts treated with monensin demonstrated that the membrane-bound α1,2-mannosidase is processed by Kex2 protease into E-I, recognizing an atypical cleavage site of the precursor. Analysis of cytosolic free N-oligosaccharides revealed that cytosolic α1,2-mannosidase E-I trims free Man8GlcNAc2 isomer B into Man7GlcNAc2 isomer B. This is believed to be the first report demonstrating the presence of soluble α1,2-mannosidase from the glycosyl hydrolases family 47 in a cytosolic compartment of the cell. PMID:19047746

  8. Multiple forms of soluble monophenol, dihydroxyphenylalanine: oxygen oxidoreductase (EC 1.14.18.1) from potato tubers (Solanum tuberosum). IV. Association and dissociation phenomena.

    PubMed

    Matheis, G; Belitz, H D

    1979-10-01

    The soluble phenol oxidase of various potato juices (adjusted from physiological pH to pH 4.5, 7.0 and 7.8) was separated by gel chromatography into multiple molecular forms. In acid or neutral and alkaline potato juices, low-mol.-wt. (less than 150,000 daltons) or high-mol.-wt. (greater than 150,000 daltons) enzyme forms predominate, respectively. Conversion of the low-mol.-wt. enzyme forms into high-mol.-wt. enzyme forms, and vice versa, was achieved by changing the pH values from acidic to neutral or alkaline pH, and vice versa. This substantiated our previous idea that the enzyme multiplicity arises from association of various subunits. In alkaline potato juice, considerable loss of monophenol oxidase activity (assayed at pH 6.0) occurred. This confirmed our previous findings that o-diphenol oxidase is more alkali-stable than monophenol oxidase. PMID:42235

  9. Soluble guanylate cyclase is activated differently by excess NO and by YC-1: Resonance Raman spectroscopic evidence†

    PubMed Central

    Ibrahim, Mohammed; Derbyshire, Emily R.; Soldatova, Alexandra V.; Marletta, Michael A.; Spiro, Thomas G.

    2010-01-01

    Modulation of soluble guanylate cyclase (sGC) activity by nitric oxide (NO) involves two distinct steps. Low level activation of sGC is achieved by the stoichiometric binding of NO (1-NO) to the heme cofactor, while much higher activation is achieved by the binding of additional NO (xsNO) at a non-heme site. Addition of the allosteric activator YC-1 to the 1-NO form leads to activity comparable to xsNO state. In this study the mechanisms of sGC activation were investigated using electronic absorption and resonance Raman (RR) spectroscopic methods. RR spectroscopy confirmed that the 1-NO form contains 5-coordinate NO-heme and showed that the addition of NO to the 1-NO form has no significant effect on the spectrum. In contrast, addition of YC-1 to either the 1-NO or xsNO forms alters the RR spectrum significantly, indicating a protein-induced change in the heme geometry. This change in the heme geometry was also observed when BAY 41-2272 was added to the xsNO form. Bands assigned to bending and stretching motions of the vinyl and propionate substituents change intensity in a pattern suggesting altered tilting of the pyrrole rings to which they are attached. In addition, the N-O stretching frequency increases, with no change in the Fe-NO frequency, an effect modeled via DFT calculations as resulting from a small opening of the Fe-N-O angle. These spectral differences demonstrate different mechanisms of activation by synthetic activators, such as YC-1 and BAY 41-2272, and excess NO. PMID:20459051

  10. Thioredoxin fusion construct enables high-yield production of soluble, active matrix metalloproteinase-8 (MMP-8) in Escherichia coli.

    PubMed

    McNiff, M L; Haynes, E P; Dixit, N; Gao, F P; Laurence, J S

    2016-06-01

    Matrix metalloproteinases (MMPs) are crucial proteases in maintaining the health and integrity of many tissues, however their dysregulation often facilitates disease progression. In disease states these remodeling and repair functions support, for example, metastasis of cancer by both loosening the matrix around tumors to enable cellular invasion and by affecting proliferation and apoptosis, and they promote degradation of biological restorations by weakening the substrate to which the restoration is attached. As such, MMPs are important therapeutic targets. MMP-8 participates in cancer, arthritis, asthma and failure of dental fillings. MMP-8 differs from other MMPs in that it has an insertion that enlarges its active site. To elucidate the unique features of MMP-8 and develop selective inhibitors to this therapeutic target, a stable and active form of the enzyme is needed. MMP-8 has been difficult to express at high yield in a soluble, active form. Typically recombinant MMPs accumulate in inclusion bodies and complex methods are applied to refold and purify protein in acceptable yield. Presented here is a streamlined approach to produce in Escherichia coli a soluble, active, stable MMP-8 fusion protein in high yield. This fusion shows much greater retention of activity when stored refrigerated without glycerol. A variant of this construct that contains the metal binding claMP Tag was also examined to demonstrate the ability to use this tag with a metalloprotein. SDS-PAGE, densitometry, mass spectrometry, circular dichroism spectroscopy and an activity assay were used to analyze the chemical integrity and function of the enzyme. PMID:26923061

  11. Identification of small-molecule binding pockets in the soluble monomeric form of the Aβ42 peptide

    PubMed Central

    Zhu, Maximillian; Simone, Alfonso De; Schenk, Dale; Toth, Gergely; Dobson, Christopher M.; Vendruscolo, Michele

    2016-01-01

    The aggregation of intrinsically disordered peptides and proteins is associated with a wide range of highly debilitating neurological and systemic disorders. In this work we explored the potential of a structure-based drug discovery procedure to target one such system, the soluble monomeric form of the Aβ42 peptide. We utilised for this purpose a set of structures of the Aβ42 peptide selected from clusters of conformations within an ensemble generated by molecular dynamics simulations. Using these structures we carried out fragment mapping calculations to identify binding ‘hot spots’ on the monomeric form of the Aβ42 peptide. This procedure provided a set of hot spots with ligand efficiencies comparable to those observed for structured proteins, and that are clustered into binding pockets. We verified that such pockets exhibit a propensity to bind small molecules known to interact with the Aβ42 peptide. Taken together these results provide an initial indication that fragment-based drug discovery may represent a potential therapeutic strategy for diseases associated with the aggregation of intrinsically disordered proteins. PMID:23883055

  12. Antipneumocystis activity of water-soluble lipopeptide L-693,989 in rats.

    PubMed Central

    Schmatz, D M; Powles, M A; McFadden, D C; Pittarelli, L; Balkovec, J; Hammond, M; Zambias, R; Liberator, P; Anderson, J

    1992-01-01

    Water-soluble lipopeptide L-693,989 was evaluated for its antipneumocystis activity in rats. Rats from colonies with latent Pneumocystis carinii infections were immunosuppressed with dexamethasone for 6 weeks to facilitate the development of acute P. carinii pneumonia (PCP). After 6 weeks, the rats were maintained on dexamethasone and were treated twice daily for 4 days with various concentrations of L-693,989. At a dose of 0.15 mg/kg of body weight, the compound effectively eliminated 90% of the cysts in 4 days. Trophozoite forms of P. carinii were still present in these animals, as determined by using a P. carinii-specific DNA probe. A 3-week therapy study showed that the trophozoite load did not expand during treatment and that the trophozoites already present at the initiation of therapy appeared to persist. This may be a consequence of the stage specificity of the compound for cyst development and the severe immunosuppressive effects of dexamethasone on rats. When evaluated as a daily parenteral prophylactic agent, L-693,989 was effective in preventing the development of both P. carinii cysts and trophozoites, demonstrating its potential for use in prophylaxis and implying that the cyst stage of P. carinii is an obligatory step in trophozoite multiplication. The foamy exudate commonly associated with P. carinii infections was absent in the lungs of rats on prophylaxis. The compound was also evaluated via oral administration and was found to have a 90% effective dose of 32 mg/kg for therapy of acute infections and 5 mg/kg for daily prophylaxis. Images PMID:1416888

  13. Effects of soluble dietary fibers on lipid metabolism and activities of intestinal disaccharidases in rats.

    PubMed

    Choi, Y S; Cho, S H; Kim, H J; Lee, H J

    1998-10-01

    The present study was aimed to investigate the effects of indigestible dextrin and polydextrose, soluble dietary fibers with low molecular weight, on lipid metabolism and disaccharidase activities of intestinal mucosa in rats fed a high sucrose diet. Their effects were compared with those of well-known soluble fibers, pectin, and guar gum, and also with an insoluble fiber, cellulose. Dietary fibers added to diets at the 5% (w/w) level were alpha-cellulose, pectin, guar gum, indigestible dextrin, and polydextrose. Male Sprague-Dawley rats were given free access to test diets for 6 weeks. Body weight gain was the lowest in rats fed guar gum, the highest in rats fed cellulose, and in-between in rats fed other diets. Although guar gum, pectin, and indigestible feeding dextrin had lower plasma lipid values than cellulose feeding did, the differences were statistically insignificant. Liver triglyceride of the guar gum-fed group was about a third that of the cellulose-fed group, but although those of rats fed polydextrose, indigestible dextrin, and pectin were lower than that of cellulose, the differences were insignificant. Liver cholesterol and phospholipid concentrations were similar among groups. Daily fecal excretion of total lipid, cholesterol, and bile acids were highest in rats fed guar gum, followed by pectin-fed and cellulose-fed rats, and the lowest in rats fed indigestible dextrin and polydextrose. Jejunal sucrase activity was low in the order of guar-gum, polydextrose, indigestible dextrin, pectin, and cellulose. The results indicate that the hypolipidemic effect of soluble dietary fibers would be lessened with reduction in molecular weight, but that the lower sucrase activity by soluble fibers with low molecular weight might be beneficial for hypoglycemic effect. PMID:9919480

  14. The Soluble Form of the EIAV Receptor Encoded by an Alternative Splicing Variant Inhibits EIAV Infection of Target Cells

    PubMed Central

    Zhang, Shu-Qin; Sun, Liu-Ke; Wang, Xue-Feng; Du, Cheng; Zhou, Jian-Hua

    2013-01-01

    Equine lentivirus receptor 1 (ELR1) has been identified as the sole receptor for equine infectious anemia virus (EIAV) and is a member of the tumor necrosis factor receptor (TNFR) superfamily. In addition to the previously described membrane-associated form of ELR1, two other major alternative splicing variant mRNAs were identified in equine monocyte-derived macrophages (eMDMs). One major spliced species (ELR1-IN) contained an insertion of 153 nt, which resulted in a premature stop codon situated 561 nt upstream of the predicted membrane spanning domain. The other major species (ELR1-DE) has a deletion of 109 nt that causes a shift of the open reading frame and generates a stop codon 312 nt downstream. Because ELR1-DE presumably encodes a peptide of a mere 23 residues, only ELR1-IN was further analyzed. The expression of a soluble form of ELR1 (sELR1) by ELR1-IN was confirmed by Western blot and immunofluorescence analyses. Similar to ELR1, the transcription level of ELR1-IN varied among individual horses and at different time points in the same individuals. The ratio of ELR1-IN mRNA species to ELR1 mRNA was approximately 1∶2.5. Pre-incubation of the recombinant sELR1 with EIAV significantly inhibited EIAV infection in equine macrophages, the primary in vivo target cell of the virus. Fetal equine dermal (FED) cells are susceptible to EIAV in vitro, and the replication of EIAV in FED cells transiently transfected with ELR1-IN was markedly reduced when compared with replication in cells transfected with the empty vector. Finally, the expression levels of both forms of the EIAV receptor were significantly regulated by infection with this virus. Taken together, our data indicate that sELR1 acts as a secreted cellular factor that inhibits EIAV infection in host cells. PMID:24278125

  15. Improved estimation of solubility and partitioning through correction of UNIFAC-derived activity coefficients

    SciTech Connect

    Banerjee, S.; Howard, P.H.

    1988-07-01

    Octanol-water partition coefficients (K/sub ow/) of 75 compounds ranging over 9 orders of magnitude are correlated by log K/sub ow/ = -0.40 + 0.73 log (..gamma../sub W/)/sub U/ -0.39 log (..gamma../sub 0/)/sub U/ (r = 0.98), where (..gamma..//sub W/)/sub U/ and (..gamma../sub 0/)/sub U/ are UNIFAC-derived activity coefficients in water and octanol, respectively. The constants 0.73 and -0.39 are obtained empirically and are intended to compensate for group nonadditivity. Correction factors of similar magnitude are obtained in independent correlations of water solubility with (..gamma../sub W/)/sub U/ and of octanol solubility with (..gamma../sub 0/)/sub U/, thereby confirming the validity of the approach.

  16. Activation of rat complement by soluble and insoluble rat IgA immune complexes.

    PubMed

    Rits, M; Hiemstra, P S; Bazin, H; Van Es, L A; Vaerman, J P; Daha, M R

    1988-12-01

    The ability of rat monoclonal IgA, specific for 2,4-dinitrophenyl (DNA), to activate the complement (C) system of the rat was investigated using aggregated IgA or IgA immune complexes (IC). IgA was coated onto a solid phase, and tested for its capacity to bind C3 upon incubation at 37 degrees C in normal rat serum (NRS) in the presence of Mg-EGTA. Binding of C3 was observed dependent on the dose of dimeric (d-), polymeric (p-) and secretory IgA tested. In contrast, little C3 fixation was observed in this system with monomeric (m-) rat IgA or with mouse m- and d-IgA (MOPC315). Soluble and insoluble rat IgA IC were prepared using dinitrophenylated rat serum albumin (DNP8RSA) as antigen (Ag), and assessed for C activation. It was shown that insoluble IC (immune precipitates; IP) containing m-, d- or pIgA of rat origin activate the alternative pathway of rat C, as demonstrated by their capacity to induce C consumption in NRS in the presence of Mg-EGTA. When p- and m-IgA IP were compared for their capacity to activate C, it was found that p-IgA activated C four times as efficiently as m-IgA IP (at 2 mg/ml). Soluble rat IgA IC were prepared in an excess of DNP8RSA, fractionated by gel filtration on Sepharose 6B, and analyzed for C activation and antibody (Ab)/Ag ratio. In contrast to m-IgA IP, soluble m-IgA did not activate C. On the other hand soluble d-IgA IC activated C dependent on their concentration and size: at a concentration of 0.1 mg/ml high-molecular weight d-IgA IC with a high Ab/Ag ratio were four times as efficient as low-molecular weight IC with a low Ab/Ag ratio, and twice as efficient as IP prepared at equivalence. To demonstrate the induction by IgA of the assembly of the terminal membrane attack complex, trinitrophenyl (TNP)-conjugated rat red blood cells (TNP-RRBC) coated with d- or p-IgA were shown to be lysed in NRS in the presence of Mg-EGTA. No lysis of m-IgA-coated TNP-RRBC was observed. The results in this study demonstrate that both soluble and

  17. Enhanced photocatalytic activity of nano titanium dioxide coated on ethanol-soluble carbon nanotubes

    SciTech Connect

    Fu, Xiaofei; Yang, Hanpei; He, Kuanyan; Zhang, Yingchao; Wu, Junming

    2013-02-15

    Graphical abstract: Homogenous and dense spreading of TiO{sub 2} on surface modified CNTs and improved photocatalytic performance of TiO{sub 2} was achieved by coupling TiO{sub 2} with ethanol-soluble CNTs. Display Omitted Highlights: ► Ethanol-soluble CNTs were acquired by surface modification. ► Enhanced photoactivity of TiO{sub 2} coated on modified CNTs was obtained. ► Improved activity of TiO{sub 2} is attributed to the intimate contact between TiO{sub 2} and CNTs. ► Dense heterojunctions through Ti–O–CNTs at the interface is proposed. -- Abstract: Surface functionalized carbon nanotubes (CNTs) with ethanol solubility were synthesized and the CNTs–TiO{sub 2} nanocomposites were prepared by coupling of TiO{sub 2} with modified CNTs through a sol–gel method. The as-prepared CNTs and composites were characterized and the composite samples were evaluated for their photocatalytic activity toward the degradation of aqueous methyl orange. It is showed that the acid oxidation of CNTs leads to the embedding of oxygenated functional groups, and as a result, the acid-treated CNTs in turn may serve as chemical reactors for subsequent covalent grafting of octadecylamine. Improved photocatalytic performance of CNTs–TiO{sub 2} composites was obtained, which is mainly attributed to the high dispersion of TiO{sub 2} on ethanol-soluble CNTs and the intimate contact between TiO{sub 2} and CNTs resulted from the dense heterojunctions through the Ti-O-C structure at the interface between TiO{sub 2} and CNTs.

  18. Induction of classical activation of macrophage in vitro by water soluble chitin

    NASA Astrophysics Data System (ADS)

    Jeon, Dong-Won; Ahn, Woong Shick; You, Su Jung; Chae, Gue Tae; Shim, Young Bock; Chun, Heung Jae

    2012-12-01

    The purpose of this study is to understand the effect of chitin on macrophage mediated immunity, which is a significant factor to wound healing and tissue regeneration. In this work, water soluble chitin (WSC) was prepared by re-acetylation of chitosan and was treated with the murine RAW 264.7 macrophage cell lines (ATCC TIB-71). WSC induced classical activation in the RAW 264.7 cells, accompanied by the induction of associated genes. The results suggest that WSC is one of the functional chitin molecules that are responsible for the immune response, especially present in macrophage classical activation.

  19. Distribution and Solubility of Radionuclides and Neutron Absorbers in Waste Forms for Disposition of Plutonium Ash and Scraps, Excess Plutonium, and Miscellaneous Spent Nuclear Fuels

    SciTech Connect

    Dr. Denis M. Strachan; Dr. David K. Shuh; Dr. Rodney C. Ewing; Dr. Eric R. Vance

    2002-09-23

    The initial goal of this project was to investigate the solubility of radionuclides in glass and other potential waste forms for the purpose of increasing the waste loading in glass and ceramic waste forms. About one year into the project, the project decided to focus on two potential waste forms - glass at PNNL and itianate ceramics at the Australian Nuclear Science and Technology Organisation (ANSTO).

  20. Measurement of FeO activity and solubility of MgO in smelting slags

    NASA Astrophysics Data System (ADS)

    Liu, Shih-Hsien; Fruehan, R. J.; Morales, A.; Ozturk, B.

    2001-02-01

    In bath smelting, the FeO activity of the slag must be known to predict the equilibrium of slag-metal reactions and for effective control of the rate of reduction in the system. Also, knowledge of the solubility of MgO in these slags is useful for reducing refractory consumption. A series of measurements of the FeO activity in simulated bath smelting slags (CaO-SiO2-Al2O3-MgOsat-FeO) were conducted by the electromotive force (EMF) technique. The influence of the slag composition on the relationship between the FeO activity coefficient and FeO content was studied. It has been found that the measured FeO activity coefficient decreases with increasing FeO content in the slag and increases slightly with increasing slag basicity, which is defined as (CaO + MgO)/(SiO2 + Al2O3) on a mole fraction basis. The measured values of the FeO activity coefficient are in reasonable agreement with previously published data. The solubility of MgO was also measured and found to rang from 16 to 30 pct and decrease with increasing basicity.

  1. Multiple active forms of thrombin. IV. Relative activities of meizothrombins

    SciTech Connect

    Doyle, M.F.; Mann, K.G. )

    1990-06-25

    The prothrombin activation intermediates meizothrombin and meizothrombin(desF1) (meizothrombin that has been autoproteolyzed to remove fragment 1) have been obtained in a relatively pure, active form with minimal autolysis, making them suitable for enzymatic characterization. When compared at equimolar concentrations, alpha-thrombin, fragment 1.2+ alpha-thrombin, meizothrombin(desF1), and meizothrombin have approximately 100, 100, 10, and 1% activity, respectively, toward the macromolecular substrates factor V, fibrinogen, and platelets. The difference in activity of these four enzymes cannot be attributed to alterations in the catalytic triad, as all four enzymes have nearly identical catalytic efficiency toward the chromogenic substrate S2238. Further, the ability of meizothrombin and meizothrombin(desF1) to activate protein C was 75% of the activity exhibited by alpha-thrombin or fragment 1.2+ alpha-thrombin. All four enzymes bind to thrombomodulin, as judged by the enhanced rate of protein C activation upon preincubation of the enzymes with thrombomodulin. The extent of rate enhancement varied, with meizothrombin/thrombomodulin exhibiting only 50% of the alpha-thrombin/thrombomodulin rate. This difference in rate is not due to a decreased affinity of the meizothrombin for thrombomodulin since the apparent dissociation constants for the alpha-thrombin-thrombomodulin complex and the meizothrombin-thrombomodulin complex are virtually identical. The difference in the observed rate is due in part to the higher Km for protein C exhibited by the meizothrombin-thrombomodulin complex. Incubation of the thrombomodulin-enzyme complex with phospholipid vesicles caused an increase in the protein C activation rates. The kinetic constants for protein C activation in the presence of phospholipid are virtually identical for these enzyme-thrombomodulin complexes.

  2. The contribution of acidulant to the antibacterial activity of acid soluble α- and β-chitosan solutions and their films.

    PubMed

    Jung, Jooyeoun; Cavender, George; Zhao, Yanyun

    2014-01-01

    This study evaluated individual contributions of dissolving acids (acetic acid, lactic acid, and hydrochloric acid) or acid solubilized chitosan to the antibacterial activity against Listeria innocua and Escherichia coli as solutions and dried films. Solutions containing chitosan showed significantly (P < 0.05) different inhibitory activity (measured as percentage of inhibition (PI), in percent) against L. innocua and E. coli, compared to equivalent acid solutions. This increase was calculated as additional inhibition (AI, in percent), which could be as high as 65% in solutions containing 300-320 kDa chitosan depending on the acid type, bacterial species, and the chitosan form (α or β). Solutions containing 4-5 kDa chitosan had lower AI and showed much greater variability among the different chitosan forms, acid types, and bacterial species. Higher molecular weight (Mw) chitosan also showed significantly higher levels of adsorption to bacterial cells than that of lower Mw samples, suggesting that the observed increase in inhibition was the result of surface phenomena. The contribution of acids to the antibacterial activity of chitosan films was assessed by comparing non-rinsed and rinsed films (rinsed in the appropriate broth to remove residual acids and active fragments formed on the dried film). Rinsing β-chitosan films has reduced PI by as much as 28% compared with non-rinsed films, indicating that part of the antibacterial activity of chitosan films is due to the presence of soluble acid compounds and/or other active fragments. Overall, both acidulant and chitosan were found to contribute to the antibacterial activity of acid solubilized α- and β-chitosan, with the exact antibacterial activity of chitosan varying based on the solution and film properties, suggesting a complex interaction. PMID:24196584

  3. Inhibition of Coxsackie B Virus Infection by Soluble Forms of Its Receptors: Binding Affinities, Altered Particle Formation, and Competition with Cellular Receptors

    PubMed Central

    Goodfellow, Ian G.; Evans, David J.; Blom, Anna M.; Kerrigan, Dave; Miners, J. Scott; Morgan, B. Paul; Spiller, O. Brad

    2005-01-01

    We previously reported that soluble decay-accelerating factor (DAF) and coxsackievirus-adenovirus receptor (CAR) blocked coxsackievirus B3 (CVB3) myocarditis in mice, but only soluble CAR blocked CVB3-mediated pancreatitis. Here, we report that the in vitro mechanisms of viral inhibition by these soluble receptors also differ. Soluble DAF inhibited virus infection through the formation of reversible complexes with CVB3, while binding of soluble CAR to CVB induced the formation of altered (A) particles with a resultant irreversible loss of infectivity. A-particle formation was characterized by loss of VP4 from the virions and required incubation of CVB3-CAR complexes at 37°C. Dimeric soluble DAF (DAF-Fc) was found to be 125-fold-more effective at inhibiting CVB3 than monomeric DAF, which corresponded to a 100-fold increase in binding affinity as determined by surface plasmon resonance analysis. Soluble CAR and soluble dimeric CAR (CAR-Fc) bound to CVB3 with 5,000- and 10,000-fold-higher affinities than the equivalent forms of DAF. While DAF-Fc was 125-fold-more effective at inhibiting virus than monomeric DAF, complement regulation by DAF-Fc was decreased 4 fold. Therefore, while the virus binding was a cooperative event, complement regulation was hindered by the molecular orientation of DAF-Fc, indicating that the regions responsible for complement regulation and virus binding do not completely overlap. Relative contributions of CVB binding affinity, receptor binding footprint on the virus capsid, and induction of capsid conformation alterations for the ability of cellular DAF and CAR to act as receptors are discussed. PMID:16140777

  4. Soluble suppressor supernatants elaborated by concanavalin A-activated human mononuclear cells. Characterization of a soluble suppressor of B cell immunoglobulin production

    SciTech Connect

    Fleisher, T.A.; Greene, W.C.; Blaese, R.M.; Waldmann, T.A.

    1981-03-01

    Human peripheral blood mononuclear cells (PBMC) activated with the mitogenic lectin concanavalin A (Con A) elaborate a soluble immune suppressor supernatant (SISS) that contains at least 2 distinct suppressor factors. One of these, SISS-B, inhibits polyclonal B cell immunoglobulin production, whereas the other, SISS-T, suppresses T cell proliferation to both mitogens and antigens. The latter mediator is discussed in the companion paper. Characteristics of the human soluble suppressor of B cell immunoglobulin production (SISS-B) include: 1) inhibition by a noncytotoxic mechanism, 2) loss of activity in the presence of the monosaccharide L-rhamnose, 3) appearance within 8 to 16 hr after the addition of Con A, 4) elaboration by cells irradiated with 500 or 2000 rads, 5) production by highly purified T cells, 6) stability at pH 2.5 but instability at 56/sup o/C, and 7) m.w. of 60 to 80,000. These data indicate that after Con A activation, selected T cells not only become potent suppressor cells, but also generate a soluble saccharide-specific factor(s) that inhibits polyclonal immunoglobulin production by human B cells.

  5. Agglutinating activity of alcohol-soluble proteins from quinoa seed flour in celiac disease.

    PubMed

    De Vincenzi, M; Silano, M; Luchetti, R; Carratù, B; Boniglia, C; Pogna, N E

    1999-01-01

    The edible seeds of the quinoa plant contain small quantities of alcohol-soluble protein which, after peptic-tryptic digestion, are unable to agglutinate K562(s) cells. When separated by affinity chromatography on sepharose-6B coupled with mannan, peptic-tryptic digest separated in two fractions. Fraction B peptides (about 1% of total protein) were shown to agglutinate K562(s) cells at a very low concentration, whereas peptides in fraction A and in the mixed fraction A+B were inactive, suggesting that fraction A contains protective peptides that interfere with the agglutinating activity of toxic peptides in fraction B. PMID:10646556

  6. In vitro and in vivo antioxidant activity of a water-soluble polysaccharide from dendrobium denneanum

    USGS Publications Warehouse

    Luo, A.; Ge, Z.; Fan, Y.; Chun, Z.; Jin, He X.

    2011-01-01

    The water-soluble crude polysaccharide (DDP) obtained from the aqueous extracts of the stem of Dendrobium denneanum through hot water extraction followed by ethanol precipitation, was found to have an average molecular weight (Mw) of about 484.7 kDa. Monosaccharide analysis revealed that DDP was composed of arabinose, xylose, mannose, glucose and galactose in a molar ratio of 1.00:2.66:8.92:34.20:10.16. The investigation of antioxidant activity both in vitro and in vivo showed that DDP is a potential antioxidant. ?? 2011.

  7. Solubility parameter and activity coefficient of HDEHP dimer in select organic diluents by vapor pressure osmometry

    SciTech Connect

    Gray, M.; Nilsson, M.; Zalupski, P.

    2013-07-01

    A thorough understanding of the non-ideal behavior of the chemical components utilized in solvent extraction contributes to the success of any large-scale spent nuclear fuel treatment. To address this, our current work uses vapor pressure osmometry to characterize the non-ideal behavior of the solvent extraction agent di-(2-ethylhexyl) phosphoric acid (HDEHP), a common extractant in proposed separation schemes. Solubility parameters were fit to data on HDEHP at four temperatures using models based on Scatchard Hildebrand regular solution theory with Flory Huggins entropic corrections. The results are comparable but not identical to the activity coefficients from prior slope analysis in the literature. (authors)

  8. Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS.

    PubMed Central

    Jarvis, B W; Lichenstein, H; Qureshi, N

    1997-01-01

    An early event in septic shock is the activation of macrophages by a complex consisting of lipopolysaccharide (LPS), LPS-binding protein (LBP), and the cell surface antigen CD14. The complexes that form between [3H]ReLPS (ReLPS is deep-rough-chemotype hexacyl LPS from E. coli D31m4), soluble CD14 (sCD14), and LBP were analyzed by two independent methods, native (nondenaturing) gel electrophoresis and size-exclusion high-performance liquid chromatography (HPLC). This is the first reported use of HPLC to purify and study LPS-protein complexes. The binding of [3H]ReLPS to LBP and sCD14 was inhibited by preincubation with diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA), a potent LPS antagonist. In addition, [3H]ReLPS bound to LBP and to a truncated form of sCD14 [sCD14(1-152)] that contained the LPS binding domain. Binding to both proteins was blocked by RsDPLA. Thus, RsDPLA competes in a 1:1 ratio for the same or nearby binding sites on ReLPS complexes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aggregated ReLPS eluting from the HPLC indicated that only LBP, not sCD14, was bound to the aggregated ReLPS. This finding supports the binary model of LPS complex formation with LBP and sCD14. PMID:9234747

  9. Water Soluble Bioactives of Nacre Mediate Antioxidant Activity and Osteoblast Differentiation

    PubMed Central

    Chaturvedi, Ratna; Singha, Prajjal Kanti; Dey, Satyahari

    2013-01-01

    The water soluble matrix of nacre is a proven osteoinductive material. In spite of the differences in the biomolecular compositions of nacre obtained from multiple species of oysters, the common biochemical properties of those principles substantiate their biological activity. However, the mechanism by which nacre stimulates bone differentiation remains largely unknown. Since the positive impact of antioxidants on bone metabolism is well acknowledged, in this study we investigated the antioxidant potential of a water soluble matrix (WSM) obtained from the nacre of the marine oyster Pinctada fucata, which could regulate its osteoblast differentiation activity. Enhanced levels of ALP activity observed in pre-osteoblast cells upon treatment with WSM, suggested the induction of bone differentiation events. Furthermore, bone nodule formation and up-regulation of bone differentiation marker transcripts, i.e. collagen type-1 and osteocalcin by WSM confirmed its ability to induce differentiation of the pre-osteoblasts into mature osteoblasts. Remarkably, same WSM fraction upon pre-treatment lowered the H2O2 and UV-B induced oxidative damages in keratinocytes, thus indicating the antioxidant potential of WSM. This was further confirmed from the in vitro scavenging of ABTS and DPPH free radicals and inhibition of lipid peroxidation by WSM. Together, these results indicate that WSM poses both antioxidant potential and osteoblast differentiation property. Thus, bioactivities associated with nacre holds potential in the development of therapeutics for bone regeneration and against oxidative stress induced damages in cells. PMID:24367677

  10. Toward understanding the effect of low-activity waste glass composition on sulfur solubility

    SciTech Connect

    Vienna, John D.; Kim, Dong -Sang; Muller, Isabelle S.; Piepel, Greg F.; Kruger, Albert A.; Jantzen, C.

    2014-07-24

    The concentration of sulfur in nuclear waste glass melter feed must be maintained below the point where salt accumulates on the melt surface. The allowable concentrations may range from 0.37 to over 2.05 weight percent (of SO3 on a calcined oxide basis) depending on the composition of the melter feed and processing conditions. If the amount of sulfur exceeds the melt tolerance level, a molten salt will accumulate, which may upset melter operations and potentially shorten the useful life of the melter. At the Hanford site, relatively conservative limits have been placed on sulfur loading in melter feed, which in turn significantly increases the amount of glass that will be produced. Crucible-scale sulfur solubility data and scaled melter sulfur tolerance data have been collected on simulated Hanford waste glasses over the last 15 years. These data were compiled and analyzed. A model was developed to predict the solubility of SO3 in glass based on 252 simulated Hanford low-activity waste (LAW) glass compositions. This model represents the data well, accounting for over 85% of the variation in data, and was well validated. The model was also found to accurately predict the tolerance for sulfur in melter feed for 13 scaled melter tests of simulated LAW glasses. The model can be used to help estimate glass volumes and make informed decisions on process options. The model also gives quantitative estimates of component concentration effects on sulfur solubility. The components that most increase sulfur solubility are Li2O > V2O5> CaO ≈ P2O5 > Na2O ≈ B2O3 > K2O. The components that most decrease sulfur solubility are Cl > Cr2O3 > Al2O3 > ZrO2 ≈ SnO2 > Others ≈ SiO2. As a result, the order of component effects is similar to previous literature data, in most cases.

  11. Toward understanding the effect of low-activity waste glass composition on sulfur solubility

    DOE PAGESBeta

    Vienna, John D.; Kim, Dong -Sang; Muller, Isabelle S.; Piepel, Greg F.; Kruger, Albert A.; Jantzen, C.

    2014-07-24

    The concentration of sulfur in nuclear waste glass melter feed must be maintained below the point where salt accumulates on the melt surface. The allowable concentrations may range from 0.37 to over 2.05 weight percent (of SO3 on a calcined oxide basis) depending on the composition of the melter feed and processing conditions. If the amount of sulfur exceeds the melt tolerance level, a molten salt will accumulate, which may upset melter operations and potentially shorten the useful life of the melter. At the Hanford site, relatively conservative limits have been placed on sulfur loading in melter feed, which inmore » turn significantly increases the amount of glass that will be produced. Crucible-scale sulfur solubility data and scaled melter sulfur tolerance data have been collected on simulated Hanford waste glasses over the last 15 years. These data were compiled and analyzed. A model was developed to predict the solubility of SO3 in glass based on 252 simulated Hanford low-activity waste (LAW) glass compositions. This model represents the data well, accounting for over 85% of the variation in data, and was well validated. The model was also found to accurately predict the tolerance for sulfur in melter feed for 13 scaled melter tests of simulated LAW glasses. The model can be used to help estimate glass volumes and make informed decisions on process options. The model also gives quantitative estimates of component concentration effects on sulfur solubility. The components that most increase sulfur solubility are Li2O > V2O5> CaO ≈ P2O5 > Na2O ≈ B2O3 > K2O. The components that most decrease sulfur solubility are Cl > Cr2O3 > Al2O3 > ZrO2 ≈ SnO2 > Others ≈ SiO2. As a result, the order of component effects is similar to previous literature data, in most cases.« less

  12. NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential

    PubMed Central

    Evgenov, Oleg V.; Pacher, Pál; Schmidt, Peter M.; Haskó, György; Schmidt, Harald H. H. W.; Stasch, Johannes-Peter

    2008-01-01

    Soluble guanylate cyclase (sGC) is a key signal-transduction enzyme activated by nitric oxide (NO). Impaired bioavailability and/or responsiveness to endogenous NO has been implicated in the pathogenesis of cardiovascular and other diseases. Current therapies that involve the use of organic nitrates and other NO donors have limitations, including non-specific interactions of NO with various biomolecules, lack of response and the development of tolerance following prolonged administration. Compounds that activate sGC in an NO-independent manner might therefore provide considerable therapeutic advantages. Here we review the discovery, biochemistry, pharmacology and clinical potential of haem-dependent sGC stimulators (including YC-1, BAY 41-2272, BAY 41-8543, CFM-1571 and A-350619) and haem-independent sGC activators (including BAY 58-2667 and HMR-1766). PMID:16955067

  13. Designing a highly active soluble PQQ-glucose dehydrogenase for efficient glucose biosensors and biofuel cells

    SciTech Connect

    Durand, Fabien; Stines-Chaumeil, Claire; Flexer, Victoria; Andre, Isabelle; Mano, Nicolas

    2010-11-26

    Research highlights: {yields} A new mutant of PQQ-GDH designed for glucose biosensors application. {yields} First mutant of PQQ-GDH with higher activity for D-glucose than the Wild type. {yields} Position N428 is a key point to increase the enzyme activity. {yields} Molecular modeling shows that the N428 C mutant displays a better interaction for PQQ than the WT. -- Abstract: We report for the first time a soluble PQQ-glucose dehydrogenase that is twice more active than the wild type for glucose oxidation and was obtained by combining site directed mutagenesis, modelling and steady-state kinetics. The observed enhancement is attributed to a better interaction between the cofactor and the enzyme leading to a better electron transfer. Electrochemical experiments also demonstrate the superiority of the new mutant for glucose oxidation and make it a promising enzyme for the development of high-performance glucose biosensors and biofuel cells.

  14. Characterization of soluble cyclic AMP phosphodiesterases and partial purification of a major form in human leiomyoma of the uterus.

    PubMed

    Robinson, M F; Levin, J; Savage, N

    1987-01-01

    Human leiomyoma of the uterus contained seven forms of cyclic AMP phosphodiesterase in the crude cytosol as revealed by a specific activity stain on non-denaturing polyacrylamide gel electrophoresis. The enzymes from human myometrium and normal uterus showed an identical activity pattern. Ferguson plot analysis showed four different molecular weight species of Mr 229,000 +/- 4,000, 186,000 +/- 4,000, 174,000 +/- 4,000 and 162,000 +/- 4,000. The Mr 174,000 species comprised four differently charged forms. Sucrose density gradient centrifugation of the crude cytosol revealed the presence of three molecular weight species sedimenting at 11.8S, 8.1S and 3.6S. The Michaelis constant (Km) for the band 1 form which displayed linear kinetics was 5 microM and the band 2 form which produced non-linear kinetics had Km values of 5.8 and 37 microM. PMID:2820644

  15. Infrared Laser Activation of Soluble and Membrane Protein Assemblies in the Gas Phase.

    PubMed

    Mikhailov, Victor A; Liko, Idlir; Mize, Todd H; Bush, Matthew F; Benesch, Justin L P; Robinson, Carol V

    2016-07-19

    Collision-induced dissociation (CID) is the dominant method for probing intact macromolecular complexes in the gas phase by means of mass spectrometry (MS). The energy obtained from collisional activation is dependent on the charge state of the ion and the pressures and potentials within the instrument: these factors limit CID capability. Activation by infrared (IR) laser radiation offers an attractive alternative as the radiation energy absorbed by the ions is charge-state-independent and the intensity and time scale of activation is controlled by a laser source external to the mass spectrometer. Here we implement and apply IR activation, in different irradiation regimes, to study both soluble and membrane protein assemblies. We show that IR activation using high-intensity pulsed lasers is faster than collisional and radiative cooling and requires much lower energy than continuous IR irradiation. We demonstrate that IR activation is an effective means for studying membrane protein assemblies, and liberate an intact V-type ATPase complex from detergent micelles, a result that cannot be achieved by means of CID using standard collision energies. Notably, we find that IR activation can be sufficiently soft to retain specific lipids bound to the complex. We further demonstrate that, by applying a combination of collisional activation, mass selection, and IR activation of the liberated complex, we can elucidate subunit stoichiometry and the masses of specifically bound lipids in a single MS experiment. PMID:27328020

  16. New water-soluble ruthenium(II) cytotoxic complex: biological activity and cellular distribution.

    PubMed

    Morais, Tânia S; Santos, Filipa C; Jorge, Tiago F; Côrte-Real, Leonor; Madeira, Paulo J Amorim; Marques, Fernanda; Robalo, M Paula; Matos, António; Santos, Isabel; Garcia, M Helena

    2014-01-01

    A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=η(5)-cyclopentadienyl, mTPPMS=diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy=2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. PMID:24145065

  17. The effect of net charge on the solubility, activity, and stability of ribonuclease Sa

    PubMed Central

    Shaw, Kevin L.; Grimsley, Gerald R.; Yakovlev, Gennady I.; Makarov, Alexander A.; Pace, C. Nick

    2001-01-01

    The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not. PMID:11369859

  18. Formation of distinct soluble microbial products by activated sludge: kinetic analysis and quantitative determination.

    PubMed

    Ni, Bing-Jie; Fang, Fang; Xie, Wen-Ming; Xu, Juan; Yu, Han-Qing

    2012-02-01

    Soluble microbial products (SMP) released by microorganisms in bioreactors are classified into two distinct groups according to their different chemical and degradation kinetics: utilization-associated products (UAP) and biomass-associated products (BAP). SMP are responsible for effluent chemical oxygen demand or for membrane fouling of membrane bioreactor. Here an effective and convenient approach, other than the complicated chemical methods or complex models, is developed to quantify the formation of UAP and BAP together with their kinetics in activated sludge process. In this approach, an integrated substrate utilization equation is developed and used to determine UAP and their production kinetics. On the basis of total SMP measurements, BAP formation is determined with an integrated BAP formation equation. The fraction of substrate electrons diverted to UAP, and the content of BAP derived from biomass can then be calculated. Dynamic quantification data are obtained for UAP and BAP separately and conveniently. The obtained kinetic parameters are found to be reasonable as they are generally bounded and comparable to the literature values. The validity of this approach is confirmed by independent SMP production tests in six different activated sludge systems, which demonstrates its applicability in a wide range of engineered system regarding SMP production. This work provides a widely applied approach to determine the formation of UAP and BAP conveniently, which may offer engineers with basis to optimize bioreactor operation to avoid a high effluent soluble organics from SMP or SMP-based membrane fouling in membrane bioreactors. PMID:22185635

  19. A functional soluble form of CTLA-4 is present in the serum of celiac patients and correlates with mucosal injury.

    PubMed

    Simone, Rita; Brizzolara, Renata; Chiappori, Alessandra; Milintenda-Floriani, Francesca; Natale, Clelia; Greco, Luigi; Schiavo, Mara; Bagnasco, Marcello; Pesce, Giampaola; Saverino, Daniele

    2009-09-01

    Celiac disease (CD) is a multifactorial disorder influenced by environmental, genetic and immunological factors. Increasing evidence showed CTLA-4 gene as an important susceptibility locus for autoimmune disorders. A native soluble cytotoxic T-lymphocyte-associated protein-4 (sCTLA-4), lacking of transmembrane sequence, has been described in several autoimmune diseases. We aimed to evaluate the presence of increased sCTLA-4 concentration in the serum of patients with CD and the possible immunoregulatory function. Blood samples were collected from 160 CD patients; sCTLA-4 levels were evaluated by ELISA, western blot and reverse transcription-PCR. The capability of serum sCTLA-4 to modulate T-lymphocyte proliferation in vitro was evaluated by two-way mixed leukocyte reaction assay. We demonstrated high levels of sCTLA-4 in serum of untreated celiac patients. Additionally, we observed that sCTLA-4 concentrations are related to gluten intake and that a correlation between autoantibodies to tissue transglutaminase and sCTLA-4 concentration exists. Moreover, sCTLA-4 levels correlate with the degree of mucosal damage. Conversely, no correlation between sCTLA4 levels and the HLA-related risk was observed. Finally, we show that sCTLA-4 from sera of CD patients displays functional activities. These results strongly suggest a regulation of sCTLA-4 synthesis depending on the presence or absence of dietary gluten and imply a possible immunomodulatory effect on cytotoxic T lymphocyte functions. In gluten-exposed patients, serum sCTLA-4 levels might provide insight about mucosal injury. PMID:19625381

  20. Inflammatory response of lung macrophages and epithelial cells after exposure to redox active nanoparticles: effect of solubility and antioxidant treatment.

    PubMed

    Urner, Martin; Schlicker, Andreas; Z'graggen, Birgit Roth; Stepuk, Alexander; Booy, Christa; Buehler, Karl P; Limbach, Ludwig; Chmiel, Corinne; Stark, Wendelin J; Beck-Schimmer, Beatrice

    2014-12-01

    The effects of an exposure to three mass-produced metal oxide nanoparticles-similar in size and specific surface area but different in redox activity and solubility-were studied in rat alveolar macrophages (MAC) and epithelial cells (AEC). We hypothesized that the cell response depends on the particle redox activity and solubility determining the amount of reactive oxygen species formation (ROS) and subsequent inflammatory response. MAC and AEC were exposed to different amounts of Mn3O4 (soluble, redox-active), CeO2 (insoluble, redox-active), and TiO2 (insoluble, redox-inert) up to 24 h. Viability and inflammatory response were monitored with and without coincubation of a free-radical scavenger (trolox). In MAC elevated ROS levels, decreased metabolic activity and attenuated inflammatory mediator secretion were observed in response to Mn3O4. Addition of trolox partially resolved these changes. In AEC, decreased metabolic activity and an attenuated inflammatory mediator secretion were found in response to CeO2 exposure without increased production of ROS, thus not sensitive to trolox administration. Interestingly, highly redox-active soluble particles did not provoke an inflammatory response. The data reveal that target and effector cells of the lung react in different ways to particle exposure making a prediction of the response depending on redox activity and intracellular solubility difficult. PMID:25343230

  1. 76 FR 42129 - Agency Information Collection Activities: Case Submission Form, Case Assistance Form

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-18

    ... FY2010. We are requesting a two year approval for the form anticipating Government Paperwork Elimination... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND SECURITY Agency Information Collection Activities: Case Submission Form, Case Assistance Form (Form...

  2. Qrtzgeotherm: An ActiveX component for the quartz solubility geothermometer

    NASA Astrophysics Data System (ADS)

    Verma, Mahendra P.

    2008-12-01

    An ActiveX component, QrtzGeotherm, to calculate temperature and vapor fraction in a geothermal reservoir using quartz solubility geothermometry was written in Visual Basic 6.0. Four quartz solubility equations along the liquid-vapor saturation curve: (i) a quadratic equation of 1/ T and pressure, (ii) a linear equation relating log SiO 2 to the inverse of absolute temperature ( T), (iii) a polynomial of T including logarithmic terms and (iv) temperature as a polynomial of SiO 2 including logarithmic terms are programmed. The QrtzGeotherm has input parameters: (i) HRes—the reservoir enthalpy (kJ/kg), (ii) SiO2TD—silica concentration in total discharge (ppm), (iii) GeoEq—number of quartz solubility equation and (iv) TempGuess—a guess value of the reservoir temperature (°C). The reservoir enthalpy Hres is assumed to be the same as the total discharge enthalpy HR. The output parameters are (i) TempRes—reservoir temperature (°C) and (ii) VapRes—reservoir vapor fraction. The first step is to calculate the total discharge concentration of silica SiO2TD from the concentration of silica SiO2Col of separated water, sampled after N-separations of vapor and water. To use QrtzGeotherm in MS-Excel, three functions SiO2TD, GeoResTemp and GeoResVap for an N-stage separation of geothermal reservoir fluid are written in Visual Basic for Application (VBA). Similarly, a demonstration program, QrtzGeothrm, is written in Visual Basic 6.0.

  3. Antitumor and immunomodulatory activity of water-soluble polysaccharide from Inonotus obliquus.

    PubMed

    Fan, Liuping; Ding, Shaodong; Ai, Lianzhong; Deng, Kequan

    2012-10-01

    The medicinal mushroom Inonotus obliquus has been used as a folk remedy for a long time in Russia and East-European countries to treat gastrointestinal cancer, cardiovascular disease and diabetes. In our study, a water-soluble polysaccharide (ISP2a) was successfully purified from I. obliquus by DEAE-Sepharose CL-6B and Sepharose CL-6B column chromatography. In vivo ISP2a had not only shown antitumor activity, but also could significantly enhance the immune response of tumor-bearing mice. In addition, ISP2a significantly enhanced the lymphocyte proliferation and increased the production of TNF-α. Results of these studies demonstrated that ISP2a had a potential application as natural antitumor agent with immunomodulatory activity. PMID:22840014

  4. Neural network with formed dynamics of activity

    SciTech Connect

    Dunin-Barkovskii, V.L.; Osovets, N.B.

    1995-03-01

    The problem of developing a neural network with a given pattern of the state sequence is considered. A neural network structure and an algorithm, of forming its bond matrix which lead to an approximate but robust solution of the problem are proposed and discussed. Limiting characteristics of the serviceability of the proposed structure are studied. Various methods of visualizing dynamic processes in a neural network are compared. Possible applications of the results obtained for interpretation of neurophysiological data and in neuroinformatics systems are discussed.

  5. 75 FR 26782 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-12

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of an Existing Information Collection; Comment Request ACTION: 60-Day Notice of Information Collection Under Review; Form I- 864, Affidavit of...

  6. Peroxidase-like activity of water-soluble cupric oxide nanoparticles and its analytical application for detection of hydrogen peroxide and glucose.

    PubMed

    Chen, Wei; Chen, Juan; Feng, Ye-Bin; Hong, Lei; Chen, Qi-Ying; Wu, Ling-Feng; Lin, Xin-Hua; Xia, Xing-Hua

    2012-04-01

    Water-soluble cupric oxide nanoparticles are fabricated via a quick-precipitation method and used as peroxidase mimetics for ultrasensitive detection of hydrogen peroxide and glucose. The water-soluble CuO nanoparticles show much higher catalytic activity than that of commercial CuO nanoparticles due to their higher affinity to hydrogen peroxide. In addition, the as-prepared CuO nanoparticles are stable over a wide range of pH and temperature. This excellent stability in the form of aqueous colloidal suspensions makes the application of the water-soluble CuO nanoparticles easier in aqueous systems. A colorimetric assay for hydrogen peroxide and glucose has been established based on the catalytic oxidation of phenol coupled with 4-amino-atipyrine by the action of hydrogen peroxide. This analytical platform not only confirms the intrinsic peroxidase-like activity of the water-soluble cupric oxide nanoparticles, but also shows its great potential applications in environmental chemistry, biotechnology and medicine. PMID:22349179

  7. Silk microgels formed by proteolytic enzyme activity.

    PubMed

    Samal, Sangram K; Dash, Mamoni; Chiellini, Federica; Kaplan, David L; Chiellini, Emo

    2013-09-01

    The proteolytic enzyme α-chymotrypsin selectively cleaves the amorphous regions of silk fibroin protein (SFP) and allows the crystalline regions to self-assemble into silk microgels (SMGs) at physiological temperature. These microgels consist of lamellar crystals in the micrometer scale, in contrast to the nanometer-scaled crystals in native silkworm fibers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zeta potential results demonstrated that α-chymotrypsin utilized only the non-amorphous domains or segments of the heavy chain of SFP to form negatively charged SMGs. The SMGs were characterized in terms of size, charge, structure, morphology, crystallinity, swelling kinetics, water content and thermal properties. The results suggest that the present technique of preparing SMGs by α-chymotrypsin is simple and efficient, and that the prepared SMGs have useful features for studies related to biomaterial and pharmaceutical needs. This process is also an easy way to obtain the amorphous peptide chains for further study. PMID:23756227

  8. Iodine solubility in a low-activity waste borosilicate glass at 1000 °C

    NASA Astrophysics Data System (ADS)

    Riley, Brian J.; Schweiger, Michael J.; Kim, Dong-Sang; Lukens, Wayne W.; Williams, Benjamin D.; Iovin, Cristian; Rodriguez, Carmen P.; Overman, Nicole R.; Bowden, Mark E.; Dixon, Derek R.; Crum, Jarrod V.; McCloy, John S.; Kruger, Albert A.

    2014-09-01

    The purpose of this study was to determine the solubility of iodine in a low-activity waste borosilicate glass when heated inside an evacuated and sealed fused quartz ampoule. The iodine was added to glass frit as KI in quantities of 99.4-24,005 ppm iodine (by mass). Each mixture was added to an ampoule, heated at 1000 °C for 2 h, and then air quenched. In samples with ⩾11,999 ppm iodine, low viscosity salt phases were observed on the surface of the melts that solidified into a white coating upon cooling. These salts were identified by X-ray diffraction as mixtures of KI, NaI, and Na2SO4. Iodine concentrations in glass specimens were analyzed with inductively-coupled plasma mass spectrometry, and the overall iodine solubility was determined to be 10,000 ppm. Several crystalline inclusions of iodine sodalite, Na8(AlSiO4)6I2, were observed in the 24,005 ppm specimen.

  9. Iodine Solubility in Low-Activity Waste Borosilicate Glass at 1000 °C

    SciTech Connect

    Riley, Brian J.; Schweiger, Michael J.; Kim, Dong-Sang; Lukens, Wayne W.; Williams, Benjamin D.; Iovin, Cristian; Rodriguez, Carmen P.; Overman, Nicole R.; Bowden, Mark E.; Dixon, Derek R.; Crum, Jarrod V.; Mccloy, John S.; Kruger, Albert A.

    2014-04-30

    The purpose of this study was to determine the solubility of iodine in a low-activity waste borosilicate glass when heated inside an evacuated and sealed fused quartz ampoule. The iodine was added to glass frit as KI in quantities of 100–24000 ppm iodine (by mass), each mixture was added to an ampoule, the ampoule was heated at 1000 °C for 2 h and then air quenched. In samples with ≥12000 ppm iodine, low viscosity salt phases were observed on the surface of the melts during cooling that solidified into a white coating upon cooling. These salts were identified as mixtures of KI, NaI, and Na2SO4 with X-ray diffraction (XRD). The iodine concentrations in glass specimens were analyzed with inductively-coupled plasma mass spectrometry and the overall iodine solubility was determined to be 10000 ppm by mass. Several crystalline inclusions of iodine sodalite, Na8(AlSiO4)6I2, were observed in the 24000 ppm specimen and were verified with micro-XRD and wavelength dispersive spectroscopy.

  10. Heme-Dependent and Independent Soluble Guanylate Cyclase Activators and Vasodilation

    PubMed Central

    Priviero, Fernanda B. M.; Webb, R. Clinton

    2010-01-01

    Since the discovery of nitric oxide (NO), which is released from endothelial cells as the main mediator of vasodilation, its target, the soluble guanylyl cyclase (sGC), has become a focus of interest for the treatment of diseases associated with endothelial dysfunction. NO donors were developed to suppress NO deficiency; however, tolerance to organic nitrates was reported. Non-NO-based drugs targeting sGC were developed to overcome the problem of tolerance. In this review, we briefly describe the process of sGC activation by its main physiological activator NO and the advances in the development of drugs capable of activating sGC in a NO-independent manner. sGC stimulators, as some of these drugs are called, require the integrity of the reduced heme moiety of the prosthetic group within the sGC and therefore are called heme-dependent stimulators. Other drugs are able to activate sGC independent of heme moiety and are hence called heme-independent activators. Because pathologic conditions modulate sGC and oxidize the heme moiety, the heme-independent sGC activators could potentially become drugs of choice because of their higher affinity to the oxidized enzyme. However, these drugs are still undergoing clinical trials and are not available for clinical use. PMID:20571429

  11. Mice transgenic for a soluble form of murine CTLA-4 show enhanced expansion of antigen-specific CD4+ T cells and defective antibody production in vivo.

    PubMed

    Ronchese, F; Hausmann, B; Hubele, S; Lane, P

    1994-03-01

    CD4+ T cell responses were analyzed in transgenic mice expressing a soluble form of murine CTLA-4, mCTLA4-H gamma 1, which blocks the interaction of the T cell activation molecules CD28 and CTLA-4 with their costimulatory ligands. Consistent with previous reports (Linsley, P. S., P. M. Wallace, J. Johnson, M. G. Gibson, J. L. Greene, J. A. Ledbetter, C. Singh, and M. A. Tepper. 1992. Science (Wash. DC). 257:792), T cell-dependent antibody production was profoundly inhibited in mCTLA4-H gamma 1 transgenic mice immunized with a protein antigen. Surprisingly, however, transgenic mice could generate quantitatively and qualitatively normal primary T cell responses, as measured by limiting dilution assays and lymphokine production. In addition, in vivo expansion of antigen-specific T cells after secondary or tertiary immunization was enhanced in mCTLA4-H gamma 1 transgenics as compared with normal mice. Although unable to deliver cognate help to B cells in vivo, T cells from mCTLA4-H gamma 1 transgenic mice were not anergic as they could help B cells to produce specific antibodies when adoptively transferred into nude hosts. Taken together, these data suggest that the engagement of CD28 and/or CTLA-4 may not be required for the induction of T cell responses, as is currently understood, but rather for the expression of T cell effector function such as the delivery of T cell help to B cells. PMID:8113677

  12. 3'-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli.

    PubMed

    Song, Ji-Won; Woo, Ji-Min; Jung, Gyoo Yeol; Bornscheuer, Uwe T; Park, Jin-Byung

    2016-01-01

    3'-Untranslated region (3'UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3'UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3'UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3'UTR. In summary, 3'UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells. PMID:27406241

  13. 76 FR 61725 - Agency Information Collection Activities: Case Submission Form, Case Assistance Form; (Form DHS...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-10-05

    ... Federal Register on July 18, 2011 at 76 FR 42129, for a 60-day public comment period. No comments were... From the Federal Register Online via the Government Publishing Office DEPARTMENT OF HOMELAND...-7001), Online Ombudsman Form DHS-7001 AGENCY: Office of the Citizenship and Immigration...

  14. Irreversible Catalyst Activation Enables Hyperpolarization and Water Solubility for NMR Signal Amplification by Reversible Exchange

    PubMed Central

    2015-01-01

    Activation of a catalyst [IrCl(COD)(IMes)] (IMes = 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; COD = cyclooctadiene)] for signal amplification by reversible exchange (SABRE) was monitored by in situ hyperpolarized proton NMR at 9.4 T. During the catalyst-activation process, the COD moiety undergoes hydrogenation that leads to its complete removal from the Ir complex. A transient hydride intermediate of the catalyst is observed via its hyperpolarized signatures, which could not be detected using conventional nonhyperpolarized solution NMR. SABRE enhancement of the pyridine substrate can be fully rendered only after removal of the COD moiety; failure to properly activate the catalyst in the presence of sufficient substrate can lead to irreversible deactivation consistent with oligomerization of the catalyst molecules. Following catalyst activation, results from selective RF-saturation studies support the hypothesis that substrate polarization at high field arises from nuclear cross-relaxation with hyperpolarized 1H spins of the hydride/orthohydrogen spin bath. Importantly, the chemical changes that accompanied the catalyst’s full activation were also found to endow the catalyst with water solubility, here used to demonstrate SABRE hyperpolarization of nicotinamide in water without the need for any organic cosolvent—paving the way to various biomedical applications of SABRE hyperpolarization methods. PMID:25372972

  15. Autoinhibition of Bruton's tyrosine kinase (Btk) and activation by soluble inositol hexakisphosphate

    PubMed Central

    Wang, Qi; Vogan, Erik M; Nocka, Laura M; Rosen, Connor E; Zorn, Julie A; Harrison, Stephen C; Kuriyan, John

    2015-01-01

    Bruton's tyrosine kinase (Btk), a Tec-family tyrosine kinase, is essential for B-cell function. We present crystallographic and biochemical analyses of Btk, which together reveal molecular details of its autoinhibition and activation. Autoinhibited Btk adopts a compact conformation like that of inactive c-Src and c-Abl. A lipid-binding PH-TH module, unique to Tec kinases, acts in conjunction with the SH2 and SH3 domains to stabilize the inactive conformation. In addition to the expected activation of Btk by membranes containing phosphatidylinositol triphosphate (PIP3), we found that inositol hexakisphosphate (IP6), a soluble signaling molecule found in both animal and plant cells, also activates Btk. This activation is a consequence of a transient PH-TH dimerization induced by IP6, which promotes transphosphorylation of the kinase domains. Sequence comparisons with other Tec-family kinases suggest that activation by IP6 is unique to Btk. DOI: http://dx.doi.org/10.7554/eLife.06074.001 PMID:25699547

  16. Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine.

    PubMed

    Szymczak, Mariusz

    2016-08-01

    This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency-high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology. PMID:27351340

  17. Crystal structure of a soluble form of human monoglyceride lipase in complex with an inhibitor at 1.35 Å resolution

    SciTech Connect

    Schalk-Hihi, Céline; Schubert, Carsten; Alexander, Richard; Bayoumy, Shariff; Clemente, Jose C.; Deckman, Ingrid; DesJarlais, Renee L.; Dzordzorme, Keli C.; Flores, Christopher M.; Grasberger, Bruce; Kranz, James K.; Lewandowski, Frank; Liu, Li; Ma, Hongchang; Maguire, Diane; Macielag, Mark J.; McDonnell, Mark E.; Haarlander, Tara Mezzasalma; Miller, Robyn; Milligan, Cindy; Reynolds, Charles

    2011-12-22

    A high-resolution structure of a ligand-bound, soluble form of human monoglyceride lipase (MGL) is presented. The structure highlights a novel conformation of the regulatory lid-domain present in the lipase family as well as the binding mode of a pharmaceutically relevant reversible inhibitor. Analysis of the structure lacking the inhibitor indicates that the closed conformation can accommodate the native substrate 2-arachidonoyl glycerol. A model is proposed in which MGL undergoes conformational and electrostatic changes during the catalytic cycle ultimately resulting in its dissociation from the membrane upon completion of the cycle. In addition, the study outlines a successful approach to transform membrane associated proteins, which tend to aggregate upon purification, into a monomeric and soluble form.

  18. Formation and catalytic activity of high molecular weight soluble polymers produced by heating amino acids in a modified sea medium

    NASA Astrophysics Data System (ADS)

    Okihana, Hiroyuki

    1982-06-01

    Eighteen protein amino acids with milk casein composition were heated in a modified sea medium. Marigranules were formed in the precipitates and soluble polymers were formed in the supernatant. Time course of the reaction (ultraviolet spectra, the concentration of metal ions, and the concentration of amino acids in the supernatant) were measured. The time course of the formation of the soluble polymers was also studied by Bio-Gel P-2 column. High molecular weight soluble polymers (HMWSP) were separated from low molecular weight ones by dialysis. It was shown that these polymers catalyzed the dehydrogenation of NADH. These polymers also catalyzed the coupled reaction between dehydrogenation of NADH and reduction of resazurin. This coupled reaction was accelerated by the light.

  19. Investigation of Water-Soluble X-ray Luminescence Nanoparticles for Photodynamic Activation

    SciTech Connect

    Liu, Yuanfang; Chen, Wei; Wang, Shaopeng; Joly, Alan G.

    2008-01-28

    In this letter, we report the synthesis of LaF3:Tb3+-MTCP (meso-Tetra(4-carboxyphenyl) porphine) nanoparticle conjugates and investigate the energy transfer as well as singlet oxygen generation following X-ray irradiation. Our observations indicate that LaF3:Tb3+-MTCP nanoparticle conjugates are efficient photodynamic agents that can be initiated by X-rays at a reasonably low dose. The addition of folic acid to facilitate targeting to folate receptors on tumor cells has no effect on the quantum yield of singlet oxygen in the nanoparticle-MTCP conjugates. Our pilot studies indicate that water-soluble scintillation nanoparticles can be potentially used to activate photodynamic therapy as a promising deep cancer treatment.

  20. Chemical constituents from the root of Polygonum multiflorum and their soluble epoxide hydrolase inhibitory activity.

    PubMed

    Sun, Ya Nan; Li, Wei; Kim, Jang Hoon; Yan, Xi Tao; Kim, Ji Eun; Yang, Seo Young; Kim, Young Ho

    2015-06-01

    Fourteen compounds were isolated from a methanol extract of Polygonum multiflorum roots, and their structures were elucidated by comparing spectroscopic data to published spectra. The inhibitory effects of the isolated compounds on soluble epoxide hydrolase (sEH) were then evaluated. Compounds 1-7 inhibited sEH activity potently, with IC50 values ranging from 6.2 ± 0.5 to 48.6 ± 3.1 μM. Moreover, a kinetic analysis of compounds 1-7 revealed that the inhibitory actions of compounds 1, 3 and 4 were non-competitive, whereas those of compounds 2 and 5-7 were mixed-type. PMID:25413971

  1. Soluble alpha-APP (sAPPalpha) regulates CDK5 expression and activity in neurons.

    PubMed

    Hartl, Daniela; Klatt, Stephan; Roch, Manfred; Konthur, Zoltan; Klose, Joachim; Willnow, Thomas E; Rohe, Michael

    2013-01-01

    A growing body of evidence suggests a role for soluble alpha-amyloid precursor protein (sAPPalpha) in pathomechanisms of Alzheimer disease (AD). This cleavage product of APP was identified to have neurotrophic properties. However, it remained enigmatic what proteins, targeted by sAPPalpha, might be involved in such neuroprotective actions. Here, we used high-resolution two-dimensional polyacrylamide gel electrophoresis to analyze proteome changes downstream of sAPPalpha in neurons. We present evidence that sAPPalpha regulates expression and activity of CDK5, a kinase that plays an important role in AD pathology. We also identified the cytoprotective chaperone ORP150 to be induced by sAPPalpha as part of this protective response. Finally, we present functional evidence that the sAPPalpha receptor SORLA is essential to mediate such molecular functions of sAPPalpha in neurons. PMID:23776568

  2. Soluble alpha-enolase activates monocytes by CD14-dependent TLR4 signalling pathway and exhibits a dual function

    PubMed Central

    Guillou, Clément; Fréret, Manuel; Fondard, Emeline; Derambure, Céline; Avenel, Gilles; Golinski, Marie-Laure; Verdet, Mathieu; Boyer, Olivier; Caillot, Frédérique; Musette, Philippe; Lequerré, Thierry; Vittecoq, Olivier

    2016-01-01

    Rheumatoid arthritis (RA) is the most common form of chronic inflammatory rheumatism. Identifying auto-antigens targeted by RA auto-antibodies is of major interest. Alpha-enolase (ENO1) is considered to be a pivotal auto-antigen in early RA but its pathophysiologic role remains unknown. The main objective of this study was to investigate the in vitro effects of soluble ENO1 on peripheral blood mononuclear cells (PBMC) from healthy donors and RA patients in order to determine the potential pathogenic role of ENO1. ELISA, transcriptomic analysis, experiments of receptor inhibition and flow cytometry analysis were performed to determine the effect, the target cell population and the receptor of ENO1. We showed that ENO1 has the ability to induce early production of pro-inflammatory cytokines and chemokines with delayed production of IL-10 and to activate the innate immune system. We demonstrated that ENO1 binds mainly to monocytes and activates the CD14-dependent TLR4 pathway both in healthy subjects and in RA patients. Our results establish for the first time that ENO1 is able to activate in vitro the CD14-dependent TLR4 pathway on monocytes involving a dual mechanism firstly pro-inflammatory and secondly anti-inflammatory. These results contribute to elucidating the role of this auto-antigen in the pathophysiologic mechanisms of RA. PMID:27025255

  3. A novel green synthesis of silver nanoparticles using soluble starch and its antibacterial activity

    PubMed Central

    Yakout, Sobhy M; Mostafa, Ashraf A

    2015-01-01

    A green method of Silver nanoparticles (AgNPs) preparation has been established. This method depends on reduction of silver nitrate with soluble starch. The formation of AgNPs was observed by the color change from colorless to dark brown through the starch addition into silver nitrate solution. It was observed that use of starch makes convenient method for the synthesis of silver nanoparticles and can reduce silver ions into the produced silver nanoparticles within one hour of reaction time without using any harsh conditions. The prepared silver nanoparticles were characterized by using UV-visible spectroscopy and evaluated for its antimicrobial activity. The synthesized green AgNPs showed a potential antibacterial activity that was stronger against Gram positive pathogenic bacteria (Staphylococus aureus and Streptococus pyogenes) than against Gram negative pathogenic bacteria (Salmonella typhi, Shigellasonnei and Pseudomonas aeruginosa). Inhibition zones diameter of antibacterial activity depends upon nanoparticles concentration as AgNPs exhibited greater inhibition zone for S.aureus (16.4 mm) followed by P. aeruginosa and S. pyogenes while the least activity was observed for S. typhi (10.4 mm) at 40 μl/ disc. These results suggested that AgNPs can be used as an effective antiseptic agents in medical fields and process of synthesis creates new opportunities in process development for the synthesis of safe and eco-friendly AgNPs. PMID:26064246

  4. Chemical properties and antioxidant activity of a water-soluble polysaccharide from Dendrobium officinale.

    PubMed

    Luo, Qiu-Lian; Tang, Zhuan-Hui; Zhang, Xue-Feng; Zhong, Yong-Hong; Yao, Su-Zhi; Wang, Li-Sheng; Lin, Cui-Wu; Luo, Xuan

    2016-08-01

    In this report, a water-soluble polysaccharide was obtained from the dried stems of Dendrobium officinale Kimura et Migo by hot-water (70-75°C) extraction and 85% ethanol precipitation, and successively purification by DEAE-cellulose anion-exchange chromatography and gel-permeation chromatography. The D. officinale polysaccharide (DOP) has a molecular weight of 8500Da. Monosaccharide composition analysis reveals that DOP is composed of mannose, glucose, and arabinose with a trace of galacturonic acid in a molar ratio of 6.2:2.3:2.1:0.1. Periodate oxidation-smith degradation and 1D and 2D NMR spectroscopy analysis suggest the predominance of mannose and glucose, and it contains a 2-O-acetylglucomannan and (1→4)-linked-β-d-mannopyranosyl and (1→4)-linked-β-d-glucopyranosyl residues. Atomic force microscope shows that DOP mainly exists as rod-shaped chains, supporting high degrees of polymerization. The antioxidant activities of the polysaccharide in vitro assay indicate that DOP has good scavenging activity of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, higher scavenging activity of hydroxyl radical, and metal chelating activities. PMID:27131730

  5. Antioxidative activity of bound-form phenolics in potato peel.

    PubMed

    Nara, Kazuhiro; Miyoshi, Takayuki; Honma, Tamaki; Koga, Hidenori

    2006-06-01

    Free and bound-form phenolics were isolated from potato (cv. Toyoshiro) flesh and peel. The free and bound-form phenolics in the peel showed high DPPH radical scavenging activity, while those in the flesh showed low activity. The total amount of chlorogenic acid and caffeic acid in the free-form phenolics from the peel was highly correlated with the DPPH radical scavenging activity. Ferulic acid was identified as the active radical scavenging compound in the bound-form phenolics from the peel. The potato peel may therefore offer an effective source of an antioxidative. PMID:16794331

  6. Characterization and antioxidant activities of marine pepsin soluble collagen from the skin of yellow goosefish Lophius litulon

    NASA Astrophysics Data System (ADS)

    Zheng, Bin; Xiang, Xingwei; Zhou, Yufang; Yang, Huicheng; Luo, Hongyu; Liao, Miaofei; Wen, Zhengshun

    2016-06-01

    Characteristics and antioxidant activities of pepsin-soluble collagen (PSC) from yellow goosefish (Lophius litulon) skins were investigated. PSC was characterized as a type I collagen, and its imino acid content was 193 residues/1 000 residues. PSC's denaturation temperature was ~17.56°C and Fourier transform infrared spectra confirmed the presence of triple helices. Solubility analysis showed good solubility at acidic pH (1-6) or low NaCl concentrations (≤2%). PSC showed scavenging activity against hydroxyl radicals and superoxide anions in a concentration-dependent manner. Furthermore, PSC could protect D-galactose-induced skin aging by significantly controlling malondialdehyde formation and improving the activity of superoxide dismutase, glutathione peroxidase, catalase, glutathione, and hydroxyproline. PSC may be a promising antioxidant in appropriate applications.

  7. 76 FR 41279 - Agency Information Collection Activities; Form I-864, Form I-864A, Form I-864EZ, and Form I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-07-13

    ... was previously published in the Federal Register on May 4, 2011, at 76 FR 25364, allowing for a 60-day... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities; Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of an Existing Information Collection;...

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    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-18

    ... on May 12, 2010, at 75 FR 26782, allowing for a 60-day public comment period. USCIS received 2... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and Form I-864W; Extension of a Currently Approved Information...

  9. Soluble CD40 ligand induces β3 integrin tyrosine phosphorylation and triggers platelet activation by outside-in signaling

    PubMed Central

    Prasad, K. S. Srinivasa; Andre, Patrick; He, Ming; Bao, Ming; Manganello, Jeanne; Phillips, David R.

    2003-01-01

    We earlier reported that the soluble form of the CD40 ligand (sCD40L), is involved in thrombosis by stabilizing platelet thrombi. In this article, we have determined the mechanism by which this protein affects platelet biology. Addition of sCD40L to washed platelets was found to activate the receptor function of αIIbβ3 as measured by the induction of fibrinogen binding and the formation of platelet microparticles. Mutation in the KGD sequence (D117E) of sCD40L, the αIIbβ3-binding domain in the N terminus of the protein resulted in a loss of the platelet-stimulatory activity of this protein. Integrilin, a αIIbβ3 antagonist, but not an antibody to CD40 that blocked the ligand-binding activity, inhibited these platelet-stimulatory events. CD40-/- platelets bound fibrinogen and formed microparticles similar to WT platelets, again indicating that CD40 is not involved in sCD40L-induced platelet activation. Exposure of platelets to sCD40L, but not D117E-sCD40L-coated surfaces, induced platelet thrombi formation under arterial shear rate. sCD40L-induced platelet stimulation resulted in the phosphorylation of tyrosine-759 in the cytoplasmic domain of β3. Platelets from the diYF mouse strain, expressing β3 in which both cytoplasmic tyrosines are mutated to phenylalanine, were defective in sCD40L-induced platelet stimulation. These data indicate that sCD40L is a primary platelet agonist and that platelet stimulation is induced by the binding of the KGD domain of sCD40L to αIIbβ3, triggering outside-in signaling by tyrosine phosphorylation of β3. PMID:14519852

  10. Epidermal keratinocyte-derived basophil promoting activity. Role of interleukin 3 and soluble CD23.

    PubMed Central

    Dalloul, A H; Arock, M; Fourcade, C; Béranger, J Y; Jaffray, P; Debré, P; Mossalayi, M D

    1992-01-01

    Human epidermal keratinocytes (EK) secrete factors able to sustain the proliferation of early myeloid cells and, in particular, the generation of basophils. This activity was previously attributed to IL-3, although no definitive in situ demonstration of this cytokine was provided. In regard to the possible physiological relevance of these data, we investigated herein the nature of EK-derived factors responsible for basophil promotion. Our data show that EK-derived supernatants (EK-sup) contain IL-3 as well as soluble CD23 (sCD23), both known for their colony stimulating activity. Messenger RNA for IL-3 and CD23 were also detected in EK. Blocking experiments using specific neutralizing monoclonal antibodies (mAb) further indicate that EK-derived basophil promoting activity is mainly due to the presence of IL-3 and sCD23 in EK-sup. Furthermore, by contrast to IL-3, sCD23 secretion by EK is cortisone sensitive and highly enhanced by IL-4, suggesting distinct regulatory mechanisms for their production. Images PMID:1401061

  11. Composition and antioxidant activity of water-soluble oligosaccharides from Hericium erinaceus.

    PubMed

    Hou, Yiling; Ding, Xiang; Hou, Wanru

    2015-05-01

    Oligosaccharide are carbohydrate molecules, comprising repeating units joined together by glycosidic bonds. In recent years, an increasing number of oligosaccharides have been reported to exhibit various biological activities, including antitumor, immune-stimulation and antioxidation effects. In the present study, crude water‑soluble oligosaccharides were extracted from the fruiting bodies of Hericium erinaceus with water and then successively purified by diethylaminoethyl‑cellulose 52 and Sephadex G‑100 column chromatography, yielding one major oligosaccharide fraction: Hericium erinaceus oligosaccharide (HEO‑A). The structural features of HEO‑A were investigated by a combination of monosaccharide component analysis by thin layer chromatography, infrared spectroscopy, nuclear magnetic resonance spectroscopy, scanning electron microscopy and high‑performance gel permeation chromatography. The results indicated that HEO‑A was composed of D‑xylose and D‑glucose, and the average molecular size was ~1,877 Da. The antioxidant activity of HEO‑A was evaluated using three biochemical methods to determine the scavenging activity of HEO‑A on 1,1‑diphenyl‑2‑picrylhydrazyl, hydrogen peroxide and 2,2'‑azino‑bis(3‑ethylbenzthiazoline‑6‑sufonic acid) diammonium radicals. The results indicated that HEO‑A may serve as an effective healthcare food and source of natural antioxidant compounds. PMID:25529054

  12. Removal of anaerobic soluble microbial products in a biological activated carbon reactor.

    PubMed

    Dong, Xiaojing; Zhou, Weili; He, Shengbing

    2013-09-01

    The soluble microbial products (SMP) in the biological treatment effluent are generally of great amount and are poorly biodegradable. Focusing on the biodegradation of anaerobic SMP, the biological activated carbon (BAC) was introduced into the anaerobic system. The experiments were conducted in two identical lab-scale up-flow anaerobic sludge blanket (UASB) reactors. The high strength organics were degraded in the first UASB reactor (UASB1) and the second UASB (UASB2, i.e., BAC) functioned as a polishing step to remove SMP produced in UASB1. The results showed that 90% of the SMP could be removed before granular activated carbon was saturated. After the saturation, the SMP removal decreased to 60% on the average. Analysis of granular activated carbon adsorption revealed that the main role of SMP removal in BAC reactor was biodegradation. A strain of SMP-degrading bacteria, which was found highly similar to Klebsiella sp., was isolated, enriched and inoculated back to the BAC reactor. When the influent chemical oxygen demand (COD) was 10,000 mg/L and the organic loading rate achieved 10 kg COD/(m3 x day), the effluent from the BAC reactor could meet the discharge standard without further treatment. Anaerobic BAC reactor inoculated with the isolated Klebsiella was proved to be an effective, cheap and easy technical treatment approach for the removal of SMP in the treatment of easily-degradable wastewater with COD lower than 10,000 mg/L. PMID:24520716

  13. Inhibitory activities of soluble and bound millet seed phenolics on free radicals and reactive oxygen species.

    PubMed

    Chandrasekara, Anoma; Shahidi, Fereidoon

    2011-01-12

    Oxidative stress, caused by reactive oxygen species (ROS), is responsible for modulating several pathological conditions and aging. Soluble and bound phenolic extracts of commonly consumed millets, namely, kodo, finger (Ravi), finger (local), foxtail, proso, little, and pearl, were investigated for their phenolic content and inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and ROS, namely, hydroxyl radical, peroxyl radical, hydrogen peroxide (H(2)O(2)), hypochlorous acid (HOCl), and singlet oxygen ((1)O(2)). Inhibition of DPPH and hydroxyl radicals was detrmined using electron paramagnetic resonance (EPR) spectroscopy. The peroxyl radical inhibitory activity was measured using the oxygen radical absorbance capacity (ORAC) assay. The scavenging of H(2)O(2), HOCl, and (1)O(2) was evaluated using colorimetric methods. The results were expressed as micromoles of ferulic acid equivalents (FAE) per gram of grain on a dry weight basis. In addition, major hydroxycinnamic acids were identified and quantified using high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (MS). All millet varieties displayed effective radical and ROS inhibition activities, which generally positively correlated with phenolic contents, except for hydroxyl radical. HPLC analysis revealed the presence of ferulic and p-coumaric acids as major hydroxycinnamic acids in phenolic extract and responsible for the observed effects. Bound extracts of millet contributed 38-99% to ROS scavenging, depending on the variety and the test system employed. Hence, bound phenolics must be included in the evaluation of the antioxidant activity of millets and other cereals. PMID:21133411

  14. Production of soluble and active microbial transglutaminase in Escherichia coli for site-specific antibody drug conjugation.

    PubMed

    Rickert, Mathias; Strop, Pavel; Lui, Victor; Melton-Witt, Jody; Farias, Santiago Esteban; Foletti, Davide; Shelton, David; Pons, Jaume; Rajpal, Arvind

    2016-02-01

    Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis. PMID:26481561

  15. Pu-238 fuel form activities, January 1-31, 1983

    SciTech Connect

    Not Available

    1983-03-01

    This monthly report for /sup 238/Pu Fuel Form Activities has two main sections: SRP-PuFF facility and SRL Fuel Form Activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  16. Pu-238 fuel form activities, January 1-31, 1981

    SciTech Connect

    Not Available

    1981-02-01

    This monthly report for /sup 238/Pu Fuel Form Activities has two main sections: SRP-PuFF facility and SRL Fuel Form Activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  17. Pu-238 fuel form activities, January 1-31, 1982

    SciTech Connect

    Not Available

    1982-03-01

    This monthly report for /sup 238/Pu fuel form activities has two main sections: SRP-PuFF facility and SRL fuel form activities. The program status, budget information, and milestone schedules are discussed in each main section. The Work Breakdown Structure (WBS) for this program is shown. Only one monthly report per year is processed for EDB.

  18. Synthesis of ZnS:Ag,Co water-soluble blue afterglow nanoparticles and application in photodynamic activation.

    PubMed

    Ma, Lun; Zou, Xiaoju; Hossu, Marius; Chen, Wei

    2016-08-01

    Silver and cobalt co-doped ZnS (ZnS:Ag,Co) water-soluble afterglow nanoparticles were synthesized using a wet chemistry method followed by aging at room temperature. The nanoparticles had a cubic zinc blende structure with average sizes of approximately 4 nm and emitted a blue fluorescence emission centered at 441 nm due to radiative transitions from surface defects to Ag(+) luminescent centers. Intense afterglow emission peaking at 475 nm from the obtained nanoparticles was observed and was red-shifted compared to the fluorescence emission peak. X-ray photoelectron spectroscopy revealed a large increase of O/S ratio, indicating a surface oxidation process during aging. The S vacancies produced accordingly may contribute to form more electron traps and enhance afterglow. The ZnS:Ag,Co afterglow nanoparticles have a very low dark-toxicity and are applied as a light source for photodynamic therapy activation by conjugating with protoporphyrin together. Our preliminary study has shown that the ZnS:Ag,Co afterglow nanoparticles can significantly reduce the x-ray dosage used in activation and thus may be a very promising candidate for future x-ray excited photodynamic therapy in deep cancer treatment. PMID:27345100

  19. Synthesis of ZnS:Ag,Co water-soluble blue afterglow nanoparticles and application in photodynamic activation

    NASA Astrophysics Data System (ADS)

    Ma, Lun; Zou, Xiaoju; Hossu, Marius; Chen, Wei

    2016-08-01

    Silver and cobalt co-doped ZnS (ZnS:Ag,Co) water-soluble afterglow nanoparticles were synthesized using a wet chemistry method followed by aging at room temperature. The nanoparticles had a cubic zinc blende structure with average sizes of approximately 4 nm and emitted a blue fluorescence emission centered at 441 nm due to radiative transitions from surface defects to Ag+ luminescent centers. Intense afterglow emission peaking at 475 nm from the obtained nanoparticles was observed and was red-shifted compared to the fluorescence emission peak. X-ray photoelectron spectroscopy revealed a large increase of O/S ratio, indicating a surface oxidation process during aging. The S vacancies produced accordingly may contribute to form more electron traps and enhance afterglow. The ZnS:Ag,Co afterglow nanoparticles have a very low dark-toxicity and are applied as a light source for photodynamic therapy activation by conjugating with protoporphyrin together. Our preliminary study has shown that the ZnS:Ag,Co afterglow nanoparticles can significantly reduce the x-ray dosage used in activation and thus may be a very promising candidate for future x-ray excited photodynamic therapy in deep cancer treatment.

  20. "Gone" into Solution: Assessing the Effect of Hands-On Activity on Students' Comprehension of Solubility

    ERIC Educational Resources Information Center

    Bruck, Laura B.; Bruck, Aaron D.; Phelps, Amy J.

    2010-01-01

    Solubility is challenging for many general chemistry students, and the interactions of aqueous species are difficult to conceptualize. Derived from the pedagogies of Johnstone, Bloom, and Piaget, our primary research questions probe whether students' conceptual understandings of solubility could be enhanced by participation in a concept-building,…

  1. Characterization of two water-soluble lignin metabolites with antiproliferative activities from Inonotus obliquus.

    PubMed

    Wang, Qingjie; Mu, Haibo; Zhang, Lin; Dong, Dongqi; Zhang, Wuxia; Duan, Jinyou

    2015-03-01

    The chaga mushroom, Inonotus obliquus has long been recognized as a remedy for cancer, gastritis, ulcers, and tuberculosis of the bones since the 16th century. Herein we reported the identification of two homogenous biological macromolecules, designated as IOW-S-1 and IOW-S-2 with anti-tumor activities from the hot-water extract of I. obliquus. Their molecular weights were determined to be 37.9 and 24.5kDa by high performance gel permeation chromatography (HPGPC) respectively. Chemical and spectral analysis indicated that both IOW-S-1 and IOW-S-2 were predominant in lignin, along with ∼20% carbohydrates. Examination of cytotoxicity showed that these two lignin-carbohydrate complexes induced cell death in a concentration dependent manner, while this apoptosis induction was largely cell-cycle independent. Further investigation demonstrated that IOW-S-1 or IOW-S-2 inhibited the activation of the nuclear transcription factor in cancer cells. These findings implied that soluble lignin derivatives were one of bioactive components in I. obliquus, and further provided insights into the understanding of molecular basis for diverse medicinal and nutritional values of this mushroom. PMID:25583019

  2. NO and CO Differentially Activate Soluble Guanylyl Cyclase via a Heme Pivot-bend Mechanism

    SciTech Connect

    Ma,X.; Sayed, N.; Beuve, A.; van den Akker, F.

    2007-01-01

    Diatomic ligand discrimination by soluble guanylyl cyclase (sGC) is paramount to cardiovascular homeostasis and neuronal signaling. Nitric oxide (NO) stimulates sGC activity 200-fold compared with only four-fold by carbon monoxide (CO). The molecular details of ligand discrimination and differential response to NO and CO are not well understood. These ligands are sensed by the heme domain of sGC, which belongs to the heme nitric oxide oxygen (H-NOX) domain family, also evolutionarily conserved in prokaryotes. Here we report crystal structures of the free, NO-bound, and CO-bound H-NOX domains of a cyanobacterial homolog. These structures and complementary mutational analysis in sGC reveal a molecular ruler mechanism that allows sGC to favor NO over CO while excluding oxygen, concomitant to signaling that exploits differential heme pivoting and heme bending. The heme thereby serves as a flexing wedge, allowing the N-terminal subdomain of H-NOX to shift concurrent with the transition of the six- to five-coordinated NO-bound state upon sGC activation. This transition can be modulated by mutations at sGC residues 74 and 145 and corresponding residues in the cyanobacterial H-NOX homolog.

  3. Formation of soluble microbial products (SMP) by activated sludge at various salinities.

    PubMed

    Li, Yan; Li, Ai-Min; Xu, Juan; Li, Wen-Wei; Yu, Han-Qing

    2013-02-01

    Soluble microbial products (SMP) present a significant component of effluent organic matter from biological wastewater treatment reactors, and can affect the membrane fouling and formation of disinfection by-products. Thus, SMP have attracted increasing concerns in wastewater treatment and reclamation. In this work, the formation of SMP by activated sludge at various NaCl concentrations is investigated by using fluorescence excitation-emission matrix (EEM) spectroscopy with parallel factor analysis (PARAFAC) and fluorescence regional integration (FRI). The results show that a high level of salinity decreases substrate removal efficiency and leads to an accumulation of SMP, especially proteins. Three components of SMP, one protein-like and two humic-acid-like components, are identified by PARAFAC, which exhibit different trends with the variation of NaCl concentration. FRI analysis reveals that the majority of protein fluorescence is attributed to tryptophan and tryptophan-like proteins, rather than tyrosine and tyrosine-like proteins. With an increase in NaCl concentration, the normalized volume percentages of tyrosine and tryptophan region increase, while those of humic- and fulvic-acid-like region decrease significantly. This work demonstrates that salinity affects the formation of SMP, and that EEM with PARAFAC in combination with FRI analysis is a useful tool to get insight into the formation of SMP by activated sludge. PMID:22622691

  4. Water-soluble Co(III) complexes of substituted phenanthrolines with cell selective anticancer activity.

    PubMed

    Jagadeesan, Sivaraman; Balasubramanian, Vimalkumar; Baumann, Patric; Neuburger, Markus; Häussinger, Daniel; Palivan, Cornelia G

    2013-11-01

    Transition metal complexes with substituted phenanthrolines as ligands represent potential anticancer products without the drawbacks of platinum complexes that are currently marketed. Here, we report the synthesis and cell selective anticancer activity of five new water-soluble Co(III) complexes with methyl substituted phenanthroline ligands. The complexes were characterized by elemental analysis, NMR, FAB-mass spectrometry, FTIR, electronic spectroscopy, and single crystal X-ray diffraction. Possible interaction of these complexes with DNA was assessed by a combination of circular dichroism, UV-vis spectroscopy titration, and ethidium bromide displacement assay, and the results indicated that DNA interaction is weak for these complexes. Cellular uptake and cytotoxicity of complexes at low concentrations were assessed by flow cytometry on PC-3 cells, while their effect on intracellular mitochondrial function was measured by MTS assay on HeLa and PC-3 cell lines. These complexes showed selective cytotoxicity with a significantly higher effect on intracellular mitochondrial function in PC-3 cells than in HeLa cells. At low concentrations, complex 2 had the highest cytotoxic effect on PC-3 cells, inducing around 38% cell death, and the correlation of cytotoxicity of these complexes to their hydrophobicity indicates that an appropriate value of the hydrophobicity is essential for high antitumor activity. PMID:24127683

  5. Soluble microbial products (SMPs) release in activated sludge systems: a review

    PubMed Central

    2012-01-01

    This review discusses the characterization, production and implications of soluble microbial products (SMPs) in biological wastewater treatment. The precise definition of SMPs is open to talk about, but is currently regarded as “the pool of organic compounds that are released into solution from substrate metabolism and biomass decay”'. Some of the SMPs have been identified as humic acids, polysaccharides, proteins, amino acids, antibiotics, extracellular enzymes and structural components of cells and products of energy metabolism. They adversely affect the kinetic activity, flocculating and settling properties of sludge. This review outlines some important findings with regard to biodegradability and treatability of SMPs and also the effect of process parameters on their production. As SMPs are produced during biological treatment process, their trace amounts normally remain in the effluent that defines the highest COD removal efficiency. Their presence in effluent represents a high potential risk of toxic by-product formation during chlorine disinfection. Studies have indicated that among all wastewater post-treatment processes, the adsorption by granular activated carbon combined with biologically induced degradation is the most effective method for removal of SMPs. However, it may be concludes that the knowledge regarding SMPs is still under progress and more work is required to fully understand their contribution to the treatment process. PMID:23369231

  6. Soluble microbial products (SMPs) release in activated sludge systems: a review.

    PubMed

    Azami, Hamed; Sarrafzadeh, Mohammad Hossein; Mehrnia, Mohammad Reza

    2012-01-01

    This review discusses the characterization, production and implications of soluble microbial products (SMPs) in biological wastewater treatment. The precise definition of SMPs is open to talk about, but is currently regarded as "the pool of organic compounds that are released into solution from substrate metabolism and biomass decay"'. Some of the SMPs have been identified as humic acids, polysaccharides, proteins, amino acids, antibiotics, extracellular enzymes and structural components of cells and products of energy metabolism. They adversely affect the kinetic activity, flocculating and settling properties of sludge. This review outlines some important findings with regard to biodegradability and treatability of SMPs and also the effect of process parameters on their production. As SMPs are produced during biological treatment process, their trace amounts normally remain in the effluent that defines the highest COD removal efficiency. Their presence in effluent represents a high potential risk of toxic by-product formation during chlorine disinfection. Studies have indicated that among all wastewater post-treatment processes, the adsorption by granular activated carbon combined with biologically induced degradation is the most effective method for removal of SMPs. However, it may be concludes that the knowledge regarding SMPs is still under progress and more work is required to fully understand their contribution to the treatment process. PMID:23369231

  7. A study on antifungal activity of water-soluble chitosan against Macrophomina phaseolina.

    PubMed

    Chatterjee, Sudipta; Chatterjee, Bishnu P; Guha, Arun K

    2014-06-01

    The objective of this study was to evaluate antifungal effect of water-soluble chitosan (s-chitosan) on Macrophomina phaseolina (M. phaseolina) causing jute seedling infection and monitor the change in activity of released enzymes during infection. The minimum inhibitory concentration (MIC) of s-chitosan for M. phaseolina was found at 12.5g/l and s-chitosan exhibited fungistatic mode of action against this pathogen. The application of s-chitosan (12.5g/l) during infection of jute seedlings by M. phaseolina inhibited fungal infection and length of the seedlings was found almost similar to seedlings without infection. M. phaseolina infected jute seedlings showed length of 22mm over 10 days of incubation and it increased to 58mm in presence of s-chitosan (12.5g/l) during incubation for 10 days. TEM study indicated presence of hyphae in the cortical and epidermal cells of fungus infected jute seedlings indicating colonization by the fungus and it disappeared after treatment with s-chitosan. The changes in enzyme profiles of jute seedling during prevention of fungal infection using s-chitosan helped in proper understanding of mode of action of s-chitosan as antifungal agent. The activity of defense related enzymes like chitosanase and peroxidase in infected seedlings was observed to be enhanced after treatment with s-chitosan. PMID:24747381

  8. Extraction, characterization and antioxidant activity of water-soluble polysaccharides from Tuber huidongense.

    PubMed

    Chen, Guangjing; Zhang, Shiqi; Ran, Chunxia; Wang, Lisha; Kan, Jianquan

    2016-10-01

    Single-factor experiment and Box-Behnken design were employed to optimize the ultrasonic-assisted extraction (UAE) of water-soluble polysaccharides from Chinese truffle Tuber huidongense (THWP). The optimal extraction conditions with an extraction yield of 7.17±0.22% crude THWP were determined as follows: ultrasonic power 99.65W, extraction time 40.39min, ratio of water to raw material 24.65mL/g, and extraction temperature 70.1°C. Two purified fractions, THWP-1 and THWP-2 with molecular weights of 128kDa and 729kDa, respectively, were obtained from crude THWP by Cellulose DEAE-52 and Sephadex G-100 chromatography. Monosaccharide component analysis by high performance liquid chromatography (HPLC) indicated that THWP-1 was composed of Glc, Man, and Gal with their corresponding mole percentages of 60.56%, 20.12% and 19.32%, respectively and that THWP-2 contained only Glc. Evaluation of the antioxidant activity in vitro suggested that THWP-1 and THWP-2 had effective scavenging activity of 2,2'-azinobis-(3-ethylbenzothiazolin-6-sulfonicacid) (ABST), hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. Accordingly, the polysaccharides from Chinese truffle might serve as a natural antioxidant in functional foods. PMID:27259646

  9. The murine neutrophil NLRP3 inflammasome is activated by soluble but not particulate or crystalline agonists.

    PubMed

    Chen, Kaiwen W; Bezbradica, Jelena S; Groß, Christina J; Wall, Adam A; Sweet, Matthew J; Stow, Jennifer L; Schroder, Kate

    2016-04-01

    Neutrophils express pattern recognition receptors (PRRs) and regulate immune responses via PRR-dependent cytokine production. An emerging theme is that neutrophil PRRs often exhibit cell type-specific adaptations in their signalling pathways. This prompted us to examine inflammasome signalling by the PRR NLRP3 in murine neutrophils, in comparison to well-established NLRP3 signalling pathways in macrophages. Here, we demonstrate that while murine neutrophils can indeed signal via the NLRP3 inflammasome, neutrophil NLRP3 selectively responds to soluble agonists but not to the particulate/crystalline agonists that trigger NLRP3 activation in macrophages via phagolysosomal rupture. In keeping with this, alum did not trigger IL-1β production from human PMN, and the lysosomotropic peptide Leu-Leu-OMe stimulated only weak NLRP3-dependent IL-1β production from murine neutrophils, suggesting that lysosomal rupture is not a strong stimulus for NLRP3 activation in neutrophils. We validated our in vitro findings for poor neutrophil NLRP3 responses to particles in vivo, where we demonstrated that neutrophils do not significantly contribute to alum-induced IL-1β production in mice. In all, our studies highlight that myeloid cell identity and the nature of the danger signal can strongly influence signalling by a single PRR, thus shaping the nature of the resultant immune response. PMID:27062120

  10. Development of clinical dosage forms for a poorly water soluble drug I: Application of polyethylene glycol-polysorbate 80 solid dispersion carrier system.

    PubMed

    Dannenfelser, Rose-Marie; He, Handan; Joshi, Yatindra; Bateman, Simon; Serajuddin, Abu T M

    2004-05-01

    Different formulation approaches were evaluated to ensure that the formulation of a poorly water soluble compound chosen during early development achieves optimum bioavailability. The insoluble compound has an aqueous solubility of 0.17 micro g/mL at 25 +/- 1 degrees C, a relatively high permeability (Caco2 P(app) = 6.1 x 10(-4) cm/min), and poor bioavailability in dogs (dry blend formulation). Based on the prediction by GastroPlus, the oral absorption of this compound is sensitive to its apparent solubility and particle size. The oral bioavailability of three different formulations was compared in a dog model: a cosolvent-surfactant solution, a solid dispersion in a mixture of polyethylene glycol 3350 and polysorbate 80, and a dry blend of micronized drug with microcrystalline cellulose. In absence of a parenteral injection, the bioavailability of the solution was considered to be 100%, and the relative oral bioavailability of the three formulations was 100, 99.1, 9.8, respectively. Comparable bioavailability was obtained with the solid dispersion and the cosolvent-surfactant solution, both of which showed a 10-fold higher bioavailability than the dry blend. Thus, a 20 mg dose strength capsule containing the solid dispersion formulation was selected for clinical development. The selected solid dispersion system was physically and chemically stable for at least 16 months at 25 degrees C/60% RH. In conclusion, the bioavailability of a poorly water soluble drug was greatly enhanced using the solid dispersion formulation containing a water soluble polymer with a surface active agent. PMID:15067693

  11. Autophosphorylation Activity of a Soluble Hexameric Histidine Kinase Correlates with the Shift in Protein Conformational Equilibrium

    PubMed Central

    Wojnowska, Marta; Yan, Jun; Sivalingam, Ganesh N.; Cryar, Adam; Gor, Jayesh; Thalassinos, Konstantinos; Djordjevic, Snezana

    2013-01-01

    Summary In a commonly accepted model, in response to stimuli, bacterial histidine kinases undergo a conformational transition between an active and inactive form. Structural information on histidine kinases is limited. By using ion mobility-mass spectrometry (IM-MS), we demonstrate an exchange between two conformational populations of histidine kinase ExsG that are linked to different levels of kinase activity. ExsG is an atypical signaling protein that incorporates an uncommon histidine kinase catalytic core at the C terminus preceded by an N-terminal “receiver domain” that is normally associated with the response regulator proteins in two-component signal transduction systems. IM-MS analysis and enzymatic assays indicate that phosphorylation of the ExsG receiver domain stabilizes the “compact” form of the protein and inhibits kinase core activity; in contrast, nucleotide binding required for kinase activity is associated with the more open conformation of ExsG. PMID:24210218

  12. Bithionol Potently Inhibits Human Soluble Adenylyl Cyclase through Binding to the Allosteric Activator Site.

    PubMed

    Kleinboelting, Silke; Ramos-Espiritu, Lavoisier; Buck, Hannes; Colis, Laureen; van den Heuvel, Joop; Glickman, J Fraser; Levin, Lonny R; Buck, Jochen; Steegborn, Clemens

    2016-04-29

    The signaling molecule cAMP regulates functions ranging from bacterial transcription to mammalian memory. In mammals, cAMP is synthesized by nine transmembrane adenylyl cyclases (ACs) and one soluble AC (sAC). Despite similarities in their catalytic domains, these ACs differ in regulation. Transmembrane ACs respond to G proteins, whereas sAC is uniquely activated by bicarbonate. Via bicarbonate regulation, sAC acts as a physiological sensor for pH/bicarbonate/CO2, and it has been implicated as a therapeutic target, e.g. for diabetes, glaucoma, and a male contraceptive. Here we identify the bisphenols bithionol and hexachlorophene as potent, sAC-specific inhibitors. Inhibition appears mostly non-competitive with the substrate ATP, indicating that they act via an allosteric site. To analyze the interaction details, we solved a crystal structure of an sAC·bithionol complex. The structure reveals that the compounds are selective for sAC because they bind to the sAC-specific, allosteric binding site for the physiological activator bicarbonate. Structural comparison of the bithionol complex with apo-sAC and other sAC·ligand complexes along with mutagenesis experiments reveals an allosteric mechanism of inhibition; the compound induces rearrangements of substrate binding residues and of Arg(176), a trigger between the active site and allosteric site. Our results thus provide 1) novel insights into the communication between allosteric regulatory and active sites, 2) a novel mechanism for sAC inhibition, and 3) pharmacological compounds targeting this allosteric site and utilizing this mode of inhibition. These studies provide support for the future development of sAC-modulating drugs. PMID:26961873

  13. Targeted water soluble copper-tetrazolate complexes: interactions with biomolecules and catecholase like activities.

    PubMed

    Saha, Manideepa; Das, Mriganka; Nasani, Rajendar; Choudhuri, Indrani; Yousufuddin, Muhammed; Nayek, Hari Pada; Shaikh, Mobin M; Pathak, Biswarup; Mukhopadhyay, Suman

    2015-12-14

    Two new mononuclear water soluble copper(II) complexes, [Cu{(5-pyrazinyl)tetrazolate}2(1,10-phenanthroline)] 1 and [Cu{(5-pyrazinyl)tetrazolate}(1,10-phenanthroline)2](NO3)0.5(N3)0.5 2, have been synthesized using the metal mediated [2 + 3] cycloaddition reaction between copper bound azide and pyrazinecarbonitrile. The interactions of these copper tetrazolate complexes 1 and 2 with biomolecules like DNA and bovine serum albumin (BSA) are studied and the catecholase like catalytic activity of compound 2 is also explored. Structural determination reveals that both compounds 1 and 2 are octahedral in nature. Screening tests were conducted to quantify the binding ability of complexes (1 and 2) towards DNA and it was revealed that complex 2 has a stronger affinity to bind to CT-DNA. DFT studies indicated that a lower HOMO-LUMO energy gap between the DNA fragment and metal complexes might be the reason for this type of stronger interaction. DNA cleavage activity was explored by gel-electrophoresis and moderate to strong DNA cleavage properties were observed in the presence and absence of co-reagents. Inhibition of cleavage in the presence of sodium azide indicates the propagation of the activity through the production of singlet molecular oxygen. Furthermore enzyme kinetic studies reflect that complex 2 is also effective in mimicking catecholase like activities. An ESI-MS spectral study indicates the probable involvement of dimeric species [(phen)2Cu-(OH)2-Cu(phen)2](2+) in the catalytic cycle. PMID:26530012

  14. A Soluble Guanylate Cyclase Activator Inhibits the Progression of Diabetic Nephropathy in the ZSF1 Rat.

    PubMed

    Boustany-Kari, Carine M; Harrison, Paul C; Chen, Hongxing; Lincoln, Kathleen A; Qian, Hu Sheng; Clifford, Holly; Wang, Hong; Zhang, Xiaomei; Gueneva-Boucheva, Kristina; Bosanac, Todd; Wong, Diane; Fryer, Ryan M; Richman, Jeremy G; Sarko, Chris; Pullen, Steven S

    2016-03-01

    Therapies that restore renal cGMP levels are hypothesized to slow the progression of diabetic nephropathy. We investigated the effect of BI 703704, a soluble guanylate cyclase (sGC) activator, on disease progression in obese ZSF1 rats. BI 703704 was administered at doses of 0.3, 1, 3, and 10 mg/kg/d to male ZSF1 rats for 15 weeks, during which mean arterial pressure (MAP), heart rate (HR), and urinary protein excretion (UPE) were determined. Histologic assessment of glomerular and interstitial lesions was also performed. Renal cGMP levels were quantified as an indicator of target modulation. BI 703704 resulted in sGC activation, as evidenced by dose-dependent increases in renal cGMP levels. After 15 weeks of treatment, sGC activation resulted in dose-dependent decreases in UPE (from 463 ± 58 mg/d in vehicle controls to 328 ± 55, 348 ± 23, 283 ± 45, and 108 ± 23 mg/d in BI 703704-treated rats at 0.3, 1, 3, and 10 mg/kg, respectively). These effects were accompanied by a significant reduction in the incidence of glomerulosclerosis and interstitial lesions. Decreases in MAP and increases in HR were only observed at the high dose of BI 703704. These results are the first demonstration of renal protection with sGC activation in a nephropathy model induced by type 2 diabetes. Importantly, beneficial effects were observed at doses that did not significantly alter MAP and HR. PMID:26729306

  15. Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-β-Glucanase Heterologously Expressed in Pichia pastoris1

    PubMed Central

    Mølhøj, Michael; Ulvskov, Peter; Dal Degan, Florence

    2001-01-01

    The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-β-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-β-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine1-lysine70), a hydrophobic transmembrane domain (isoleucine71-valine93), and a periplasmic catalytic core (lysine94-proline621). Here, we report the functional analysis of Δ1-90Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Δ1-90Cel16 in a pure form. The molecular mass of Δ1-90Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Δ1-90Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Δ1-90Cel16 had a pH optimum of 6.0. The activity of Δ1-90Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Δ1-90Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1→3),(1→4)-β-d-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Δ1-90Cel16-hydrolyzed carboxymethylcellulose showed that Δ1-90Cel16 is a true endo-acting glucanase. PMID:11598241

  16. Improved Aqueous Solubility and Antihypercholesterolemic Activity of Ezetimibe on Formulating with Hydroxypropyl-β-Cyclodextrin and Hydrophilic Auxiliary Substances.

    PubMed

    Srivalli, Kale Mohana Raghava; Mishra, Brahmeshwar

    2016-04-01

    The purpose of this study was to improve the aqueous solubility, dissolution, and pharmacodynamic properties of a BCS class II drug, ezetimibe (Eze) by preparing ternary cyclodextrin complex systems. We investigated the potential synergistic effect of two novel hydrophilic auxiliary substances, D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) and L-ascorbic acid-2-glucoside (AA2G) on hydroxypropyl-β-cyclodextrin (HPBCD) solubilization of poorly water-soluble hypocholesterolemic drug, Eze. In solution state, the binary and ternary systems were analyzed by phase solubility studies and Job's plot. The solid complexes prepared by freeze-drying were characterized by Fourier transform infrared (FTIR), differential scanning calorimetry (DSC), powder X-ray diffraction (XRD), nuclear magnetic resonance (NMR), and scanning electron microscopy (SEM). The log P values, aqueous solubility, dissolution, and antihypercholesterolemic activity of all systems were studied. The analytical techniques confirmed the formation of inclusion complexes in the binary and ternary systems. HPBCD complexation significantly (p < 0.05) reduced the log P and improved the solubility, dissolution, and hypocholesterolemic properties of Eze, and the addition of ternary component produced further significant improvement (p < 0.05) even compared to binary system. The remarkable reduction in log P and enhancement in solubility, dissolution, and antihypercholesterolemic activity due to the addition of TPGS or AA2G may be attributed to enhanced wetting, dispersibility, and complete amorphization. The use of TPGS or AA2G as ternary hydrophilic auxiliary substances improved the HPBCD solubilization and antihypercholesterolemic activity of Eze. PMID:26077622

  17. Soluble porous coordination polymers by mechanochemistry: from metal-containing films/membranes to active catalysts for aerobic oxidation.

    PubMed

    Zhang, Pengfei; Li, Haiying; Veith, Gabriel M; Dai, Sheng

    2015-01-14

    Soluble porous coordination polymers from mechanochemical synthesis are presented through a coordination polymerization between highly contorted, rigid tetraphenol and a broad variety of transition metal ions. These polymers can be easily cast as metal-containing films or freestanding membranes. Importantly, as-made coordination polymers are highly active and stable in the aerobic oxidation of allylic C-H bonds. PMID:25389070

  18. Synthesis and biological evaluation of novel pyrazoles and indazoles as activators of the nitric oxide receptor, soluble guanylate cyclase.

    PubMed

    Selwood, D L; Brummell, D G; Budworth, J; Burtin, G E; Campbell, R O; Chana, S S; Charles, I G; Fernandez, P A; Glen, R C; Goggin, M C; Hobbs, A J; Kling, M R; Liu, Q; Madge, D J; Meillerais, S; Powell, K L; Reynolds, K; Spacey, G D; Stables, J N; Tatlock, M A; Wheeler, K A; Wishart, G; Woo, C K

    2001-01-01

    Database searching and compound screening identified 1-benzyl-3-(3-dimethylaminopropyloxy)indazole (benzydamine, 3) as a potent activator of the nitric oxide receptor, soluble guanylate cyclase. A comprehensive structure-activity relationship study surrounding 3 clearly showed that the indazole C-3 dimethylaminopropyloxy substituent was critical for enzyme activity. However replacement of the indazole ring of 3 by appropriately substituted pyrazoles maintained enzyme activity. Compounds were evaluated for inhibition of platelet aggregation and showed a general lipophilicity requirement. Aryl-substituted pyrazoles 32, 34, and 43 demonstrated potent activation of soluble guanylate cyclase and potent inhibition of platelet aggregation. Pharmacokinetic studies in rats showed that compound 32 exhibits modest oral bioavailability (12%). Furthermore 32 has an excellent selectivity profile notably showing no significant inhibition of phosphodiesterases or nitric oxide synthases. PMID:11141091

  19. Synthesis and structure-activity relationship of piperidine-derived non-urea soluble epoxide hydrolase inhibitors

    SciTech Connect

    Pecic, Stevan; Pakhomova, Svetlana; Newcomer, Marcia E.; Morisseau, Christophe; Hammock, Bruce D.; Zhu, Zhengxiang; Rinderspacher, Alison; Deng, Shi-Xian

    2013-09-27

    A series of potent amide non-urea inhibitors of soluble epoxide hydrolase (sEH) is disclosed. The inhibition of soluble epoxide hydrolase leads to elevated levels of epoxyeicosatrienoic acids (EETs), and thus inhibitors of sEH represent one of a novel approach to the development of vasodilatory and anti-inflammatory drugs. Structure–activities studies guided optimization of a lead compound, identified through high-throughput screening, gave rise to sub-nanomolar inhibitors of human sEH with stability in human liver microsomal assay suitable for preclinical development.

  20. Active, soluble recombinant melittin purified by extracting insoluble lysate of Escherichia coli without denaturation

    PubMed Central

    Buhrman, Jason S.; Cook, Laura C.; Rayahin, Jamie E.; Federle, Michael J.; Gemeinhart, Richard A.

    2013-01-01

    Cell lytic peptides are a class of drugs that can be used to selectively kill invading organisms or diseased cells. Several of these peptides have been identified as potential therapeutics. Herein, we report a novel process for purifying recombinant melittin, a cell lytic peptide that inserts into the membranes of cells causing cell lysis, from Escherichia coli. The process involves surfactant and low pH to solubilize melittin fusion proteins from the insoluble fraction of bacterial lysates. We are able to significantly improve purity of the final product and confirm the activity of the peptide. The process yields recombinant melittin that is effective when used to treat U-87 MG glioma cells and inhibits growth of the Gram-positive pathogenic bacterium Streptococcus pyogenes. We demonstrate a method of repeated extraction of the insoluble protein fraction with mild detergent at a low pH that is able to generate a yield of pure, soluble melittin of approximately 0.5 to 1 mg/L of E. coli culture. PMID:23926061

  1. Antitumor and immunomodulatory activities of a water-soluble polysaccharide from Chaenomeles speciosa.

    PubMed

    Xie, Xianfei; Zou, Guolin; Li, Chenghai

    2015-11-01

    In this study, a water-soluble polysaccharide (CSP) was successfully purified from Chaenomeles speciosa by DEAE-Sepharose and Sephadex G-100 column chromatography. CSP had a weight-average molecular weight of about 6.3 × 10(4)Da and was composed of glucose (Glc), galactose (Gal), rhamnose (Rha) and arabinose (Ara) with a relative molar ratio of 4.6:1.3:0.8:0.5. CSP could not only inhibit the growth of S180 tumor transplanted in mice, but also increase the relative spleen index and body weight of tumor bearing mice. Moreover, concanavalin A (ConA) and lipopolysaccharide (LPS) induced splenocyte proliferation and peritoneal macrophage phagocytosis were also enhanced after CSP administration. Furthermore, CSP treatment could improve delayed type hypersensitivity (DTH) and promote the secretion of IL-2, TNF-α and IFN-γ in serum. The overall findings suggest that the antitumor effect of CSP is might be associated with its potent immunostimulatory activity. PMID:26256355

  2. Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes.

    PubMed

    Alfano, Massimo; Cinque, Paola; Giusti, Guido; Proietti, Silvia; Nebuloni, Manuela; Danese, Silvio; D'Alessio, Silvia; Genua, Marco; Portale, Federica; Lo Porto, Manuela; Singhal, Pravin C; Rastaldi, Maria Pia; Saleem, Moin A; Mavilio, Domenico; Mikulak, Joanna

    2015-01-01

    Increased plasma level of soluble urokinase-type plasminogen activator receptor (suPAR) was associated recently with focal segmental glomerulosclerosis (FSGS). In addition, different clinical studies observed increased concentration of suPAR in various glomerular diseases and in other human pathologies with nephrotic syndromes such as HIV and Hantavirus infection, diabetes and cardiovascular disorders. Here, we show that suPAR induces nephrin down-modulation in human podocytes. This phenomenon is mediated only by full-length suPAR, is time-and dose-dependent and is associated with the suppression of Wilms' tumor 1 (WT-1) transcription factor expression. Moreover, an antagonist of αvβ3 integrin RGDfv blocked suPAR-induced suppression of nephrin. These in vitro data were confirmed in an in vivo uPAR knock out Plaur(-/-) mice model by demonstrating that the infusion of suPAR inhibits expression of nephrin and WT-1 in podocytes and induces proteinuria. This study unveiled that interaction of full-length suPAR with αvβ3 integrin expressed on podocytes results in down-modulation of nephrin that may affect kidney functionality in different human pathologies characterized by increased concentration of suPAR. PMID:26380915

  3. Inhibition of soluble epoxide hydrolase activity by compounds isolated from the aerial parts of Glycosmis stenocarpa.

    PubMed

    Kim, Jang Hoon; Morgan, Abubaker M A; Tai, Bui Huu; Van, Doan Thi; Cuong, Nguyen Manh; Kim, Young Ho

    2016-08-01

    The aim of this study is to search for soluble epoxide hydrolase (sEH) inhibitors from natural plants, bioassay-guided fractionation of lipophilic n-hexane and chloroform layers of an extract of the aerial parts of Glycosmis stenocarpa led to the isolation of 12 compounds (1-12) including murrayafoline-A (1), isomahanine (2), bisisomahanine (3), saropeptate (4), (24 S)-ergost-4-en-3,6-dione (5), stigmasta-4-en-3,6-dion (6), stigmast-4-en-3-one (7), β-sitosterol (8), 24-methylpollinastanol (9), trans-phytol (10), neosarmentol III (11) and (+)-epiloliolide (12). Their structures were elucidated on the basis of spectroscopic data. Among them, neosarmentol III (11) was isolated from nature for the first time. All the isolated compounds were evaluated for their inhibitory activity against sEH. Among isolated carbazole-type compounds, isomahanine (2) and bisisomahanine (3) were identified as a potent inhibitor of sEH, with IC50 values of 22.5 ± 1.7 and 7.7 ± 1.2 µM, respectively. Moreover, the inhibitory action of 2 and 3 represented mixed-type enzyme inhibition. PMID:26444316

  4. Bismuth(III) 5-sulfosalicylate complexes: structure, solubility and activity against Helicobacter pylori.

    PubMed

    Andrews, Philip C; Deacon, Glen B; Ferrero, Richard L; Junk, Peter C; Karrar, Abdulgader; Kumar, Ish; MacLellan, Jonathan G

    2009-08-28

    Treatment of 5-sulfosalicylic acid (H(3)Ssal) with BiPh(3) results in the formation of the first dianionic carboxylate-sulfonate bismuth complex, [PhBi(HSsal)H(2)O](infinity) 1a, and its ethanol analogue [PhBi(HSsal)EtOH](infinity) 1b (space group P2(1)/c), while Bi(OAc)(3) gives the mixed monoanionic and dianionic complex, {[Bi(HSsal)(H(2)Ssal)(H(2)O)(3)](2) x 2 H(2)O}(infinity) 2 (space group P1). The three complexes are all polymeric in the solid state as determined by single crystal X-ray diffraction, with extended frameworks constructed from dimeric [Bi(HSsal)](2), 1a and 1b, or from [Bi(HSsal)(H(2)Ssal)](2) units, 2. The heteroleptic bismuth complexes 1a and 2 display remarkable aqueous solubility, 10 and 2.5 mg ml(-1) respectively, resulting in a clear solution of pH 1.5. In contrast, 1b is essentially insoluble in aqueous environments. All three complexes show significant activity against the bacterium Helicobacter pylori of <6.25 microg ml(-1). PMID:19655072

  5. Isolation and prebiotic activity of water-soluble polysaccharides fractions from the bamboo shoots (Phyllostachys praecox).

    PubMed

    He, Shudong; Wang, Xin; Zhang, Yi; Wang, Jing; Sun, Hanju; Wang, Junhui; Cao, Xiaodong; Ye, Yongkang

    2016-10-20

    The water-soluble polysaccharides from bamboo shoots (Phyllostachys praecox) (WBP) were isolated, and the characterizations as well as prebiotic activities were investigated. The yield of WBP was 7.58±0.31% under optimal hot-water extraction conditions. Two fractions, i.e., WBP-1 and WBP-2 with molecular weight of 83.50kDa and 80.08kDa, respectively, were purified by chromatography. Both the polysaccharides fractions were identified as heteropolysaccharides-protein complexes composed of 15 kinds of common amino acids in protein part and rhamnose, arabinose, xylose, mannose, glucose and galactose in different molar ratios in polysaccharide part. The existence of α- and β-glycosidic linkages between the sugar units was confirmed by FTIR and NMR spectra. Compared with the blank control and the reference of FOS, WBP-1 and WBP-2 significantly increased the numbers of Bifidobacterium adolescentis and Bifidobacterium bifidum (P<0.05), which contributed to the production of organic acids, suggesting that the polysaccharides have potential prebiotic properties. PMID:27474570

  6. Functional characterization of a chimeric soluble Fas ligand polymer with in vivo anti-tumor activity.

    PubMed

    Daburon, Sophie; Devaud, Christel; Costet, Pierre; Morello, Aurore; Garrigue-Antar, Laure; Maillasson, Mike; Hargous, Nathalie; Lapaillerie, Delphine; Bonneu, Marc; Dechanet-Merville, Julie; Legembre, Patrick; Capone, Myriam; Moreau, Jean-François; Taupin, Jean-Luc

    2013-01-01

    Binding of ligand FasL to its receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the homotrimeric state. We generated a series of FasL chimeras by fusing FasL to domains of the Leukemia Inhibitory Factor receptor gp190 which confer homotypic oligomerization, and analyzed the capacity of these soluble chimeras to trigger cell death. We observed that the most efficient FasL chimera, called pFasL, was also the most polymeric, as it reached the size of a dodecamer. Using a cellular model, we investigated the structure-function relationships of the FasL/Fas interactions for our chimeras, and we demonstrated that the Fas-mediated apoptotic signal did not solely rely on ligand-mediated receptor aggregation, but also required a conformational adaptation of the Fas receptor. When injected into mice, pFasL did not trigger liver injury at a dose which displayed anti-tumor activity in a model of human tumor transplanted to immunodeficient animals, suggesting a potential therapeutic use. Therefore, the optimization of the FasL conformation has to be considered for the development of efficient FasL-derived anti-cancer drugs targeting Fas. PMID:23326557

  7. A new water soluble MAPK activator exerts antitumor activity in melanoma cells resistant to the BRAF inhibitor vemurafenib.

    PubMed

    Graziani, Grazia; Artuso, Simona; De Luca, Anastasia; Muzi, Alessia; Rotili, Dante; Scimeca, Manuel; Atzori, Maria Grazia; Ceci, Claudia; Mai, Antonello; Leonetti, Carlo; Levati, Lauretta; Bonanno, Elena; Tentori, Lucio; Caccuri, Anna Maria

    2015-05-01

    Recovery of mitogen activated protein kinase (MAPK) or activation of alternative pathways, such as the PI3K/AKT/mTOR, are involved in acquired resistance to BRAF inhibitors which represent the first-line treatment of BRAF-mutated metastatic melanoma. We recently demonstrated that 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX) and its water soluble analog 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)ethoxy)ethoxy)ethanol (MC3181) trigger apoptosis in BRAF V600E mutated melanoma cells through activation of the MAPK c-Jun N-terminal kinase (JNK). Herein, we investigated whether NBDHEX and MC3181 might exert antitumor activity against BRAF V600E mutated human melanoma cells rendered resistant to the BRAF inhibitor vemurafenib. To this aim we generated a subline of A375 melanoma resistant in vitro and in vivo to vemurafenib (A375-VR8) and characterized by NRAS G13R mutation, high basal levels of CRAF protein and phospho-activation of AKT. In these cells ERK phosphorylation was not significantly down-modulated by vemurafenib concentrations capable of abrogating ERK phosphorylation in sensitive A375 cells. Both NBDHEX and MC3181 induced marked antiproliferative and apoptotic effects in A375-VR8 cells and, at equitoxic concentrations, caused a strong phosphorylation of JNK, p38, and of the downstream mediators of apoptosis ATF2 and p53. Drug treatment further increased ERK phosphorylation, which was required for the cellular response to the NBD derivatives, as apoptosis was antagonized by the ERK inhibitor FR180204. Finally, in vivo administration of MC3181 provoked JNK activation at the tumor site and markedly reduced A375-VR8 growth. These evidences strongly suggest that the activation of multiple pro-apoptotic MAPK pathways by MC3181 might represent a new strategy for the treatment of melanoma resistant to BRAF inhibitors. PMID:25795251

  8. Increased Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) Levels in Plasma of Suicide Attempters

    PubMed Central

    Ventorp, Filip; Gustafsson, Anna; Träskman-Bendz, Lil; Westrin, Åsa; Ljunggren, Lennart

    2015-01-01

    The soluble form of the urokinase receptor, suPAR, has been suggested as a novel biomarker of low-grade inflammation. Activation of the immune system has been proposed to contribute to the development of depression and suicidal behavior. In order to identify depressed and suicidal individuals who could benefit from an anti-inflammatory treatment, a reliable biomarker of low-grade inflammation is vital. This study evaluates plasma suPAR levels as a biomarker of low-grade inflammation in patients with major depressive disorder and in patients who recently attempted suicide. The plasma suPAR and an established biomarker, C reactive protein (CRP) of suicide attempters (n = 54), depressed patients (n = 19) and healthy controls (n = 19) was analyzed with enzyme-linked immunosorbent assays. The biomarker attributes of sensitivity and sensibility were evaluated using ROC curve analysis. Both the depressed patients and suicide attempters had increased plasma suPAR. The levels of suPAR discriminated better between controls and suicide attempters than did CRP. In the future, plasma suPAR might be a superior prognosticator regarding outcome of treatment applying conventional antidepressants in conjunction with anti-inflammatory drugs. PMID:26451727

  9. Synthesis of water soluble chitosan derivatives with halogeno-1,2,3-triazole and their antifungal activity.

    PubMed

    Li, Qing; Tan, Wenqiang; Zhang, Caili; Gu, Guodong; Guo, Zhanyong

    2016-10-01

    Chitosan is an abundant and renewable polysaccharide, which exhibits attractive bioactivities and natural properties. Improvement such as chemical modification of chitosan is often performed prior to further utilization. Three novel water soluble chitosan derivatives containing 1,2,3- triazole with or without halogen was designed and synthesized. Their antifungal activity against three kinds of phytopathogens was estimated by hyphal measurement in vitro. The inhibitory property and water solubility of the synthesized chitosan derivatives exhibited a remarkable improvement over chitosan. It is hypothesized that thiazolyl groups enable the synthesized chitosan to possess obviously better antifungal activity. Moreover, CTCTS and BTCTS, which have halogens at the periphery of polymers, inhibited the growth of tested phytopathogens more effectively with inhibitory indices of 81-93% at 1.0mg/mL. The halogens could have a synergistic effect with triazole as they exhibited antifungal activity and electron-withdrawing capacity, which improve the antifungal activity of chitosan derivatives. PMID:27267573

  10. Lower Critical Solubility Temperature Behavior in Membranes Formed from a 2-(2-Methoxyethoxy)ethoxy-Containing Polyphosphazene

    SciTech Connect

    Stewart, F.F.; Lash, R.P.

    2002-03-26

    A phosphazene polymer with three pendant groups was synthesized and characterized as a membrane material. Substitution of the phosphazene with 64% 2-(2-methoxyethoxy)ethanol (MEE), 27% 4-methoxyphenol, and 9% 2-allyphenol yielded a hydrophilic elastomer with considerable flow at room temperature. Solution behavior showed significant aging effects where, using fresh solutions, membranes could not cast on porous ceramic supports (0.2-micron pore size) without significant polymer penetration into the pores. Solutions aged for two weeks were found to readily penetrate into the pores of the ceramic support. Analysis of fresh and aged solutions by laser light scattering showed significant loss in molecular weight with time. Pervaporation of water-dye solutions using dimensionally stabilized membranes revealed in inverse correlation between flux and temperature, suggesting thermally induced morphological changes within the polymer. This polymer was found to exhibit, in the bulk state, lower critical solubility temperature (LCST) behavior where the material becomes less hydrophilic with increasing temperature. LCST behavior was probed thermally and gravimetrically and has been attributed to the anomalous pervaporation results. The degree to which LCST effects membrane transport was influenced by changes in the crosslink density and permeate side pressure.

  11. Improvement of the antitumor activity of poorly soluble sapacitabine (CS-682) by using Soluplus® as a surfactant.

    PubMed

    Obata, Tohru; Suzuki, Yuka; Ogawa, Noriko; Kurimoto, Ippei; Yamamoto, Hiromitsu; Furuno, Tadahide; Sasaki, Takuma; Tanaka, Motohiro

    2014-01-01

    Sapacitabine (CS-682 or CYC682; 1-[2-C-cyano-2-deoxy-β-D-arabino-pentfuranosyl]N4-palmitoyl cytosine), a novel antitumor 2'-deoxycytidine analogue, shows a marked reduction in the water solubility because of the fatty acid side chain on the N4 group of the cytosine moiety. Poor water solubility is one of the important reasons why sapacitabine does not exert maximum antitumor activity. Therefore, we attempted to improve the water solubility of sapacitabine using a novel surfactant, Soluplus®, which consisted of a polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer. In this study, we examined whether Soluplus® increased the water solubility and an antitumor activity of sapacitabine. The cytotoxicity of Soluplus® alone was lower than that of Tween 80 and Kolliphor® D-α-tocopherylpolyethylene glycol 1000 succinate (TPGS). The water solubility and the chemosensitivity of sapacitabine against several tumor cell lines to sapacitabine markedly increased upon using Soluplus®. In addition, the potential of Soluplus® including sapacitabine in increasing the antitumor activity was compared with sapacitabine alone in vivo. Although the total dose in the experimental period was considerably lower than the effective dose of sapacitabine alone, the life span of mice treated with sapacitabine containing 40 mg/mL Soluplus® increased by 150%. If Soluplus® was used as the solubilizing agent in clinical trials of sapacitabine, a low administration dose was appeared to require, and thus side effects might be prevented. PMID:24790002

  12. Complexation of synthetic CDM-AM copolymer with natamycin and carbendazim to improve solubility and fungicidal activity.

    PubMed

    Li, Yong-Fu; Jin, Jing; Guo, Qin; Ha, Yi-Ming; Li, Qing-Peng

    2015-07-10

    The β-cyclodextrin-acrylamide (CDM-AM) copolymer was prepared from acrylamide and β-CD maleate (CDM) using K2S2O8 as initiator. The effects of the CDM-AM copolymer on the solubility and fungicidal activity of natamycin (NM) and carbendazim (MBC) were investigated. The stability constant of NM·CDM-AM and MBC·CDM-AM complexes at 303 K were of 10,725.45 M(-1) and 3000.89 M(-1), respectively. The complexes were characterized using phase solubility diagrams, NMR spectra and FT-IR spectra. The analysis of the biological activities of these two complexes indicated that they possessed enhancing fungicidal activities compared to NM and MBC alone. PMID:25857986

  13. Thrombomodulin Binding Selects the Catalytically Active Form of Thrombin.

    PubMed

    Handley, Lindsey D; Treuheit, Nicholas A; Venkatesh, Varun J; Komives, Elizabeth A

    2015-11-01

    Human α-thrombin is a serine protease with dual functions. Thrombin acts as a procoagulant, cleaving fibrinogen to make the fibrin clot, but when bound to thrombomodulin (TM), it acts as an anticoagulant, cleaving protein C. A minimal TM fragment consisting of the fourth, fifth, and most of the sixth EGF-like domain (TM456m) that has been prepared has much improved solubility, thrombin binding capacity, and anticoagulant activity versus those of previous TM456 constructs. In this work, we compare backbone amide exchange of human α-thrombin in three states: apo, D-Phe-Pro-Arg-chloromethylketone (PPACK)-bound, and TM456m-bound. Beyond causing a decreased level of amide exchange at their binding sites, TM and PPACK both cause a decreased level of amide exchange in other regions including the γ-loop and the adjacent N-terminus of the heavy chain. The decreased level of amide exchange in the N-terminus of the heavy chain is consistent with the historic model of activation of serine proteases, which involves insertion of this region into the β-barrel promoting the correct conformation of the catalytic residues. Contrary to crystal structures of thrombin, hydrogen-deuterium exchange mass spectrometry results suggest that the conformation of apo-thrombin does not yet have the N-terminus of the heavy chain properly inserted for optimal catalytic activity, and that binding of TM allosterically promotes the catalytically active conformation. PMID:26468766

  14. Form-Focused Discovery Activities in English Classes

    ERIC Educational Resources Information Center

    Ogeyik, Muhlise Cosgun

    2011-01-01

    Form-focused discovery activities allow language learners to grasp various aspects of a target language by contributing implicit knowledge by using discovered explicit knowledge. Moreover, such activities can assist learners to perceive and discover the features of their language input. In foreign language teaching environments, they can be used…

  15. Soluble Forms of Intercellular and Vascular Cell Adhesion Molecules Independently Predict Progression to Type 2 Diabetes in Mexican American Families

    PubMed Central

    Kulkarni, Hemant; Mamtani, Manju; Peralta, Juan; Almeida, Marcio; Dyer, Thomas D.; Goring, Harald H.; Johnson, Matthew P.; Duggirala, Ravindranath; Mahaney, Michael C.; Olvera, Rene L.; Almasy, Laura; Glahn, David C.; Williams-Blangero, Sarah; Curran, Joanne E.; Blangero, John

    2016-01-01

    Objective While the role of type 2 diabetes (T2D) in inducing endothelial dysfunction is fairly well-established the etiological role of endothelial dysfunction in the onset of T2D is still a matter of debate. In the light of conflicting evidence in this regard, we conducted a prospective study to determine the association of circulating levels of soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vessel cell adhesion molecule 1 (sVCAM-1) with incident T2D. Methods Data from this study came from 1,269 Mexican Americans of whom 821 initially T2D-free individuals were longitudinally followed up in the San Antonio Family Heart Study. These individuals were followed for 9752.95 person-years for development of T2D. Prospective association of sICAM-1 and sVCAM-1 with incident T2D was studied using Kaplan-Meier survival plots and mixed effects Cox proportional hazards modeling to account for relatedness among study participants. Incremental value of adhesion molecule biomarkers was studied using integrated discrimination improvement (IDI) and net reclassification improvement (NRI) indexes. Results Decreasing median values for serum concentrations of sICAM-1 and sVCAM-1 were observed in the following groups in this order: individuals with T2D at baseline, individuals who developed T2D during follow-up, individuals with prediabetes at baseline and normal glucose tolerant (NGT) individuals who remained T2D-free during follow-up. Top quartiles for sICAM-1 and sVCAM-1 were strongly and significantly associated with homeostatic model of assessment—insulin resistance (HOMA-IR). Mixed effects Cox proportional hazards modeling revealed that after correcting for important clinical confounders, high sICAM-1 and sVCAM-1 concentrations were associated with 2.52 and 1.99 times faster progression to T2D as compared to low concentrations, respectively. Individuals with high concentrations for both sICAM-1 and sVCAM-1 progressed to T2D 3.42 times faster than those with low

  16. Enhanced Glutathione Peroxidase Activity of Water-Soluble and Polyethylene Glycol-Supported Selenides, Related Spirodioxyselenuranes, and Pincer Selenuranes.

    PubMed

    McNeil, Nicole M R; Press, David J; Mayder, Don M; Garnica, Pablo; Doyle, Lisa M; Back, Thomas G

    2016-09-01

    Diaryl selenides containing o-hydroxymethylene substituents function as peroxide-destroying mimetics of the antioxidant selenoenzyme glutathione peroxidase (GPx), via oxidation to the corresponding spirodioxyselenuranes with hydrogen peroxide and subsequent reduction back to the original selenides with glutathione. Parent selenides with 3-hydroxypropyl or 2,3-dihydroxypropyl groups produced the novel compounds 10 and 11, respectively, with greatly improved aqueous solubility and catalytic activity. The phenolic derivative 28 displayed similarly ameliorated properties and also modest radical-inhibiting antioxidant activity, as evidenced by an assay based on phenolic hydrogen atom transfer to the stable free radical DPPH. In contrast, several selenides that afford pincer selenuranes (e.g., 20 and 21) instead of spiroselenuranes upon oxidation showed inferior catalytic activity. Several selenide analogues were attached to polyethylene glycol (PEG) oligomers, as PEG substituents can improve water solubility and bioavailability, while retarding clearance. Again, the PEG derivatives afforded remarkable activity when oxidation generated spirodioxyselenuranes and diminished activity when pincer compounds were produced. Several such compounds proved to be ca. 10- to 100-fold catalytically superior to the diaryl selenides and their spirodioxyselenurane counterparts investigated previously. Finally, an NMR-based assay employing glutathione in D2O was designed to accommodate the faster reacting water-soluble mimetics and to more closely duplicate in vivo conditions. PMID:27525346

  17. pH Effects on solubility, zeta potential, and correlation between antibacterial activity and molecular weight of chitosan.

    PubMed

    Chang, Shun-Hsien; Lin, Hong-Ting Victor; Wu, Guan-James; Tsai, Guo Jane

    2015-12-10

    Six chitosans with molecular weights (MWs) of 300, 156, 72.1, 29.2, 7.1, and 3.3 kDa were prepared by cellulase degradation of chitosan (300 kDa) and ultrafiltration techniques. We examined the correlation between activity against Escherichia coli and Staphylococcus aureus and chitosan MW, and provided the underlying explanation. In acidic pH conditions, the chitosan activity increased with increasing MW, irrespective of the temperature and bacteria tested. However, at neutral pH, chitosan activity increased as the MW decreased, and little activity was observed for chitosans with MW >29.2 kDa. At pH 5.0 and 6.0, chitosans exhibited good water solubility and zeta potential (ZP) decreased with the MW, whereas the solubility and ZP of the chitosans decreased with increasing MW at pH 7.0. Particularly, low solubility and negative ZP values were determined for chitosans with MW >29.2 kDa, which may explain the loss of their antibacterial activity at pH 7.0. PMID:26428102

  18. Site occupancy and glycan compositional analysis of two soluble recombinant forms of the attachment glycoprotein of Hendra virus.

    PubMed

    Colgrave, Michelle L; Snelling, Hayley J; Shiell, Brian J; Feng, Yan-Ru; Chan, Yee-Peng; Bossart, Katharine N; Xu, Kai; Nikolov, Dimitar B; Broder, Christopher C; Michalski, Wojtek P

    2012-04-01

    Hendra virus (HeV) continues to cause morbidity and mortality in both humans and horses with a number of sporadic outbreaks. HeV has two structural membrane glycoproteins that mediate the infection of host cells: the attachment (G) and the fusion (F) glycoproteins that are essential for receptor binding and virion-host cell membrane fusion, respectively. N-linked glycosylation of viral envelope proteins are critical post-translation modifications that have been implicated in roles of structural integrity, virus replication and evasion of the host immune response. Deciphering the glycan composition and structure on these glycoproteins may assist in the development of glycan-targeted therapeutic intervention strategies. We examined the site occupancy and glycan composition of recombinant soluble G (sG) glycoproteins expressed in two different mammalian cell systems, transient human embryonic kidney 293 (HEK293) cells and vaccinia virus (VV)-HeLa cells, using a suite of biochemical and biophysical tools: electrophoresis, lectin binding and tandem mass spectrometry. The N-linked glycans of both VV and HEK293-derived sG glycoproteins carried predominantly mono- and disialylated complex-type N-glycans and a smaller population of high mannose-type glycans. All seven consensus sequences for N-linked glycosylation were definitively found to be occupied in the VV-derived protein, whereas only four sites were found and characterized in the HEK293-derived protein. We also report, for the first time, the existence of O-linked glycosylation sites in both proteins. The striking characteristic of both proteins was glycan heterogeneity in both N- and O-linked sites. The structural features of G protein glycosylation were also determined by X-ray crystallography and interactions with the ephrin-B2 receptor are discussed. PMID:22171062

  19. Cell-free Co-expression of Functional Membrane Proteins and Apolipoprotein, Forming Soluble Nanolipoprotein Particles*S⃞

    PubMed Central

    Cappuccio, Jenny A.; Blanchette, Craig D.; Sulchek, Todd A.; Arroyo, Erin S.; Kralj, Joel M.; Hinz, Angela K.; Kuhn, Edward A.; Chromy, Brett A.; Segelke, Brent W.; Rothschild, Kenneth J.; Fletcher, Julia E.; Katzen, Federico; Peterson, Todd C.; Kudlicki, Wieslaw A.; Bench, Graham; Hoeprich, Paul D.; Coleman, Matthew A.

    2008-01-01

    Here we demonstrate rapid production of solubilized and functional membrane protein by simultaneous cell-free expression of an apolipoprotein and a membrane protein in the presence of lipids, leading to the self-assembly of membrane protein-containing nanolipoprotein particles (NLPs). NLPs have shown great promise as a biotechnology platform for solubilizing and characterizing membrane proteins. However, current approaches are limited because they require extensive efforts to express, purify, and solubilize the membrane protein prior to insertion into NLPs. By the simple addition of a few constituents to cell-free extracts, we can produce membrane proteins in NLPs with considerably less effort. For this approach an integral membrane protein and an apolipoprotein scaffold are encoded by two DNA plasmids introduced into cell-free extracts along with lipids. For this study reported here we used plasmids encoding the bacteriorhodopsin (bR) membrane apoprotein and scaffold protein Δ1–49 apolipoprotein A-I fragment (Δ49A1). Cell free co-expression of the proteins encoded by these plasmids, in the presence of the cofactor all-trans-retinal and dimyristoylphosphatidylcholine, resulted in production of functional bR as demonstrated by a 5-nm shift in the absorption spectra upon light adaptation and characteristic time-resolved FT infrared difference spectra for the bR → M transition. Importantly the functional bR was solubilized in discoidal bR·NLPs as determined by atomic force microscopy. A survey study of other membrane proteins co-expressed with Δ49A1 scaffold protein also showed significantly increased solubility of all of the membrane proteins, indicating that this approach may provide a general method for expressing membrane proteins enabling further studies. PMID:18603642

  20. Soluble ST2: A new and promising activity marker in ulcerative colitis

    PubMed Central

    Díaz-Jiménez, David; Núñez, Lucía E; Beltrán, Caroll J; Candia, Enzo; Suazo, Cristóbal; Álvarez-Lobos, Manuel; González, María-Julieta; Hermoso, Marcela A; Quera, Rodrigo

    2011-01-01

    AIM: To correlate circulating soluble ST2 (sST2) levels with the severity of ulcerative colitis (UC) and serum levels of pro-inflammatory cytokines, and to demonstrate the predictive power of sST2 levels for differentiation between active and inactive UC. METHODS: We recruited 153 patients: 82 with UC, 26 with Crohn’s disease (CD) and 43 disease controls [non-inflammatory bowel disease (IBD)]. Subjects were excluded if they had diagnosis of asthma, autoimmune diseases or hypertension. The serum levels of sST2 and pro-inflammatory cytokines [pg/mL; median (25th-75th)] as well as clinical features, endoscopic and histological features, were subjected to analyses. The sST2 performance for discrimination between active and inactive UC, non-IBD and healthy controls (HC) was determined with regard to sensitivity and specificity, and Spearman’s rank correlation coefficient (r). To validate the method, the area under the curve (AUC) of receiver-operator characteristic (ROC) was determined (AUC, 95% CI) and the total ST2 content of the colonic mucosa in UC patients was correlated with circulating levels of sST2. RESULTS: The serum sST2 value was significantly higher in patients with active [235.80 (90.65-367.90) pg/mL] rather than inactive UC [33.19 (20.04-65.32) pg/mL], based on clinical, endoscopic and histopathological characteristics, as well as compared with non-IBD and HC (P < 0.001). The median level of sST2 in CD patients was 54.17 (35.02-122.0) pg/mL, significantly higher than that of the HC group only (P < 0.01). The cutoff was set at 74.87 pg/mL to compare active with inactive UC in a multicenter cohort of patients. Values of sensitivity, specificity, and ability to correctly classify UC, according to activity, were 83.33%, 83.33% and 83.33%, respectively. The AUC of the ROC curve to assess the ability of this molecule to discriminate between active vs inactive UC was 0.92 (0.86-0.97, P < 0.0001). The serum levels of sST2 in patients with UC significantly

  1. Stability of cytokines, chemokines and soluble activation markers in unprocessed blood stored under different conditions

    PubMed Central

    Aziz, Najib; Detels, Roger; Quint, Joshua J.; Li, Qian; Gjertson, David; Butch, Anthony W.

    2016-01-01

    Background Biomarkers such as cytokines, chemokines, and soluble activation markers can be unstable when processing of blood is delayed. The stability of various biomarkers in serum and plasma was investigated when unprocessed blood samples were stored for up to 24 h at room and refrigerator temperature. Methods Blood was collected from 16 healthy volunteers. Unprocessed serum, EDTA and heparinized blood was stored at room (20–25 °C) and refrigerator temperature (4–8 °C) for 0.5, 2, 4, 6, 8, and 24 h after collection before centrifugation and separation of serum and plasma. Samples were batch tested for various biomarkers using commercially available immunoassays. Statistically significant changes were determined using the generalized estimating equation. Results IFN-γ, sIL-2Rα, sTNF-RII and β2-microglobulin were stable in unprocessed serum, EDTA and heparinized blood samples stored at either room or refrigerator temperature for up to 24 h. IL-6, TNF-α, MIP-1β and RANTES were unstable in heparinized blood at room temperature; TNF-α, and MIP-1β were unstable in unprocessed serum at room temperature; IL-12 was unstable in unprocessed serum at refrigerator temperature; and neopterin was unstable in unprocessed EDTA blood at room temperature. IL-1ra was stable only in unprocessed serum at room temperature. Conclusion All the biomarkers studied, with the exception of IL-1ra, were stable in unprocessed EDTA blood stored at refrigerator temperature for 24 h. This indicates that blood for these biomarkers should be collected in EDTA and if delays in processing are anticipated the unseparated blood should be stored at refrigerator temperature until processing. PMID:27208752

  2. Isolation and antiviral activity of water-soluble Cynomorium songaricum Rupr. polysaccharides.

    PubMed

    Tuvaanjav, Suvdmaa; Shuqin, Han; Komata, Masashi; Ma, Chunjie; Kanamoto, Taisei; Nakashima, Hideki; Yoshida, Takashi

    2016-01-01

    The plant, Cynomorium songaricum Rupr., is used as a traditional medicine in China and Mongolia. In the present study, two new water-soluble polysaccharides isolated from C. songaricum Rupr. were purified by successive Sephadex G-75 and G-50 column chromatographies and then characterized by high resolution NMR and IR spectroscopies. The molecular weights of two polysaccharides were determined by an aqueous GPC to be [Formula: see text] = 3.7 × 10(4) and 1.0 × 10(4), respectively. In addition, it was found that the polysaccharide with the larger molecular weight was an acidic polysaccharide. It was found that the iodine-starch reaction of both isolated polysaccharides was negative and the methylation analysis gave 2, 4, 6-tri-O-methyl alditol acetate as a main product. NMR and IR measurements and sugar analysis revealed that both polysaccharides had a (1 → 3)-α-d-glucopyranosidic main chain with a small number of branches. After sulfation, the sulfated C. songaricum Rupr. polysaccharides were found to have a potent inhibitory effect on HIV infection of MT-4 cells at a 50% effective concentration of 0.3-0.4 μg/ml, a concentration that has almost the same high activity as standard dextran and curdlan sulfates, EC50 = 0.35 and 0.14 μg/ml, respectively. The 50% cytotoxic concentration was low, CC50>1000 μg/ml. In addition, the interaction between the sulfated polysaccharides and poly-l-lysine as a model protein compound was investigated by a surface plasmon resonance to reveal the anti-HIV mechanism. PMID:26838028

  3. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S

    2013-02-19

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  4. Activated carbon fibers and engineered forms from renewable resources

    DOEpatents

    Baker, Frederick S.

    2010-06-01

    A method of producing activated carbon fibers (ACFs) includes the steps of providing a natural carbonaceous precursor fiber material, blending the carbonaceous precursor material with a chemical activation agent to form chemical agent-impregnated precursor fibers, spinning the chemical agent-impregnated precursor material into fibers, and thermally treating the chemical agent-impregnated precursor fibers. The carbonaceous precursor material is both carbonized and activated to form ACFs in a single step. The method produces ACFs exclusive of a step to isolate an intermediate carbon fiber.

  5. Cellulase activity screening using pure carboxymethylcellulose: application to soluble cellulolytic samples and to plant tissue prints.

    PubMed

    Johnsen, Hanne R; Krause, Kirsten

    2014-01-01

    Reliable, rapid and inexpensive detection of cellulolytic enzymes that can be used for a wide variety of biological and environmental samples are currently in high demand. Here, a new cellulase detection protocol is described that circumvents problems observed with popular agar-based methods by exploiting the ability of carboxymethylcellulose (CMC) to form gel-like surfaces on its own. These pure CMC-layers are sensitive to cellulolytic degradation and stainable by Gram's iodine without showing unwelcome reactions with other enzymes. The staining intensity negatively correlates with the enzyme activity and can be used for quantification. Cellulase activities are not obstructed by high sugar contents (e.g., in plant material) which limit the applicability of other quantification methods, making our new method particularly attractive for screening of plant extracts. A useful variant of this new method is its applicability to plant tissue prints for spatial mapping of the cellulolytic activity in a zymogram-like fashion. PMID:24413752

  6. Temperature-dependent solubilities and mean ionic activity coefficients of alkali halides in water from molecular dynamics simulations.

    PubMed

    Mester, Zoltan; Panagiotopoulos, Athanassios Z

    2015-07-28

    The mean ionic activity coefficients of aqueous KCl, NaF, NaI, and NaCl solutions of varying concentrations have been obtained from molecular dynamics simulations following a recently developed methodology based on gradual insertions of salt molecules [Z. Mester and A. Z. Panagiotopoulos, J. Chem. Phys. 142, 044507 (2015)]. The non-polarizable ion models of Weerasinghe and Smith [J. Chem. Phys. 119, 11342 (2003)], Gee et al. [J. Chem. Theory Comput. 7, 1369 (2011)], Reiser et al. [J. Chem. Phys. 140, 044504 (2014)], and Joung and Cheatham [J. Phys. Chem. B 112, 9020 (2008)] were used along with the extended simple point charge (SPC/E) water model [Berendsen et al., J. Phys. Chem. 91, 6269 (1987)] in the simulations. In addition to the chemical potentials in solution used to obtain the activity coefficients, we also calculated the chemical potentials of salt crystals and used them to obtain the solubility of these alkali halide models in SPC/E water. The models of Weerasinghe and Smith [J. Chem. Phys. 119, 11342 (2003)] and Gee et al. [J. Chem. Theory Comput. 7, 1369 (2011)] provide excellent predictions of the mean ionic activity coefficients at 298.15 K and 1 bar, but significantly underpredict or overpredict the solubilities. The other two models generally predicted the mean ionic activity coefficients only qualitatively. With the exception of NaF for which the solubility is significantly overpredicted, the model of Joung and Cheatham predicts salt solubilities that are approximately 40%-60% of the experimental values. The models of Reiser et al. [J. Chem. Phys. 140, 044504 (2014)] make good predictions for the NaCl and NaI solubilities, but significantly underpredict the solubilities for KCl and NaF. We also tested the transferability of the models to temperatures much higher than were used to parametrize them by performing simulations for NaCl at 373.15 K and 1 bar, and at 473.15 K and 15.5 bar. All models overpredict the drop in the values of mean ionic

  7. Temperature-dependent solubilities and mean ionic activity coefficients of alkali halides in water from molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Mester, Zoltan; Panagiotopoulos, Athanassios Z.

    2015-07-01

    The mean ionic activity coefficients of aqueous KCl, NaF, NaI, and NaCl solutions of varying concentrations have been obtained from molecular dynamics simulations following a recently developed methodology based on gradual insertions of salt molecules [Z. Mester and A. Z. Panagiotopoulos, J. Chem. Phys. 142, 044507 (2015)]. The non-polarizable ion models of Weerasinghe and Smith [J. Chem. Phys. 119, 11342 (2003)], Gee et al. [J. Chem. Theory Comput. 7, 1369 (2011)], Reiser et al. [J. Chem. Phys. 140, 044504 (2014)], and Joung and Cheatham [J. Phys. Chem. B 112, 9020 (2008)] were used along with the extended simple point charge (SPC/E) water model [Berendsen et al., J. Phys. Chem. 91, 6269 (1987)] in the simulations. In addition to the chemical potentials in solution used to obtain the activity coefficients, we also calculated the chemical potentials of salt crystals and used them to obtain the solubility of these alkali halide models in SPC/E water. The models of Weerasinghe and Smith [J. Chem. Phys. 119, 11342 (2003)] and Gee et al. [J. Chem. Theory Comput. 7, 1369 (2011)] provide excellent predictions of the mean ionic activity coefficients at 298.15 K and 1 bar, but significantly underpredict or overpredict the solubilities. The other two models generally predicted the mean ionic activity coefficients only qualitatively. With the exception of NaF for which the solubility is significantly overpredicted, the model of Joung and Cheatham predicts salt solubilities that are approximately 40%-60% of the experimental values. The models of Reiser et al. [J. Chem. Phys. 140, 044504 (2014)] make good predictions for the NaCl and NaI solubilities, but significantly underpredict the solubilities for KCl and NaF. We also tested the transferability of the models to temperatures much higher than were used to parametrize them by performing simulations for NaCl at 373.15 K and 1 bar, and at 473.15 K and 15.5 bar. All models overpredict the drop in the values of mean ionic

  8. Urine soluble urokinase-type plasminogen activator receptor levels correlate with proteinuria in Puumala hantavirus infection

    PubMed Central

    Outinen, Tuula K.; Mäkelä, Satu; Huttunen, Reetta; Mäenpää, Niina; Libraty, Daniel; Vaheri, Antti; Mustonen, Jukka; Aittoniemi, Janne

    2014-01-01

    Objectives Urokinase-type plasminogen activator receptor (uPAR) is upregulated during inflammation and known to bind to β3-integrins, receptors used by pathogenic hantaviruses to enter endothelial cells. It has been proposed that soluble uPAR (suPAR) is a circulating factor that causes focal segmental glomerulosclerosis and proteinuria by activating β3-integrin in kidney podocytes. Proteinuria is also a characteristic feature of hantavirus infections. The aim of this study was to evaluate the relation between urine suPAR levels and disease severity in acute Puumala hantavirus (PUUV) infection. Design A single-centre, prospective cohort study. Subjects and methods Urinary suPAR levels were measured twice during the acute phase and once during convalescence in 36 patients with serologically confirmed PUUV infection. Fractional excretion of suPAR (FE suPAR) and of albumin (FE alb) were calculated. Results The FE suPAR was significantly elevated during the acute phase of PUUV infection compared to the convalescent phase (median 3.2%, range 0.8–52.0%, vs. median 1.9%, range 1.0–5.8%, P = 0.005). Maximum FE suPAR was correlated markedly with maximum FE alb (r = 0.812, P < 0.001), and with several other variables that reflect disease severity. There was a positive correlation with the length of hospitalization (r = 0.455, P = 0.009) and maximum plasma creatinine level (r = 0.780, P < 0.001), and an inverse correlation with minimum urinary output (r = −0.411, P = 0.030). There was no correlation between FE suPAR and plasma suPAR (r = 0.180, P = 0.324). Conclusion Urinary suPAR is markedly increased during acute PUUV infection and is correlated with proteinuria. High urine suPAR level may reflect local production of suPAR in the kidney during the acute infection. PMID:24717117

  9. Effects of the soluble guanylyl cyclase activator, YC-1, on vascular tone, cyclic GMP levels and phosphodiesterase activity

    PubMed Central

    Galle, Jan; Zabel, Ulrike; Hübner, Ulrich; Hatzelmann, Armin; Wagner, Birgit; Wanner, Christoph; Schmidt, Harald H H W

    1999-01-01

    The vasomotor and cyclic GMP-elevating activity of YC-1, a novel NO-independent activator of soluble guanylyl cyclase (sGC), was studied in isolated rabbit aortic rings and compared to that of the NO donor compounds sodium nitroprusside (SNP) and NOC 18.Similarly to SNP and NOC 18, YC-1 (0.3–300 μM) caused a concentration-dependent, endothelium-independent relaxation that was greatly reduced by the sGC inhibitor 1-H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ 10 μM; 59% inhibition of dilation induced by 100 μM YC-1) suggesting the activation of sGC as one mechanism of action.Preincubation with YC-1 (3 and 30 μM) significantly increased the maximal dilator responses mediated by endogenous NO in aortic rings that was released upon exposure to acetylcholine, and enhanced the dilator response to the exogenous NO-donors, SNP and NOC 18, by almost two orders of magnitude.Vasoactivity induced by SNP and YC-1 displayed different kinetics as evidenced by a long-lasting inhibition by YC-1 (300 μM) on the phenylephrine (PE)-induced contractile response, which was not fully reversible even after extensive washout (150 min) of YC-1, and was accompanied by a long-lasting elevation of intracellular cyclic GMP content. In contrast, SNP (30 μM) had no effect on the vasoconstrictor potency of PE, and increases in intravascular cyclic GMP levels were readily reversed after washout of this NO donor compound.Surprisingly, YC-1 not only activated sGC, but also affected cyclic GMP metabolism, as it inhibited both cyclic GMP break down in aortic extracts and the activity of phosphodiesterase isoforms 1–5 in vitro.In conclusion, YC-1 caused persistent elevation of intravascular cyclic GMP levels in vivo by activating sGC and inhibiting cyclic GMP break down. Thus, YC-1 is a highly effective vasodilator compound with a prolonged duration of action, and mechanisms that are unprecedented for any previously known sGC activator. PMID:10369473

  10. An ELISA method detecting the active form of suPAR.

    PubMed

    Zhou, Xiaolei; Xu, Mingming; Huang, Hailong; Mazar, Andrew; Iqbal, Zafar; Yuan, Cai; Huang, Mingdong

    2016-11-01

    Urokinase plasminogen activator receptor (uPAR) exists in a number of formats in human plasma, including soluble uPAR (suPAR) and uPAR fragments. We developed an ELISA method to detect specifically the active form suPAR, which binds to its natural ligand uPA. The intra CV and inter CV of this ELISA assay is 8.5% and 9.6% respectively, and the assay can recover 99.74% of added recombinant suPAR from 10% plasma. This assay is quite sensitive, capable of detecting down to 15pg/ml of suPAR, and can measure suPAR concentrations in the range of 0.031-8ng/ml with high linear relationship. Plasma samples from pregnant women were also measured for the active form of suPAR with this assay, giving an averaged level of 1.39ng/ml, slightly higher than the level of pooled plasma from healthy donors (0.96ng/ml). This study demonstrates the feasibility to measure the active form of suPAR, which will likely have value in clinical applications. PMID:27591605

  11. Toward Understanding the Effect of Low-Activity Waste Glass Composition on Sulfur Solubility

    SciTech Connect

    Vienna, John D.; Kim, Dong-Sang; Muller, Isabelle S.; Piepel, Gregory F.; Kruger, Albert A.

    2014-10-01

    The concentration of sulfur in nuclear waste glass melter feed must be maintained below the point where salt accumulates on the melt surface. The allowable concentrations may range from 0.37 to over 2.05 weight percent (of SO3 on a calcined oxide basis). If the amount of sulfur exceeds its tolerance level a molten salt will accumulate and upset melter operations and potentially shorten melter useful life. Therefore relatively conservative limits have been placed on sulfur loading in melter feed which in-turn significantly impacts the amount of glass that will be produced, in particular at the Hanford site. Crucible-scale sulfur solubility data and scaled melter sulfur tolerance data have been collected on simulated Hanford waste glasses over the last 15 years. These data were compiled and analyzed. A model was developed to predict the solubility of SO3 in glass based on 312 individual glass compositions. This model was shown to well represent the data, accounting for over 80% of the variation in data and was well validated. The model was also found to accurately predict the tolerance for sulfur in melter feed based on 19 scaled melter tests. The model is appropriate for control of waste glass processing which includes uncertainty quantification. The model also gives quantitative estimates of component concentration effects on sulfur solubility. The components that most increase sulfur solubility are Li2O > V2O5 ≈ TiO2 < CaO < P2O5 ≈ ZnO. The components that most decrease sulfur solubility are Cl > Cr2O3 > SiO2 ≈ ZrO2 > Al2O3.

  12. Novel third-generation water-soluble noscapine analogs as superior microtubule-interfering agents with enhanced antiproliferative activity

    PubMed Central

    Henary, Maged; Narayana, Lakshminarayana; Ahad, Shazia; Gundala, Sushma R.; Mukkavilli, Rao; Sharma, Vibhuti; Owens, Eric A.; Yadav, Yogesh; Nagaraju, Mulpuri; Hamelberg, Donald; Tandon, Vibha; Panda, Dulal; Aneja, Ritu

    2015-01-01

    Noscapine, an opium-derived ‘kinder-gentler’ microtubule-modulating drug is in Phase I/II clinical trials for cancer chemotherapy. However, its limited water solubility encumbers its development into an oral anticancer drug with clinical promise. Here we report the synthesis of 9 third-generation, water-soluble noscapine analogs with negatively charged sulfonato and positively charged quaternary ammonium groups using noscapine, 9-bromonoscapine and 9-aminonoscapine as scaffolds. The predictive free energy of solvation was found to be lower for sulfonates (6a–c;8a–c) compared to the quaternary ammonium-substituted counterparts, explaining their higher water solubility. In addition, sulfonates showed higher charge dispersability, which may effectively shield the hydrophobicity of isoquinoline nucleus as indicated by hydrophobicity mapping methods. These in silico data underscore efficient net charge balancing, which may explain higher water solubility and thus enhanced antiproliferative efficacy and improved bioavailability. We observed that 6b, 8b and 8c strongly inhibited tubulin polymerization and demonstrated significant antiproliferative activity against four cancer cell lines compared to noscapine. Molecular simulation and docking studies of tubulin-drug complexes revealed that the brominated compound with a four-carbon chain (4b, 6b, 8b) showed optimal binding with tubulin heterodimers. Interestingly, 6b, 8b and 8c treated PC-3 cells resulted in preponderance of mitotic cells with multipolar spindle morphology, suggesting that they stall the cell cycle. Furthermore, in vivo pharmacokinetic evaluation of 6b, 8b and 8c revealed at least 1–2 fold improvement in their bioavailability compared to noscapine. To our knowledge, this is the first report to demonstrate novel water-soluble noscapine analogs that may pave the way for future pre-clinical drug development. PMID:25124704

  13. Novel third-generation water-soluble noscapine analogs as superior microtubule-interfering agents with enhanced antiproliferative activity.

    PubMed

    Henary, Maged; Narayana, Lakshminarayana; Ahad, Shazia; Gundala, Sushma R; Mukkavilli, Rao; Sharma, Vibhuti; Owens, Eric A; Yadav, Yogesh; Nagaraju, Mulpuri; Hamelberg, Donald; Tandon, Vibha; Panda, Dulal; Aneja, Ritu

    2014-11-15

    Noscapine, an opium-derived 'kinder-gentler' microtubule-modulating drug is in Phase I/II clinical trials for cancer chemotherapy. However, its limited water solubility encumbers its development into an oral anticancer drug with clinical promise. Here we report the synthesis of 9 third-generation, water-soluble noscapine analogs with negatively charged sulfonato and positively charged quaternary ammonium groups using noscapine, 9-bromonoscapine and 9-aminonoscapine as scaffolds. The predictive free energy of solvation was found to be lower for sulfonates (6a-c; 8a-c) compared to the quaternary ammonium-substituted counterparts, explaining their higher water solubility. In addition, sulfonates showed higher charge dispersability, which may effectively shield the hydrophobicity of isoquinoline nucleus as indicated by hydrophobicity mapping methods. These in silico data underscore efficient net charge balancing, which may explain higher water solubility and thus enhanced antiproliferative efficacy and improved bioavailability. We observed that 6b, 8b and 8c strongly inhibited tubulin polymerization and demonstrated significant antiproliferative activity against four cancer cell lines compared to noscapine. Molecular simulation and docking studies of tubulin-drug complexes revealed that the brominated compound with a four-carbon chain (4b, 6b, and 8b) showed optimal binding with tubulin heterodimers. Interestingly, 6b, 8b and 8c treated PC-3 cells resulted in preponderance of mitotic cells with multipolar spindle morphology, suggesting that they stall the cell cycle. Furthermore, in vivo pharmacokinetic evaluation of 6b, 8b and 8c revealed at least 1-2-fold improvement in their bioavailability compared to noscapine. To our knowledge, this is the first report to demonstrate novel water-soluble noscapine analogs that may pave the way for future pre-clinical drug development. PMID:25124704

  14. Structural basis for VEGF-C binding to neuropilin-2 and sequestration by a soluble splice form.

    PubMed

    Parker, Matthew W; Linkugel, Andrew D; Goel, Hira Lal; Wu, Tingting; Mercurio, Arthur M; Vander Kooi, Craig W

    2015-04-01

    Vascular endothelial growth factor C (VEGF-C) is a potent lymphangiogenic cytokine that signals via the coordinated action of two cell surface receptors, Neuropilin-2 (Nrp2) and VEGFR-3. Diseases associated with both loss and gain of VEGF-C function, lymphedema and cancer, respectively, motivate studies of VEGF-C/Nrp2 binding and inhibition. Here, we demonstrate that VEGF-C binding to Nrp2 is regulated by C-terminal proteolytic maturation. The structure of the VEGF-C C terminus in complex with the ligand binding domains of Nrp2 demonstrates that a cryptic Nrp2 binding motif is released upon proteolysis, allowing specific engagement with the b1 domain of Nrp2. Based on the identified structural requirements for Nrp2 binding to VEGF-C, we hypothesized that the endogenous secreted splice form of Nrp2, s9Nrp2, may function as a selective inhibitor of VEGF-C. We find that s9Nrp2 forms a stable dimer that potently inhibits VEGF-C/Nrp2 binding and cellular signaling. These data provide critical insight into VEGF-C/Nrp2 binding and inhibition. PMID:25752543

  15. Flow cytometry-based methods for assessing soluble scFv activities and detecting pathogen antigens in solution

    SciTech Connect

    Gray, Sean; Weigel, Kris M.; Miller, Keith D.; Ndung'u, Joseph; Buscher, Philippe; Tran, Thao N.; Baird, Cheryl L.; Cangelosi, Gerard A.

    2010-04-01

    Novel methods are reported for evaluating and utilizing single chain fragment variable (scFv) antibodies derived from yeast-display libraries. Yeast-display was used to select scFv specific to invariant surface glycoproteins (ISG) of Trypanosoma brucei. A limiting step in the isolation of scFv from nonimmune libraries is the conversion of highly active yeast-displayed scFv into soluble antibodies that can be used in standard immunoassays. Challenges include limited solubility or activity following secretion and purification of scFv. For this reason, few scFv derived from yeast-display platforms have moved into development and implementation as diagnostic reagents. To address this problem, assays were developed that employ both yeastdisplayed and secreted scFv as analytical reagents. The first is a competitive inhibition flow cytometry (CIFC) assay that detects secreted scFv by virtue of its ability to competitively inhibit the binding of biotinylated antigen to yeast-displayed scFv. The second is an epitope binning assay that uses secreted scFv toidentify additional yeast-displayed scFv that bind nonoverlapping or noncompeting epitopes on an antigen. The epitope binning assay was used not only to identify sandwich assay pairs with yeast-displayed scFv, but also to identify active soluble scFv present in low concentration in a crude expression extract. Finally, a CIFC assay was developed that bypasses entirely the need for soluble scFv expression, by using yeast displayed scFv to detect unlabeled antigen in samples. These methods will facilitate the continued development and practical implementation of scFv derived from yeast-display libraries.

  16. Characterisation of selected active agents regarding pKa values, solubility concentrations and pH profiles by SiriusT3.

    PubMed

    Schönherr, D; Wollatz, U; Haznar-Garbacz, D; Hanke, U; Box, K J; Taylor, R; Ruiz, R; Beato, S; Becker, D; Weitschies, W

    2015-05-01

    The aim of this work was to determine pKa values and solubility properties of 34active agents using the SiriusT3 apparatus. The selected drug substances belong to the groups of ACE-inhibitors, β-blockers, antidiabetics and lipid lowering substances. Experimentally obtained pKa and intrinsic solubility values were compared to calculated values (program ACD/ChemSketch) and pKa values to published data as well. Solubility-pH profiles were generated to visualise the substance solubility over the gastrointestinal pH range. The relationship between the solubility characteristic of a substance, its bioavailability and categorisation according to the Biopharmaceutics Classification System (BCS) was examined as well. The results showed a good agreement between experimentally obtained, calculated and published pKa values. The measured and calculated intrinsic solubility values indicated several major deviations. All solubility-pH profiles showed the expected shape and appearance for acids, bases or zwitterionic substances. The obtained results for the pKa and solubility measurements of the examined active agents may help to predict their physicochemical behaviour in vivo, and to understand the bioavailability of the substances according to their BCS categorisation. The easy and reproducible determination of pKa and solubility values makes the SiriusT3 apparatus a useful tool in early stages of drug and formulation development. PMID:25758123

  17. Preformed Soluble Chemoreceptor Trimers That Mimic Cellular Assembly States and Activate CheA Autophosphorylation

    PubMed Central

    2015-01-01

    Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (TarFO). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the TarFO modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The TarFO variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of TarFO to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of TarFO into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis

  18. Pore-forming activity of clostridial binary toxins.

    PubMed

    Knapp, O; Benz, R; Popoff, M R

    2016-03-01

    Clostridial binary toxins (Clostridium perfringens Iota toxin, Clostridium difficile transferase, Clostridium spiroforme toxin, Clostridium botulinum C2 toxin) as Bacillus binary toxins, including Bacillus anthracis toxins consist of two independent proteins, one being the binding component which mediates the internalization into cell of the intracellularly active component. Clostridial binary toxins induce actin cytoskeleton disorganization through mono-ADP-ribosylation of globular actin and are responsible for enteric diseases. Clostridial and Bacillus binary toxins share structurally and functionally related binding components which recognize specific cell receptors, oligomerize, form pores in endocytic vesicle membrane, and mediate the transport of the enzymatic component into the cytosol. Binding components retain the global structure of pore-forming toxins (PFTs) from the cholesterol-dependent cytotoxin family such as perfringolysin. However, their pore-forming activity notably that of clostridial binding components is more related to that of heptameric PFT family including aerolysin and C. perfringens epsilon toxin. This review focuses upon pore-forming activity of clostridial binary toxins compared to other related PFTs. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. PMID:26278641

  19. Correlation between fouling propensity of soluble extracellular polymeric substances and sludge metabolic activity altered by different starvation conditions.

    PubMed

    Wu, Siang Chen; Lee, Chi Mei

    2011-05-01

    Soluble extracellular polymeric substances (EPSs) cause membrane fouling in membrane bioreactors (MBRs), correlated with MBR sludge characteristics. Effects of F/M ratios on the evolution of soluble EPSs, fouling propensity of supernatants, and sludge metabolic activity were measured in this study in a two-period sequencing batch reactor (SBR). The experimental results show that fouling propensity was directly correlated with soluble-EPS concentration and composition. Sludge that had entirely lost active cells by long-term starvation released 64.4 ± 0.9 mg/L of humic acids, which caused a rapid increase in membrane resistance (40.67 ± 2.24 × 10(11) m(-1)) during fouling tests. During short-term starvation, induced by incubation at a normal to low F/M ratio of 0.05 d(-1), sludge can use previously secreted utilization-associated products (UAPs) to maintain endogenous respiration. Therefore, the strategies of accumulating sludge and prolonging sludge retention time in MBRs may create long-term starvation and promote membrane fouling. PMID:21163646

  20. A decision-support tool for the formulation of orally active, poorly soluble compounds.

    PubMed

    Branchu, Sébastien; Rogueda, Philippe G; Plumb, A Philip; Cook, Walter G

    2007-10-01

    Physicochemical data for a set of potentially poorly soluble compounds was analysed in relation to suitable formulations for these compounds. Physical chemistry was found to be a key determinant of formulation class expressed in terms of conventional, solid dispersion, lipidic/surfactant, and crystalline nanoparticle systems. This relationship was used to build a decision-support tool aimed to guide formulation selection for poorly soluble compounds during product development. Tool components included a user interface, a database of compound cases together with known formulations, and predictive modules based on statistics, decision trees, and case-based reasoning. The tool was tested and exhibited significant and consistent predictive ability across testing conditions. This type of tool has the potential to improve the efficiency and predictability of the formulation development process. PMID:17689226

  1. MRI-based detection of alkaline phosphatase gene reporter activity using a porphyrin solubility switch

    PubMed Central

    Westmeyer, Gil G.; Emer, Elena G.; Lintelmann, Jutta; Jasanoff, Alan

    2014-01-01

    SUMMARY The ability to map patterns of gene expression noninvasively in living animals could have impact in many areas of biology. Reporter systems compatible with magnetic resonance imaging (MRI) could be particularly valuable, but existing strategies tend to lack sensitivity or specificity. Here we address the challenge of MRI-based gene mapping using the reporter enzyme secreted alkaline phosphatase (SEAP), in conjunction with a water soluble metalloporphyrin contrast agent. SEAP cleaves the porphyrin into an insoluble product that accumulates at sites of enzyme expression and can be visualized by MRI and optical absorbance. The contrast mechanism functions in vitro, in brain slices, and in animals. The system also provides the possibility of readout both in the living animal and by post mortem histology, and it notably does not require intracellular delivery of the contrast agent. The solubility switch mechanism used to detect SEAP could be adapted for imaging of additional reporter enzymes or endogenous targets. PMID:24613020

  2. 21 CFR 520.44 - Acetazolamide sodium soluble powder.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 6 2012-04-01 2012-04-01 false Acetazolamide sodium soluble powder. 520.44... Acetazolamide sodium soluble powder. (a) Specifications. The drug is in a powder form containing acetazolamide sodium, USP equivalent to 25 percent acetazolamide activity. (b) Sponsor. See No. 053501 in §...

  3. 21 CFR 520.44 - Acetazolamide sodium soluble powder.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 6 2013-04-01 2013-04-01 false Acetazolamide sodium soluble powder. 520.44... Acetazolamide sodium soluble powder. (a) Specifications. The drug is in a powder form containing acetazolamide sodium, USP equivalent to 25 percent acetazolamide activity. (b) Sponsor. See No. 053501 in §...

  4. Water-soluble carbon monoxide-releasing molecules: helping to elucidate the vascular activity of the 'silent killer'.

    PubMed

    Chatterjee, Prabal Kumar

    2004-06-01

    Carbon monoxide (CO) is formed during the degradation of haeme by haeme oxygenase (HO). As well as being an important signalling molecule and vasodilator, CO also possesses antihypertensive, anti-inflammatory and antiapoptotic qualities and protects against ischaemic tissue injury. Several approaches have been used to investigate the therapeutic potential of CO, ranging from direct administration of CO gas to the use of prodrugs, which generate CO upon metabolism. A novel approach involves the use of specific CO carriers, which will release measurable, controllable and effective amounts of CO into biological systems. Transitional metal carbonyls based around iron, manganese or ruthenium have recently been developed as CO-releasing molecules (CO-RMs) that, under appropriate conditions, will release CO. Such molecules have been shown to provide cardioprotection in both ex vivo and in vivo experiments. To date, CO-RMs have been largely incompatible with biological systems in that they are only soluble in organic solvents or have to be preactivated either by physical or chemical stimuli. However, the recent development of water-soluble CO-RMs has provided new opportunities to investigate the pharmacological and biological features of CO without such confounding influences. CORM-3, a novel water-soluble CO-RM, has recently been used to confirm the cardioprotective actions of CO. In this issue of British Journal of Pharmacology, Foresti and co-workers report that CORM-3 delivers CO, produces aortic vasodilation ex vivo and reduces blood pressure in vivo via modulation of the same cGMP and potassium channels utilised by endogenous and exogenous CO. These findings suggest that CORM-3 has the potential for use as a modulator of vascular function and hypertension. However, the use of water-soluble CO-RMs raises several questions of their own which will need to be addressed if CO-RMs are to be of future use therapeutically. PMID:15148242

  5. Water-soluble carbon monoxide-releasing molecules: helping to elucidate the vascular activity of the ‘silent killer'

    PubMed Central

    Chatterjee, Prabal Kumar

    2004-01-01

    Carbon monoxide (CO) is formed during the degradation of haeme by haeme oxygenase (HO). As well as being an important signalling molecule and vasodilator, CO also possesses antihypertensive, anti-inflammatory and antiapoptotic qualities and protects against ischaemic tissue injury. Several approaches have been used to investigate the therapeutic potential of CO, ranging from direct administration of CO gas to the use of prodrugs, which generate CO upon metabolism. A novel approach involves the use of specific CO carriers, which will release measurable, controllable and effective amounts of CO into biological systems. Transitional metal carbonyls based around iron, manganese or ruthenium have recently been developed as CO-releasing molecules (CO-RMs) that, under appropriate conditions, will release CO. Such molecules have been shown to provide cardioprotection in both ex vivo and in vivo experiments. To date, CO-RMs have been largely incompatible with biological systems in that they are only soluble in organic solvents or have to be preactivated either by physical or chemical stimuli. However, the recent development of water-soluble CO-RMs has provided new opportunities to investigate the pharmacological and biological features of CO without such confounding influences. CORM-3, a novel water-soluble CO-RM, has recently been used to confirm the cardioprotective actions of CO. In this issue of British Journal of Pharmacology, Foresti and co-workers report that CORM-3 delivers CO, produces aortic vasodilation ex vivo and reduces blood pressure in vivo via modulation of the same cGMP and potassium channels utilised by endogenous and exogenous CO. These findings suggest that CORM-3 has the potential for use as a modulator of vascular function and hypertension. However, the use of water-soluble CO-RMs raises several questions of their own which will need to be addressed if CO-RMs are to be of future use therapeutically. PMID:15148242

  6. Cloud condensation nucleus activity comparison of dry- and wet-generated mineral dust aerosol: the significance of soluble material

    NASA Astrophysics Data System (ADS)

    Garimella, S.; Huang, Y.-w.; Seewald, J. S.; Cziczo, D. J.

    2013-11-01

    This study examines the interaction of clay mineral particles and water vapor to determine the conditions required for cloud droplet formation. Droplet formation conditions are investigated for three clay minerals: illite, sodium-rich montmorillonite, and Arizona Test Dust. Using wet and dry particle generation coupled to a differential mobility analyzer (DMA) and cloud condensation nuclei counter, the critical activation of the clay mineral particles as cloud condensation nuclei is characterized. Electron microscopy (EM) is used to determine non-sphericity in particle shape. EM is also used to determine particle surface area and account for transmission of multiply charged particles by the DMA. Single particle mass spectrometry and ion chromatography are used to investigate soluble material in wet-generated samples and demonstrate that wet and dry generation yield compositionally different particles. Activation results are analyzed in the context of both κ-Köhler theory and Frenkel, Halsey, and Hill (FHH) adsorption activation theory. This study has two main results: (1) κ-Köhler is a suitable framework, less complex than FHH theory, to describe clay mineral nucleation activity despite apparent differences in κ with respect to size. For dry-generated particles the size dependence is likely an artifact of the shape of the size distribution: there is a sharp drop-off in particle concentration at ~300 nm, and a large fraction of particles classified with a mobility diameter less than ~300 nm are actually multiply charged, resulting in a much lower critical supersaturation for droplet activation than expected. For wet-generated particles, deviation from κ-Köhler theory is likely a result of the dissolution and redistribution of soluble material. (2) Wet-generation is found to be unsuitable for simulating the lofting of fresh dry dust because it changes the size-dependent critical supersaturations by fractionating and re-partitioning soluble material.

  7. How Lipid Membranes Affect Pore Forming Toxin Activity.

    PubMed

    Rojko, Nejc; Anderluh, Gregor

    2015-12-15

    Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally

  8. Biological activity, membrane-targeting modification, and crystallization of soluble human decay accelerating factor expressed in E. coli

    PubMed Central

    White, Jennifer; Lukacik, Petra; Esser, Dirk; Steward, Michael; Giddings, Naomi; Bright, Jeremy R.; Fritchley, Sarah J.; Morgan, B. Paul; Lea, Susan M.; Smith, Geoffrey P.; Smith, Richard A.G.

    2004-01-01

    Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 Å, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential. PMID:15322283

  9. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection.

    PubMed

    Francis, Suzanna C; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R; Hayes, Richard J; Shattock, Robin J

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies. PMID:26814891

  10. Immune Activation in the Female Genital Tract: Expression Profiles of Soluble Proteins in Women at High Risk for HIV Infection

    PubMed Central

    Francis, Suzanna C.; Hou, Yanwen; Baisley, Kathy; van de Wijgert, Janneke; Watson-Jones, Deborah; Ao, Trong T.; Herrera, Carolina; Maganja, Kaballa; Andreasen, Aura; Kapiga, Saidi; Coulton, Gary R.; Hayes, Richard J.; Shattock, Robin J.

    2016-01-01

    Soluble cervicovaginal biomarkers of inflammation, immune activation and risk of HIV acquisition are needed to reliably assess the safety of new biomedical prevention strategies including vaccines and microbicides. However, a fuller understanding of expression profiles in women at high risk for HIV infection is crucial to the effective use of these potential biomarkers in Phase 3 trial settings. We have measured 45 soluble proteins and peptides in cervicovaginal lavage samples from 100 HIV negative women at high risk for HIV infection. Women were followed over one menstrual cycle to investigate modulation by hormonal contraception, menstrual cycle phase, recent sexual exposure and intravaginal practices. Women using injectable DMPA had increased concentration of several soluble proteins of the innate and adaptive immune system, including IL-1α, IL-1β, IL-2, MIP-1β, IP-10, IL-8, TGF-β, HBD4, IgA, IgG1, and IgG2. Women using combined oral contraceptives had a similar signature. There were differences in concentrations among samples from post-ovulation compared to pre-ovulation, notably increased immunoglobulins. Increased prostate-specific antigen, indicative of recent sexual exposure, was correlated with increased IL-6, MCP-1, and SLPI, and decreased GM-CSF and HBD3. The identified signature profiles may prove critical in evaluating the potential safety and impact on risk of HIV acquisition of different biomedical intervention strategies. PMID:26814891

  11. The Soluble NAD+-Reducing [NiFe]-Hydrogenase from Ralstonia eutropha H16 Consists of Six Subunits and Can Be Specifically Activated by NADPH

    PubMed Central

    Burgdorf, Tanja; van der Linden, Eddy; Bernhard, Michael; Yuan Yin, Qing; Back, Jaap W.; Hartog, Aloysius F.; Muijsers, Anton O.; de Koster, Chris G.; Albracht, Simon P. J.; Friedrich, Bärbel

    2005-01-01

    The soluble [NiFe]-hydrogenase (SH) of the facultative lithoautotrophic proteobacterium Ralstonia eutropha H16 has up to now been described as a heterotetrameric enzyme. The purified protein consists of two functionally distinct heterodimeric moieties. The HoxHY dimer represents the hydrogenase module, and the HoxFU dimer constitutes an NADH-dehydrogenase. In the bimodular form, the SH mediates reduction of NAD+ at the expense of H2. We have purified a new high-molecular-weight form of the SH which contains an additional subunit. This extra subunit was identified as the product of hoxI, a member of the SH gene cluster (hoxFUYHWI). Edman degradation, in combination with protein sequencing of the SH high-molecular-weight complex, established a subunit stoichiometry of HoxFUYHI2. Cross-linking experiments indicated that the two HoxI subunits are the closest neighbors. The stability of the hexameric SH depended on the pH and the ionic strength of the buffer. The tetrameric form of the SH can be instantaneously activated with small amounts of NADH but not with NADPH. The hexameric form, however, was also activated by adding small amounts of NADPH. This suggests that HoxI provides a binding domain for NADPH. A specific reaction site for NADPH adds to the list of similarities between the SH and mitochondrial NADH:ubiquinone oxidoreductase (Complex I). PMID:15838039

  12. Plasma Levels of Soluble Urokinase-Type Plasminogen Activator Receptor Associate with the Clinical Severity of Acute Puumala Hantavirus Infection

    PubMed Central

    Outinen, Tuula K.; Tervo, Laura; Mäkelä, Satu; Huttunen, Reetta; Mäenpää, Niina; Huhtala, Heini; Vaheri, Antti; Mustonen, Jukka; Aittoniemi, Janne

    2013-01-01

    Objectives Urokinase-type plasminogen activator receptor is a multifunctional glycoprotein, the expression of which is increased during inflammation. It is known to bind to β3-integrins, which are elementary for the cellular entry of hantaviruses. Plasma soluble form of the receptor (suPAR) levels were evaluated as a predictor of severe Puumala hantavirus (PUUV) infection and as a possible factor involved in the pathogenesis of the disease. Design A single-centre prospective cohort study. Subjects and Methods Plasma suPAR levels were measured twice during the acute phase and once during the convalescence in 97 patients with serologically confirmed acute PUUV infection using a commercial enzyme-linked immunosorbent assay (ELISA). Results The plasma suPAR levels were significantly higher during the acute phase compared to the control values after the hospitalization (median 8.7 ng/ml, range 4.0–18.2 ng/ml vs. median 4.7 ng/ml, range 2.4–12.2 ng/ml, P<0.001). The maximum suPAR levels correlated with several variables reflecting the severity of the disease. There was a positive correlation with maximum leukocyte count (r = 0.475, p<0.001), maximum plasma creatinine concentration (r = 0.378, p<0.001), change in weight during the hospitalization (r = 0.406, p<0.001) and the length of hospitalization (r = 0.325, p = 0.001), and an inverse correlation with minimum platelet count (r = −0.325, p = 0.001) and minimum hematocrit (r = −0.369, p<0.001). Conclusion Plasma suPAR values are markedly increased during acute PUUV infection and associate with the severity of the disease. The overexpression of suPAR possibly activates β3-integrin in PUUV infection, and thus might be involved in the pathogenesis of the disease. PMID:23990945

  13. Soluble extract from Moringa oleifera leaves with a new anticancer activity.

    PubMed

    Jung, Il Lae

    2014-01-01

    Moringa oleifera has been regarded as a food substance since ancient times and has also been used as a treatment for many diseases. Recently, various therapeutic effects of M. oleifera such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, and antioxidant effects have been investigated; however, most of these studies described only simple biological phenomena and their chemical compositions. Due to the increasing attention on natural products, such as those from plants, and the advantages of oral administration of anticancer drugs, soluble extracts from M. oleifera leaves (MOL) have been prepared and their potential as new anticancer drug candidates has been assessed in this study. Here, the soluble cold Distilled Water extract (4°C; concentration, 300 µg/mL) from MOL greatly induced apoptosis, inhibited tumor cell growth, and lowered the level of internal reactive oxygen species (ROS) in human lung cancer cells as well as other several types of cancer cells, suggesting that the treatment of cancer cells with MOL significantly reduced cancer cell proliferation and invasion. Moreover, over 90% of the genes tested were unexpectedly downregulated more than 2-fold, while just below 1% of the genes were upregulated more than 2-fold in MOL extract-treated cells, when compared with nontreated cells. Since severe dose-dependent rRNA degradation was observed, the abnormal downregulation of numerous genes was considered to be attributable to abnormal RNA formation caused by treatment with MOL extracts. Additionally, the MOL extract showed greater cytotoxicity for tumor cells than for normal cells, strongly suggesting that it could potentially be an ideal anticancer therapeutic candidate specific to cancer cells. These results suggest the potential therapeutic implications of the soluble extract from MOL in the treatment of various types of cancers. PMID:24748376

  14. Soluble Extract from Moringa oleifera Leaves with a New Anticancer Activity

    PubMed Central

    Jung, Il Lae

    2014-01-01

    Moringa oleifera has been regarded as a food substance since ancient times and has also been used as a treatment for many diseases. Recently, various therapeutic effects of M. oleifera such as antimicrobial, anticancer, anti-inflammatory, antidiabetic, and antioxidant effects have been investigated; however, most of these studies described only simple biological phenomena and their chemical compositions. Due to the increasing attention on natural products, such as those from plants, and the advantages of oral administration of anticancer drugs, soluble extracts from M. oleifera leaves (MOL) have been prepared and their potential as new anticancer drug candidates has been assessed in this study. Here, the soluble cold Distilled Water extract (4°C; concentration, 300 µg/mL) from MOL greatly induced apoptosis, inhibited tumor cell growth, and lowered the level of internal reactive oxygen species (ROS) in human lung cancer cells as well as other several types of cancer cells, suggesting that the treatment of cancer cells with MOL significantly reduced cancer cell proliferation and invasion. Moreover, over 90% of the genes tested were unexpectedly downregulated more than 2-fold, while just below 1% of the genes were upregulated more than 2-fold in MOL extract-treated cells, when compared with nontreated cells. Since severe dose-dependent rRNA degradation was observed, the abnormal downregulation of numerous genes was considered to be attributable to abnormal RNA formation caused by treatment with MOL extracts. Additionally, the MOL extract showed greater cytotoxicity for tumor cells than for normal cells, strongly suggesting that it could potentially be an ideal anticancer therapeutic candidate specific to cancer cells. These results suggest the potential therapeutic implications of the soluble extract from MOL in the treatment of various types of cancers. PMID:24748376

  15. Calpain-Mediated Processing of Adenylate Cyclase Toxin Generates a Cytosolic Soluble Catalytically Active N-Terminal Domain

    PubMed Central

    Ostolaza, Helena

    2013-01-01

    Bordetella pertussis, the whooping cough pathogen, secretes several virulence factors among which adenylate cyclase toxin (ACT) is essential for establishment of the disease in the respiratory tract. ACT weakens host defenses by suppressing important bactericidal activities of the phagocytic cells. Up to now, it was believed that cell intoxication by ACT was a consequence of the accumulation of abnormally high levels of cAMP, generated exclusively beneath the host plasma membrane by the toxin N-terminal catalytic adenylate cyclase (AC) domain, upon its direct translocation across the lipid bilayer. Here we show that host calpain, a calcium-dependent Cys-protease, is activated into the phagocytes by a toxin-triggered calcium rise, resulting in the proteolytic cleavage of the toxin N-terminal domain that releases a catalytically activesoluble AC”. The calpain-mediated ACT processing allows trafficking of the “soluble AC” domain into subcellular organella. At least two strategic advantages arise from this singular toxin cleavage, enhancing the specificity of action, and simultaneously preventing an indiscriminate activation of cAMP effectors throughout the cell. The present study provides novel insights into the toxin mechanism of action, as the calpain-mediated toxin processing would confer ACT the capacity for a space- and time-coordinated production of different cAMP “pools”, which would play different roles in the cell pathophysiology. PMID:23840759

  16. High-Resolution Structure of a Self-Assembly-Competent Form of a Hydrophobic Peptide Captured in a Soluble [beta]-Sheet Scaffold

    SciTech Connect

    Makabe, Koki; Biancalana, Matthew; Yan, Shude; Tereshko, Valentina; Gawlak, Grzegorz; Miller-Auer, Hélène; Meredith, Stephen C.; Koide, Shohei

    2010-02-08

    {beta}-Rich self-assembly is a major structural class of polypeptides, but still little is known about its atomic structures and biophysical properties. Major impediments for structural and biophysical studies of peptide self-assemblies include their insolubility and heterogeneous composition. We have developed a model system, termed peptide self-assembly mimic (PSAM), based on the single-layer {beta}-sheet of Borrelia outer surface protein A. PSAM allows for the capture of a defined number of self-assembly-like peptide repeats within a water-soluble protein, making structural and energetic studies possible. In this work, we extend our PSAM approach to a highly hydrophobic peptide sequence. We show that a penta-Ile peptide (Ile{sub 5}), which is insoluble and forms {beta}-rich self-assemblies in aqueous solution, can be captured within the PSAM scaffold in a form capable of self-assembly. The 1.1-{angstrom} crystal structure revealed that the Ile{sub 5} stretch forms a highly regular {beta}-strand within this flat {beta}-sheet. Self-assembly models built with multiple copies of the crystal structure of the Ile5 peptide segment showed no steric conflict, indicating that this conformation represents an assembly-competent form. The PSAM retained high conformational stability, suggesting that the flat {beta}-strand of the Ile{sub 5} stretch primed for self-assembly is a low-energy conformation of the Ile{sub 5} stretch and rationalizing its high propensity for self-assembly. The ability of the PSAM to 'solubilize' an otherwise insoluble peptide stretch suggests the potential of the PSAM approach to the characterization of self-assembling peptides.

  17. The soluble form of Alzheimer's amyloid beta protein is complexed to high density lipoprotein 3 and very high density lipoprotein in normal human plasma.

    PubMed

    Koudinov, A; Matsubara, E; Frangione, B; Ghiso, J

    1994-12-15

    The amyloid fibrils of Alzheimer's neuritic plaques and cerebral blood vessels are mainly composed of aggregated forms of a 39 to 44 amino acids peptide, named amyloid beta (A beta). A similar although soluble form of A beta (sA beta) has been identified in plasma, cerebrospinal fluid and cell culture supernatants, indicating that it is produced under physiologic conditions. We report here that sA beta in normal human plasma is associated with lipoprotein particles, in particular to the HDL3 and VHDL fractions where it is complexed to ApoJ and, to a lesser extent, to ApoAI. This was assessed by immunoprecipitation experiments of purified plasma lipoproteins and lipoprotein-depleted plasma and confirmed by means of amino acid sequence analysis. Moreover, biotinylated synthetic peptide A beta 1-40 was traced in normal human plasma in in vitro experiments. As in the case of sA beta, biotinylated A beta 1-40 was specifically recovered in the HDL3 and VHDL fractions. This data together with the previous demonstration that A beta 1-40 is taken up into the brain via a specific mechanism and possibly as an A beta 1-40-ApoJ complex indicate a role for HDL3- and VHDL-containing ApoJ in the transport of the peptide in circulation and suggest their involvement in the delivery of sA beta across the blood-brain barrier. PMID:7802646

  18. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  19. Influence of solid state fermentation by Trichoderma spp. on solubility, phenolic content, antioxidant, and antimicrobial activities of commercial turmeric.

    PubMed

    Mohamed, Saleh A; Saleh, Rashad M; Kabli, Saleh A; Al-Garni, Saleh M

    2016-05-01

    The influence of solid state fermentation (SSF) by Trichoderma spp. on the solubility, total phenolic content, antioxidant, and antibacterial activities of turmeric was determined and compared with unfermented turmeric. The solubility of turmeric was monitored by increase in its phenolic content. The total phenolic content of turmeric extracted by 80% methanol and water after SSF by six species of Trichoderma spp. increased significantly from 2.5 to 11.3-23.3 and from 0.5 to 13.5-20.4 GAE/g DW, respectively. The antioxidant activities of fermented turmeric were enhanced using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS), and ferric ion-reducing antioxidant power (FRAP) assays. The antibacterial activity of fermented turmeric against human-pathogenic bacteria Escherichia coli, Streptococcus agalactiae, Staphylococcus aureus, Entreococcus faecalis, Methicillin-Resistant S. aureus, Klebsiella pneumonia, and Pseudomonas aeruginosae showed a broad spectrum inhibitory effect. In conclusion, the results indicated the potentials of using fermented turmeric as natural antioxidant and antimicrobial material for food applications. PMID:27023794

  20. The effect of continuous Zn (II) exposure on the organic degradation capability and soluble microbial products (SMP) of activated sludge.

    PubMed

    Han, Jing-chao; Liu, Yan; Liu, Xiang; Zhang, Yi; Yan, Yang-wei; Dai, Rui-hua; Zha, Xiao-song; Wang, Cheng-shan

    2013-01-15

    This study describes the change of organic degradation capability and soluble microbial products (SMP) generated in activated sludge under continuous exposure to Zn (II) in a sequencing batch reactor (SBR). In 338 days of operation, the added Zn (II) concentrations were gradually increased from 50 to 100, 200, 400 to 600 and 800 mg/L. Results showed that after adaptation, the activated sludge could endure 400mg/L Zn (II) without showing evident reduction in organic degradation ability (92±1% of chemical oxygen demand (COD) removal in stable state). However, when 600 and 800 mg/L Zn (II) were applied, the effluent water quality significantly deteriorated. Meanwhile, under increasing Zn (II) concentrations, the SMP content in the activated sludge, together with its main biochemical constituents, first increased slightly below 400mg/L of Zn (II), then rose sharply under 600 and 800 mg/L Zn (II). Furthermore, a close correlation was found between SMP content and effluent soluble COD in both the Experimental Reactor and Control Reactor. In addition, the Zn (II) concentrations in the effluent and SMP extraction liquid were further analyzed and discussed to reveal the role that SMP constituents played in defense and resistance to the toxicity of Zn (II). PMID:23183340

  1. The mRNA expression of soluble urokinase plasminogen activator surface receptor in human adipose tissue is positively correlated with body mass index.

    PubMed

    Ng, Hien Fuh; Chin, Kin Fah; Chan, Kok-Gan; Ngeow, Yun Fong

    2015-06-01

    suPLAUR is the transcript variant that encodes the soluble form of the urokinase plasminogen activator surface receptor (suPLAUR). This soluble protein has been shown to enhance leukocyte migration and adhesion, and its circulatory level is increased in inflammatory states. In this pilot study, we used RNA-Seq to examine the splicing pattern of PLAUR in omental adipose tissues from obese and lean individuals. Of the three transcript variants of the PLAUR gene, only the proportion of suPLAUR (transcript variant 2) increases in obesity. After removing the effects of gender and age, the expression of suPLAUR is positively correlated with body mass index. This observation was validated using RT-qPCR with an independent cohort of samples. Additionally, in our RNA-Seq differential expression analysis, we also observed, in obese adipose tissues, an up-regulation of genes encoding other proteins involved in the process of chemotaxis and leukocyte adhesion; of particular interest is the integrin beta 2 (ITGB2) that is known to interact with suPLAUR in leukocyte adhesion. These findings suggest an important role for suPLAUR in the recruitment of immune cells to obese adipose tissue, in the pathogenesis of obesity. PMID:26284904

  2. Localization and environment of tryptophans in soluble and membrane-bound states of a pore-forming toxin from Staphylococcus aureus.

    PubMed Central

    Raja, S M; Rawat, S S; Chattopadhyay, A; Lala, A K

    1999-01-01

    The location and environment of tryptophans in the soluble and membrane-bound forms of Staphylococcus aureus alpha-toxin were monitored using intrinsic tryptophan fluorescence. Fluorescence quenching of the toxin monomer in solution indicated varying degrees of tryptophan burial within the protein interior. N-Bromosuccinimide readily abolished 80% of the fluorescence in solution. The residual fluorescence of the modified toxin showed a blue-shifted emission maximum, a longer fluorescence lifetime as compared to the unmodified and membrane-bound alpha-toxin, and a 5- to 6-nm red edge excitation shift, all indicating a restricted tryptophan environment and deeply buried tryptophans. In the membrane-bound form, the fluorescence of alpha-toxin was quenched by iodide, indicating a conformational change leading to exposure of some tryptophans. A shorter average lifetime of tryptophans in the membrane-bound alpha-toxin as compared to the native toxin supported the conclusions based on iodide quenching of the membrane-bound toxin. Fluorescence quenching of membrane-bound alpha-toxin using brominated and spin-labeled fatty acids showed no quenching of fluorescence using brominated lipids. However, significant quenching was observed using 5- and 12-doxyl stearic acids. An average depth calculation using the parallax method indicated that the doxyl-quenchable tryptophans are located at an average depth of 10 A from the center of the bilayer close to the membrane interface. This was found to be in striking agreement with the recently described structure of the membrane-bound form of alpha-toxin. PMID:10049328

  3. Phytase activity of lactic acid bacteria and their impact on the solubility of minerals from wholemeal wheat bread.

    PubMed

    Cizeikiene, Dalia; Juodeikiene, Grazina; Bartkiene, Elena; Damasius, Jonas; Paskevicius, Algimantas

    2015-01-01

    The objective of this study was to determinate phytase activity of bacteriocins producing lactic acid bacteria previously isolated from spontaneous rye sourdough. The results show that the highest extracellular phytase activity produces Pediococcus pentosaceus KTU05-8 and KTU05-9 strains with a volumetric phytase activity of 32 and 54 U/ml, respectively, under conditions similar to leavening of bread dough (pH 5.5 and 30 °C). In vitro studies in simulated gastrointestinal tract media pH provide that bioproducts prepared with P. pentosaceus strains used in wholemeal wheat bread preparation increase solubility of iron, zinc, manganese, calcium and phosphorus average 30%. Therefore, P. pentosaceus KTU05-9 and KTU05-8 strains could be recommended to use as a starter for sourdough preparation for increasing of mineral bioavailability from wholemeal wheat bread. PMID:26397032

  4. Solubility Database

    National Institute of Standards and Technology Data Gateway

    SRD 106 IUPAC-NIST Solubility Database (Web, free access)   These solubilities are compiled from 18 volumes (Click here for List) of the International Union for Pure and Applied Chemistry(IUPAC)-NIST Solubility Data Series. The database includes liquid-liquid, solid-liquid, and gas-liquid systems. Typical solvents and solutes include water, seawater, heavy water, inorganic compounds, and a variety of organic compounds such as hydrocarbons, halogenated hydrocarbons, alcohols, acids, esters and nitrogen compounds. There are over 67,500 solubility measurements and over 1800 references.

  5. Redox tuning of the catalytic activity of soluble fumarate reductases from Shewanella.

    PubMed

    Paquete, Catarina M; Saraiva, Ivo H; Louro, Ricardo O

    2014-06-01

    Many enzymes involved in bioenergetic processes contain chains of redox centers that link the protein surface, where interaction with electron donors or acceptors occurs, to a secluded catalytic site. In numerous cases these redox centers can transfer only single electrons even when they are associated to catalytic sites that perform two-electron chemistry. These chains provide no obvious contribution to enhance chemiosmotic energy conservation, and often have more redox centers than those necessary to hold sufficient electrons to sustain one catalytic turnover of the enzyme. To investigate the role of such a redox chain we analyzed the transient kinetics of fumarate reduction by two flavocytochromes c3 of Shewanella species while these enzymes were being reduced by sodium dithionite. These soluble monomeric proteins contain a chain of four hemes that interact with a flavin adenine dinucleotide (FAD) catalytic center that performs the obligatory two electron-two proton reduction of fumarate to succinate. Our results enabled us to parse the kinetic contribution of each heme towards electron uptake and conduction to the catalytic center, and to determine that the rate of fumarate reduction is modulated by the redox stage of the enzyme, which is defined by the number of reduced centers. In both enzymes the catalytically most competent redox stages are those least prevalent in a quasi-stationary condition of turnover. Furthermore, the electron distribution among the redox centers during turnover suggested how these enzymes can play a role in the switch between respiration of solid and soluble terminal electron acceptors in the anaerobic bioenergetic metabolism of Shewanella. PMID:24530355

  6. Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei

    PubMed Central

    Miinalainen, Ilkka J.; Hiltunen, J. Kalervo; Michels, Paul A. M.; Antonenkov, Vasily D.

    2012-01-01

    Background Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages ±150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (PK+/PCl−∼0.31), while the other two types of channels are slightly selective for cations (PK+/PCl− ratios ∼1.15 and ∼1.27 for the high- and low-conductance channels, respectively). The anion-selective channel showed an intrinsic current rectification that may suggest a functional asymmetry of the channel's pore. Conclusions/Significance These results indicate that the membrane of glycosomes apparently

  7. YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

    PubMed Central

    Martin, Emil; Lee, Yu-Chen; Murad, Ferid

    2001-01-01

    YC-1 [3-(5′-hydroxymethyl-2′furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme. PMID:11687640

  8. YC-1 activation of human soluble guanylyl cyclase has both heme-dependent and heme-independent components

    NASA Technical Reports Server (NTRS)

    Martin, E.; Lee, Y. C.; Murad, F.

    2001-01-01

    YC-1 [3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole] is an allosteric activator of soluble guanylyl cyclase (sGC). YC-1 increases the catalytic rate of the enzyme and sensitizes the enzyme toward its gaseous activators nitric oxide or carbon monoxide. In other studies the administration of YC-1 to experimental animals resulted in the inhibition of the platelet-rich thrombosis and a decrease of the mean arterial pressure, which correlated with increased cGMP levels. However, details of YC-1 interaction with sGC and enzyme activation are incomplete. Although evidence in the literature indicates that YC-1 activation of sGC is strictly heme-dependent, this report presents evidence for both heme-dependent and heme-independent activation of sGC by YC-1. The oxidation of the sGC heme by 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one completely inhibited the response to NO, but only partially attenuated activation by YC-1. We also observed activation by YC-1 of a mutant sGC, which lacks heme. These findings indicate that YC-1 activation of sGC can occur independently of heme, but that activation is substantially increased when the heme moiety is present in the enzyme.

  9. Structure-activity relationships of the plasminogen modulator SMTP with respect to the inhibition of soluble epoxide hydrolase.

    PubMed

    Matsumoto, Naoki; Suzuki, Eriko; Tsujihara, Kota; Nishimura, Yuuichi; Hasumi, Keiji

    2015-11-01

    A family of fungal metabolites, SMTP, is a small-molecule plasminogen modulator that enhances plasminogen activation, leading to thrombolysis. We recently demonstrated that SMTP-7 effectively treats ischemic stroke due to its thrombolytic activity as well as anti-inflammatory action, which is attributable to soluble epoxide hydrolase (sEH) inhibition. In this paper, we studied detailed structure-activity relationships of plasminogen modulation and sEH inhibition using 25 SMTP congeners including six newly synthesized ones. The results clearly demonstrate that the structure of the N-linked side chain of SMTP congeners markedly affect their activities toward plasminogen modulation and inhibitions of the two activities of sEH (C-terminal epoxide hydrolase and N-terminal phosphatase). A slight change in the N-linked side chain results in affording selectivity of SMTP congeners. Many congeners, which lacked plasminogen modulation activity, differently inhibited the two sEH activities depending on the structures of the N-linked side chain. Some congeners were active in plasminogen modulation and inhibition of both activities of sEH. These results help comprehensive understanding of ideal design of a drug useful for ischemic diseases that are associated with inflammation, such as stroke. PMID:25966853

  10. NUCLEAR ACTIVITY IS MORE PREVALENT IN STAR-FORMING GALAXIES

    SciTech Connect

    Rosario, D. J.; Lutz, D.; Berta, S.; Popesso, P.; Genzel, R.; Saintonge, A.; Tacconi, L.; Wuyts, S. E-mail: lutz@mpe.mpg.de E-mail: popesso@mpe.mpg.de E-mail: amelie@mpe.mpg.de E-mail: swuyts@mpe.mpg.de; and others

    2013-07-01

    We explore the question of whether low and moderate luminosity active galactic nuclei (AGNs) are preferentially found in galaxies that are undergoing a transition from active star formation (SF) to quiescence. This notion has been suggested by studies of the UV-optical colors of AGN hosts, which find them to be common among galaxies in the so-called Green Valley, a region of galaxy color space believed to be composed mostly of galaxies undergoing SF quenching. Combining the deepest current X-ray and Herschel/PACS far-infrared (FIR) observations of the two Chandra Deep Fields with redshifts, stellar masses, and rest-frame photometry derived from the extensive and uniform multi-wavelength data in these fields, we compare the rest-frame U - V color distributions and star formation rate distributions of AGNs and carefully constructed samples of inactive control galaxies. The UV-to-optical colors of AGNs are consistent with equally massive inactive galaxies at redshifts out to z {approx} 2, but we show that such colors are poor tracers of SF. While the FIR distributions of both star-forming AGNs and star-forming inactive galaxies are statistically similar, we show that AGNs are preferentially found in star-forming host galaxies, or, in other words, AGNs are less likely to be found in weakly star-forming or quenched galaxies. We postulate that, among X-ray-selected AGNs of low and moderate accretion luminosities, the supply of cold gas primarily determines the accretion rate distribution of the nuclear black holes.

  11. Ruthenium-based olefin metathesis catalysts bearing pH-responsive ligands: External control of catalyst solubility and activity

    NASA Astrophysics Data System (ADS)

    Balof, Shawna Lynn

    2011-12-01

    Sixteen novel, Ru-based olefin metathesis catalysts bearing pH responsive ligands were synthesized. The pH-responsive groups employed with these catalysts included dimethylamino (NMe2) modified NHC ligands as well as N-donor dimethylaminopyridine (DMAP) and 3-(o-pyridyl)propylidene ligands. These pH-responsive ligands provided the means by which the solubility and/or activity profiles of the catalysts produced could be controlled via acid addition. The main goal of this dissertation was to design catalyst systems capable of performing ring opening metathesis (ROMP) and ring closing metathesis (RCM) reactions in both organic and aqueous media. In an effort to quickly gain access to new catalyst structures, a template synthesis for functionalized NHC ligand precursors was designed, in addition to other strategies, to obtain ligand precursors with ancillary NMe2 groups. Kinetic studies for the catalysts produced from these precursors showed external control of catalyst solubility was afforded via protonation of the NMe2 groups of their NHC ligands. Additionally, this protonation afforded external control of catalyst propagation rates for several catalysts. This is the first known independent external control for the propagation rates of ROMP catalysts. The incorporation of pH-responsive N-donor ligands into catalyst structures also provided the means for the external control of metathesis activity, as the protonation of these ligands resulted in an increased initiation rate based on their fast and irreversible dissociation from the metal center. The enhanced external control makes these catalysts applicable to a wide range of applications, some of which have been explored by us and/or through collaboration. Three of the catalysts designed showed remarkable metathesis activity in aqueous media. These catalysts displayed comparable RCM activity in aqueous media to a class of water-soluble catalysts reported by Grubbs et al., considered to be the most active catalyst for

  12. Application of the soluble salt-assisted route to scalable synthesis of ZnO nanopowder with repeated photocatalytic activity.

    PubMed

    Lv, Yingying; Yu, Leshu; Huang, Heyong; Feng, Yuying; Chen, Dongzhen; Xie, Xin

    2012-02-17

    In this paper, the soluble salt-assisted route has been extended to the low-cost and scalable preparation of ZnO nanostructures via the simple oxidation of Zn-Na2SO4 mixture followed by washing with water. The as-prepared ZnO nanopowders are of nanoscaled size, hexagonal phase, and pure, without being stained by Na2SO4. Their optical band gap is 3.22 eV, exhibiting a red-shift of 0.15 eV in comparison with pure ZnO bulk, and their optical absorbance is strong in the region of 200-400 nm, suggesting their full utilization of most of the UV light in sunlight. The product shows evident photocatalytic activity in degradation of RhB under solar light irradiation, and then its solar light degradation efficiency is close to that under UV irradiation, indicating that there is a possibility of practical application. More importantly, the obtained ZnO nanoparticles, because of the quick precipitation by themselves in solution with no stirring, could be easily recycled without any accessorial means such as high-speed centrifuge. The low-cost and scalable preparation, high photocatalytic activity, and convenient recycling of this ZnO nanomaterial gives it potential in purifying waste water. Hence the interesting results in this study indicate the wide range of the soluble salt-assisted route for the industrial preparation of many other advanced nanomaterials. PMID:22248758

  13. Laccase-gum Arabic conjugate for preparation of water-soluble oligomer of catechin with enhanced antioxidant activity.

    PubMed

    Jadhav, Swati B; Singhal, Rekha S

    2014-05-01

    Catechin was oligomerized using free laccase and laccase-gum Arabic conjugate. The process of oligomerization was optimized with respect to solvent, ratio of solvent to buffer (0.2:10 to 1:10), pH of buffer (3-10), enzyme (575-18,400 U/mg) and substrate concentration (1-7mM). Maximum production of oligomer was observed in methanol at ratio 0.6:10 of methanol:buffer of pH 5 using 2300 U/mg of laccase and 5mM of catechin. The laccase-gum Arabic conjugate showed lower activity but higher stability than free laccase in methanol. Free laccase produced cross linked water-insoluble oligomer, whereas conjugated laccase produced linear water-soluble oligomer. The linear water-soluble oligomer showed higher antioxidant activity, as determined by the DPPH assay, and reducing power as compared to monomer making it suitable for biological applications. The molecular weight of the linear oligomer was found to be 13.14kDa, which suggested it to be composed of 45 monomer units. Further characterizations of linear and cross linked oligomer were done using FTIR and differential scanning calorimetry. PMID:24360412

  14. Biphasic dissolution method for quality control and assurance of drugs containing active substances in the form of weak acid salts.

    PubMed

    Franc, Aleš; Muselłk, Jan; Goněc, Roman; Vetchý, David

    2016-03-01

    Substances in the form of weak acid salts have been found to be problematic for dissolution testing. Their absorption can start only after they are turned into the form of an acid following the gastric passage although they were administered in the form of a salt. Due to poor solubility, they cannot be tested in acidic gastric environment for a biased dissolution profile. The biphasic dissolution method is promising for overcoming this obstacle. Tablets with warfarin clathrate sodium salt in two concentrations and two different particle size distributions were tested as a suitable model for finding the medium and process conditions of dissolution. The dissolution method based on the use of the upper organic layer (1-octanol) and the lower aqueous layer 0.1 mol L(-1) HCl) was found suitable and discriminatory for tablets containing active substances in the form of salts of weak acids. The method also reflects physical differences in the quality of used substances. PMID:26959550

  15. The hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) exists in several forms as shown by electrospray-ionisation mass spectrometry.

    PubMed

    Buzy, A; Millar, A L; Legros, V; Wilkins, P C; Dalton, H; Jennings, K R

    1998-06-15

    The hydroxylase of the soluble methane monooxygenase from the bacterium Methylococcus capsulatus (Bath) has been investigated by means of electrospray-ionisation mass spectrometry (ESI-MS) and liquid chromatography ESI-MS (LC/ESI-MS). The hydroxylase is a non-heme diiron protein consisting of three pairs of non-identical subunits (alpha approximately 60 kDa, beta approximately 45 kDa and gamma approximately 20 kDa). Liquid chromatographic separation of the hydroxylase subunits was required before MS analysis in order to detect the alpha-subunit. The masses measured for the three subunits were found to disagree with those calculated from their gene sequences. Experiments involving the use of CNBr and trypsin cleavage followed by LC/ESI-MS and MS/MS analyses permitted the location and correction of errors in the sequences deduced from the use of cDNA. The ESI-MS results also showed that the alpha-subunit of the hydroxylase exists in multiple forms which result from cleavage of the protein. This observation explains a number of enigmatic features of the protein previously reported in the literature and illustrates the pivotal role of ESI-MS in complementing data obtained from molecular biology for the characterisation of the primary sequence of proteins. PMID:9688272

  16. 76 FR 25364 - Agency Information Collection Activities: Form I-864, Form I-864A, Form I-864EZ, and From I-864W...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-04

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form I-864, Form I- 864A, Form I-864EZ, and From I-864W; Extension of an Existing Information Collection; Comment Request. ACTION: 60-Day Notice of Information Collection Under Review; Form I- 864, Affidavit of...

  17. Soluble FGFR4 extracellular domain inhibits FGF19-induced activation of FGFR4 signaling and prevents nonalcoholic fatty liver disease

    SciTech Connect

    Chen, Qiang; Jiang, Yuan; An, Yuan; Zhao, Na; Zhao, Yang; Yu, Chundong

    2011-06-17

    Highlights: {yields} Soluble FGFR4 extracellular domain (FGFR4-ECD) was effectively expressed. {yields} FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling. {yields} FGFR4-ECD reduced palmitic acid-induced steatosis of HepG2 cells. {yields} FGFR4-ECD reduced tetracycline-induced fatty liver in mice. {yields} FGFR4-ECD partially restored tetracycline-repressed PPAR{alpha} expression. -- Abstract: Fibroblast growth factor receptor 4 (FGFR4) is a transmembrane tyrosine kinase receptor that plays a crucial role in the regulation of hepatic bile acid and lipid metabolism. FGFR4 underlies high-fat diet-induced hepatic steatosis, suggesting that inhibition of FGFR4 activation may be an effective way to prevent or treat nonalcoholic fatty liver disease (NAFLD). To determine whether neutralization of FGFR4 ligands by soluble FGFR4 extracellular domain (FGFR4-ECD) can inhibit the activation of FGFR4, we constructed FGFR4-ECD expression vector and showed that FGFR4-ECD was effectively expressed in cells and secreted into culture medium. FGFR4-ECD inhibited FGF19-induced activation of FGFR4 signaling and reduced steatosis of HepG2 induced by palmitic acid in vitro. Furthermore, in a tetracycline-induced fatty liver model, expression of FGFR4-ECD in mouse liver reduced the accumulation of hepatic lipids and partially restored the expression of peroxisome proliferator-activated receptor {alpha} (PPAR{alpha}), which promotes the mitochondrial fatty acid beta-oxidation but is repressed by tetracycline. Taken together, these results demonstrate that FGFR4-ECD can block FGFR4 signaling and prevent hepatic steatosis, highlighting the potential value of inhibition of FGFR4 signaling as a method for therapeutic intervention against NAFLD.

  18. Cloud condensation nucleus activity comparison of dry- and wet-generated mineral dust aerosol: the significance of soluble material

    NASA Astrophysics Data System (ADS)

    Garimella, S.; Huang, Y.-W.; Seewald, J. S.; Cziczo, D. J.

    2014-06-01

    This study examines the interaction of clay mineral particles and water vapor for determining the conditions required for cloud droplet formation. Droplet formation conditions are investigated for two common clay minerals, illite and sodium-rich montmorillonite, and an industrially derived sample, Arizona Test Dust. Using wet and dry particle generation coupled to a differential mobility analyzer (DMA) and cloud condensation nuclei counter, the critical activation of the clay mineral particles as cloud condensation nuclei is characterized. Electron microscopy (EM) is used in order to determine non-sphericity in particle shape. It is also used in order to determine particle surface area and account for transmission of multiply charged particles by the DMA. Single particle mass spectrometry and ion chromatography are used to investigate soluble material in wet-generated samples and demonstrate that wet and dry generation yield compositionally different particles. Activation results are analyzed in the context of both κ-Köhler theory (κ-KT) and Frenkel-Halsey-Hill (FHH) adsorption activation theory. This study has two main results: (1) κ-KT is the suitable framework to describe clay mineral nucleation activity. Apparent differences in κ with respect to size arise from an artifact introduced by improper size-selection methodology. For dust particles with mobility sizes larger than ~300 nm, i.e., ones that are within an atmospherically relevant size range, both κ-KT and FHH theory yield similar critical supersaturations. However, the former requires a single hygroscopicity parameter instead of the two adjustable parameters required by the latter. For dry-generated particles, the size dependence of κ is likely an artifact of the shape of the size distribution: there is a sharp drop-off in particle concentration at ~300 nm, and a large fraction of particles classified with a mobility diameter less than ~300 nm are actually multiply charged, resulting in a much

  19. Are soluble and membrane-bound rat brain acetylcholinesterase different

    SciTech Connect

    Andres, C.; el Mourabit, M.; Stutz, C.; Mark, J.; Waksman, A. )

    1990-11-01

    Salt-soluble and detergent-soluble acetylcholinesterases (AChE) from adult rat brain were purified to homogeneity and studied with the aim to establish the differences existing between these two forms. It was found that the enzymatic activities of the purified salt-soluble AChE as well as the detergent-soluble AChE were dependent on the Triton X-100 concentration. Moreover, the interaction of salt-soluble AChE with liposomes suggests amphiphilic behaviour of this enzyme. Serum cholinesterase (ChE) did not bind to liposomes but its activity was also detergent-dependent. Detergent-soluble AChE remained in solution below critical micellar concentrations of Triton X-100. SDS polyacrylamide gel electrophoresis of purified, Biobeads-treated and iodinated detergent-soluble 11 S AChE showed, under non reducing conditions, bands of 69 kD, 130 kD and greater than 250 kD corresponding, respectively, to monomers, dimers and probably tetramers of the same polypeptide chain. Under reducing conditions, only a 69 kD band was detected. It is proposed that an amphiphilic environment stabilizes the salt-soluble forms of AChE in the brain in vivo and that detergent-soluble Biobeads-treated 11 S AChE possess hydrophobic domain(s) different from the 20 kD peptide already described.

  20. Could both vitamin D and geomagnetic activity impact serum levels of soluble cell adhesion molecules in young men?

    NASA Astrophysics Data System (ADS)

    Bleizgys, Andrius; Šapoka, Virginijus

    2015-11-01

    Vitamin D might have a role in diminishing endothelial dysfunction (ED). The initial aim was to test the hypothesis of reciprocity between levels of 25-hydroxyvitamin D (25(OH)D) and levels of soluble endothelial cell adhesion molecules (CAMs) that could serve as biomarkers of ED. Randomly selected men of age 20-39 were examined at February or March (cold season) and reexamined at August or September (warm season). Some lifestyle and anthropometrical data were recorded. Laboratory measurements, including those for serum levels of soluble CAMs—sICAM-1, sVCAM-1, sE-selectin and sP-selectin—were also performed. As some of the results were rather unexpected, indices of geomagnetic activity (GMA), obtained from the online database, were included in further analysis as a confounder. In 2012-2013, 130 men were examined in cold season, and 125 of them were reexamined in warm season. 25(OH)D levels were found to be significantly negatively associated with sVCAM-1 levels (β = -0.15, p = 0.043 in warm season; β = -0.19, p = 0.007 for changes). Levels of sVCAM-1 and sICAM-1 from the same seasons were notably different between years and have changed in an opposite manner. Soluble P-selectin levels were higher at warm season in both years. GMA was positively associated with sVCAM-1 (β = 0.17, p = 0.039 in cold season; β = 0.22, p = 0.002 for changes) and negatively with sICAM-1 (β = -0.30. p < 0.001 in cold season) levels. Vitamin D might play a role in diminishing sVCAM-1 levels. Levels of sVCAM-1 and sICAM-1 were associated with the GMA; this implies a need for further research.

  1. Could both vitamin D and geomagnetic activity impact serum levels of soluble cell adhesion molecules in young men?

    NASA Astrophysics Data System (ADS)

    Bleizgys, Andrius; Šapoka, Virginijus

    2016-07-01

    Vitamin D might have a role in diminishing endothelial dysfunction (ED). The initial aim was to test the hypothesis of reciprocity between levels of 25-hydroxyvitamin D (25(OH)D) and levels of soluble endothelial cell adhesion molecules (CAMs) that could serve as biomarkers of ED. Randomly selected men of age 20-39 were examined at February or March (cold season) and reexamined at August or September (warm season). Some lifestyle and anthropometrical data were recorded. Laboratory measurements, including those for serum levels of soluble CAMs—sICAM-1, sVCAM-1, sE-selectin and sP-selectin—were also performed. As some of the results were rather unexpected, indices of geomagnetic activity (GMA), obtained from the online database, were included in further analysis as a confounder. In 2012-2013, 130 men were examined in cold season, and 125 of them were reexamined in warm season. 25(OH)D levels were found to be significantly negatively associated with sVCAM-1 levels ( β = -0.15, p = 0.043 in warm season; β = -0.19, p = 0.007 for changes). Levels of sVCAM-1 and sICAM-1 from the same seasons were notably different between years and have changed in an opposite manner. Soluble P-selectin levels were higher at warm season in both years. GMA was positively associated with sVCAM-1 ( β = 0.17, p = 0.039 in cold season; β = 0.22, p = 0.002 for changes) and negatively with sICAM-1 ( β = -0.30. p < 0.001 in cold season) levels. Vitamin D might play a role in diminishing sVCAM-1 levels. Levels of sVCAM-1 and sICAM-1 were associated with the GMA; this implies a need for further research.

  2. Soluble tissue factor has unique angiogenic activities that selectively promote migration and differentiation but not proliferation of endothelial cells

    SciTech Connect

    He Yingbo; Chang Guodong; Zhan Shunli; Song Xiaomin; Wang Xiaofeng; Luo Yongzhang

    2008-06-06

    The level of circulating tissue factor (TF) is up-regulated in human angiogenesis-related malignancies. However, whether circulating TF has angiogenic activities has not been determined. Soluble TF (sTF) is the main domain of circulating TF. Here, using cell migration, wound healing, and tubule formation assays, human recombinant sTF was found to significantly promote the migration and differentiation of endothelial cells. The stress fiber formation and rearrangement induced by sTF observed through immunofluorescence microscope may be responsible for the stimulatory migration effect of sTF. Nevertheless, sTF had no effects on endothelial cell proliferation. Interestingly, sTF can be internalized by endothelial cells, which implies a novel mechanism for sTF in angiogenesis. These results suggest that sTF has unique angiogenic activities and may serve as a potential therapeutic target to treat diseases associated with angiogenesis such as cancer and rheumatoid arthritis.

  3. A recombinant, soluble, single-chain class I major histocompatibility complex molecule with biological activity.

    PubMed Central

    Mage, M G; Lee, L; Ribaudo, R K; Corr, M; Kozlowski, S; McHugh, L; Margulies, D H

    1992-01-01

    Heterodimeric class I major histocompatibility complex molecules, which consist of a 45-kDa heavy-chain and a 12-kDa beta 2-microglobulin (beta 2m) light chain, bind endogenously synthesized peptides for presentation to antigen-specific T cells. We have synthesized a gene encoding a single-chain, soluble class I molecule derived from mouse H-2Dd, in which the carboxyl terminus of beta 2m is linked via a peptide spacer to the amino terminus of the heavy chain. The chimeric protein is secreted efficiently from transfected L cells, is thermostable, and when loaded with an appropriate antigenic peptide, stimulates an H-2Dd-restricted antigen-specific T-cell hybridoma. Thus, functional binding of peptide does not require the complete dissociation of beta 2m, implying that a heavy chain/peptide complex is not an obligate intermediate in the assembly of the heavy-chain/beta 2m/peptide heterotrimer. Single-chain major histocompatibility complex molecules uniformly loaded with peptide have potential uses for structural studies, toxin or fluor conjugates, and vaccines. Images PMID:1438262

  4. Phototoxic Activity and DNA Interactions of Water-Soluble Porphyrins and Their Rhenium(I) Conjugates.

    PubMed

    Mion, Giuliana; Gianferrara, Teresa; Bergamo, Alberta; Gasser, Gilles; Pierroz, Vanessa; Rubbiani, Riccardo; Vilar, Ramon; Leczkowska, Anna; Alessio, Enzo

    2015-11-01

    In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water-soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivative, in which either four or one [1,4,7]-triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker; we also report their corresponding tetracationic Re(I) conjugates. The in vitro (photo)toxic effects of the compounds toward the human cell lines HeLa (cervical cancer), H460M2 (non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic epithelial cells) are reported. Three of the compounds are not cytotoxic in the dark up to 100 μm, and the fourfold-symmetric couple revealed very good phototoxic indexes (PIs). The intracellular localization of all derivatives was studied in HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA; we observed significant selectivity in the tris-methylpyridinium derivatives for G-quadruplex interactions. PMID:26332425

  5. A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease

    PubMed Central

    Spinale, Joann M.; Mariani, Laura H.; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X.; Wong, Hetty N.; Troost, Jonathan P.; Gadegbeku, Crystal A.; Gipson, Debbie S.; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B.

    2014-01-01

    It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 hours. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multi-center observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared to other diagnoses. Thus, these results do not support a pathological role for suPAR in FSGS. PMID:25354239

  6. A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease.

    PubMed

    Spinale, Joann M; Mariani, Laura H; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X; Wong, Hetty N; Troost, Jonathan P; Gadegbeku, Crystal A; Gipson, Debbie S; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B

    2015-03-01

    It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 h. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multicenter observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria, and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared with other diagnoses. Thus these results do not support a pathological role for suPAR in FSGS. PMID:25354239

  7. Bactericidal Activity of N-Chlorotaurine against Biofilm-Forming Bacteria Grown on Metal Disks

    PubMed Central

    Ammann, Christoph G.; Fille, Manfred; Hausdorfer, Johann; Nogler, Michael

    2014-01-01

    Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies. PMID:24492358

  8. Effect of Vanadate, Molybdate, and Azide on Membrane-Associated ATPase and Soluble Phosphatase Activities of Corn Roots 1

    PubMed Central

    Gallagher, Sean R.; Leonard, Robert T.

    1982-01-01

    The effects of vanadate, molybdate, and azide on ATP phosphohydrolase (ATPase) and acid phosphatase activities of plasma membrane, mitochondrial, and soluble supernatant fractions from corn (Zea mays L. WF9 × MO17) roots were investigated. Azide (0.1-10 millimolar) was a selective inhibitor of pH 9.0-ATPase activity of the mitochondrial fraction, while molybdate (0.01-1.0 millimolar) was a relatively selective inhibitor of acid phosphatase activity in the supernatant fraction. The pH 6.4-ATPase activity of the plasma membrane fraction was inhibited by vanadate (10-500 micromolar), but vanadate, at similar concentrations, also inhibited acid phosphatase activity. This result was confirmed for oat (Avena sativa L.) root and coleoptile tissues. While vanadate does not appear to be a selective inhibitor, it can be used in combination with molybdate and azide to distinguish the plasma membrane ATPase from mitochondrial ATPase or supernatant acid phosphatase. Vanadate appeared to be a noncompetitive inhibitor of the plasma membrane ATPase, and its effectiveness was increased by K+. K+-stimulated ATPase activity was inhibited by 50% at about 21 micromolar vanadate. The rate of K+ transport in excised corn root segments was inhibited by 66% by 500 micromolar vanadate. PMID:16662676

  9. Immunomodulatory effect of prednisolone (PRD) induced soluble suppressor factor(s) (PRD-SSF) on natural killer (NK) cell activity

    SciTech Connect

    Nair, M.P.N.; Cilik, J.M.; Schwartz, S.A.

    1986-03-01

    The authors have previously reported that peripheral blood lymphocytes precultured for 24 hrs with PRD showed significant suppression of their NK activity. Purified HNK-1/sup +/ lymphocytes were treated either directly with PRD or with supernates from allogeneic lymphocytes precultured with 10/sup -6/ to 10/sup -9/M PRD and examined for any inhibition of NK activity. For the NK assay K562 and U937 cell lines were used as targets in a 4 hr /sup 51/Cr release assay. HNK-1/sup +/ lymphocytes precultured with PRD showed significantly lower level of NK activity. In a single cell assay, both HNK-1/sup +/ and HNK-1/sup -/ subpopulations of PBL precultured with PRD also suppressed the target binding and lytic capacity of allogeneic fresh large granular lymphocytes, suggesting that NK cells/T cells or their precursors can be stimulated by PRD to inhibit NK activity. PBL precultured with increasing concentrations of culture supernates containing PRD-SSF showed a dose dependent inhibitory effect of their NK activity. This data suggest that PRD activated suppressor cells function through the release of soluble mediators. These findings may be of clinical significance to patients receiving corticosteroids for a variety of disorders including malignant, autoimmune and atopic diseases.

  10. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein.

    PubMed

    Hötzel, Isidro; Cheevers, William P

    2005-09-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain beta-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding. PMID

  11. Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

    SciTech Connect

    Hoetzel, Isidro . E-mail: ihotzel@gene.com; Cheevers, William P.

    2005-09-01

    The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain {beta}-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding.

  12. Partial proteolysis as a probe of the conformation of the gamma subunit in activated soluble and membrane-bound chloroplast coupling factor 1.

    PubMed

    Schumann, J; Richter, M L; McCarty, R E

    1985-09-25

    Treatments that enhance the latent ATPase activity of the chloroplast coupling factor (CF1) also induce hypersensitivity of the gamma subunit toward trypsin. A number of different gamma subunit cleavage products are formed (Moroney, J. V., and McCarty, R. E. (1982) J. Biol. Chem. 257, 5910-5914). We have compared the gamma cleavage products of membrane-bound and isolated CF1, activated either by reduction of the gamma disulfide bond or by removal of the epsilon subunit. The gamma subunit of isolated CF1 lacking the epsilon subunit was cleaved to a 27,000-Da species. The same cleavage site became exposed following energy-dependent conformational changes in the membrane-bound enzyme. Activation by reduction of the gamma disulfide bond also exposed this site. However, the gamma subunit of reduced CF1 was cleaved rapidly at an additional site and trypsin treatment gave rise to a 25,000-Da gamma species. The small peptide generated by the second cleavage contains one of the cysteinyl residues of the reduced disulfide bridge of gamma. This peptide dissociates from the enzyme and can be isolated by gel filtration. The close proximity of the trypsin cleavage sites to the disulfide bond of gamma is discussed with respect to the effects of tryptic cleavage on the ATPase activity of CF1. The data indicate that structural changes in a limited region of the gamma subunit strongly influence the catalytic properties of both soluble and membrane-bound CF1. PMID:2864336

  13. Inclusion bodies and purification of proteins in biologically active forms.

    PubMed

    Mukhopadhyay, A

    1997-01-01

    Even though recombinant DNA technology has made possible the production of valuable therapeutic proteins, its accumulation in the host cell as inclusion body poses serious problems in the recovery of functionally active proteins. In the last twenty years, alternative techniques have been evolved to purify biologically active proteins from inclusion bodies. Most of these remain only as inventions and very few are commercially exploited. This review summarizes the developments in isolation, refolding and purification of proteins from inclusion bodies that could be used for vaccine and non-vaccine applications. The second section involves a discussion on inclusion bodies, how they are formed, and their physicochemical properties. In vivo protein folding in Escherichia coli and kinetics of in vitro protein folding are the subjects of the third and fourth sections respectively. The next section covers the recovery of bioactive protein from inclusion bodies: it includes isolation of inclusion body from host cell debris, purification in denatured state alternate refolding techniques, and final purification of active molecules. Since purity and safety are two important issues in therapeutic grade proteins, the following three sections are devoted to immunological and biological characterization of biomolecules, nature, and type of impurities normally encountered, and their detection. Lastly, two case studies are discussed to demonstrate the sequence of process steps involved. PMID:8939059

  14. Water soluble laser dyes

    DOEpatents

    Hammond, Peter R.; Feeman, James F.; Field, George F.

    1998-01-01

    Novel water soluble dyes of the formula I are provided ##STR1## wherein R.sup.1 and R.sup.4 are alkyl of 1 to 4 carbon atoms or hydrogen; or R.sup.1 -R.sup.2 or R.sup.2 -R.sup.4 form part of aliphatic heterocyclic rings; R.sup.2 is hydrogen or joined with R.sup.1 or R.sup.4 as described above; R.sup.3 is --(CH.sub.2).sub.m --SO.sub.3.sup.-, where m is 1 to 6; X is N, CH or ##STR2## where Y is 2 --SO.sub.3.sup.- ; Z is 3, 4, 5 or 6 --SO.sub.3.sup.-. The novel dyes are particularly useful as the active media in water solution dye lasers.

  15. Water soluble laser dyes

    DOEpatents

    Hammond, P.R.; Feeman, J.F.; Field, G.F.

    1998-08-11

    Novel water soluble dyes of the formula 1 are provided by the formula described in the paper wherein R{sup 1} and R{sup 4} are alkyl of 1 to 4 carbon atoms or hydrogen; or R{sup 1}--R{sup 2} or R{sup 2}--R{sup 4} form part of aliphatic heterocyclic rings; R{sup 2} is hydrogen or joined with R{sup 1} or R{sup 4} as described above; R{sup 3} is --(CH{sub 2}){sub m}--SO{sub 3}{sup {minus}}, where m is 1 to 6; X is N, CH or formula 2 given in paper where Y is 2 --SO{sub 3}{sup {minus}} ; Z is 3, 4, 5 or 6 --SO{sub 3}{sup {minus}}. The novel dyes are particularly useful as the active media in water solution dye lasers.

  16. Chemical Potentials, Activity Coefficients, and Solubility in Aqueous NaCl Solutions: Prediction by Polarizable Force Fields.

    PubMed

    Moučka, Filip; Nezbeda, Ivo; Smith, William R

    2015-04-14

    We describe a computationally efficient molecular simulation methodology for calculating the concentration dependence of the chemical potentials of both solute and solvent in aqueous electrolyte solutions, based on simulations of the salt chemical potential alone. We use our approach to study the predictions for aqueous NaCl solutions at ambient conditions of these properties by the recently developed polarizable force fields (FFs) AH/BK3 of Kiss and Baranyai (J. Chem. Phys. 2013, 138, 204507) and AH/SWM4-DP of Lamoureux and Roux (J. Phys. Chem. B 2006, 110, 3308 - 3322) and by the nonpolarizable JC FF of Joung and Cheatham tailored to SPC/E water (J. Phys. Chem. B 2008, 112, 9020 - 9041). We also consider their predictions of the concentration dependence of the electrolyte activity coefficient, the crystalline solid chemical potential, the electrolyte solubility, and the solution specific volume. We first highlight the disagreement in the literature concerning calculations of solubility by means of molecular simulation in the case of the JC FF and provide strong evidence of the correctness of our methodology based on recent independently obtained results for this important test case. We then compare the predictions of the three FFs with each other and with experiment and draw conclusions concerning their relative merits, with particular emphasis on the salt chemical potential and activity coefficient vs concentration curves and their derivatives. The latter curves have only previously been available from Kirkwood-Buff integrals, which require approximate numerical integrations over system pair correlation functions at each concentration. Unlike the case of the other FFs, the AH/BK3 curves are nearly parallel to the corresponding experimental curves at moderate and higher concentrations. This leads to an excellent prediction of the water chemical potential via the Gibbs-Duhem equation and enables the activity coefficient curve to be brought into excellent agreement

  17. Hydrothermal ore-forming processes in the light of studies in rock- buffered systems: I. Iron-copper-zinc-lead sulfide solubility relations

    USGS Publications Warehouse

    Hemley, J.J.; Cygan, G.L.; Fein, J.B.; Robinson, G.R.; d'Angelo, W. M.

    1992-01-01

    Experimental studies, using cold-seal and extraction vessel techniques, were conducted on Fe, Pb, Zn, and Cu sulfide solubilities in chloride soultions at temperatures from 300?? to 700??C and pressures from 0.5 to 2 kbars. The solutions were buffered in pH by quartz monzonite and the pure potassium feldspar-muscovite-quartz assemblage and in fS2-fO2 largely by the assemblage pyrite-pyrrhotite-magnetite. Solubilities increase with increasing temperature and total chloride, and decrease with increasing pressure. The effect of increasing chloride concentration on solubility reflects primarily a shift to lower pH via the silicate buffer reactions. Similarity in behaviour with respect to the temperature and pressure of Fe, Zn, and Pb sulfide solubilities points to similarity in chloride speciation, and the neutral species appear to be dominant in the high-temperature region. -from Authors

  18. Absorption improvement of tranilast by forming highly soluble nano-size composite structures associated with α-glucosyl rutin via spray drying.

    PubMed

    Sato, Hideyuki; Fujimori, Miki; Suzuki, Hiroki; Kadota, Kazunori; Shirakawa, Yoshiyuki; Onoue, Satomi; Tozuka, Yuichi

    2015-05-01

    Tranilast (TL) composite particles with α-glucosyl rutin (Rutin-G) were developed to improve the solubility and oral bioavailability of TL. Composite formulation of TL/Rutin-G was prepared using the spray-drying method, and their physicochemical properties were evaluated with respect to the morphology, particle size distribution, solubility and crystallinity. The nanostructure formation of Rutin-G was characterized by dynamic light scattering and transmission electron microscopy when Rutin-G or TL/Rutin-G spray-dried particles (SDPs) were introduced into water. A pharmacokinetic study was also performed to assess the improvement of oral absorption in rats. TL/Rutin-G SDPs were spherical particles with a diameter of 5.5μm. Even in the acidic condition, the remarkable improvement in solubility of TL was achieved, as evidenced by a 32.2-fold increase in solubility compared with untreated TL. The median size of Rutin-G nanostructures in water was 2nm. The formation of Rutin-G nanostructures and their drug inclusion properties may enhance the solubility and dissolution behavior of TL. A drastic increase was found in the exposure of TL in rats, with an increase in Cmax and AUC values of 114- and 36.4-fold, respectively, compared with those of untreated TL. These findings indicated that a TL formulation spray-dried with Rutin-G could enhance its solubility and absorption and thus its therapeutic properties. PMID:25725261

  19. Optically active surfaces formed by ion implantation and thermal treatment

    SciTech Connect

    Gea, L.A.; Boatner, L.A.; Evans, H.M.; Zuhr, R.

    1996-08-01

    Embedded VO{sub 2} precipitates have been formed in single-crystal sapphire by the ion co-implantation of vanadium and oxygen and subsequent thermal annealing. The embedded VO{sub 2} particles have been shown to exhibit an optical switching behavior that is comparable to that of continuous thin films. In this work, the mechanisms of formation of these optically active particles are investigated. It is shown that precipitation of the vanadium dioxide phase is favored when the thermal treatment is performed on an ion-damaged but still crystalline (rather than amorphized) Al{sub 2}O{sub 3} substrate. The best optical switching behavior is observed in this case, and this behavior is apparently correlated with a more-favorable dispersion of VO{sub 2} small particles inside the matrix.

  20. Crystal structures of human soluble adenylyl cyclase reveal mechanisms of catalysis and of its activation through bicarbonate

    PubMed Central

    Kleinboelting, Silke; Diaz, Ana; Moniot, Sebastien; van den Heuvel, Joop; Weyand, Michael; Levin, Lonny R.; Buck, Jochen; Steegborn, Clemens

    2014-01-01

    cAMP is an evolutionary conserved, prototypic second messenger regulating numerous cellular functions. In mammals, cAMP is synthesized by one of 10 homologous adenylyl cyclases (ACs): nine transmembrane enzymes and one soluble AC (sAC). Among these, only sAC is directly activated by bicarbonate (HCO3−); it thereby serves as a cellular sensor for HCO3−, carbon dioxide (CO2), and pH in physiological functions, such as sperm activation, aqueous humor formation, and metabolic regulation. Here, we describe crystal structures of human sAC catalytic domains in the apo state and in complex with substrate analog, products, and regulators. The activator HCO3− binds adjacent to Arg176, which acts as a switch that enables formation of the catalytic cation sites. An anionic inhibitor, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, inhibits sAC through binding to the active site entrance, which blocks HCO3− activation through steric hindrance and trapping of the Arg176 side chain. Finally, product complexes reveal small, local rearrangements that facilitate catalysis. Our results provide a molecular mechanism for sAC catalysis and cellular HCO3− sensing and a basis for targeting this system with drugs. PMID:24567411

  1. Chemical Fingerprints of Star Forming Regions and Active Galaxies

    NASA Astrophysics Data System (ADS)

    Pérez-Beaupuits, Juan-Pablo

    2010-10-01

    This thesis is devoted to the study of the physical conditions of the interstellar medium (ISM) in active galactic nuclei (AGNs) and Galactic star-forming regions, using mostly single-dish millimeter observations. I first study the excitation conditions of dense gas in a group of Seyfert galaxies using radiative transfer models (Chapter 2). I then study the galaxy NGC 1068, and try to distinguish signatures of the contributions from the AGN and the starburst ring by incorporating observations of high-J transitions of dense gas tracers (Chapter 3). Later, I venture into the mid-infrared spectral region to study different aspects of the AGN and starburst components in the galaxy NGC 4945 (Chapter 4). In Chapter 5 I delve into theoretical aspects of the dynamical evolution of gas in an AGN torus. I use a 3D hydrodynamic simulation with chemical abundances driven by X-rays. The aim is to understand the effects of X-ray irradiation by the AGN on the temperature, formation and destruction of the molecular gas. I finally explore a Galactic star-forming region, the Omega Nebula, with high resolution single dish observations, to study the properties of the warm gas and to constrain chemical models (Chapters 6 and 7).

  2. 75 FR 16492 - Agency Information Collection Activities: Form G-28, and Form G-28I, Revision of an Existing...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-01

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form G-28, and Form G- 28I, Revision of an Existing Information Collection; Comment Request ACTION: 60-Day Notice of Information Collection under Review: Form G- 28, Notice of Entry of Appearance as Attorney or...

  3. Generation of a soluble recombinant trimeric form of bovine CD40L and its potential use as a vaccine adjuvant in cows.

    PubMed

    Pujol, Julien; Bouillenne, Fabrice; Farnir, Frédéric; Dufrasne, Isabelle; Mainil, Jacques; Galleni, Moreno; Lekeux, Pierre; Bureau, Fabrice; Fiévez, Laurence

    2015-11-15

    Vaccination is the most cost-effective way to control infectious diseases in cattle. However, many infectious diseases leading to severe economical losses worldwide still remain for which a really effective and safe vaccine is not available. These diseases are most often due to intracellular pathogens such as bacteria or viruses, which are, by their localization, protected from antibiotics and/or CD4(+) T cell-dependent humoral responses. We therefore postulated that strategies leading to induction of not only CD4(+) T cell responses but also CD8(+) cytotoxic T lymphocyte (CTL) responses against infected cells should be privileged in the development of new vaccines against problematic intracellular pathogens in bovines. CD40 signaling in antigen-presenting cells may lead to the induction of robust CD4-independent CTL responses and several studies, especially in mice, have used CD40 stimulation to promote CD8(+) T cell-mediated immunity. For example, we have recently shown that immunization of mice with heat-killed Staphylococcus aureus (HKSA) and agonistic anti-CD40 monoclonal antibodies elicits strong CTL responses capable of protecting mice from subsequent staphylococcal mastitis. Unfortunately, there is at present no tool available to efficiently stimulate CD40 in cattle. In this study, we therefore first produced a soluble recombinant trimeric form of the natural bovine CD40 ligand (sboCD40LT). We then observed that sboCD40LT was able to potently stimulate bovine cells in vitro. Finally, we provide evidence that immunization of cows with sboCD40LT combined with HKSA was able to significantly increase the number of both HKSA-specific CD4(+) and CD8(+) T cells in the draining lymph nodes. In conclusion, we suggest that this new molecular tool could help in the development of vaccine strategies against bovine diseases caused by intracellular pathogens. PMID:26553560

  4. Long-term fungal inhibitory activity of water-soluble extract from Amaranthus spp. seeds during storage of gluten-free and wheat flour breads.

    PubMed

    Giuseppe Rizzello, Carlo; Coda, Rossana; De Angelis, Maria; Di Cagno, Raffaella; Carnevali, Paola; Gobbetti, Marco

    2009-05-31

    This study aimed at investigating the use of the water-soluble extract of amaranth seeds for extending the shelf-life of gluten-free and wheat flour breads. The antifungal activity of the amaranth water-soluble extract was shown by agar diffusion, conidia germination and dry biomass assays, using Penicillium roqueforti DPPMAF1 as the indicator fungus. The crude water-soluble extract had minimal inhibitory concentration (MIC) of 5 mg of peptides/ml and showed inhibition towards a large number of fungal species isolated from bakeries. Four novel antifungal peptides, encrypted in amaranth agglutinin sequences, were identified from the water-soluble extract by nano-Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra (nano-LC-ESI-MS/MS). The water-soluble extract of amaranth was used as an ingredient for the manufacture of gluten-free and wheat flour breads and the inhibitory activity was confirmed during long-term shelf-life under pilot plant conditions. The effect of the water-soluble extract on gluten-free bread rheology and sensory properties was also shown. PMID:19328576

  5. Influence of extraction parameters on physico-chemical characters and antioxidant activity of water soluble polysaccharides from Macrocybe gigantea (Massee) Pegler & Lodge.

    PubMed

    Khatua, Somanjana; Acharya, Krishnendu

    2016-04-01

    Polysaccharides from mushrooms are potentially active pharmaceutical ingredients and their action is dependent upon conformation, composition, size etc. In this context, three water soluble crude polysaccharide rich fractions viz. hot water extracted polysaccharide (HWP), cold alkaline extracted polysaccharide (CAP) and hot alkaline extracted polysaccharide (HAP) have been isolated using varying extraction parameters from Macrocybe gigantea, a well-known edible mushroom collected from Gangetic plain of West Bengal and authenticated by DNA barcoding of nrDNA ITS region. Physico-chemical investigation revealed that the fractions were mainly composed of β-configuration in pyranose form of sugars conjugated with small amount of protein. Further analysis presented that polysaccharides were composed of same monosaccharide even in similar order of ratio (D-glucose > D-galactose > D-mannose > D-xylose). However, D-glucose as well as β-glucan were found to be in the highest amount in CAP. The helical structure was determined by Congo red assay which indicated that polysaccharides were in aggregate forms except HWP which consisted of tertiary structure. These diverse structural features may have imparted effect on free radical scavenging activity of polysaccharides where HWP was the most active in all assays. HWP was proved to be a good scavenger of free radicals, strong chelator of ferrous ion and had high reducing power. Thus it can be inferred that HWP may foster further studies for searching active compound which might be used as ingredients of functional foods, nutraceuticals and pharmaceuticals. Moreover, to the best of our knowledge this is the first report on chemical composition and antioxidant activity of different crude polysaccharides from M. gigantea. PMID:27413214

  6. Soluble Phosphatidylserine Binds to Two Sites on Human Factor IXa in a Ca2+ Dependent Fashion to Specifically Regulate Structure and Activity

    PubMed Central

    Majumder, Rinku; Cole, Daud; Chattopadhyay, Rima; Biswas, Subir; Monroe, Dougald; Lentz, Barry R.

    2014-01-01

    Clinical studies have demonstrated a correlation between elevated levels of FIX and the risk of coronary heart disease, while reduced plasma FIX causes hemophilia B. FIXa interacts with FVIIIa in the presence of Ca2+ and phosphatidylserine (PS)-containing membranes to form a factor X-activating complex (Xase) that is key to propagation of the initiated blood coagulation process in human. We test the hypothesis that PS in these membranes up-regulates the catalytic activity of this essential enzyme. We used a soluble form of phosphatidylserine, 1, 2-dicaproyl-sn-glycero-3-phospho-L-serine (C6PS), as a tool to do so. C6PS and PS in membranes are reported to regulate the homologous FXa nearly identically. FIXa binds a molecule of C6PS at each of with two sites with such different affinities (∼100-fold) that these appear to be independent. A high affinity C6PS binding site (Kd∼1.4 µM) regulates structure, whereas a low-affinity binding site (Kd∼140 µM) regulates activity. Equilibrium dialysis experiments were analyzed globally with four other data sets (proteolytic and amidolytic activities, intrinsic fluorescence, ellipticity) to unequivocally demonstrate stoichiometries of one for both sites. Michaelis-Menten parameters for FIXa proteolytic activity were the same in the presence of C6PS or PS/PC membranes. We conclude that the PS molecule and not a membrane surface is the key regulator of both factors Xa and IXa. Despite some minor differences in the details of regulation of factors Xa and IXa, the similarities we found suggest that lipid regulation of these two proteases may be similar, a hypothesis that we continue to test. PMID:24979705

  7. A highly active water-soluble cross-coupling catalyst based on dendritic polyglycerol N-heterocyclic carbene palladium complexes.

    PubMed

    Meise, Markus; Haag, Rainer

    2008-01-01

    A new water-soluble polyglycerol derivative functionalized with N-heterocyclic carbene palladium complexes was prepared and applied as catalyst for Suzuki cross-coupling reactions in water. The complex displays a metal loading of around 65 metal centers per dendrimeric molecule, which is estimated to contain 130 chelating groups and thus corresponds approximately to the formation of 2:1 NHC/metal complexes. Monomeric analogues were also synthesized to validate the reactivity of the dendritic catalyst. Both types of catalysts were tested with various aryl bromides and arylboronic acids. Turnover frequencies of up to 2586 h(-1) at 80 degrees C were observed with the dendritic catalyst along with turnover numbers of up to 59 000, which are among the highest turnover numbers reported for polymer-supported catalysts in neat water. The dendritic catalyst could be used (reused) in five consecutive reactions without loss in activity. PMID:18702166

  8. Enhanced hydrolysis of soluble cellulosic substrates by a metallocellulase with veratryl alcohol-oxidase activity

    SciTech Connect

    Evans, B.R.; Margalt, R.; Woodward, J.

    1995-12-31

    A cellulose enzyme fraction was separated from Trichoderma reesei Pulpzyme HA{trademark}, and its characteristics suggested that it was mainly composed of cellobiohydrolase II (CBH II). The covalent attachment of pentaammineruthenium (III) to this enzyme resulted in threefold and fourfold enhancements of its hydrolytic activity on carboxymethyl cellulose (CMC) and barley {beta}-glucan, respectively, as well as endowing it with veratryl alcohol-oxidase activity. Enhancement of hydrolysis was not affected by addition of tartrate or hydrogen peroxide to the reaction mixture. Both native and pentaammineruthenium modified enzymes had negligible activity on cellobiose and p-nitrophenyl {beta}-cellobioside (PNPC).

  9. The fibrate gemfibrozil is a NO- and haem-independent activator of soluble guanylyl cyclase: in vitro studies

    PubMed Central

    Sharina, I G; Sobolevsky, M; Papakyriakou, A; Rukoyatkina, N; Spyroulias, G A; Gambaryan, S; Martin, E

    2015-01-01

    Background and Purpose Fibrates are a class of drugs widely used to treat dyslipidaemias. They regulate lipid metabolism and act as PPARα agonists. Clinical trials demonstrate that besides changes in lipid profiles, fibrates decrease the incidence of cardiovascular events, with gemfibrozil exhibiting the most pronounced benefit. This study aims to characterize the effect of gemfibrozil on the activity and function of soluble guanylyl cyclase (sGC), the key mediator of NO signalling. Experimental Approach High-throughput screening of a drug library identified gemfibrozil as a direct sGC activator. Activation of sGC is unique to gemfibrozil and is not shared by other fibrates. Key Results Gemfibrozil activated purified sGC, induced endothelium-independent relaxation of aortic rings and inhibited platelet aggregation. Gemfibrozil-dependent activation was absent when the sGC haem domain was deleted, but was significantly enhanced when sGC haem was lacking or oxidized. Oxidation of sGC haem enhanced the vasoactive and anti-platelet effects of gemfibrozil. Gemfibrozil competed with the haem-independent sGC activators ataciguat and cinaciguat. Computational modelling predicted that gemfibrozil occupies the space of the haem group and interacts with residues crucial for haem stabilization. This is consistent with structure-activity data which revealed an absolute requirement for gemfibrozil's carboxyl group. Conclusions and Implications These data suggest that in addition to altered lipid and lipoprotein state, the cardiovascular preventive benefits of gemfibrozil may derive from direct activation and protection of sGC function. A sGC-directed action may explain the more pronounced cardiovascular benefit of gemfibrozil observed over other fibrates and some of the described side effects of gemfibrozil. PMID:25536881

  10. Carcinoma cells induce lumen filling and EMT in epithelial cells through soluble E-cadherin-mediated activation of EGFR.

    PubMed

    Patil, Pratima U; D'Ambrosio, Julia; Inge, Landon J; Mason, Robert W; Rajasekaran, Ayyappan K

    2015-12-01

    In epithelial cancers, carcinoma cells coexist with normal cells. Although it is known that the tumor microenvironment (TME) plays a pivotal role in cancer progression, it is not completely understood how the tumor influences adjacent normal epithelial cells. In this study, a three-dimensional co-culture system comprising non-transformed epithelial cells (MDCK) and transformed carcinoma cells (MSV-MDCK) was used to demonstrate that carcinoma cells sequentially induce preneoplastic lumen filling and epithelial-mesenchymal transition (EMT) in epithelial cysts. MMP-9 secreted by carcinoma cells cleaves cellular E-cadherin (encoded by CDH1) from epithelial cells to generate soluble E-cadherin (sE-cad), a pro-oncogenic protein. We show that sE-cad induces EGFR activation, resulting in lumen filling in MDCK cysts. Long-term sE-cad treatment induced EMT. sE-cad caused lumen filling by induction of the ERK signaling pathway and triggered EMT through the sustained activation of the AKT pathway. Although it is known that sE-cad induces MMP-9 release and consequent EGFR activation in tumor cells, our results, for the first time, demonstrate that carcinoma cells can induce sE-cad shedding in adjacent epithelial cells, which leads to EGFR activation and the eventual transdifferentiation of the normal epithelial cells. PMID:26483386

  11. Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1β and tumor necrosis factor-α.

    PubMed

    Hwang, Ji-Sun; Jung, Eun-Hye; Kwon, Mi-Youn; Han, Inn-Oc

    2016-09-15

    We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1β than primary astrocyte cells. Neutralization of TNF-α and IL-1β in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment. PMID:27609291

  12. Uptake and degradation of soluble aggregates of IgG by monocytes of patients with rheumatoid arthritis: relation to disease activity.

    PubMed Central

    Heurkens, A H; Westedt, M L; Breedveld, F C; Jonges, E; Cats, A; Stijnen, T; Daha, M R

    1991-01-01

    Monocytes from patients with rheumatoid arthritis (RA) and rheumatoid vasculitis have a diminished ability to degrade soluble complexes of aggregated IgG in the absence (mediated by Fc receptors) as well as in the presence of complement (C) (mediated by (Fc + C) receptors). To investigate whether a relation exists between the receptor mediated degradation of aggregated IgG by adherent monocytes and disease activity a longitudinal study was performed in 79 patients with RA and rheumatoid vasculitis over a period of 16 months. Adherent monocytes were incubated in vitro with 125I labelled IgG aggregates of restricted size in the absence or presence of fresh serum and the percentage of catabolised IgG aggregates was measured. Cross sectionally the degradation of aggregated IgG by monocytes, mediated by Fc and (Fc + C) receptors, correlated significantly with disease activity as scored by the Ritchie articular index, the presence of extra-articular features, and circulating immune complexes. A high number of Fc receptors on monocytes correlated with diminished degradation, whereas high numbers of complement receptors 1 and 3 correlated with enhanced degradation of aggregated IgG mediated by both Fc and (Fc + C) receptors. The degradation of aggregated IgG by monocytes did not correlate with disease activity in individual patients followed up longitudinally. When patient groups were formed according to the results of longitudinal studies, however, degradation of aggregated IgG mediated by Fc and (Fc + C) receptors was significantly decreased in patients with rheumatoid vasculitis and in patients with active RA in comparison with patients with inactive RA and healthy controls. Patients with active RA and rheumatoid vasculitis also expressed significantly more Fc receptors and less complement receptors on the monocytes than patients with inactive RA. Drug treatment did not correlate with receptor expression or the degradation of aggregated IgG by monocytes either in cross

  13. Design, synthesis and biological activity of a novel Rutin analogue with improved lipid soluble properties.

    PubMed

    Baldisserotto, Anna; Vertuani, Silvia; Bino, Alessia; De Lucia, Daniela; Lampronti, Ilaria; Milani, Roberta; Gambari, Roberto; Manfredini, Stefano

    2015-01-01

    Recent interest in flavonoids has increased greatly due to their biological and pharmacological activities. Flavonoids, consist of a large group of low molecular weight polyphenolic substances, naturally occurring in fruits, vegetables, tea, and wine, and are an integral part of the human diet. Rutin is a common dietary flavonoid that is widely consumed worldwide from plant-derived beverages and foods as traditional and folk medicine remedy as well. Rutin exhibit important pharmacological activities, including anti-oxidation, anti-inflammation, anti-diabetic, anti-adipogenic, neuroprotective and hormone therapy. Here, we present the synthesis, antimicrobial, antiproliferative and pro-apoptotic effect on human leukemic K562 cells of compound R2, a new semi-synthetic derivative of Rutin as compared to Rutin itself. The new derivative was also included in finished topical formulations to evaluate a potential application to the dermatology field in view of the antioxidant/antimicrobial/antiinflammatory properties. Stability studies were performed by HPLC; PCL assay and ORAC tests were used to determine the antioxidant activity. R2 presented an antioxidant activity very close to that of the parent Rutin while bearing much better lipophilic character. Regarding antiproliferative effects on the human K562 cell line, R2 was found to be more effective than parent Rutin. Preliminary experiments demonstrated that R2 inhibits NF-kB activity and promotes cellular apoptosis. PMID:25496805

  14. Antitumor and immunomodulatory activity of a water-soluble polysaccharide from Grifola frondosa.

    PubMed

    Mao, Guang-Hua; Ren, Yi; Feng, Wei-Wei; Li, Qian; Wu, Hui-Yu; Jin, Dun; Zhao, Ting; Xu, Cai-Quan; Yang, Liu-Qing; Wu, Xiang-Yang

    2015-12-10

    Grifola frondosa has long been known and respected as a medically important fungus. This study investigated the characterization, antitumor and immunomodulatory activity of a polysaccharide named GP11 purified from G. frondosa. The results revealed that GP11 was composed of → 1)-D-Manp-(6 →,→ 1)-D-Glcp-(4 →,→ 1)-D-Galp-(6 → and → 2,3,6)-D-Glcp-(1 →, with branches attached at O-2,3 of 1,2,3,6-linked Glcp residues and terminal T-Glcp. GP11 exhibited indirect cytotoxic activity against HepG-2 cells in vitro, and it significantly inhibited the growth of Heps cells in vivo. GP11 increased the relative thymus and spleen weights as well as serum tumor necrosis factor-alpha and interleukin-2 levels. GP11 stimulated tumoricidal activity and the production of nitric oxide (NO), TNF-α and interleukin-1β, and it also stimulated the protein expression of iNOS and mRNA expression of iNOS and TNF-α. TLR-4 is a potential receptor for GP11-mediated macrophage activation. The results suggested that the antitumor activity of GP11 may be due to the improvement of immune functions through the TLR-4-mediated up-regulation of NO and TNF-α. PMID:26428141

  15. Granzyme B Cleaves Decorin, Biglycan and Soluble Betaglycan, Releasing Active Transforming Growth Factor-β1

    PubMed Central

    Boivin, Wendy A.; Shackleford, Marlo; Vanden Hoek, Amanda; Zhao, Hongyan; Hackett, Tillie L.; Knight, Darryl A.; Granville, David J.

    2012-01-01

    Objective Granzyme B (GrB) is a pro-apoptotic serine protease that contributes to immune-mediated target cell apoptosis. However, during inflammation, GrB accumulates in the extracellular space, retains its activity, and is capable of cleaving extracellular matrix (ECM) proteins. Recent studies have implicated a pathogenic extracellular role for GrB in cardiovascular disease, yet the pathophysiological consequences of extracellular GrB activity remain largely unknown. The objective of this study was to identify proteoglycan (PG) substrates of GrB and examine the ability of GrB to release PG-sequestered TGF-β1 into the extracellular milieu. Methods/Results Three extracellular GrB PG substrates were identified; decorin, biglycan and betaglycan. As all of these PGs sequester active TGF-β1, cytokine release assays were conducted to establish if GrB-mediated PG cleavage induced TGF-β1 release. Our data confirmed that GrB liberated TGF-β1 from all three substrates as well as from endogenous ECM and this process was inhibited by the GrB inhibitor 3,4-dichloroisocoumarin. The released TGF-β1 retained its activity as indicated by the induction of SMAD-3 phosphorylation in human coronary artery smooth muscle cells. Conclusion In addition to contributing to ECM degradation and the loss of tissue structural integrity in vivo, increased extracellular GrB activity is also capable of inducing the release of active TGF-β1 from PGs. PMID:22479366

  16. Soluble CD163 masks fibronectin-binding protein A-mediated inflammatory activation of Staphylococcus aureus infected monocytes.

    PubMed

    Kneidl, Jessica; Mysore, Vijayashree; Geraci, Jennifer; Tuchscherr, Lorena; Löffler, Bettina; Holzinger, Dirk; Roth, Johannes; Barczyk-Kahlert, Katarzyna

    2014-03-01

    Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation. PMID:24118665

  17. [The effect of a water-soluble vitamins on the activity of some enzymes in diabetes].

    PubMed

    Petrov, S A; Danilova, A O; Karpov, L M

    2014-01-01

    Intramuscular injections of the vitamin complex containing: thiamine chloride (B1), riboflavin (B2), lipoic acid (N), calcium pantothenate (B5), pyridoxine hydrochloride (B6), folic acid (B9), ascorbic acid (C) can reduce the blood glucose level in serum of rats with alloxan diabetes, stabilize activity of some enzymes of energy metabolism, lactate dehydrogenase and pyruvate dehydrogenase complex. PMID:25552500

  18. Could both vitamin D and geomagnetic activity impact serum levels of soluble cell adhesion molecules in young men?

    PubMed

    Bleizgys, Andrius; Šapoka, Virginijus

    2016-07-01

    Vitamin D might have a role in diminishing endothelial dysfunction (ED). The initial aim was to test the hypothesis of reciprocity between levels of 25-hydroxyvitamin D (25(OH)D) and levels of soluble endothelial cell adhesion molecules (CAMs) that could serve as biomarkers of ED. Randomly selected men of age 20-39 were examined at February or March (cold season) and reexamined at August or September (warm season). Some lifestyle and anthropometrical data were recorded. Laboratory measurements, including those for serum levels of soluble CAMs-sICAM-1, sVCAM-1, sE-selectin and sP-selectin-were also performed. As some of the results were rather unexpected, indices of geomagnetic activity (GMA), obtained from the online database, were included in further analysis as a confounder. In 2012-2013, 130 men were examined in cold season, and 125 of them were reexamined in warm season. 25(OH)D levels were found to be significantly negatively associated with sVCAM-1 levels (β = -0.15, p = 0.043 in warm season; β = -0.19, p = 0.007 for changes). Levels of sVCAM-1 and sICAM-1 from the same seasons were notably different between years and have changed in an opposite manner. Soluble P-selectin levels were higher at warm season in both years. GMA was positively associated with sVCAM-1 (β = 0.17, p = 0.039 in cold season; β = 0.22, p = 0.002 for changes) and negatively with sICAM-1 (β = -0.30. p < 0.001 in cold season) levels. Vitamin D might play a role in diminishing sVCAM-1 levels. Levels of sVCAM-1 and sICAM-1 were associated with the GMA; this implies a need for further research. PMID:26546313

  19. Soluble form of complement C3b/C4b receptor (CR1) results from a proteolytic cleavage in the C-terminal region of CR1 transmembrane domain.

    PubMed Central

    Hamer, I; Paccaud, J P; Belin, D; Maeder, C; Carpentier, J L

    1998-01-01

    The complement C3b/C4b receptor (CR1) is an integral protein, anchored in the plasma membrane through a hydrophobic domain of 25 amino acids, but is also found in the plasma in soluble form (sCR1). A recombinant, soluble form of CR1 has been demonstrated to reduce complement-dependent tissue injury in animal models of ischaemia/reperfusion. In view of the important pathophysiological relevance of sCR1, we have investigated the mechanisms governing CR1 release by using various mutated and chimaeric receptors transiently expressed in COS cells. Pulse-chase experiments revealed that (1) sCR1 is produced by a proteolytic process, (2) the cleavage site lies within the C-terminus of CR1 transmembrane domain, (3) the proteolytic process involves a fully glycosylated CR1 form and (4) this process takes place in late secretory vesicles or at the plasma membrane. PMID:9405292

  20. Simplified sample treatment for the determination of total concentrations and chemical fractionation forms of Ca, Fe, Mg and Mn in soluble coffees.

    PubMed

    Pohl, Pawel; Stelmach, Ewelina; Szymczycha-Madeja, Anna

    2014-11-15

    A simpler, and faster than wet digestion, sample treatment was proposed prior to determination of total concentrations for selected macro- (Ca, Mg) and microelements (Fe, Mn) in soluble coffees by flame atomic absorption spectrometry. Samples were dissolved in water and acidified with HNO3. Precision was in the range 1-4% and accuracy was better than 2.5%. The method was used in analysis of 18 soluble coffees available on the Polish market. Chemical fractionation patterns for Ca, Fe, Mg and Mn in soluble coffees, as consumed, using a two-column solid-phase extraction method, determined Ca, Mg and Mn were present predominantly as cations (80-93% of total content). This suggests these elements are likely to be highly bioaccessible. PMID:24912692

  1. Soluble cytokine receptors in biological therapy.

    PubMed

    Fernandez-Botran, Rafael; Crespo, Fabian A; Sun, Xichun

    2002-08-01

    Due to their fundamental involvement in the pathogenesis of many diseases, cytokines constitute key targets for biotherapeutic approaches. The discovery that soluble forms of cytokine receptors are involved in the endogenous regulation of cytokine activity has prompted substantial interest in their potential application as immunotherapeutic agents. As such, soluble cytokine receptors have many advantages, including specificity, low immunogenicity and high affinity. Potential disadvantages, such as low avidity and short in vivo half-lifes, have been addressed by the use of genetically-designed receptors, hybrid proteins or chemical modifications. The ability of many soluble cytokine receptors to inhibit the binding and biological activity of their ligands makes them very specific cytokine antagonists. Several pharmaceutical companies have generated a number of therapeutic agents based on soluble cytokine receptors and many of them are undergoing clinical trials. The most advanced in terms of clinical development is etanercept (Enbrel, Immunex), a fusion protein between soluble TNF receptor Type II and the Fc region of human IgG1. This TNF-alpha; antagonist was the first soluble cytokine receptor to receive approval for use in humans. In general, most agents based on soluble cytokine receptors have been safe, well-tolerated and have shown only minor side effects in the majority of patients. Soluble cytokine receptors constitute a new generation of therapeutic agents with tremendous potential for applications in a wide variety of human diseases. Two current areas of research are the identification of their most promising applications and characterisation of their long-term effects. PMID:12171504

  2. Analysis of a soluble calmodulin binding protein from fava bean roots: identification of glutamate decarboxylase as a calmodulin-activated enzyme.

    PubMed

    Ling, V; Snedden, W A; Shelp, B J; Assmann, S M

    1994-08-01

    The identity of a soluble 62-kD Ca(2+)-dependent calmodulin binding protein (CaM-BP) from fava bean seedlings was determined. Using 125I-CaM overlay assays, a class of soluble CaM-BPs was detected in extracts of tissues comprising the axis of 1.5-week-old seedlings, excluding the root tip and emergent leaves. The size of these CaM-BPs was not uniform within all parts of the plant; the apparent molecular masses were 62 kD in roots, 60 kD in stems, and 64 kD in nodules. The root 62-kD CaM-BP was purified, and internal microsequence analysis was performed on the protein. A tryptic peptide derived from the CaM-BP consisted of a 13-residue sequence corresponding to a highly conserved region of glutamate decarboxylase (GAD), an enzyme that catalyzes the alpha-decarboxylation of glutamate to form the stress-related metabolite gamma-aminobutyrate. Activity assays of partially purified, desalted, root GAD revealed a 50% stimulation by the addition of 100 microM Ca2+, a 100% stimulation by the addition of 100 microM Ca2+ plus 100 nM CaM, and no appreciable stimulation by CaM in the absence of added Ca2+. The demonstration that plant GAD is a Ca(2+)-CaM-stimulated enzyme provides a model in which stress-linked metabolism is modulated by a Ca(2+)-mediated signal transduction pathway. PMID:7919983

  3. Soluble FasR ligand-binding domain: high-yield production of active fusion and non-fusion recombinant proteins using the baculovirus/insect cell system.

    PubMed

    Mahiou, J; Abastado, J P; Cabanie, L; Godeau, F

    1998-03-01

    We used the recombinant baculovirus/insect cell system to express two soluble forms of the mouse Fas receptor (mFasR) extracellular domain (ECD): a monomer comprising the entire ligand-binding portion of mFasR followed by a carboxy-terminal hexa-histidine extension aiding purification by immobilized metal affinity chromatography and an immunoadhesin in which the same 148 residues were fused to the Fc portion of a truncated human IgG1 immunoglobulin heavy chain. Both constructs harboured a 24 base pairs insertion placed upstream of the initiating ATG [Peakman, Charles, Sydenham, Gewert, Page, and Makoff (1992) Nucleic Acids Res. 20, 6111-6112]. Despite its hexa-histidine extension, the monovalent recombinant protein from crude culture media failed to bind immobilized Ni2+ unless proteins were first precipitated twice by ammonium sulphate. The overall procedure then yielded approximately 10mg/l of protein which could be purified to near homogeneity using two additional chromatographic steps. The glycosylated polypeptide migrated as a band of Mr=(21-31) x 10(3) in SDS/PAGE and was monomeric in physiological buffers. Under non-reducing conditions, denaturation in 6 M guanidinium chloride was reversible after slow removal of the denaturing agent. The mFasR immunoadhesin was secreted (approximately 5-10 mg/l) as a disulphide-linked homodimer, and endowed with ligand-binding activity since it could bind FasL on the surface of D11S, FasL-expressing cells. When tested for their ability to inhibit FasR-dependent cell lysis, the soluble dimeric immunoadhesin markedly inhibited FasL-mediated cytotoxicity (IC50 approximately 30 nM), and was approximately 6 times as effective as its monomeric counterpart. PMID:9480929

  4. A Water-Soluble Polysaccharide from the Fruit Bodies of Bulgaria inquinans (Fries) and Its Anti-Malarial Activity

    PubMed Central

    Bi, Hongtao; Han, Han; Li, Zonghong; Ni, Weihua; Chen, Yan; Zhu, Jingjing; Gao, Tingting; Hao, Miao; Zhou, Yifa

    2011-01-01

    A water-soluble polysaccharide (BIWS-4b) was purified from the fruit bodies of Bulgaria inquinans (Fries). It is composed of mannose (27.2%), glucose (15.5%) and galactose (57.3%). Its molecular weight was estimated to be 7.4 kDa (polydispersity index, Mw/Mn: 1.35). Structural analyses indicated that BIWS-4b mainly contains (1 → 6)-linked, (1 → 5)-linked and (1 → 5,6)-linked β-Galf units; (1 → 4)-linked and non-reducing terminal β-Glcp units; and (1 → 2)-linked, (1 → 6)-linked, (1 → 2,6)-linked and non-reducing terminal α-Manp units. When examined by the 4-day method and in a prophylactic assay in mice, BIWS-4b exhibited markedly suppressive activity against malaria while enhancing the activity of artesunate. Immunological tests indicated that BIWS-4b significantly enhanced macrophage phagocytosis and splenic lymphocyte proliferation in malaria-bearing mice and normal mice. The anti-malarial activity of BIWS-4b might be intermediated by enhancing immune competence and restoring artesunate-suppressed immune function. Thus, BIWS-4b is a potential adjuvant of anti-malaria drugs. PMID:21785644

  5. Preparation and X-ray structures of metal-free, dicobalt and dimanganese forms of soluble methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

    PubMed

    Sazinsky, Matthew H; Merkx, Maarten; Cadieux, Elisabeth; Tang, Sonya; Lippard, Stephen J

    2004-12-28

    A three-component soluble methane monooxygenase (sMMO) enzyme system catalyzes the hydroxylation of methane to methanol at a carboxylate-bridged diiron center housed in the alpha-subunit of the hydroxylase (MMOH). Catalysis is facilitated by the presence of a regulatory protein (MMOB) and inhibited by MMOD, a protein of unknown function encoded in the sMMO operon. Both MMOB and MMOD are presumed to bind to the same region of the MMOH alpha-subunit. A colorimetric method for monitoring removal of Fe(II) from MMOH was developed using 1,10-phenanthroline and yields apo MMOH with <0.1 Fe/homodimer. With the use of this method, it was possible to investigate the X-ray structure of the apoenzyme and to perform metal reconstitution studies. Using MMOH from Methylococccus capsulatus (Bath), the effects of MMOB and MMOD on metal binding were studied and structural perturbations relevant to the function of this enzyme were identified. X-ray crystal structures of the apo, Mn(II)-soaked, and Co(II)-grown MMOH, determined to 2.3 A or greater resolution, reveal that the presence of metal ions is essential for the proper folding of helices E, F, and H of the alpha-subunit. The active sites of Mn(II)-soaked and Co(II)-grown MMOH are similar to that of reduced, native MMOH with notable differences in the metal-metal distances and ligand coordination sphere that may reflect how this dinuclear metal center might change in the presence of MMOB. MMOB and MMOD decrease the rate of removal of Fe(II) from the enzyme by 22- and 16-fold, respectively. On the basis of previous studies, it is hypothesized that MMOB, and perhaps MMOD, function to block solvent access to the MMOH active site. Finally, ITC studies and the observed disorder in helices E, F, and H in the apo and Mn(II)-soaked structures suggest that these regions of MMOH are critical for MMOB and MMOD binding. PMID:15610020

  6. Serodiagnosis of bovine trypanosomosis caused by non-tsetse transmitted Trypanosoma (Duttonella) vivax parasites using the soluble form of a Trypanozoon variant surface glycoprotein antigen.

    PubMed

    Uzcanga, Graciela L; Pérez-Rojas, Yenis; Camargo, Rocío; Izquier, Adriana; Noda, José A; Chacín, Ronny; Parra, Nereida; Ron, Lenin; Rodríguez-Hidalgo, Richar; Bubis, José

    2016-03-15

    Previous studies have shown that a 64-kDa antigen (p64) that was purified from the Venezuelan TeAp-N/D1 isolate of Trypanosoma (Trypanozoon) equiperdum corresponds to the soluble form of its predominant variant surface glycoprotein (VSG), and exhibited cross-reactivity with Trypanosoma (Duttonella) vivax. The course of experimental acute infections of bovines with T. vivax were followed by measuring whole anti-p64 antibodies and specific anti-p64 IgG and IgM antibodies in animal sera by indirect enzyme-linked immunosorbent assay (ELISA). The value of p64 to diagnose bovine trypanosomosis was also examined using 350 sera from healthy and T. vivax-infected cows living in a trypanosomosis-endemic and enzootic stable area, and 48 sera obtained during a trypanosomosis outbreak. Serological assays showed that ∼ 70-80% of the infected sera contained anti-p64 antibodies, based on the comparative immunodetection of the T. equiperdum clarified antigenic fraction used as a reference test. In the absence of a gold standard, Bayesian analysis for multiple testing estimated a sensitivity and specificity of 71.6% and 98.8%, respectively, for the indirect ELISA using p64 as antigen. An apparent prevalence of 37.7% for bovine trypanosomosis infection was also estimated with a Bayesian approach when the p64 ELISA test was used. Employing blood from acute infected cows, the indirect ELISA response against p64 was contrasted with the microhematocrit centrifuge method and analyses by polymerase chain reaction (PCR) using specific primers targeting the inter-specific length variation of the internal transcribed spacer 1 region of the 18S ribosomal gene. The efficiency of p64 for the detection of anti-trypanosome antibodies in acute infected bovines was also corroborated serologically by comparing its response to that of the Indonesian Trypanosoma evansi Rode Trypanozoon antigen type (RoTat) 1.2 VSG, which possesses high specificity and sensitivity. As expected, PCR was the best

  7. Alkylphloroglucinol derivatives and triterpenoids with soluble epoxide hydrolase inhibitory activity from Callistemon citrinus.

    PubMed

    Khanh, Pham Ngoc; Duc, Ho Viet; Huong, Tran Thu; Son, Ninh The; Ha, Vu Thi; Van, Doan Thi; Tai, Bui Huu; Kim, Ji Eun; Jo, Ah Reum; Kim, Young Ho; Cuong, Nguyen Manh

    2016-03-01

    Phytochemical analysis of the leaves and stems of Callistemon citrinus (Curtis) Skeels led to the isolation of two new alkylphloroglucinols, gallomyrtucommulone E and F (1 and 2), along with four other known alkylphloroglucinol derivatives, gallomyrtucommulone A (3), endoperoxide G3 (4), myrtucommulone B (5), callistenone B (6) and five known triterpenoids, including betulinic acid (7), 3β-acetylmorolic acid (8), 3β-hydroxy-urs-11-en-13(28)-olide (9), diospyrolide (10) and ursolic acid (11). The structures of the natural compounds were determined from the spectroscopic evidences including 1D-/2D-NMR and HR-MS spectrometry. All the isolated compounds were assessed for the effects on the sEH inhibitory activity. The acylphloroglucinols myrtucommulone B (5)/callistenone B (6) (in mixture), and two triterpenoids, ursolic acid (11) and 3β-hydroxy-urs-11-en-13(28)-olide (9) displayed strong inhibition of sEH activity, with IC50 values of 0.7, 11.2 and 24.8 μM, respectively. PMID:26548595

  8. Second generation bisheteroarylpiperazine (BHAP) HIV-1 reverse transcriptasae inhibitors: Enhancement of antiviral activity and aqueous solubility via 5- and 6-substitution of the indole ring

    SciTech Connect

    Poel, T.; Thomas, R.C.; Romero, D.L.; Hosley, M.J.; Morge, R.A.; Biles, C.; Reusser, F.; Althaus, I.W.; Schinzer, W.C.; Platzer, D.J.

    1993-12-31

    U-87201E, a potent HIV-1 reverse transcriptase inhibitor (RTI) discovered at Upjohn, is currently in Phase II clinical trials. Additional structure-activity studies have identified second-generation BHAPs with enhanced antiviral activity and improved pharmaceutical properties, notably increased aqueous solubility. Capitalizing on initial SAR studies which demonstrated a tolerance for substitution in the indole ring, a series of BHAPs bearing 5- and 6-substituted indoles was evaluated. Substituents such as ethers, sulfonamides, ureas, and sulfamides containing water-solubilizing groups such as polyethers or basic amines provided highly potent BHAPs with greatly enhanced solubility, such as U-93923. The synthesis, antiviral evaluation and solubility properties of these potent HIV-1 RTIs will be detailed.

  9. Production of concentrates of mono- and dihydric phenols from the total water-soluble phenols formed in the high-speed pyrolysis of brown coals

    SciTech Connect

    Kazakov, E.I.; Belov, P.S.; Korenev, K.P.; Molchanova, I.V.

    1982-01-01

    The results are given of the separation of the water-soluble phenols obtained in the high-speed pyrolysis of Kansk-Achinsk coals into uniform concentrates of mono- and dihydric phenols. It has been shown that the concentrate of monhydric phenols is suitable for working up by a known technology into phenolic products.

  10. Effective Energy Transfer via Plasmon-Activated High-Energy Water Promotes Its Fundamental Activities of Solubility, Ionic Conductivity, and Extraction at Room Temperature

    NASA Astrophysics Data System (ADS)

    Yang, Chih-Ping; Chen, Hsiao-Chien; Wang, Ching-Chiung; Tsai, Po-Wei; Ho, Chia-Wen; Liu, Yu-Chuan

    2015-12-01

    Water is a ubiquitous solvent in biological, physical, and chemical processes. Unique properties of water result from water’s tetrahedral hydrogen-bonded (HB) network (THBN). The original THBN is destroyed when water is confined in a nanosized environment or localized at interfaces, resulting in corresponding changes in HB-dependent properties. In this work, we present an innovative idea to validate the reserve energy of high-energy water and applications of high-energy water to promote water’s fundamental activities of solubility, ionic conductivity, and extraction at room temperature. High-energy water with reduced HBs was created by utilizing hot electrons with energies from the decay of surface plasmon excited at gold (Au) nanoparticles (NPs). Compared to conventional deionized (DI) water, solubilities of alkali metal-chloride salts in high-energy water were significantly increased, especially for salts that release heat when dissolved. The ionic conductivity of NaCl in high-energy water was also markedly higher, especially when the electrolyte’s concentration was extremely low. In addition, antioxidative components, such as polyphenols and 2,3,5,4’-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG) from teas, and Polygonum multiflorum (PM), could more effectively be extracted using high-energy water. These results demonstrate that high-energy water has emerged as a promising innovative solvent for promoting water’s fundamental activities via effective energy transfer.

  11. Effective Energy Transfer via Plasmon-Activated High-Energy Water Promotes Its Fundamental Activities of Solubility, Ionic Conductivity, and Extraction at Room Temperature.

    PubMed

    Yang, Chih-Ping; Chen, Hsiao-Chien; Wang, Ching-Chiung; Tsai, Po-Wei; Ho, Chia-Wen; Liu, Yu-Chuan

    2015-01-01

    Water is a ubiquitous solvent in biological, physical, and chemical processes. Unique properties of water result from water's tetrahedral hydrogen-bonded (HB) network (THBN). The original THBN is destroyed when water is confined in a nanosized environment or localized at interfaces, resulting in corresponding changes in HB-dependent properties. In this work, we present an innovative idea to validate the reserve energy of high-energy water and applications of high-energy water to promote water's fundamental activities of solubility, ionic conductivity, and extraction at room temperature. High-energy water with reduced HBs was created by utilizing hot electrons with energies from the decay of surface plasmon excited at gold (Au) nanoparticles (NPs). Compared to conventional deionized (DI) water, solubilities of alkali metal-chloride salts in high-energy water were significantly increased, especially for salts that release heat when dissolved. The ionic conductivity of NaCl in high-energy water was also markedly higher, especially when the electrolyte's concentration was extremely low. In addition, antioxidative components, such as polyphenols and 2,3,5,4'-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG) from teas, and Polygonum multiflorum (PM), could more effectively be extracted using high-energy water. These results demonstrate that high-energy water has emerged as a promising innovative solvent for promoting water's fundamental activities via effective energy transfer. PMID:26658304

  12. Effective Energy Transfer via Plasmon-Activated High-Energy Water Promotes Its Fundamental Activities of Solubility, Ionic Conductivity, and Extraction at Room Temperature

    PubMed Central

    Yang, Chih-Ping; Chen, Hsiao-Chien; Wang, Ching-Chiung; Tsai, Po-Wei; Ho, Chia-Wen; Liu, Yu-Chuan

    2015-01-01

    Water is a ubiquitous solvent in biological, physical, and chemical processes. Unique properties of water result from water’s tetrahedral hydrogen-bonded (HB) network (THBN). The original THBN is destroyed when water is confined in a nanosized environment or localized at interfaces, resulting in corresponding changes in HB-dependent properties. In this work, we present an innovative idea to validate the reserve energy of high-energy water and applications of high-energy water to promote water’s fundamental activities of solubility, ionic conductivity, and extraction at room temperature. High-energy water with reduced HBs was created by utilizing hot electrons with energies from the decay of surface plasmon excited at gold (Au) nanoparticles (NPs). Compared to conventional deionized (DI) water, solubilities of alkali metal-chloride salts in high-energy water were significantly increased, especially for salts that release heat when dissolved. The ionic conductivity of NaCl in high-energy water was also markedly higher, especially when the electrolyte’s concentration was extremely low. In addition, antioxidative components, such as polyphenols and 2,3,5,4’-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG) from teas, and Polygonum multiflorum (PM), could more effectively be extracted using high-energy water. These results demonstrate that high-energy water has emerged as a promising innovative solvent for promoting water’s fundamental activities via effective energy transfer. PMID:26658304

  13. Water soluble polysaccharides from Spirulina platensis: extraction and in vitro anti-cancer activity.

    PubMed

    Kurd, Forouzan; Samavati, Vahid

    2015-03-01

    Polysaccharides from Spirulina platensis algae (SP) were extracted by ultrasound-assisted extraction procedure. The optimal conditions for ultrasonic extraction of SP were determined by response surface methodology. The four parameters were, extraction time (X1), extraction temperature (X2), ultrasonic power (X3) and the ratio of water to raw material (X4), respectively. The experimental data obtained were fitted to a second-order polynomial equation. The optimum conditions were extraction time of 25 min, extraction temperature 85°C, ultrasonic power 90 W and ratio of water to raw material 20 mL/g. Under these optimal conditions, the experimental yield was 13.583±0.51%, well matched with the predicted models with the coefficients of determination (R2) of 0.9971. Then, we demonstrated that SP polysaccharides had strong scavenging activities in vitro on DPPH and hydroxyl radicals. Overall, SP may have potential applications in the medical and food industries. PMID:25583023

  14. Experimental and Computational Study of Steric and Electronic Effects on the Coordination of Bulky, Water-Soluble Alkylphosphines to Palladium under Reducing Conditions: Correlation to Catalytic Activity

    SciTech Connect

    DeVasher, Rebecca B.; Spruell, Jason M.; Dixon, David A.; Broker, Grant A.; Griffin, Scott T.; Rogers, Robin D.; Shaughnessy, Kevin H.

    2005-02-28

    The research described in this product was performed in part in the Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy's Office of Biological and Environmental Research and located at Pacific Northwest National Laboratory. Sterically demanding, water-soluble alkylphosphine ligands 2-(di-tert-butylphosphino)- ethyltrimethylammonium chloride (t-Bu-Amphos) and 4-(di-tert-butylphosphino)-N,N-dimethylpiperidinium chloride (t-Bu-Pip-phos) in combination with palladium salts provided active catalysts for the cross-coupling of aryl halides under mild conditions in aqueous solvents, whereas 4-(dicyclohexylphosphino)-N,N-dimethylpiperidinium chloride (Cy-Pipphos) gave a less active catalyst. Catalyst activity increased with increasing cone angle of the ligands, but the ø electronic parameter determined from the symmetric C-O stretching frequency of LNi(CO)₃ did not correlate with catalyst activity. Catalyst activity correlated with other calculated electronic parameters, such as the HOMO-LUMO energy gap of the ligand and the HOMO energy level of the LPd(0) species. Multinuclear NMR spectroscopic studies showed that t-Bu-Amphos and t-Bu-Pip-phos rapidly form L₂Pd(0) (L= t-Bu-Amphos or t-Bu-Pip-phos) complexes when reacted with Pd(OAc)₂ under reducing conditions over a range of L:Pd ratios. In contrast, the coordination chemistry of Cy-Pip-phos depended on the Cy-Pip-phos:Pd ratio. At ≤1:1 Cy-Pip-phos:Pd ratio, rapid formation of L₂Pd(0) occurred. At higher L:Pd ratios, initial formation of trans-(Cy-Pip-phos)₂PdCl₂ was observed followed by slow reduction to the Pd(0) complex.

  15. Rutile solubility in H2O-NaAlSi3O8 fluids at High T and P: Implications form HFSE mobility in Subduction zones

    NASA Astrophysics Data System (ADS)

    Antignano, A.; Manning, C. E.

    2005-12-01

    The trace element signatures of arc magmas are characterized by HFSE depletion relative to the LILE. Rutile, a common accessory phase in high-pressure assemblages, is an important reservoir for the HFSE and is often invoked to explain the HFSE depletion of arc magmas. This model is in part based on experimental studies, which show that rutile has very low solubility in pure H2O. However, rutile is also a common accessory to eclogite-facies vein assemblages of albite, paragonite and quartz, which likely precipitated from slab-derived fluids. This observation requires either that fluid fluxes were unrealistically high, or that current estimates of Ti solubility are too low. A possible solution to this problem is that dissolved silicate components can enhance Ti solubility via complexing. To test this, we measured the solubility of rutile in H2O-NaAlSi3O8 (albite) bearing fluids at high T and P. Experiments were conducted using a piston-cylinder apparatus with NaCl-graphite furnaces. A single synthetic rutile crystal was loaded into a 1.6 mm OD Pt inner capsule, which was lightly crimped and then placed in a 3.5 OD Pt outer capsule with ultra pure H2O and powdered Amelia albite. Solubility was determined by the weight loss of the rutile grain after 10 hrs. A time series demonstrates that equilibrium is achieved after 8-10 hrs. Preliminary results at 800°C, 1.0 GPa, show that rutile solubility rises with increasing NaAlSi3O8 concentration from 1.15(12) millimolal at 2.18 wt% NaAlSi3O8 to 3.77(13) at 8.80 wt% NaAlSi3O8. Corundum mats + fluid are observed in 3.4-8.80 wt% NaAlSi3O8 and are interpreted to be the result of incongruent dissolution of albite. Quenched melt spheres where observed in an experiment containing ~15 wt% NaAlSi3O8, but not at 8.80 wt%. At 8.80 wt% NaAlSi3O8, rutile solubility is higher by a factor of 6 relative to that in pure H2O. Our results suggest that TiO2 solubility is increased by complexing with Na-Al-Si-bearing fluid components. It has

  16. Identification and Characterization of CD44RC, a Novel Alternatively Spliced Soluble CD44 Isoform that can Potentiate the Hyaluronan Binding Activity of Cell Surface CD44

    PubMed Central

    Chiu, Roland K; Carpenito, Carmine; Dougherty, Shona T; Hayes, Gregory M; Dougherty, Graeme J

    1999-01-01

    Abstract Soluble CD44 proteins generated by proteolytic cleavage or aberrant intron retention have been shown to antagonize the ligand binding activity of the corresponding cell surface receptor, inducing apoptosis and inhibiting tumor growth. Interestingly, such findings appear to contradict recent studies demonstrating a correlation between the presence of high levels of soluble CD44 in the serum of cancer patients and poor prognosis. In the present study, we report the cloning of a novel, naturally occurring, differentially expressed, soluble CD44 isoform, designated CD44RC, which, in contrast to previously described soluble CD44 proteins, can dramatically enhance the hyaluronan binding activity of cell surface CD44. Sequence analysis suggests that CD44RC is generated by an alternative splicing event in which the 3′ end of CD44 exon 2 is spliced into an internal splice acceptor site present within exon 18, altering reading frame and giving rise to a soluble protein with a unique COOH terminus. Functional studies suggest that CD44RC enhances hyaluronan binding by adhering to chondroitin sulfate side-chains attached to cell surface CD44, generating a multivalent complex with increased avidity for hyaluronan. PMID:10933060

  17. Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms.

    PubMed Central

    Alvarez, L; Mingorance, J; Pajares, M A; Mato, J M

    1994-01-01

    A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3). A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside. This protein was distributed in both the soluble and insoluble fractions and accounted for approx. 30% of the total bacterial protein. The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity. In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine. Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography. It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers. Images Figure 1 PMID:8043003

  18. Activation of murine peritoneal macrophages by water-soluble extracts of Bursaphelenchus xylophilus, a pine wood nematode.

    PubMed

    Kaji, Hiroaki; Tai, Akihiro; Matsushita, Kazufumi; Kanzaki, Hiroshi; Yamamoto, Itaru

    2006-01-01

    In our previous study, water-soluble extracts from Bursaphelenchus xylophilus (B. xylophilus), a pine wood nematode, were shown to enhance interleukin (IL)-4 plus lipopolysaccharide-induced polyclonal immunoglobulin (Ig) E production in vitro in mice and to increase serum levels of an antigen-nonspecific IgE in vivo. Here we examined whether the nematode extracts stimulate immunofunctions of murine peritoneal macrophages. In both resident and inflammatory macrophages, Fcgamma receptor-mediated phagocytosis was markedly activated by B. xylophilus extracts, while non-specific phagocytosis was not. The enhancement of specific phagocytosis was accompanied by an increase in the formation of IgG-Fcgamma receptor rosettes. B. xylophilus extracts also stimulated IL-1beta production in both types of macrophages, and enhanced NO production and mRNA expression of inflammatory cytokines in inflammatory macrophages. These results indicate that the extracts of B. xylophilus contain an activating substance(s) for immunofunctions in macrophages, besides an enhancing factor for polyclonal IgE production. PMID:16428838

  19. Development of new active packaging film made from a soluble soybean polysaccharide incorporating ZnO nanoparticles.

    PubMed

    Salarbashi, Davoud; Mortazavi, Seyed Ali; Noghabi, Mostafa Shahidi; Fazly Bazzaz, Bibi Sedigheh; Sedaghat, Naser; Ramezani, Mohammad; Shahabi-Ghahfarrokhi, Iman

    2016-04-20

    This study aimed to develop a soluble soybean polysaccharide (SSPS) nanocomposite incorporating ZnO nanoparticles. The nanocomposites were prepared using the solvent-casting method. SEM, AFM, DSC and X-ray diffraction methods were applied to characterize the resulting films. Furthermore, the antibacterial and anti-mold activities of SSPS/ZN films were assessed against the selected microorganisms. The results indicated that incorporating ZNs into the SSPS film affected the tensile strength and elongation at break significantly. In addition, the antibacterial, antifungal and yeasticidal activities of ZnO/SSPS films have been approved. XRD results showed a crystal plane of hexagonal ZN, while SEM showed that there was not a good affinity between ZN and SSPS. Mono-dispersed particles with clearly spherical morphology and with no voids on the surface were observed using AFM. Fluctuation in Tg and Tm resulted from incorporating ZN. In summary, the potential of ZNs as a functional filler in SSPS film has been demonstrated. PMID:26876847

  20. Risk factors associated with serum levels of the inflammatory biomarker soluble urokinase plasminogen activator receptor in a general population.

    PubMed

    Haupt, Thomas H; Kallemose, Thomas; Ladelund, Steen; Rasmussen, Line Jh; Thorball, Christian W; Andersen, Ove; Pisinger, Charlotta; Eugen-Olsen, Jesper

    2014-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers. PMID:25574132

  1. Risk Factors Associated with Serum Levels of the Inflammatory Biomarker Soluble Urokinase Plasminogen Activator Receptor in a General Population

    PubMed Central

    Haupt, Thomas H; Kallemose, Thomas; Ladelund, Steen; Rasmussen, Line JH; Thorball, Christian W; Andersen, Ove; Pisinger, Charlotta; Eugen-Olsen, Jesper

    2014-01-01

    The soluble urokinase plasminogen activator receptor (suPAR) is a biomarker of mortality risk in various patient populations. However, little is known about the implications of lifestyle for suPAR levels in the general population. Lifestyle, demographic, and cardiovascular disease (CVD) risk factor data were collected from 5,538 participants in the Danish population-based Inter99 study. Their suPAR levels were measured using a sandwich enzyme-linked immunosorbent assay. In the final adjusted model, smoking and morbid obesity were strongly associated with higher suPAR levels (P < 0.001). An unhealthy diet and alcohol abstinence in men were also associated with higher suPAR levels. Physical activity in leisure time had a modest impact on suPAR levels in univariate analysis, but not in the final adjusted model. In conclusion, smoking and morbid obesity were strongly associated with higher serum suPAR levels in this general population. Diet and alcohol consumption also seemed to impact suPAR levels. Lifestyle changes are likely to affect suPAR since ex-smokers had suPAR levels comparable to those of never-smokers. PMID:25574132

  2. 76 FR 30738 - Agency Information Collection Activities: Form G-845 and Form G-845 Supplement, Revision of a...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-05-26

    ... the Federal Register on February 22, 2011, at 76 FR 9805, allowing for a 60-day public comment period... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form G-845 and Form G- 845 Supplement, Revision of a Currently Approved Information Collection; Comment Request...

  3. An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities.

    PubMed

    Moss, Marcia L; Minond, Dmitriy; Yoneyama, Toshie; Hansen, Hinrich P; Vujanovic, Nikola; Rasmussen, Fred H

    2016-08-15

    A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, -10, and -9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH2, has specificity constants of 6.3 (±0.3) × 10(4) M(-1) s(-1) and 2.4 (±0.3) × 10(3) M(-1) s(-1) for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH2 and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH2. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, -2, -3, -8, -9, -12, and -14. This substrate provides a unique tool in which to assess ADAM17, -10, and -9 activities. PMID:27177841

  4. Antioxidant, antitumor and immunomodulatory activities of water-soluble polysaccharides in Abrus cantoniensis.

    PubMed

    Wu, Shaowei; Fu, Xiong; You, Lijun; Abbasi, Arshad Mehmood; Meng, Hecheng; Liu, Dong; Aadil, Rana Muhammad

    2016-08-01

    Abrus cantoniensis is a vegetative food in tropical areas of Asia and claimed as folk beverages and soups consumed for cleansing liver toxicants and preventing liver diseases. Polysaccharides (ACP-І and ACP-II) were extracted with hot water from A. cantoniensis, and isolated by DEAE cellulose chromatography. The chemical properties as well as antioxidant, antitumor and immunomodulatory activities of ACP-I and ACP-II were investigated. The results showed that the ACP-I (9.09kDa) contained only glucose and ACP-II (38.45kDa) consisted of rhamnose, arabinose, galactose and glucose. ACP-II exhibited higher oxygen radical absorbance capacity (ORAC) and hydroxyl radical prevention capacity (HRPC) than ACP-I with ORAC values and HRPC values of 53.42±3.32μmol Trolox equiv./g DW and 34.84±5.07μmol Trolox equiv./g DW. Besides, in the wound healing assay, ACP-II exhibited potent migration inhibitory effects on MCF-7 cells. ACP-II could also significantly stimulate the proliferation of splenocytes and thymocytes, and enhanced NO production of peritoneal macrophages. These findings suggest that the polysaccharide ACP-II in A. cantoniensis could be served as a novel potential functional food. PMID:27057623

  5. Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

    PubMed

    Crofts, Naoko; Abe, Natsuko; Oitome, Naoko F; Matsushima, Ryo; Hayashi, Mari; Tetlow, Ian J; Emes, Michael J; Nakamura, Yasunori; Fujita, Naoko

    2015-08-01

    Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein-protein interactions in maize and wheat amyloplasts. This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200-400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs-BEs, and, among BE isozymes, BEIIa-Pho1, and pullulanase-type DBE-BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. PMID:25979995

  6. Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes

    PubMed Central

    Crofts, Naoko; Abe, Natsuko; Oitome, Naoko F.; Matsushima, Ryo; Hayashi, Mari; Tetlow, Ian J.; Emes, Michael J.; Nakamura, Yasunori; Fujita, Naoko

    2015-01-01

    Amylopectin is a highly branched, organized cluster of glucose polymers, and the major component of rice starch. Synthesis of amylopectin requires fine co-ordination between elongation of glucose polymers by soluble starch synthases (SSs), generation of branches by branching enzymes (BEs), and removal of misplaced branches by debranching enzymes (DBEs). Among the various isozymes having a role in amylopectin biosynthesis, limited numbers of SS and BE isozymes have been demonstrated to interact via protein–protein interactions in maize and wheat amyloplasts. This study investigated whether protein–protein interactions are also found in rice endosperm, as well as exploring differences between species. Gel permeation chromatography of developing rice endosperm extracts revealed that all 10 starch biosynthetic enzymes analysed were present at larger molecular weights than their respective monomeric sizes. SSIIa, SSIIIa, SSIVb, BEI, BEIIb, and PUL co-eluted at mass sizes >700kDa, and SSI, SSIIa, BEIIb, ISA1, PUL, and Pho1 co-eluted at 200–400kDa. Zymogram analyses showed that SSI, SSIIIa, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1 eluted in high molecular weight fractions were active. Comprehensive co-immunoprecipitation analyses revealed associations of SSs–BEs, and, among BE isozymes, BEIIa–Pho1, and pullulanase-type DBE–BEI interactions. Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes. These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes. Detailed analyses of these complexes will shed light on the mechanisms controlling the unique branch and cluster structure of amylopectin, and the physicochemical properties of starch. PMID:25979995

  7. The adjuvant activity of a non-toxic, water-soluble glycopeptide present in large quantities in the culture filtrate of Mycobacterium tuberculosis strain DT.

    PubMed Central

    Stewart-Tull, D E; Shimono, T; Kotani, S; Kato, M; Ogawa, Y; Yamamura, Y; Koga, T; Pearson, C M

    1975-01-01

    A water-soluble mycobacterial glycopeptide was obtained in large quantities from the culture supernatant fluid of M. tuberculosis strain DT. This glycopeptide was strongly adjuvant-active when injected, in a water-in-oil emulsion contianing ovalbumin, into guinea-pigs. In addition, it was devoid of cord factor toxicity in mice, polyarthritogenic activity in rats and cavity stimulating activity in rabbit lungs. Images FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 8 PMID:806515

  8. Activation of soluble guanylyl cyclase by BAY 58-2667 improves bladder function in cyclophosphamide-induced cystitis in mice.

    PubMed

    de Oliveira, Mariana G; Calmasini, Fabiano B; Alexandre, Eduardo C; De Nucci, Gilberto; Mónica, Fabíola Z; Antunes, Edson

    2016-07-01

    Activators of soluble guanylyl cyclase (sGC) interact directly with its prosthetic heme group, enhancing the enzyme responsiveness in pathological conditions. This study aimed to evaluate the effects of the sGC activator BAY 58-2667 on voiding dysfunction, protein expressions of α1 and β1 sGC subunits and cGMP levels in the bladder tissues after cyclophosphamide (CYP) exposure. Female C57BL/6 mice (20-25 g) were injected with CYP (300 mg/kg ip) to induce cystitis. Mice were pretreated or not with BAY 58-2667 (1 mg/kg, gavage), given 1 h before CYP injection. The micturition patterns and in vitro bladder contractions were evaluated at 24 h. In freely moving mice, the CYP injection produced reduced the micturition volume and increased the number of urine spots. Cystometric recordings in CYP-injected mice revealed significant increases in basal pressure, voiding frequency, and nonvoiding contractions (NVCs), along with decreases in bladder capacity, intercontraction interval, and compliance. BAY 58-2667 significantly prevented the micturition alterations observed in both freely moving mice and cystometry and normalized the reduced in vitro carbachol-induced contractions in the CYP group. Reduced protein expressions of α1 and β1 sGC subunits and of cGMP levels were observed in the CYP group, all of which were prevented by BAY 58-2667. CYP exposure significantly increased reactive-oxygen species (ROS) generation in both detrusor and urothelium, and this was normalized by BAY 58-2667. The increased myeloperoxidase and cyclooxygenase-2 activities in the bladders of the CYP group remained unchanged by BAY 58-2667. Activators of sGC may constitute a novel and promising therapeutic approach for management of interstitial cystitis. PMID:27122537

  9. Investigation of DNA binding, DNA photocleavage, topoisomerase I inhibition and antioxidant activities of water soluble titanium(IV) phthalocyanine compounds.

    PubMed

    Özel, Arzu; Barut, Burak; Demirbaş, Ümit; Biyiklioglu, Zekeriya

    2016-04-01

    The binding mode of water soluble peripherally tetra-substituted titanium(IV) phthalocyanine (Pc) compounds Pc1, Pc2 and Pc3 with calf thymus (CT) DNA was investigated by using UV-Vis spectroscopy and thermal denaturation studies in this work. The results of DNA binding constants (Kb) and the changes in the thermal denaturation profile of DNA with the addition of Pc compounds indicated that Pc1, Pc2 and Pc3 are able to bind to CT-DNA with different binding affinities. DNA photocleavage studies of Pc compounds were performed in the absence and presence of oxidizing agents such as hydrogen peroxide (H2O2), ascorbic acid (AA) and 2-mercaptoethanol (ME) using the agarose gel electrophoresis method at irradiation 650nm. According to the results of electrophoresis studies, Pc1, Pc2 and Pc3 cleaved of supercoiled pBR322 DNA via photocleavage pathway. The Pc1, Pc2 and Pc3 compounds were examined for topoisomerase I inhibition by measuring the relaxation of supercoiled pBR322 DNA. The all of Pc compounds inhibited topoisomerase I at 20μM concentration. A series of antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging (SOD) assay and metal chelating effect assay were performed for Pc1, Pc2 and Pc3 compounds. The results of antioxidant assays indicated that Pc1, Pc2 and Pc3 compounds have remarkable superoxide radical scavenging activities, moderate 2,2-diphenyl-1-picrylhydrazyl activities and metal chelating effect activities. All the experimental studies showed that Pc1, Pc2 and Pc3 compounds bind to CT-DNA via minor groove binding, cleave of supercoiled pBR322 DNA via photocleavage pathway, inhibit topoisomerase I and have remarkable superoxide radical scavenging activities. Thanks to these properties the Pc1, Pc2 and Pc3 compounds are suitable agents for photo dynamic therapy. PMID:26882290

  10. Batch cultivation of Methylosinus trichosporium OB3B: IV. Production of hydrogen-driven soluble or particulate methane monooxygenase activity.

    PubMed

    Shah, N N; Hanna, M L; Jackson, K J; Taylor, R T

    1995-02-01

    Batch culture conditions were established for the formation of H(2)-driven whole-cell soluble or particulate methane monooxygenase (sMMO or pMMO) activity in the obligate methanotroph, Methylosinus trichosporum Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H(2) to a nitrate-containing minimal salts growth medium or Ni and Mo to a nitrate-lacking growth medium (induces a nitrogenase that generates intracellular H(2)) markedly enhanced both the hydrogenase and the accompanying washed-cell H(2)-driven MMO activities of shake-flask cultured cells. For sMMO containing cells, H(2) provided in vitro reducing power for the oxidation of chlorinated solvents such as chloroform and trichloroethylene. Cell cultivations under N(2)-fixing conditions in a 5-L bioreactor, however, required an initial nitrate concentration of at least 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H(2)-driven sMMO or pMMO activity. Elevation of the initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalent growth yield. High nitrate also shortened the culture time for sMMO containing cells by approximately 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting-state cells retained 90% and 70% of their H(2)-driven sMMO and pMMO activity, respectively. This makes their practical use quite feasible. (c) 1995 John Wiley & Sons, Inc. PMID:18623142

  11. Biological activity of designed photolabile metal nitrosyls: light-dependent activation of soluble guanylate cyclase and vasorelaxant properties in rat aorta.

    PubMed

    Madhani, Melanie; Patra, Apurba K; Miller, Thomas W; Eroy-Reveles, Aura A; Hobbs, Adrian J; Fukuto, Jon M; Mascharak, Pradip K

    2006-12-14

    The biological and pharmacological utility of nitric oxide (NO) has led to the development of many classes of NO-donor compounds as both research tools and therapeutic agents. Many donors currently in use rely on thermal decomposition or bioactivation for the release of NO. We have developed several photolabile metal-nitrosyl donors that release NO when exposed to either visible or UV light. Herein, we show that these donors are capable of activating the primary "NO receptor", soluble guanylate cyclase (sGC), in a light-dependent fashion leading to increases in cGMP. Moreover, we demonstrate that these donors are capable of eliciting light-dependent increases of cGMP in smooth muscle cells and vasorelaxation of rat aortic smooth muscle tissue, all effects that are attributed to activation of sGC. The potential utility of these compounds as drugs and/or research tools is discussed. PMID:17149862

  12. The Essay: Theory and Pedagogy for an Active Form.

    ERIC Educational Resources Information Center

    Heilker, Paul

    Calling for a radical reexamination of the traditional foundation of composition instruction--the thesis/support form, this book argues that the essay, with its informality, conversational tone, meditative mood, and integration of form and content, is better suited to developmental, epistemological, ideological, and feminist rhetorical…

  13. Pulmonary and systemic vasodilator responses to the soluble guanylyl cyclase activator, BAY 60–2770, are not dependent on endogenous nitric oxide or reduced heme

    PubMed Central

    Pankey, Edward A.; Bhartiya, Manish; Badejo, Adeleke M.; Haider, Umair; Stasch, Johannes-Peter; Murthy, Subramanyam N.; Nossaman, Bobby D.

    2011-01-01

    4-({(4-Carboxybutyl)[2-(5-fluoro-2-{[4′-(trifluoromethyl)biphenyl-4-yl]methoxy}phenyl)ethyl]amino}methyl)benzoic acid (BAY 60–2770) is a nitric oxide (NO)-independent activator of soluble guanylyl cyclase (sGC) that increases the catalytic activity of the heme-oxidized or heme-free form of the enzyme. In this study, responses to intravenous injections of the sGC activator BAY 60–2770 were investigated under baseline and elevated tone conditions induced by the thromboxane mimic U-46619 when NO synthesis was inhibited by Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME), when sGC activity was inhibited by 1H-[1,2,4]-oxadizaolo[4,3]quinoxaline-1-one (ODQ), an agent that oxidizes sGC, and in animals with monocrotaline-induced pulmonary hypertension. The intravenous injections of BAY 60–2770 under baseline conditions caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and no change or small increases in cardiac output. Under elevated tone conditions during infusion of U-46619, intravenous injections of BAY 60–2770 caused larger decreases in pulmonary arterial pressure, smaller decreases in systemic arterial pressure, and increases in cardiac output. Pulmonary vasodilator responses to BAY 60–2770 were enhanced by l-NAME or by ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside. ODQ had no significant effect on baseline pressures and attenuated pulmonary and systemic vasodilator responses to the sGC stimulator BAY 41–8543 2-{1-[2-(fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5(4-morpholinyl)-4,6-pyrimidinediamine. BAY 60–2770 and sodium nitroprusside decreased pulmonary and systemic arterial pressures in monocrotaline-treated rats in a nonselective manner. The present data show that BAY 60–2770 has vasodilator activity in the pulmonary and systemic vascular beds that is enhanced by ODQ and NOS inhibition, suggesting that the heme-oxidized form of sGC can be

  14. Pulmonary and systemic vasodilator responses to the soluble guanylyl cyclase activator, BAY 60-2770, are not dependent on endogenous nitric oxide or reduced heme.

    PubMed

    Pankey, Edward A; Bhartiya, Manish; Badejo, Adeleke M; Haider, Umair; Stasch, Johannes-Peter; Murthy, Subramanyam N; Nossaman, Bobby D; Kadowitz, Philip J

    2011-03-01

    4-({(4-Carboxybutyl)[2-(5-fluoro-2-{[4'-(trifluoromethyl)biphenyl-4-yl]methoxy}phenyl)ethyl]amino}methyl)benzoic acid (BAY 60-2770) is a nitric oxide (NO)-independent activator of soluble guanylyl cyclase (sGC) that increases the catalytic activity of the heme-oxidized or heme-free form of the enzyme. In this study, responses to intravenous injections of the sGC activator BAY 60-2770 were investigated under baseline and elevated tone conditions induced by the thromboxane mimic U-46619 when NO synthesis was inhibited by N(ω)-nitro-L-arginine methyl ester hydrochloride (L-NAME), when sGC activity was inhibited by 1H-[1,2,4]-oxadizaolo[4,3]quinoxaline-1-one (ODQ), an agent that oxidizes sGC, and in animals with monocrotaline-induced pulmonary hypertension. The intravenous injections of BAY 60-2770 under baseline conditions caused small decreases in pulmonary arterial pressure, larger decreases in systemic arterial pressure, and no change or small increases in cardiac output. Under elevated tone conditions during infusion of U-46619, intravenous injections of BAY 60-2770 caused larger decreases in pulmonary arterial pressure, smaller decreases in systemic arterial pressure, and increases in cardiac output. Pulmonary vasodilator responses to BAY 60-2770 were enhanced by L-NAME or by ODQ in a dose that attenuated responses to the NO donor sodium nitroprusside. ODQ had no significant effect on baseline pressures and attenuated pulmonary and systemic vasodilator responses to the sGC stimulator BAY 41-8543 2-{1-[2-(fluorophenyl)methyl]-1H-pyrazolo[3,4-b]pyridin-3-yl}-5(4-morpholinyl)-4,6-pyrimidinediamine. BAY 60-2770 and sodium nitroprusside decreased pulmonary and systemic arterial pressures in monocrotaline-treated rats in a nonselective manner. The present data show that BAY 60-2770 has vasodilator activity in the pulmonary and systemic vascular beds that is enhanced by ODQ and NOS inhibition, suggesting that the heme-oxidized form of sGC can be activated in vivo in an

  15. Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

    PubMed

    Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

    2016-05-01

    Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. PMID:26849963

  16. Activation of lipid catabolism by the water-soluble fraction of petroleum in the crustacean Macrobrachium borellii.

    PubMed

    Lavarías, S; Pollero, R J; Heras, H

    2006-05-01

    Little is known about the effect of the water-soluble fraction of crude oil (WSF) on lipid metabolism in invertebrates. The effect of the WSF on the triacylglycerol (TAG) mobilization, fatty acid activation and degradation was evaluated in the decapod Macrobrachium borellii, exposing adult and eggs at different stages of development for 7 days to a sublethal concentration of WSF. Using radioactive tracers, mitochondrial palmitoyl-CoA synthetase (ACS), triacylglycerol lipase (TAG-lipase) and fatty acid beta-oxidation system activities were assayed. Before studying the effect of WSF, the kinetic parameters of ACS were determined in purified mitochondria. Its optimal temperature and pH were 32 degrees C and 8.0, respectively, the apparent K(m) 2.48 micromol l(-1), and its V(max) of 1.93 nmol min(-1) mg protein(-1). These kinetic parameters differed significantly from this shrimp's microsomal isoform. After 7 days exposure to a sublethal concentration of WSF (0.6 mg/l), changes were observed in the enzymatic activity of all enzymes or enzymatic system assayed in adult midgut gland as well as in stage 5 eggs, a period of active organogenesis. An increase in the mobilization of energy stores was detected as early as stage 4, where TAG-lipase activity increased by 27% in exposed eggs. The increase was even more marked in exposed eggs at stage 5 where a three-fold rise (154%) was determined. Exposed adult shrimp also showed an augmented lipase activity by 38%. Fatty acid beta-oxidation increased by 51.0 and 35.5% in midgut gland and eggs at stage 5, respectively, but no changes were observed at less-developed stages. Mitochondrial fatty acid activation by ACS also increased in adults and stage 5 eggs by 7.4 and 52.0%, respectively. A similar response of the lipid catabolic pathways to WSF contamination in both adult and eggs, suggests that the exposure to this pollutant causes an increase in the energy needs of this shrimp. When validated by field studies, these catabolic

  17. Promoter activity of the 5'-flanking regions of medaka fish soluble guanylate cyclase alpha1 and beta1 subunit genes.

    PubMed Central

    Yamamoto, Takehiro; Suzuki, Norio

    2002-01-01

    We examined the spatial expression pattern of medaka fish (Oryzias latipes) soluble guanylate cyclase alpha(1) and beta(1) subunit genes, OlGCS-alpha(1) and OlGCS-beta(1), and characterized the 5'-flanking region required for expression of both genes by introducing various promoter-luciferase fusion-gene constructs into COS-1 cells and medaka fish embryos. The OlGCS-alpha(1) and OlGCS-beta(1) gene transcripts were detected in whole brain and kidney in 7-day and 9-day embryos. Primer-extension analysis demonstrated that there were no differences among various adult organs (brain, eye, kidney, ovary and testis) in the transcription start site of the OlGCS-alpha(1) and OlGCS-beta(1) genes. Neither gene contained the functional TATA box within its 5'-flanking region, and the basal promoter activity was found between nucleotides +33 and +42 in the OlGCS-alpha(1) gene and between nucleotides +146 and +155 in the OlGCS-beta(1) gene. In the assay of medaka fish embryos, the 5'-flanking region of the OlGCS-beta(1) gene exhibited lower promoter activity than that of the OlGCS-alpha(1) gene. In the experiments on dual-luciferase fusion-gene constructs, the 5'-flanking region of the OlGCS-alpha(1) gene connected to the 5'-flanking region of the OlGCS-beta(1) gene was introduced into medaka fish embryos, and the 5'-flanking regions of both subunit genes were shown to mutually influence each other's promoter activity. PMID:11772405

  18. Effect of soluble guanylyl cyclase activator and stimulator therapy on nitroglycerin-induced nitrate tolerance in rats.

    PubMed

    Jabs, A; Oelze, M; Mikhed, Y; Stamm, P; Kröller-Schön, S; Welschof, P; Jansen, T; Hausding, M; Kopp, M; Steven, S; Schulz, E; Stasch, J-P; Münzel, T; Daiber, A

    2015-08-01

    Chronic nitroglycerin (GTN) anti-ischemic therapy induces side effects such as nitrate tolerance and endothelial dysfunction. Both phenomena could be based on a desensitization/oxidation of the soluble guanylyl cyclase (sGC). Therefore, the present study aims at investigating the effects of the therapy with the sGC activator BAY 60-2770 and the sGC stimulator BAY 41-8543 on side effects induced by chronic nitroglycerin treatment. Male Wistar rats were treated with nitroglycerin (100mg/kg/d for 3.5days, s.c. in ethanol) and BAY 60-2770 (0.5 or 2.5mg/kg/d) or BAY 41-8543 (1 and 5mg/kg/d) for 6days. Therapy with BAY 60-2770 but not with BAY 41-8543 improved nitroglycerin-triggered endothelial dysfunction and nitrate tolerance, corrected the decrease in aortic nitric oxide levels, improved the cGMP dependent activation of protein kinase I in aortic tissue and reduced vascular, cardiac and whole blood oxidative stress (fluorescence and chemiluminescence assays; 3-nitrotyrosine staining). In contrast to BAY 41-8543, the vasodilator potency of BAY 60-2770 was not impaired in isolated aortic ring segments from nitrate tolerant rats. sGC activator therapy improves partially the adverse effects of nitroglycerin therapy whereas sGC stimulation has only minor beneficial effects pointing to a nitroglycerin-dependent sGC oxidation/inactivation mechanism contributing to nitrate tolerance. PMID:25869522

  19. Fed-batch production of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in soluble form in Escherichia coli and its purification and characterization.

    PubMed

    Li, Ping; Gu, Qing; Wu, Xuechang

    2016-10-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent. The aim of this study is to produce large quantities of highly pure and bioactive recombinant human TRAIL. Here, TRAIL was expressed in soluble form by pH-stat fed-batch cultivation and purified using a rapid and simple two-step chromatographic procedure. To improve the soluble yield, expression of TRAIL in Escherichia coli was induced with low IPTG concentration (0.1 mM) at low temperature (28 °C) supplemented with ZnSO4 (0.5 mM), using glycerol as carbon source. Under the optimized conditions, 4.14 ± 0.19 g/L of TRAIL in soluble form was achieved at 19 h without pure oxygen. To purify the recombinant TRAIL, we developed an efficient two-step chromatographic procedure including affinity chromatography and cation-exchange chromatography, especially improved the cation-exchange chromatography using a combination of pH and NaCl gradients strategy. Consequently, 4313.5 mg of target protein with high purity (98.1%) was obtained from 2.3 L of cell broth. Our results also showed that the purified TRAIL was with ordered secondary and tertiary structures, in homogeneous form and with strong cytotoxicity. PMID:27335160

  20. Cavitating ultrasound hydrogenation of water-soluble olefins employing inert dopants: Studies of activity, selectivity and reaction mechanisms

    SciTech Connect

    Disselkamp, Robert S.; Chajkowski, Sarah M.; Boyles, Kelly R.; Hart, Todd R.; Peden, Charles HF

    2006-12-07

    Here we discuss results obtained as part of a three-year investigation at Pacific Northwest National Laboratory of ultrasound processing to effect selectivity and activity in the hydrogenation of water-soluble olefins on transition metal catalysts. We have shown previously that of the two regimes for ultrasound processing, high-power cavitating and high-power non-cavitating, only the former can effect product selectivity dramatically (> 1000%) whereas the selectivity of the latter was comparable with those obtained in stirred/silent control experiments [R.S. Disselkamp, Y.-H. Chin, C.H.F. Peden, J. Catal., 227, 552 (2005)]. As a means of ensuring the benefits of cavitating ultrasound processing, we introduced the concept of employing inert dopants into the reacting solution. These inert dopants do not partake in solution chemistry but enable a more facile transition from high-power non-cavitating to cavitating conditions during sonication treatment. With cavitation processing conditions ensured, we discuss here results of isotopic H/D substitution for a variety of substrates and illustrate how such isotope dependent chemistries during substrate hydrogenation elucidate detailed mechanistic information about these reaction systems.

  1. Soluble inhibitors generated during hydrothermal pretreatment of oil palm mesocarp fiber suppressed the catalytic activity of Acremonium cellulase.

    PubMed

    Zakaria, Mohd Rafein; Hirata, Satoshi; Fujimoto, Shinji; Ibrahim, Izzudin; Hassan, Mohd Ali

    2016-01-01

    Oil palm mesocarp fiber was subjected to hydrothermal pretreatment under isothermal and non-isothermal conditions. The pretreated slurries were separated by filtration, pretreated liquids and solids were characterized. An enzymatic digestibility study was performed for both pretreated slurries and solids to understand the effect of soluble inhibitors generated during the pretreatment process. The highest glucose yield obtained from pretreated slurries was 70.1%, and gradually decreased with higher pretreatment severities. The highest glucose yield obtained in pretreated solids was 100%, after pretreatment at 210°C for 20min. In order to study the inhibitory effects of compounds generated during pretreatment with cellulase, technical grade solutions that mimic the pretreated liquid were prepared and their effect on Acremonium cellulase activity was monitored using Avicel. Xylo-oligomers and tannic acid were identified as powerful inhibitors of Acremonium cellulase, and the lowest hydrolysis rate of Avicel of 0.18g/g-glucose released/L/h was obtained from tannic acid. PMID:26524253

  2. DNA binding and topoisomerase II inhibitory activity of water-soluble ruthenium(II) and rhodium(III) complexes.

    PubMed

    Singh, Sanjay Kumar; Joshi, Shweta; Singh, Alok Ranjan; Saxena, Jitendra Kumar; Pandey, Daya Shankar

    2007-12-10

    Water-soluble piano-stool arene ruthenium complexes based on 1-(4-cyanophenyl)imidazole (CPI) and 4-cyanopyridine (CNPy) with the formulas [(eta6-arene)RuCl2(L)] (L = CPI, eta6-arene = benzene (1), p-cymene (2), hexamethylbenzene (3); L = CNPy, eta6-arene = benzene (4), p-cymene (5), hexamethylbenzene (6)) have been prepared by our earlier methods. The molecular structure of [(eta6-C6Me6)RuCl2(CNPy)] (6) has been determined crystallographically. Analogous rhodium(III) complex [(eta5-C5Me5)RhCl2(CPI)] (7) has also been prepared and characterized. DNA interaction with the arene ruthenium complexes and the rhodium complex has been examined by spectroscopic and gel mobility shift assay; condensation of DNA and B-->Z transition have also been described. Arene ruthenium(II) and EPh3 (E = P, As)-containing arene ruthenium(II) complexes exhibited strong binding behavior, however, rhodium(III) complexes were found to be Topo II inhibitors with an inhibition percentage of 70% (7) and 30% (7a). Furthermore, arene ruthenium complexes containing polypyridyl ligands also act as mild Topo II inhibitors (10%, 3c and 40%, 3d) in contrast to their precursor complexes. Complexes 4-6 also show significant inhibition of beta-hematin/hemozoin formation activity. PMID:18001110

  3. Production and anti-diabetic activity of soluble dietary fiber from apricot pulp by Trichoderma viride fermentation.

    PubMed

    Cui, Jie; Gu, Xin; Zhang, Qiaohui; Ou, Yangjie; Wang, Jianzhong

    2015-05-01

    Soluble dietary fiber (SDF) was prepared by Trichoderma viride fermentation by using apricot pulp as the raw material. A four-factor and three-level response surface methodology was applied to optimize the fermentation conditions affecting the extraction rate of SDF. The optimum fermentation conditions were listed: crude enzyme volume, 9.59 mL g(-1); fermentation temperature, 43 °C; initial pH, 5.36; fermentation time, 6.47 h. Under these conditions, 15.69% yield was obtained and its relative error with the predicted theoretical value (15.87%) was 1.14%. The dietary fiber content of SDF was 84.0% whereas it was found to be 43.1% in apricot pulp flour. The anti-diabetic effect of apricot pulp SDF on rat models of diabetes was investigated. Both the blood glucose level and body weight were significantly changed in apricot pulp SDF-treated groups compared with the diabetic group (p < 0.01) after intragastric administration for 28 days. In addition, SDF elicited inhibitory effects on the α-glucosidase activity with an IC50 of 17.458 mg mL(-1). These results implied that apricot pulp SDF relieved the symptoms of diabetic rats. PMID:25882161

  4. Affinity to bovine serum albumin and anticancer activity of some new water-soluble metal Schiff base complexes

    NASA Astrophysics Data System (ADS)

    Asadi, Mozaffar; Asadi, Zahra; Zarei, Leila; Sadi, Somaye Barzegar; Amirghofran, Zahra

    2014-12-01

    Metal Schiff-base complexes show biological activity but they are usually insoluble in water so four new water-soluble metal Schiff base complexes of Na2[M(5-SO3-1,2-salben]; (5-SO3-1,2-salben denoted N,N";-bis(5-sulphosalicyliden)-1,2-diaminobenzylamine and M = Mg, Mn, Cu, Zn) were synthesized and characterized. The formation constants of the metal complexes were determined by UV-Vis absorption spectroscopy. The interaction of these complexes with bovine serum albumin (BSA) was studied by fluorescence spectroscopy. Type of quenching, binding constants, number of binding sites and binding stoichiometries were determined by fluorescence quenching method. The results showed that the mentioned complexes strongly bound to BSA. Thermodynamic parameters indicated that hydrophobic association was the major binding force and that the interaction was entropy driven and enthalpically disfavoured. The displacement experiment showed that these complexes could bind to the subdomain IIA (site I) of albumin. Furthermore the synchronous fluorescence spectra showed that the microenvironment of the tryptophan residues was not apparently changed. Based on the Förster theory of non-radiation energy transfer, the distance between the donor (Trp residues) and the acceptor metal complexes was obtained. The growth inhibitory effect of complexes toward the K562 cancer cell line was measured.

  5. Extensive sugar modification improves triple helix forming oligonucleotide activity in vitro but reduces activity in vivo.

    PubMed

    Alam, Md Rowshon; Majumdar, Alokes; Thazhathveetil, Arun Kalliat; Liu, Su-Ting; Liu, Ji-Lan; Puri, Nitin; Cuenoud, Bernard; Sasaki, Shigeki; Miller, Paul S; Seidman, Michael M

    2007-09-01

    We are developing triple helix forming oligonucleotides (TFOs) for gene targeting. Previously, we synthesized bioactive TFOs containing 2'-O-methylribose (2'-OMe) and 2'-O-aminoethylribose (2'-AE) residues. Active TFOs contained four contiguous 2'-AE residues and formed triplexes with high thermal stability and rapid association kinetics. In an effort to further improve bioactivity, we synthesized three series of TFOs containing the 2'-AE patch and additional ribose modifications distributed throughout the remainder of the oligonucleotide. These were either additional 2'-AE residues, the conformationally locked BNA/LNA ribose with a 2'-O,4'-C-methylene bridge, or the 2'-O,4'-C-ethylene analogue (ENA). The additionally modified TFOs formed triplexes with greater thermal stability than the reference TFO, and some had improved association kinetics. However, the most active TFOs in the biochemical and biophysical assays were the least active in the bioassay. We measured the thermal stability of triplexes formed by the TFOs in each series on duplex targets containing a change in sequence at a single position. The Tm value of the variant sequence triplexes increased as the number of all additional modifications increased. A simple explanation for the failure of the improved TFOs in the bioassay was that the increased affinity for nonspecific targets lowered the effective nuclear concentration. Enhancement of TFO bioactivity will require chemical modifications that improve interaction with the specific targets while retaining selectivity against mismatched sequences. PMID:17691818

  6. Water-soluble aluminium phthalocyanine-polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice.

    PubMed

    Brasseur, N; Ouellet, R; La Madeleine, C; van Lier, J E

    1999-07-01

    The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13,000-23,000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc-PEG and AlPc-PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75-100% of animals at a low drug dose (0.25 micromol kg(-1)) following PDT (400 J cm(-2), 650-700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 micromol kg(-1). In the non-cured animals, AlPc-PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc-PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this

  7. Water-soluble aluminium phthalocyanine–polymer conjugates for PDT: photodynamic activities and pharmacokinetics in tumour-bearing mice

    PubMed Central

    Brasseur, N; Ouellet, R; Madeleine, C La; Lier, J E van

    1999-01-01

    The potential use of unsubstituted aluminium phthalocyanine (AlClPc) as a sensitizer for photodynamic therapy (PDT) of cancer has not been fully exploited in spite of its higher efficiency as compared to the sulphonated derivatives. This is largely due to the strong hydrophobic character of AlClPc which renders the material difficult to formulate for in vivo administration. We prepared two water-soluble derivatives of AlClPc by axial coordination of polyethyleneglycol (PEG, MW 2000) or polyvinylalcohol (PVA, MW 13 000–23 000) to the central aluminium ion. Their photodynamic activities were evaluated in vitro against the EMT-6 mouse mammary tumour cells and in vivo against the EMT-6 and the colon carcinoma Colo-26 tumours implanted intradermally in Balb/c mice. Pharmacokinetics were studied in the EMT-6 tumour-bearing mice. After 1 h incubation, the light dose required to kill 90% of cells (LD90) was at least three times less for AlClPc (Cremophor emulsion) as compared to AlPc–PEG and AlPc–PVA, while after 24 h incubation all three preparations were highly phototoxic. All three dye preparations induced complete EMT-6 tumour regression in 75–100% of animals at a low drug dose (0.25 μmol kg−1) following PDT (400 J cm−2, 650–700 nm) at 24 h pi. Complete tumour regression in the Colo-26 tumour model was obtained in 30% of mice at a dose of 2 μmol kg−1. In the non-cured animals, AlPc–PVA induced the most significant tumour growth delay. This dye showed a prolonged plasma half-life (6.8 h) as compared to AlClPc (2.6 h) and AlPc–PEG (23 min), lower retention by liver and spleen and higher tumour-to-skin and tumour-to-muscle ratios. Our data demonstrate that addition of hydrophilic axial ligands to AlPc, while modifying in vitro and in vivo kinetics, does not reduce the PDT efficiency of the parent molecule. Moreover, in the case of the polyvinylalcohol derivative, axial coordination confers advantageous pharmacokinetics to AlPc, which makes this

  8. Odorants with Multiple Oxygen-Containing Functional Groups and Other Odorants with High Water Solubility Preferentially Activate Posterior Olfactory Bulb Glomeruli

    PubMed Central

    Johnson, Brett A.; Arguello, Spart; Leon, Michael

    2008-01-01

    In past studies in which we mapped 2-deoxyglucose uptake evoked by systematically different odorant chemicals across the entire rat olfactory bulb, glomerular responses could be related to each odorant's particular oxygen-containing functional group. In the present study, we tested whether aliphatic odorants containing two such functional groups (esters, ketones, acids, alcohols, and ethers) would stimulate the combination of glomerular regions that are associated with each of the functional groups separately, or whether they would evoke unique responses in different regions of the bulb. We found that these very highly water-soluble molecules rarely evoked activity in the regions responding to the individual functional groups; instead, they activated posterior glomeruli located about halfway between the dorsal and ventral extremes in both the lateral and the medial aspects of the bulb. Additional highly water-soluble odorants, including very small molecules with single oxygenic groups, also strongly stimulated these posterior regions, resulting in a statistically significant correlation between posterior 2-deoxyglucose uptake and molecular properties associated with water solubility. By showing that highly water-soluble odorants stimulate a part of the bulb associated with peripheral and ventral regions of the epithelium, our results challenge a prevalent notion that such odorants would activate class I odorant receptors located in zone 1 of the olfactory epithelium, which projects to the dorsal aspect of the bulb. PMID:17366613

  9. A constitutively activated mutant of human soluble guanylyl cyclase (sGC): implication for the mechanism of sGC activation

    NASA Technical Reports Server (NTRS)

    Martin, Emil; Sharina, Iraida; Kots, Alexander; Murad, Ferid

    2003-01-01

    Heterodimeric alphabeta soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the beta subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type alphabeta enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant alphabetaCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrim idin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the beta subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer.

  10. A constitutively activated mutant of human soluble guanylyl cyclase (sGC): Implication for the mechanism of sGC activation

    PubMed Central

    Martin, Emil; Sharina, Iraida; Kots, Alexander; Murad, Ferid

    2003-01-01

    Heterodimeric αβ soluble guanylyl cyclase (sGC) is a recognized receptor for nitric oxide (NO) and mediates many of its physiological functions. Although it has been clear that the heme moiety coordinated by His-105 of the β subunit is crucial for mediating the activation of the enzyme by NO, it is not understood whether the heme moiety plays any role in the function of the enzyme in the absence of NO. Here we analyze the effects of biochemical and genetic removal of heme and its reconstitution on the activity of the enzyme. Detergent-induced loss of heme from the wild-type αβ enzyme resulted in several-fold activation of the enzyme. This activation was inhibited after hemin reconstitution. A heme-deficient mutant αβCys-105 with Cys substituted for His-105 was constitutively active with specific activity approaching the activity of the wild-type enzyme activated by NO. However, reconstitution of mutant enzyme with heme and/or DTT treatment significantly inhibited the enzyme. Mutant enzyme reconstituted with ferrous heme was activated by NO and CO alone and showed additive effects between gaseous effectors and the allosteric activator 5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine. We propose that the heme moiety through its coordination with His-105 of the β subunit acts as an endogenous inhibitor of sGC. Disruption of the heme-coordinating bond induced by binding of NO releases the restrictions imposed by this bond and allows the formation of an optimally organized catalytic center in the heterodimer. PMID:12883009

  11. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway.

    PubMed

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  12. Soluble egg antigens of Schistosoma japonicum induce senescence in activated hepatic stellate cells by activation of the STAT3/p53/p21 pathway

    PubMed Central

    Chen, Jinling; Pan, Jing; Wang, Jianxin; Song, Ke; Zhu, Dandan; Huang, Caiqun; Duan, Yinong

    2016-01-01

    Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs). Recent findings suggest that senescence of activated HSCs might limit the development of liver fibrosis. Based on previously observed anti-fibrotic effects of soluble egg antigens from Schistosoma japonicum in vitro, we hypothesized that SEA might play a crucial role in alleviating liver fibrosis through promoting senescence of activated HSCs. We show here that SEA inhibited expression of α-SMA and pro-collagen I and promoted senescence of activated HSCs in vitro. In addition, SEA induced an increased expression of P-p53 and p21. Knockdown of p53 inhibited the expression of p21 and failed to induce senescence of activated-HSCs. Phosphorylated STAT3 was elevated upon SEA stimulation, while loss of STAT3 decreased the level of p53 and senescence of HSCs. Results from immunoprecipitation analysis demonstrated that SOCS3 might be involved in the SEA-induced senescence in HSCs through its interaction with p53. This study demonstrates the potential capacity of SEA in restricting liver fibrosis through promoting senescence in HSCs. Furthermore, a novel STAT3-p53-p21 pathway might participate in the observed SEA-mediated senescence of HSCs. Our results suggest that SEA might carry potential therapeutic effects of restraining liver fibrosis through promoting senescence. PMID:27489164

  13. Structure elucidation and anti-tumor activities of water-soluble oligosaccharides from Lactarius deliciosus (L. ex Fr.) Gray

    PubMed Central

    Ding, Xiang; Hou, Yiling; Hou, Wanru; Zhu, Yuanxiu; Fu, Lei; Zhu, Hongqing

    2015-01-01

    Background: Oligosaccharides are composed of a variable number of monosaccharide units and very important in the biologically diverse of biological systems. Materials and Methods: Crude water-soluble oligosaccharide was extracted from the fruiting bodies with water and then successively purified by DEAE–cellulose 52 and Sephadex G-100 column chromatography, yielding one major oligosaccharides fractions: LES-A. Structural features of Lactarius deliciosus (L. ex Fr.) Gray oligosaccharide (LDGO-A) were investigated by a combination of monosaccharide component analysis by thin layer chromatography, infrared spectra, nuclear magnetic resonance spectroscopy, scanning electron microscopy, and high-performance gel permeation chromatography analysis. Result: The results indicated that LDGO-A was composed of D-glucose and D-xylose, and the average molecular sizes was approximately 945 Da. The anti-tumor activity of LDGO-A was evaluated in vivo. The inhibitory rate in mice treated with 40 mg/kg LDGO-A can reach 40.02%, being the highest in the three doses, which may be comparable to mannatide. Histology of immune organs shows that the tissues arranged more regular and firmer, but the tumor tissue arranged looser in LDGO-A group than those in the control group. Meanwhile, there is no obvious damage to other organs, such as heart. The anti-tumor activity of the LDGO-A was usually believed to be a consequence of the stimulation of the cell-mediated immune response because it can significantly promote the lymphocyte and macrophage cells in the dose range of 100–400 μg/mL in vitro. LDGO-A also effected the expression of some housekeeping genes mRNA in S180 tumor. Conclusion: Accordingly, the LDGO-A might serve as an effective healthcare food and source of natural anti-tumor compounds. PMID:26600715

  14. Soluble Factors Released by Endogenous Viable Cells Enhance the Antioxidant and Chemoattractive Activities of Cryopreserved Amniotic Membrane

    PubMed Central

    Duan-Arnold, Yi; Gyurdieva, Alexandra; Johnson, Amy; Jacobstein, Douglas A.; Danilkovitch, Alla

    2015-01-01

    Objective: Regulation of oxidative stress and recruitment of key cell types are activities of human amniotic membrane (hAM) that contribute to its benefits for wound treatment. Progress in tissue preservation has led to commercialization of hAM. The majority of hAM products are devitalized with various degrees of matrix alteration. Data show the importance of hAM matrix preservation, but little is known about the advantages of retaining viable endogenous cells. In this study, we compared the antioxidant and chemoattractive properties of viable intact cryopreserved hAM (int-hAM) and devitalized cryopreserved hAM (dev-hAM) to determine the benefits of cell preservation. Approach: We evaluated the ability of int-hAM and dev-hAM to protect fibroblasts from oxidant-induced cell damage, to suppress oxidants, and to recruit fibroblasts and keratinocytes in vitro. Results: Both the int-hAM–derived conditioned medium (CM) and the int-hAM tissue rescued significantly more fibroblasts from oxidant-induced damage than dev-hAM (844% and 93% more, respectively). The int-hAM CM showed a 202% greater antioxidant capacity than dev-hAM. The int-hAM CM enhanced the recruitment of fibroblasts and normal and diseased keratinocytes to a greater extent than dev-hAM (1,555%, 315%, and 151% greater, respectively). Innovation and Conclusion: Int-hAM, in which all native components are preserved, including endogenous viable cells, demonstrated a significantly greater antioxidant and fibroblast and keratinocyte chemoattractive potential compared to dev-hAM, in which viable cells are destroyed. The release of soluble factors that protect fibroblasts from oxidative injury by hAM containing viable cells is a mechanism of hAM antioxidant activity, which is a novel finding of this study. PMID:26029483

  15. Cinaciguat, a novel activator of soluble guanylate cyclase, protects against ischemia/reperfusion injury: role of hydrogen sulfide

    PubMed Central

    Salloum, Fadi N.; Das, Anindita; Samidurai, Arun; Hoke, Nicholas N.; Chau, Vinh Q.; Ockaili, Ramzi A.; Stasch, Johannes-Peter

    2012-01-01

    Cinaciguat (BAY 58–2667) is a novel nitric oxide (NO)-independent activator of soluble guanylate cyclase (sGC), which induces cGMP-generation and vasodilation in diseased vessels. We tested the hypothesis that cinaciguat might trigger protection against ischemia/reperfusion (I/R) in the heart and adult cardiomyocytes through cGMP/protein kinase G (PKG)-dependent generation of hydrogen sulfide (H2S). Adult New Zealand White rabbits were pretreated with 1 or 10 μg/kg cinaciguat (iv) or 10% DMSO (vehicle) 15 min before I/R or with 10 μg/kg cinaciguat (iv) at reperfusion. Additionally, adult male ICR mice were treated with either cinaciguat (10 μg/kg ip) or vehicle 30 min before I/R or at the onset of reperfusion (10 μg/kg iv). The PKG inhibitor KT5283 (KT; 1 mg/kg ip) or dl-propargylglycine (PAG; 50 mg/kg ip) the inhibitor of the H2S-producing enzyme cystathionine-γ-lyase (CSE) were given 10 and 30 min before cinaciguat. Cardiac function and infarct size were assessed by echocardiography and tetrazolium staining, respectively. Primary adult mouse cardiomyocytes were isolated and treated with cinaciguat before simulated ischemia/reoxygenation. Cinaciguat caused 63 and 41% reduction of infarct size when given before I/R and at reperfusion in rabbits, respectively. In mice, cinaciguat pretreatment caused a more robust 80% reduction in infarct size vs. 63% reduction when given at reperfusion and preserved cardiac function following I/R, which were blocked by KT and PAG. Cinaciguat also caused an increase in myocardial PKG activity and CSE expression. In cardiomyocytes, cinaciguat (50 nM) reduced necrosis and apoptosis and increased H2S levels, which was abrogated by KT. Cinaciguat is a novel molecule to induce H2S generation and a powerful protection against I/R injury in heart. PMID:22268103

  16. The activity of milk leukocytes in response to a water-soluble fraction of Mycobacterium phlei in bovine subclinical mastitis.

    PubMed

    Mukherjee, R; Ram, G C; Dash, P K; Goswami, T

    2004-01-01

    The effect of a water-soluble fraction (WSF) of a non-pathogenic strain of Mycobacterium phlei was studied in bovine subclinical mastitis (SCM) by measuring the myeloperoxidase and acid phosphatase enzyme levels in the milk leukocytes. Forty-five cows were divided into three equal groups. Group I, consisting of 15 healthy cows, served as the control, whereas groups II and III each contained 15 cows with subclinical mastitis on the basis of a positive reaction in the California mastitis test (CMT). The cows in group II received 100 microg of WSF in 5 ml sterile phosphate-buffered saline, pH 7.4 (PBS) once only, while those in group III received 5 ml sterile PBS daily for 7 days, both treatments being given by the intramammary route. Observations were made up to 30 days after treatment (AT). The CMT of the healthy milk was negative (0), whereas it ranged between 1 and 2 points in SCM. The somatic cell count (SCC) increased significantly (p < 0.05) on day 3, then fell steeply from day 7 up to day 30 AT in the cows in group II. A steady decrease in the total bacterial count (TBC) was observed in the group treated with WSF but the bacterial counts remained high in the groups treated with PBS. The mean acid phosphatase level was enhanced by 119% on day 3 AT in group II but only by 18.7% in the cows in group III. The mean myeloperoxidase level was enhanced by 100% in the cows in group II but only by 18% in those in group III on day 3 AT. This significant reduction in the bacterial load in infected cows caused by intramammary infusion of WSF may be due to activation of the microbicidal activity of the neutrophils, but this requires confirmation. PMID:14989362

  17. Preconditioning with soluble guanylate cyclase activation prevents postischemic inflammation and reduces nitrate tolerance in heme oxygenase-1 knockout mice.

    PubMed

    Wang, Walter Z; Jones, Allan W; Wang, Meifang; Durante, William; Korthuis, Ronald J

    2013-08-15

    Previously we have shown that, unlike wild-type mice (WT), heme oxygenase-1 knockout (HO-1-/-) mice developed nitrate tolerance and were not protected from inflammation caused by ischemia-reperfusion (I/R) when preconditioned with a H2S donor. We hypothesized that stimulation (with BAY 41-2272) or activation (with BAY 60-2770) of soluble guanylate cyclase (sGC) would precondition HO-1-/- mice against an inflammatory effect of I/R and increase arterial nitrate responses. Intravital fluorescence microscopy was used to visualize leukocyte rolling and adhesion to postcapillary venules of the small intestine in anesthetized mice. Relaxation to ACh and BAY compounds was measured on superior mesenteric arteries isolated after I/R protocols. Preconditioning with either BAY compound 10 min (early phase) or 24 h (late phase) before I/R reduced postischemic leukocyte rolling and adhesion to sham control levels and increased superior mesenteric artery responses to ACh, sodium nitroprusside, and BAY 41-2272 in WT and HO-1-/- mice. Late-phase preconditioning with BAY 60-2770 was maintained in HO-1-/- and endothelial nitric oxide synthase knockout mice pretreated with an inhibitor (dl-propargylglycine) of enzymatically produced H2S. Pretreatment with BAY compounds also prevented the I/R increase in small intestinal TNF-α. We speculate that increasing sGC activity and related PKG acts downstream to H2S and disrupts signaling processes triggered by I/R in part by maintaining low cellular Ca²⁺. In addition, BAY preconditioning did not increase sGC levels, yet increased the response to agents that act on reduced heme-containing sGC. Collectively these actions would contribute to increased nitrate sensitivity and vascular function. PMID:23771693

  18. Generation of soluble microbial products by bio-activated carbon filter during drinking water advanced treatment and its influence on spectral characteristics.

    PubMed

    Shen, Hong; Chen, Xin; Zhang, Dong; Chen, Hong-Bin

    2016-11-01

    In order to improve our understanding of bio-activated carbon (BAC) filter, the water quality of influent and effluent treated with BAC in a drinking water treatment plant (DWTP) of Shanghai during 2015 was valued. Combining the results from UV254, SUVA254, dissolved organic carbon (DOC) and scanning electron microscopic (SEM), it is found that performance of BAC treatment will be affected by characteristics of activated carbon (AC), which is relevant to the type of activated carbon (including shape and operating time) in this study. Fluorescence excitation-emission matrix (FEEM) shows that the humification index (HIX) and index of recent autochthonous contribution (BIX) is a reliable indicator to descript the variation of dissolved organic matter (DOM) during BAC process. The pattern of variation in BIX and HIX implies that soluble microbial products (SMPs) are formed and humic-like substances are removed during BAC treatment, which is also confirmed by the change of peaks of FEEM in BAC effluent. Large, positive correlations between SUVA254 and disinfection by-products formation potential yield (DBPFP yield) demonstrate that UV-absorbing DOM is directly related to the generation of DBPs. Poor correlations of HIX with DBPFP suggest that non-humic substances with UV-absorbing properties play an important role in the generation of DBPs in water with low SUVA254. Finally, strong but negative correlations between BIX and DBPFP suggest that vigorous microbial metabolism of BAC results in a decrease in DBPFP. However, the DBPFP yield will be enhanced for the generation of SMPs by BAC, especially in summer. PMID:27436775

  19. STYPu fuel form activities, March 1-September 30, 1985

    SciTech Connect

    Not Available

    1986-01-01

    The SRP portion of this report summarizes production STYPuO2 fuel forms for use in radioisotopic thermoelectric generators (RTG's) in the Plutonium Fuel Form (PuFF) Facility at the Savannah River Plant. The PuFF Facility began producing iridium-encapsulated, 62.5-watt STYPuO2 right circular cylinders for GPHS (General Purpose Heat Source) RTG's in June 1980; this program was completed in December 1983. The PuFF Facility has been placed in a production readiness mode of operation pending funding of additional heat source programs.

  20. New water-soluble ruthenium(II) terpyridine complexes for anticancer activity: synthesis, characterization, activation kinetics, and interaction with guanine derivatives.

    PubMed

    Rilak, Ana; Bratsos, Ioannis; Zangrando, Ennio; Kljun, Jakob; Turel, Iztok; Bugarčić, Živadin D; Alessio, Enzo

    2014-06-16

    With the aim of assessing whether ruthenium(II) compounds with meridional geometry might be utilized as potential antitumor agents, a series of new, water-soluble, monofunctional ruthenium(II) complexes of the general formula mer-[Ru(L3)(N-N)X][Y]n (where L3 = 2,2':6',2″-terpyridine (tpy) or 4'-chloro-2,2':6',2″-terpyridine (Cl-tpy), N-N = 1,2-diaminoethane (en), 1,2-diaminocyclohexane (dach), or 2,2'-bipyridine (bpy); X = Cl or dmso-S; Y = Cl, PF6, or CF3SO3; n = 1 or 2, depending on the nature of X) were synthesized. All complexes were fully characterized by elemental analysis and spectroscopic techniques (IR, UV/visible, and 1D and 2D NMR), and for three of them, i.e., [Ru(Cl-tpy)(bpy)Cl][Cl] (3Cl), [Ru(Cl-tpy)(en)(dmso-S)][Y]2 [Y = PF6 (6PF6), CF3SO3 (6OTf)] and [Ru(Cl-tpy)(bpy)(dmso-S)][CF3SO3]2 (8OTf), the X-ray structure was also determined. The new terpyridine complexes, with the exception of 8, are well soluble in water (>25 mg/mL). (1)H and (31)P NMR spectroscopy studies performed on the three selected complexes [Ru(Cl-tpy)(N-N)Cl](+) [N-N = en (1), dach (2), and bpy (3)] demonstrated that, after hydrolysis of the Cl ligand, they are capable of interacting with guanine derivatives [i.e., 9-methylguanine (9MeG) or guanosine-5'-monophosphate (5'-GMP)] through N7, forming monofunctional adducts with rates and extents that depend strongly on the nature of N-N: 1 ≈ 2 ≫ 3. In addition, compound 1 shows high selectivity toward 5'-GMP compared to adenosine-5'-monophosphate (5'-AMP), in a competition experiment. Quantitative kinetic investigations on 1 and 2 were performed by means of UV/visible spectroscopy. Overall, the complexes with bidentate aliphatic diamines proved to be superior to those with bpy in terms of solubility and reactivity (i.e., release of Cl(-) and capability to bind guanine derivatives). Contrary to the chlorido compounds, the corresponding dmso derivatives proved to be inert (viz., they do not release the monodentate ligand) in

  1. Mechanically driven activation of polyaniline into its conductive form.

    PubMed

    Baytekin, Bilge; Baytekin, H Tarik; Grzybowski, Bartosz A

    2014-07-01

    Mechanical treatment of polymers produces surface cations and anions which, as demonstrated here for the first time, can drive chemical reactions. In particular, it is shown that such a mechanical treatment transforms nonconductive polyaniline into its conductive form. These results provide a mechanical means of patterning conductive polymers and also coating small polymer objects with conductive polyaniline films preventing accumulation of static electricity. PMID:24824971

  2. Serum Soluble Urokinase-Type Plasminogen Activator Receptor Levels and Idiopathic FSGS in Children: A Single-Center Report

    PubMed Central

    Price, Heather E.; Gallon, Lorenzo; Langman, Craig B.

    2013-01-01

    Summary Background and objectives FSGS is the primary cause of childhood nephrotic syndrome leading to ESRD. Permeability factors, including circulating serum soluble urokinase-type plasminogen activator receptor (suPAR), have been postulated as putative causes in adults with primary FSGS. Similar results have yet to be proven in children. Design, setting, participants, & measurements This cross-sectional single-center study assessed the association of serum suPAR in children with FSGS or other glomerular and nonglomerular kidney diseases. Results This study examined 110 samples retrieved from 99 individuals (between January 2011 and April 2012), aged 1–21 years; of these individuals, 20 had primary FSGS, 24 had non-FSGS glomerular disease, 26 had nonglomerular kidney disease, and 29 were healthy controls. suPAR levels were not significantly different in children with FSGS, non-FSGS glomerular disease, and healthy controls (P>0.05). However, suPAR levels (median [25%–75%]) were higher in children with nonglomerular kidney disease (3385 pg/ml [2695–4392]) versus FSGS (2487 pg/ml [2191–3351]; P<0.05). Female patients with nephrotic-range proteinuria (U-Pr/Cr >2) had lower suPAR levels than those without proteinuria (2380 pg/ml [2116–2571] versus 3125 pg/ml [2516–4198], respectively; P<0.001). This trend was not seen among male participants; suPAR levels in all female participants were lower than in male participants (P=0.03). Thirty-four patients studied were kidney transplant recipients; transplant status was not associated with suPAR levels in patients with FSGS or non-FSGS diagnoses, independent of proteinuria, race, or sex (P>0.05). Conclusions On the basis of these results, circulating suPAR is unlikely the leading cause for childhood idiopathic FSGS. PMID:23620441

  3. Design of a Water Soluble Fluorescent 3-Hydroxy-4-Pyridinone Ligand Active at Physiological pH Values.

    PubMed

    Leite, Andreia; Silva, Ana M G; Coutinho, Catarina; Cunha-Silva, Luís; de Castro, Baltazar; Rangel, Maria

    2016-09-01

    In the present work we report the structure and the spectroscopic characterization of a new fluorescent 3-hydroxy-4-pyridinone ligand D-3,4-HPO. The synthesis of the compound was performed in two steps, which involve the reaction of the commercially available fluorophore dansyl chloride with a 3-hydroxy-4-pyridinone chelating unit and further deprotection. The new fluorescent chelator was characterized in the solid state by single-crystal X-ray diffraction and in solution by NMR, MS, absorption and fluorescence spectroscopies. The analysis of the variation of the absorption spectrum with pH allowed the determination of four pK a values (pK a1  = 3.50, pK a2  = 4.50, pK a3  = 9.60, pK a4  = 10.20) and establishment of the corresponding distribution diagram. The study of the fluorescence properties of the ligand show that in the pH range between 4 and 9 the fluorescence intensity is constant and has its maximum value thus allowing its further use at physiological pH values. The interaction of the ligand with copper(II) was accessed by fluorescence spectroscopy in MOPS buffer and the results show that the presence of copper(II) quenches the fluorescence of the ligand in ca 94 % at a ligand: metal ratio of 2:1. The latter result is consistent with the formation of a copper(II) complex with the bidentate ligand, as confirmed by the EPR spectroscopy. Graphical Abstract New water soluble fluorescent ligand active at physiological pH values. PMID:27357392

  4. Preparation of a Novel Form of Gelatin With a Three-Dimensional Ordered Macroporous Structure to Regulate the Release of Poorly Water-Soluble Drugs.

    PubMed

    Xu, Jie; Zhao, Zongzhe; Hao, Yanna; Zhao, Ying; Qiu, Yang; Jiang, Jie; Yu, Tong; Ji, Peng; Liu, Ying; Wu, Chao

    2016-09-01

    In this study, a novel three-dimensional ordered macroporous gelatin (3DOMG) was fabricated as a carrier for increasing the solubility of poorly water-soluble drugs, offering sustained release and a high oral bioavailability. Polymethyl methacrylate nanospheres (257 nm) were used as a colloidal plastic framework to synthesize 3DOMG. Fenofibrate (FNB) was selected as a model drug and loaded onto 3DOMG by the adsorption equilibrium method. Detailed characterization showed that the FNB absorbed onto 3DOMG was in a microcrystalline state. A fluorescence experiment and the prepared drug microcrystal network gave further information on the physical state of the drug. A degradation experiment proved that 3DOMG was readily biodegradable. In vitro release testing showed that 3DOMG increased the dissolution rate of FNB and produced a sustained release. An in vivo pharmacokinetic study confirmed that 3DOMG improved the oral bioavailability compared with that of commercial sustained-release capsules. These findings confirm that 3DOMG can be regarded as a promising carrier for an oral drug delivery system. PMID:26906173

  5. The solution structure of the soluble form of the lipid-modified azurin from Neisseria gonorrhoeae, the electron donor of cytochrome c peroxidase.

    PubMed

    Nóbrega, Cláudia S; Saraiva, Ivo H; Carreira, Cíntia; Devreese, Bart; Matzapetakis, Manolis; Pauleta, Sofia R

    2016-02-01

    Neisseria gonorrhoeae colonizes the genitourinary track, and in these environments, especially in the female host, the bacteria are subjected to low levels of oxygen, and reactive oxygen and nitrosyl species. Here, the biochemical characterization of N. gonorrhoeae Laz is presented, as well as, the solution structure of its soluble domain determined by NMR. N. gonorrhoeae Laz is a type 1 copper protein of the azurin-family based on its spectroscopic properties and structure, with a redox potential of 277±5 mV, at pH7.0, that behaves as a monomer in solution. The globular Laz soluble domain adopts the Greek-key motif, with the copper center located at one end of the β-barrel coordinated by Gly48, His49, Cys113, His118 and Met122, in a distorted trigonal geometry. The edge of the His118 imidazole ring is water exposed, in a surface that is proposed to be involved in the interaction with its redox partners. The heterologously expressed Laz was shown to be a competent electron donor to N. gonorrhoeae cytochrome c peroxidase. This is an evidence for its involvement in the mechanism of protection against hydrogen peroxide generated by neighboring lactobacilli in the host environment. PMID:26589091

  6. Monitoring the effects of chelating agents and electrical fields on active forms of Pb and Zn in contaminated soil.

    PubMed

    Tahmasbian, Iman; Safari Sinegani, Ali Akbar

    2013-11-01

    The application of electrical fields and chelating agents is an innovative hybrid technology used for the decontamination of soil polluted by heavy metals. The effects of four center-oriented electrical fields and chelating agents on active fractions of lead and zinc were investigated in this pot experiment. Ethylenediaminetetraacetic acid (EDTA) as a synthetic chelator and cow manure extract (CME) and poultry manure extract (PME) as natural chelators were applied to the pots (2 g kg(-1)) 30 days after the first irrigation. Two weeks later, four center-oriented electrical fields were applied in each pot (in three levels of 0, 10, and 30 V) for 1 h each day for 14 days. The soil near the cathode and anodes was collected and analyzed as cathodic and anodic soil, respectively. Results indicated that the soluble-exchangeable fraction of lead and zinc were decreased in the cathodic soil, while the carbonate-bound fractions were increased. In the anodic soil, however, the opposite result was observed. EDTA enhanced the soluble-exchangeable form of the metals in both anodic and cathodic soils. Furthermore, the amounts of carbonate-bound heavy metals were increased by the application of CME in both soils. The organic-bound fraction of the metals was increased by the application of natural chelators, while electrical fields had no significant impacts on this fraction. PMID:23685981

  7. Forming a Learning Culture to Promote Fracture Prevention Activities

    ERIC Educational Resources Information Center

    Hjalmarson, Helene V.; Strandmark, Margaretha

    2012-01-01

    Purpose: The purpose of this paper is to explore interprofessional experiences of incorporating fracture prevention activities in clinical practice inspired by an empowerment approach. Design/methodology/approach: Data collection consisted primarily of focus groups interviews, systematized and analyzed by the grounded theory method. The study took…

  8. A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts

    PubMed Central

    De Luca, Anastasia; Rotili, Dante; Carpanese, Debora; Lenoci, Alessia; Calderan, Laura; Scimeca, Manuel; Mai, Antonello; Bonanno, Elena; Rosato, Antonio; Geroni, Cristina; Quintieri, Luigi; Caccuri, Anna Maria

    2015-01-01

    We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo. PMID:25595904

  9. A novel orally active water-soluble inhibitor of human glutathione transferase exerts a potent and selective antitumor activity against human melanoma xenografts.

    PubMed

    De Luca, Anastasia; Rotili, Dante; Carpanese, Debora; Lenoci, Alessia; Calderan, Laura; Scimeca, Manuel; Mai, Antonello; Bonanno, Elena; Rosato, Antonio; Geroni, Cristina; Quintieri, Luigi; Caccuri, Anna Maria

    2015-02-28

    We designed and synthesized two novel nitrobenzoxadiazole (NBD) analogues of the anticancer agent 6-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)thio)hexan-1-ol (NBDHEX). The new compounds, namely MC3165 and MC3181, bear one and two oxygen atoms within the hydroxy-containing alkyl chain at the C4 position of the NBD scaffold, respectively. This insertion did not alter the chemical reactivity with reduced glutathione, while it conferred a remarkable increase in water solubility. MC3181 was more selective than NBDHEX towards the target protein, glutathione transferase P1-1, and highly effective in vitro against a panel of human melanoma cell lines, with IC50 in the submicromolar-low micromolar range. Interestingly, the cellular response to MC3181 was cell-type-specific; the compound triggered a JNK-dependent apoptosis in the BRAF-V600E-mutated A375 cells, while it induced morphological changes together with an increase in melanogenesis in BRAF wild-type SK23-MEL cells. MC3181 exhibited a remarkable therapeutic activity against BRAF-V600E-mutant xenografts, both after intravenous and oral administration. Outstandingly, no treatment-related signs of toxicity were observed both in healthy and tumor-bearing mice after single and repeated administrations. Taken together, these results indicate that MC3181 may represent a potential novel therapeutic opportunity for BRAF-mutated human melanoma, while being safe and water-soluble and thus overcoming all the critical aspects of NBDHEX in vivo. PMID:25595904

  10. A novel water-soluble benzothiazole derivative BD926 inhibits human activated T cell proliferation by down-regulating the STAT5 activation.

    PubMed

    Liu, Yang; Lai, Yi; Li, Hua; Liu, Jin; Luo, Xing-Yan; Li, Min-Hui; Yang, Tai; Wang, Yan-Tang; Yang, Shu-Xia; Li, Li-Mei; Zou, Qiang; Chen, Zheng-Liang

    2015-08-15

    Immunosuppressants are widely used for treatment of T cell-mediated autoimmune diseases and allogeneic graft rejection. However, because of the toxicity and tolerance of these drugs, novel immunosuppressants are urgently needed. We synthesized a series of novel water-soluble benzothiazole derivatives and found that BD926 [sodium 2-(benzo[d]thiazol-2-yl)-4,5,6,7-tetrahydro-2H-indazol-3-olate] had potent immunosuppressive activity. Treatment with BD926 significantly inhibited anti-CD3/anti-CD28 and alloantigen-induced human T cell proliferation as well as IL2-stimulated activated T cell proliferation in a dose-dependent manner in vitro. BD926 had no obvious cytotoxicity against human resting T cells, IL-4 treated activated T cells and fibroblast-like synoviocytes in our experimental conditions. Furthermore, BD926 induced cell cycle arrest at G0/G1 phase and inhibited the cyclin D3 and CDK 6 expression in activated T cells. BD926 inhibited the STAT5, but not Akt and p70S6K, phosphorylation in a dose-dependent manner in the IL-2-treated activated T cells. Interestingly, BD926 inhibited IFN-γ, IL-6 and IL-17, but not IL-2, IL-4 and IL-10, production in activated T cells. Finally, treatment with BD926 reduced delayed-type hypersensitivity in mice in a dose-dependent manner. Collectively, these data suggest that BD926 may be a lead compound for the design and development of new immunosuppressants for the intervention of allograft rejection and autoimmune diseases. PMID:25935419

  11. Solubility of cobalt in cement.

    PubMed

    Fregert, S; Gruvberger, B

    1978-02-01

    Unlike chromate, cobalt occurring as cobalt oxides in cement is not water-soluble in a detectable amount. Cobalt oxides are to some extent soluble in the presence of amino acids with which cobalt forms complexes. Such complexes can elicit patch test reactions. It is postulated that cobalt is more readily dissolved by forming complexes in eczematous skin than in normal skin. This may explain why cobalt sensitization in cement eczemas is secondary to chromate sensitivity. PMID:657784

  12. 3′-UTR engineering to improve soluble expression and fine-tuning of activity of cascade enzymes in Escherichia coli

    PubMed Central

    Song, Ji-Won; Woo, Ji-Min; Jung, Gyoo Yeol; Bornscheuer, Uwe T.; Park, Jin-Byung

    2016-01-01

    3′-Untranslated region (3′UTR) engineering was investigated to improve solubility of heterologous proteins (e.g., Baeyer-Villiger monooxygenases (BVMOs)) in Escherichia coli. Insertion of gene fragments containing putative RNase E recognition sites into the 3′UTR of the BVMO genes led to the reduction of mRNA levels in E. coli. Importantly, the amounts of soluble BVMOs were remarkably enhanced resulting in a proportional increase of in vivo catalytic activities. Notably, this increase in biocatalytic activity correlated to the number of putative RNase E endonucleolytic cleavage sites in the 3′UTR. For instance, the biotransformation activity of the BVMO BmoF1 (from Pseudomonas fluorescens DSM50106) in E. coli was linear to the number of RNase E cleavage sites in the 3′UTR. In summary, 3′UTR engineering can be used to improve the soluble expression of heterologous enzymes, thereby fine-tuning the enzyme activity in microbial cells. PMID:27406241

  13. Amyloid-β(1-42) protofibrils formed in modified artificial cerebrospinal fluid bind and activate microglia.

    PubMed

    Paranjape, Geeta S; Terrill, Shana E; Gouwens, Lisa K; Ruck, Benjamin M; Nichols, Michael R

    2013-03-01

    Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer's disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1-42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1-42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1-42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

  14. Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia

    PubMed Central

    Paranjape, Geeta S.; Terrill, Shana E.; Gouwens, Lisa K.; Ruck, Benjamin M.; Nichols, Michael R.

    2012-01-01

    Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia. PMID:23242692

  15. Soluble Urokinase-Type Plasminogen Activator Receptor Plasma Concentration May Predict Susceptibility to High Altitude Pulmonary Edema.

    PubMed

    Hilty, Matthias Peter; Zügel, Stefanie; Schoeb, Michele; Auinger, Katja; Dehnert, Christoph; Maggiorini, Marco

    2016-01-01

    Introduction. Acute exposure to high altitude induces inflammation. However, the relationship between inflammation and high altitude related illness such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) is poorly understood. We tested if soluble urokinase-type plasminogen activator receptor (suPAR) plasma concentration, a prognostic factor for cardiovascular disease and marker for low grade activation of leukocytes, will predict susceptibility to HAPE and AMS. Methods. 41 healthy mountaineers were examined at sea level (SL, 446 m) and 24 h after rapid ascent to 4559 m (HA). 24/41 subjects had a history of HAPE and were thus considered HAPE-susceptible (HAPE-s). Out of the latter, 10/24 HAPE-s subjects were randomly chosen to suppress the inflammatory cascade with dexamethasone 8 mg bid 24 h prior to ascent. Results. Acute hypoxic exposure led to an acute inflammatory reaction represented by an increase in suPAR (1.9 ± 0.4 at SL versus 2.3 ± 0.5 at HA, p < 0.01), CRP (0.7 ± 0.5 at SL versus 3.6 ± 4.6 at HA, p < 0.01), and IL-6 (0.8 ± 0.4 at SL versus 3.3 ± 4.9 at HA, p < 0.01) in all subjects except those receiving dexamethasone. The ascent associated decrease in PaO2 correlated with the increase in IL-6 (r = 0.46, p < 0.001), but not suPAR (r = 0.27, p = 0.08); the increase in IL-6 was not correlated with suPAR (r = 0.16, p = 0.24). Baseline suPAR plasma concentration was higher in the HAPE-s group (2.0 ± 0.4 versus 1.8 ± 0.4, p = 0.04); no difference was found for CRP and IL-6 and for subjects developing AMS. Conclusion. High altitude exposure leads to an increase in suPAR plasma concentration, with the missing correlation between suPAR and IL-6 suggesting a cytokine independent, leukocyte mediated mechanism of low grade inflammation. The correlation between IL-6 and PaO2 suggests a direct effect of hypoxia, which is not the case for suPAR. However, suPAR plasma concentration measured before hypoxic exposure may predict

  16. Soluble Urokinase-Type Plasminogen Activator Receptor Plasma Concentration May Predict Susceptibility to High Altitude Pulmonary Edema

    PubMed Central

    Zügel, Stefanie; Schoeb, Michele; Auinger, Katja; Dehnert, Christoph; Maggiorini, Marco

    2016-01-01

    Introduction. Acute exposure to high altitude induces inflammation. However, the relationship between inflammation and high altitude related illness such as high altitude pulmonary edema (HAPE) and acute mountain sickness (AMS) is poorly understood. We tested if soluble urokinase-type plasminogen activator receptor (suPAR) plasma concentration, a prognostic factor for cardiovascular disease and marker for low grade activation of leukocytes, will predict susceptibility to HAPE and AMS. Methods. 41 healthy mountaineers were examined at sea level (SL, 446 m) and 24 h after rapid ascent to 4559 m (HA). 24/41 subjects had a history of HAPE and were thus considered HAPE-susceptible (HAPE-s). Out of the latter, 10/24 HAPE-s subjects were randomly chosen to suppress the inflammatory cascade with dexamethasone 8 mg bid 24 h prior to ascent. Results. Acute hypoxic exposure led to an acute inflammatory reaction represented by an increase in suPAR (1.9 ± 0.4 at SL versus 2.3 ± 0.5 at HA, p < 0.01), CRP (0.7 ± 0.5 at SL versus 3.6 ± 4.6 at HA, p < 0.01), and IL-6 (0.8 ± 0.4 at SL versus 3.3 ± 4.9 at HA, p < 0.01) in all subjects except those receiving dexamethasone. The ascent associated decrease in PaO2 correlated with the increase in IL-6 (r = 0.46, p < 0.001), but not suPAR (r = 0.27, p = 0.08); the increase in IL-6 was not correlated with suPAR (r = 0.16, p = 0.24). Baseline suPAR plasma concentration was higher in the HAPE-s group (2.0 ± 0.4 versus 1.8 ± 0.4, p = 0.04); no difference was found for CRP and IL-6 and for subjects developing AMS. Conclusion. High altitude exposure leads to an increase in suPAR plasma concentration, with the missing correlation between suPAR and IL-6 suggesting a cytokine independent, leukocyte mediated mechanism of low grade inflammation. The correlation between IL-6 and PaO2 suggests a direct effect of hypoxia, which is not the case for suPAR. However, suPAR plasma concentration measured before hypoxic exposure may predict

  17. Investigation of solubilising effects of bile salts on an active pharmaceutical ingredient with unusual pH dependent solubility by NMR spectroscopy.

    PubMed

    Vogtherr, M; Marx, A; Mieden, A-C; Saal, C

    2015-05-01

    The interaction between an ampholytic and amphiphilic Active Pharmaceutical Ingredient (API) showing unusual pH dependent solubility and Fasted State Simulated Intestinal Fluid (FaSSIF) was studied by NMR spectroscopy. Solubility in FaSSIF was drastically increased, about 30 fold, compared to simulated gastrointestinal fluid without bile salts. Our studies aimed at understanding the mechanisms that lead to this drastic enhancement. All species present in solution at various concentrations of API were characterised by Diffusion Ordered Spectroscopy (DOSY) NMR measurements. These indicated the presence of mixed taurocholate-lecithin and pure taurocholate micelles in pure FaSSIF, and formation of mixed taurocholate-API micelles after addition of API. The formation of taurocholate-API micelles was also supported by Nuclear Overhauser Effect/Enhancement (NOE) contacts between taurocholate and the API. Formation of mixed taurocholate-API micelles took place at the expense of pure taurocholate micelles, whereas mixed taurocholate-lecithin micelles remained uninfluenced by the presence of API. Our results showed that the increase in solubility was due to similar amphiphilic properties of the API and taurocholate which enabled formation of mixed taurocholate-API micelles. From results of determination of solubility as well as NMR experiments a phase diagram comprising several micellar species was derived. PMID:25720817

  18. Redox activity and in vitro bioactivity of the water-soluble fraction of urban particulate matter in relation to particle size and chemical composition.

    PubMed

    Velali, Ekaterini; Papachristou, Eleni; Pantazaki, Anastasia; Choli-Papadopoulou, Theodora; Planou, Styliani; Kouras, Athanasios; Manoli, Evangelia; Besis, Athanasios; Voutsa, Dimitra; Samara, Constantini

    2016-01-01

    Chemical and toxicological characterization of the water-soluble fraction of size-segregated urban particulate matter (PM) (<0.49, 0.49-0.97, 0.97-1.5, 1.5-3.0, 3.0-7.2 and >7.2 μm) was carried out at two urban sites, traffic and urban background, during the cold and the warm period. Chemical analysis of the water-soluble PM fraction included ionic species (NO3(-), SO4(2-), Cl(-), Na(+), NH4(+), K(+), Mg(2+), Ca(2+)), water-soluble organic carbon (WSOC), and trace elements (Al, As, Ba, Cd, Cr, Cu, Fe, Pb, Mn, Ni, Zn, Pt, Pd, Rh, Ru, Ir, Ca, and Mg). The dithiothreitol (DTT) assay was employed for the abiotic assessment of the oxidative PM activity. Cytotoxic responses were investigated in vitro by applying the mitochondrial dehydrogenase (MTT) and the lactate dehydrogenase (LDH) bioassays on human lung cells (MRC-5), while DNA damage was estimated by the single cell gel electrophoresis assay, known as Comet assay. The correlations between the observed bioactivity responses and the concentrations of water-soluble chemical PM constituents in the various size ranges were investigated. The results of the current study corroborate that short-term bioassays using lung human cells and abiotic assays, such as the DTT assay, could be relevant to complete the routine chemical analysis and to obtain a preliminary screening of the potential effects of PM-associated airborne pollutants on human health. PMID:26586634

  19. Active Curved Polymers Form Vortex Patterns on Membranes

    NASA Astrophysics Data System (ADS)

    Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

    2016-04-01

    Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation.

  20. Active Curved Polymers Form Vortex Patterns on Membranes.

    PubMed

    Denk, Jonas; Huber, Lorenz; Reithmann, Emanuel; Frey, Erwin

    2016-04-29

    Recent in vitro experiments with FtsZ polymers show self-organization into different dynamic patterns, including structures reminiscent of the bacterial Z ring. We model FtsZ polymers as active particles moving along chiral, circular paths by Brownian dynamics simulations and a Boltzmann approach. Our two conceptually different methods point to a generic phase behavior. At intermediate particle densities, we find self-organization into vortex structures including closed rings. Moreover, we show that the dynamics at the onset of pattern formation is described by a generalized complex Ginzburg-Landau equation. PMID:27176542

  1. Alkali-Activated Fly ash-slag Cement based nuclear waste forms

    SciTech Connect

    Jiang, W.; Wu, X.; Roy, D.M.

    1993-12-31

    This paper is based on the results of an in-progress research project on Alkali-Activated Cement System at MRL. The objective of this research is to establish the potential for large volume use of fly ash and slag as main components of the cement system. Alkali-activated Fly ash-slag Cement (AFC) was studied as a matrix for immobilization of nuclear waste. AFC is characterized by high early strength, high ultimate strength, low porosity, lower solubilities of the hydrates, and high resistance to chemical corrosion as well as to freezing and thawing. All these advanced properties are particularly favorable to the immobilization the nuclear wastes.

  2. 75 FR 21013 - Agency Information Collection Activities: Form N-644; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-04-22

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-644... Collection Under Review; Form N- 644, Application for Posthumous Citizenship; OMB Control No. 1615-0059. The..., USCIS will be evaluating whether to revise the Form N-644. Should USCIS decide to revise Form N-644...

  3. 76 FR 21913 - Agency Information Collection Activities: Form N-644; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-04-19

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-644... Collection Under Review: Form N- 644, Application for Posthumous Citizenship; OMB Control No. 1615-0059. The..., USCIS will be evaluating whether to revise the Form N-644. Should USCIS decide to revise Form N-644...

  4. 75 FR 51095 - Agency Information Collection Activities: Form N-336; Extension of an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-08-18

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-336... Collection under Review; Form N- 336, Request for Hearing on a Decision in Naturalization Proceedings Under... the Form N-336. Should USCIS decide to revise Form N-336 we will advise the public when we publish...

  5. New ideas about the solubility of drugs.

    PubMed

    Box, Karl; Comer, John E; Gravestock, Tom; Stuart, Martin

    2009-11-01

    Methods are described for detecting precipitation of ionisable drugs under conditions of changing pH, estimating kinetic solubility from the onset of precipitation, and measuring solubility by chasing equilibrium. Definitions are presented for kinetic, equilibrium, and intrinsic solubility of ionisable drugs, supersaturation and subsaturation, and for chasers and non-chasers, which are two classes of ionisable drug with significantly different solubility properties. The use of Bjerrum Curves and Neutral-Species Concentration Profiles to depict solubility properties are described and illustrated with case studies showing super-dissolving behaviour, conversion between crystalline forms and enhancement of solubility through supersaturation, and the use of additives and simulated gastrointestinal fluids. PMID:19937815

  6. Preparation of water-soluble melanin from squid ink using ultrasound-assisted degradation and its anti-oxidant activity.

    PubMed

    Guo, Xin; Chen, Shiguo; Hu, Yaqin; Li, Guoyun; Liao, Ningbo; Ye, Xingqian; Liu, Donghong; Xue, Changhu

    2014-12-01

    Water-soluble squid melanin fractions were firstly prepared using ultrasound-assistant degradation method under alkaline condition, which is optimized by response surface methodology. The processing melanin fractions were divided into different molecular weight (Mw) fractions by membrane separation (below 10 kDa, among 10-50 kDa and over 50 kDa). The AFM image and particle-size analysis showed monomer units of the melanin were destroyed, and huge polymers were degraded into smaller soluble particles after ultrasound. While, UV, IR and solid (13)C NMR spectra indicated that the basic structure of melanin fraction was still retained after ultrasound process. Further analysis showed soluble melanin fractions obtained in 0.5 and 1 M NaOH, with Mw above 10 kDa exhibited much higher in vitro antioxidant potency. The IC50 of these fractions (IC50 among 19-80 μg) on scavenging O 2 ∙¯ is more efficient than carnosine (IC50 = 355 μg/ml.), a commercialized antioxidant. They (IC50 mong 115-180 μg/ml) are as efficient as carnosine (IC50 = 110 μg/ml) on scavenging ∙OH. Our research has reported a novel method for preparation of water-soluble melanin fractions from squid ink, which could be a promising free radical scavenger from nature resource. PMID:25477634

  7. Subphthalocyanines: addressing water-solubility, nano-encapsulation, and activation for optical imaging of B16 melanoma cells.

    PubMed

    Bernhard, Yann; Winckler, Pascale; Chassagnon, Remi; Richard, Philippe; Gigot, Élodie; Perrier-Cornet, Jean-Marie; Decréau, Richard A

    2014-11-21

    Water-soluble disulfonato-subphthalocyanines (SubPcs) or hydrophobic nano-encapsulated SubPcs are efficient probes for the fluorescence imaging of cells. 20 nm large liposomes (TEM and DLS) incorporated about 13% SubPc. Moreover, some of these fluorophores were found to be pH activatable. PMID:25266256

  8. Water-soluble gold nanoclusters prepared by protein-ligand interaction as fluorescent probe for real-time assay of pyrophosphatase activity.

    PubMed

    Deng, Hao-Hua; Wang, Fei-Fei; Shi, Xiao-Qiong; Peng, Hua-Ping; Liu, Ai-Lin; Xia, Xing-Hua; Chen, Wei

    2016-09-15

    This paper reports a new and facile method for the synthesis of water-soluble thiolate-protected AuNCs via protein-ligand interaction. Using 3-mercaptopropionic acid (MPA) as a model ligand and bovine serum albumin (BSA) as a model protein, water-soluble AuNCs (BSA/MPA-AuNCs) with intense orange-yellow fluorescent emission (quantum yield=16%) are obtained. Results show that AuNCs produced with this method have hydrophobic interactions with BSA. The synthetic strategy is then successfully extended to produce water-soluble AuNCs protected by other thiolates. Moreover, a sensitive and eco-friendly sensing system is established for detection of the activity of inorganic pyrophosphatase (PPase), which relies on the selective coordination of Fe(3+)with BSA/MPA-AuNCs, the higher affinity between pyrophosphate (PPi) and Fe(3+), and the hydrolysis of PPi by PPase. A good linearity between the fluorescence intensity and PPase activity within the range from 0.1 to 3U/L is found, with a detection limit down to 0.07U/L. Additionally, the fluorescent assay developed here is utilized to assay the PPase activity in real biological samples and as well as to evaluate PPase inhibitor, illustrating the great potential for biological analysis. PMID:27093483

  9. Heat of Hydration of Low Activity Cementitious Waste Forms

    SciTech Connect

    Nasol, D.

    2015-07-23

    During the curing of secondary waste grout, the hydraulic materials in the dry mix react exothermally with the water in the secondary low-activity waste (LAW). The heat released, called the heat of hydration, can be measured using a TAM Air Isothermal Calorimeter. By holding temperature constant in the instrument, the heat of hydration during the curing process can be determined. This will provide information that can be used in the design of a waste solidification facility. At the Savannah River National Laboratory (SRNL), the heat of hydration and other physical properties are being collected on grout prepared using three simulants of liquid secondary waste generated at the Hanford Site. From this study it was found that both the simulant and dry mix each had an effect on the heat of hydration. It was also concluded that the higher the cement content in the dry materials mix, the greater the heat of hydration during the curing of grout.

  10. Identification of biotransformation products of citalopram formed in activated sludge.

    PubMed

    Beretsou, Vasiliki G; Psoma, Aikaterini K; Gago-Ferrero, Pablo; Aalizadeh, Reza; Fenner, Kathrin; Thomaidis, Nikolaos S

    2016-10-15

    Citalopram (CTR) is a worldwide highly consumed antidepressant which has demonstrated incomplete removal by conventional wastewater treatment. Despite its global ubiquitous presence in different environmental compartments, little is known about its behaviour and transformation processes during wastewater treatment. The present study aims to expand the knowledge on fate and transformation of CTR during the biological treatment process. For this purpose, batch reactors were set up to assess biotic, abiotic and sorption losses of this compound. One of the main objectives of the study was the identification of the formed transformation products (TPs) by applying suspect and non-target strategies based on liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). The complementary use of reversed phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) for the identification of polar TPs, and the application of in-house developed quantitative structure-retention relationship (QSRR) prediction models, in addition to the comprehensive evaluation of the obtained MS/MS spectra, provided valuable information to support identification. In total, fourteen TPs were detected and thirteen of them were tentatively identified. Four compounds were confirmed (N-desmethylCTR, CTR amide, CTR carboxylic acid and 3-oxo-CTR) through the purchase of the corresponding reference standard. Probable structures based on diagnostic evidence were proposed for the additional nine TPs. Eleven TPs are reported for the first time. A transformation pathway for the biotransformation of CTR was proposed. The presence of the identified TPs was assessed in real wastewater samples through retrospective analysis, resulting in the detection of five compounds. Finally, the potential ecotoxicological risk posed by CTR and its TPs to different trophic levels of aquatic organisms was evaluated by means of risk quotients. PMID:27459150

  11. Wound healing activity of topical application forms based on ayurveda.

    PubMed

    Datta, Hema Sharma; Mitra, Shankar Kumar; Patwardhan, Bhushan

    2011-01-01

    The traditional Indian medicine-Ayurveda, describes various herbs, fats, oils and minerals with anti-aging as well as wound healing properties. With aging, numerous changes occur in skin, including decrease in tissue cell regeneration, decrease in collagen content, loss of skin elasticity and mechanical strength. We prepared five topical anti-aging formulations using cow ghee, flax seed oil, Phyllanthus emblica fruits, Shorea robusta resin, Yashada bhasma as study materials. For preliminary efficacy evaluation of the anti-aging activity we chose excision and incision wound healing animal models and studied the parameters including wound contraction, collagen content and skin breaking strength which in turn is indicative of the tissue cell regeneration capacity, collagenation capacity and mechanical strength of skin. The group treated with the formulations containing Yashada bhasma along with Shorea robusta resin and flax seed oil showed significantly better wound contraction (P < .01), higher collagen content (P < .05) and better skin breaking strength (P < .01) as compared to control group; thus proposing them to be effective prospective anti-aging formulations. PMID:19252191

  12. Wound Healing Activity of Topical Application Forms Based on Ayurveda

    PubMed Central

    Datta, Hema Sharma; Mitra, Shankar Kumar; Patwardhan, Bhushan

    2011-01-01

    The traditional Indian medicine—Ayurveda, describes various herbs, fats, oils and minerals with anti-aging as well as wound healing properties. With aging, numerous changes occur in skin, including decrease in tissue cell regeneration, decrease in collagen content, loss of skin elasticity and mechanical strength. We prepared five topical anti-aging formulations using cow ghee, flax seed oil, Phyllanthus emblica fruits, Shorea robusta resin, Yashada bhasma as study materials. For preliminary efficacy evaluation of the anti-aging activity we chose excision and incision wound healing animal models and studied the parameters including wound contraction, collagen content and skin breaking strength which in turn is indicative of the tissue cell regeneration capacity, collagenation capacity and mechanical strength of skin. The group treated with the formulations containing Yashada bhasma along with Shorea robusta resin and flax seed oil showed significantly better wound contraction (P < .01), higher collagen content (P < .05) and better skin breaking strength (P < .01) as compared to control group; thus proposing them to be effective prospective anti-aging formulations. PMID:19252191

  13. [L forms of Staphylococcus aureus. Behavior of coagulase, hemolytic and desoxyribonuclease activities and antibiotic sensitivity].

    PubMed

    Loschiavo, F; Giarrizzo, S

    1977-01-01

    L Forms derived from strains of coagulase positive Staphylococcus aureus, have, on the whole, preserved their DNAsic, haemolitic and coagulastic activities. L. forms showed high resistence to antibiotics acting on the bacterial cell-wall. The sensibility to other antibiotics was, roughly, analogous for the L forms as well as for the bacterial strains ones, with the exception of the clortetraciclin and the diidrostreptomicin, ehich proved to be comparatively more active on the L forms. PMID:614141

  14. Five water-soluble zwitterionic copper(II)-carboxylate polymers: role of dipyridyl coligands in enhancing the DNA-binding, cleaving and anticancer activities.

    PubMed

    Chen, Ming; Tang, Xiao-Yan; Yang, Shui-Ping; Li, Huan-Huan; Zhao, Hai-Qing; Jiang, Zhi-Hong; Chen, Jin-Xiang; Chen, Wen-Hua

    2015-08-01

    Five water-soluble zwitterionic copper-carboxylate polymers were prepared from the reaction of N-carboxymethyl-(3,5-dicarboxyl)pyridinium bromide (H3CmdcpBr) with Cu(NO3)2 in the presence of NaOH by modulating the temperature, solvent and ancillary dipyridyl ligands. These complexes include a 1D ladder-shaped polymer {[Cu3(Cmdcp)2(OH)2(H2O)2]·H2O}n () formed in H2O at room temperature, and a 2D network polymer {[Cu(Cmdcp) (H2O)2]·2H2O}n () isolated in H2O at 135 °C. At 100 °C in H2O/DMF, the same reaction in the presence of an additional 2,2'-bipyridine (bipy) gave a 2D zwitterionic complex {[Cu(Cmdcp)(bipy)]·3H2O}n () together with a 1D double-stranded polymer {[Cu(Cmdcp)(H2O)2]·H2O}n () as a minor product. The replacement of bipy with phenanthroline (phen) afforded a 1D zigzag polymer chain {[Cu(Cmdcp)(phen)(H2O)]2·9H2O}5 (). All these complexes were characterized by IR, elemental analyses and single crystal X-ray crystallography. Agarose gel electrophoresis (GE) and ethidium bromide (EB) displacement experiments indicated that complex exhibited the highest pBR322 DNA cleaving ability with the catalytic efficiency (kmax/KM) of 14.80 h(-1) mM(-1) and the highest binding affinity toward calf-thymus DNA. The MTT assay indicated that complex showed significant inhibitory activity toward the proliferation of several tumor cells. Its IC50 value was at micromolar level and lower than those of cisplatin and complexes , especially toward resistant lung adenocarcinoma cell A549. PMID:26131732

  15. Opposing Roles of Membrane and Soluble Forms of the Receptor for Advanced Glycation End Products in Primary Respiratory Syncytial Virus Infection

    PubMed Central

    Miller, Allison L.; Sims, Gary P.; Brewah, Yambasu A.; Rebelatto, Marlon C.; Kearley, Jennifer; Benjamin, Ebony; Keller, Ashley E.; Brohawn, Philip; Herbst, Ronald; Coyle, Anthony J.; Kolbeck, Roland

    2012-01-01

    Respiratory syncytial virus (RSV), a common respiratory pathogen in infants and the older population, causes pulmonary inflammation and airway occlusion that leads to impairment of lung function. Here, we have established a role for receptor for advanced glycation end products (RAGE) in RSV infection. RAGE-deficient (ager−/−) mice were protected from RSV-induced weight loss and inflammation. This protection correlated with an early increase in type I interferons, later decreases in proinflammatory cytokines, and a reduction in viral load. To assess the contribution of soluble RAGE (sRAGE) to RSV-induced disease, wild-type and ager−/− mice were given doses of sRAGE following RSV infection. Of interest, sRAGE treatment prevented RSV-induced weight loss and neutrophilic inflammation to a degree similar to that observed in ager−/− mice. Our work further elucidates the roles of RAGE in the pathogenesis of respiratory infections and highlights the opposing roles of membrane and sRAGE in modulating the host response to RSV infection. PMID:22262795

  16. A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

    SciTech Connect

    Rowe, Cynthia L.; Connolly, Sarah A.; Chen, Jia; Jardetzky, Theodore S.; Longnecker, Richard

    2013-02-05

    We investigated whether soluble EBV gH/gL (sgH/gL) functions in fusion and made a series of truncations of gH/gL domains based on the gH/gL crystal structure. We found sgH/gL failed to mediate cell-cell fusion both when co-expressed with the other entry glycoproteins and when added exogenously to fusion assays. Interestingly, sgH/gL inhibited cell-cell fusion in a dose dependent manner when co-expressed. sgH/gL from HSV was unable to inhibit EBV fusion, suggesting the inhibition was specific to EBV gH/gL. sgH/gL stably binds gp42, but not gB nor gH/gL. The domain mutants, DI/gL, DI-II/gL and DI-II-III/gL were unable to bind gp42. Instead, DI-II/gL, DI-II-III/gL and sgH/gL but not DI/gL decreased the expression of gp42, resulting in decreased overall fusion. Overall, our results suggest that domain IV may be required for proper folding and the transmembrane domain and cytoplasmic tail of EBV gH/gL are required for the most efficient fusion.

  17. DNA-damaging activity in ethanol-soluble fractions of feces from New Zealand groups at varying risks of colorectal cancer.

    PubMed

    Ferguson, L R; Alley, P G; Gribben, B M

    1985-01-01

    Using repair-proficient and repair-deficient strains of E. coli, we investigated the application of a liquid incubation assay to measure the DNA-damaging activity of ethanol-soluble fecal extracts. This method appears to be suitable for the study of a wide range of sample types. It was used to measure the DNA-modifying activity of ethanol-soluble fecal extracts from a group of European colorectal cancer patients. Data were compared with those from Europeans of similar age and sex distribution who did not have bowel cancer. We also studied groups of Maoris, Samoans, and European Seventh-Day Adventists who followed an ovo-lacto vegetarian diet. There are significant levels of DNA-modifying materials in the feces of many Europeans on a mixed diet, regardless of whether or not they have cancer. The number of positive samples was less in the Polynesian groups, and there were no samples that could be unequivocally scored as positive in the Seventh-Day Adventist groups. We conclude that diet can significantly reduce the level of ethanol-soluble mutagens, at least in New Zealand Europeans. The data may provide an explanation for the reduced incidence of bowel cancer in Seventh-Day Adventist groups. PMID:3906579

  18. Bactericidal activity of juvenile chinook salmon macrophages against Aeromonas salmonicida after exposure to live or heat-killed Renibacterium salmoninarum or to soluble proteins produced by R. salmoninarum

    USGS Publications Warehouse

    Siegel, D.C.; Congleton, J.L.

    1997-01-01

    Macrophages isolated from the anterior kidney of juvenile chinook salmon Oncorhynchus tshawytscha in 96-well microtiter plates were exposed for 72 h to 0, 105, or 106 live or heat-killed Renibacterium salmoninarum cells per well or to 0, 0.1, 1.0, or 10 ??g/mL of R. salmoninarum soluble proteins. After treatment, the bactericidal activity of the macrophages against Aerornonas salmonicida was determined by a colorimetric assay based on the reduction of the tetrazolium dye MTT to formazan by viable bacteria. The MTT assay was modified to allow estimation of the percentage of bacteria killed by reference to a standard curve relating the number of bacteria added to microtiter wells to absorbance by formazan at 600 nm. The live and heat-killed R. salmoninarum treatments significantly (P < 0.001) increased killing of A. salmonicida by chinook salmon macrophages. In each of the five trials, significantly (P < 0.05) greater increases in killing occurred after exposure to 105 R. salmoninarum cells than to 106 R. salmoninarum cells per well. In contrast, treatment of macrophages with 10 ??g/mL R. salmoninarum soluble proteins significantly (P < 0.001) decreased killing of A. salmonicida, but treatment with lower doses did not. These results show that the bactericidal activity of chinook salmon macrophages is stimulated by exposure to R. salmoninarum cells at lower dose levels but inhibited by exposure to R. salmoninarum cells or soluble proteins at higher dose levels.

  19. Functionalized poly(ethylene glycol)-stabilized water-soluble palladium nanoparticles: property/activity relationship for the aerobic alcohol oxidation in water.

    PubMed

    Feng, Bo; Hou, Zhenshan; Yang, Hanmin; Wang, Xiangrui; Hu, Yu; Li, Huan; Qiao, Yunxiang; Zhao, Xiuge; Huang, Qingfa

    2010-02-16

    The preparation, characterization, and catalytic properties of water-soluble palladium nanoparticles stabilized by the functionalized-poly(ethylene glycol) as a protective ligand were demonstrated for aerobic oxidation of alcohols in aqueous phase. UV/vis spectra and X-ray photoelectron spectroscopy (XPS) proved that there was an electronic interaction between the bidentate nitrogen ligand and palladium atoms. Transmission electron microscopy and XPS analysis showed that the particle size and surface properties of the generated palladium nanoparticles can be controlled by varying the amount of protective ligand and the kinds of reducing agents. It was found that both the size and surface properties of palladium nanoparticles played very important roles in affecting catalytic performance. The stabilized metallic palladium nanoparticles were proven to be the active centers for benzyl alcohol oxidation in the present system, and the water-soluble Pd nanocatalysts can also be extended to the selective oxidation of various alcohols. PMID:20039597

  20. Chimpanzees Immunized with Recombinant Soluble CD4 Develop Anti-Self CD4 Antibody Responses with Anti-Human Immunodeficiency Virus Activity

    NASA Astrophysics Data System (ADS)

    Watanabe, Mamoru; Boyson, Jonathan E.; Lord, Carol I.; Letvin, Norman L.

    1992-06-01

    In view of the efficiency with which human immunodeficiency virus replication can be blocked in vitro with anti-CD4 antibodies, the elicitation of an anti-CD4 antibody response through active immunization might represent a useful therapeutic strategy for AIDS. Here we demonstrate that immunization of chimpanzees with recombinant soluble human CD4 elicited an anti-CD4 antibody response. The elicited antibody bound self CD4 on digitonin-treated but not freshly isolated lymphocytes. Nevertheless, this antibody blocked human immunodeficiency virus replication in chimpanzee and human lymphocytes. These observations suggest that immunization with recombinant soluble CD4 from human immunodeficiency virus-infected humans may be feasible and therapeutically beneficial.

  1. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  2. Use of biorelevant media for assessment of a poorly soluble weakly basic drug in the form of liquisolid compacts: in vitro and in vivo study.

    PubMed

    Badawy, Mahmoud A; Kamel, Amany O; Sammour, Omaima A

    2016-01-01

    The purpose of this work is to use biorelevant media to evaluate the robustness of a poorly water soluble weakly basic drug to variations along the gastrointestinal tract (GIT) after incorporation in liquisolid compacts and to assess the success of these models in predicting the in vivo performance. Liquisolid tablets were prepared using mosapride citrate as a model drug. A factorial design experiment was used to study the effect of three factors, namely: drug concentration at two levels (5% and 10%), carriers at three levels (avicel, mannitol and lactose) and powder excipients ratio (R) of the coating material at two levels (25 and 30). The in vitro dissolution media utilized were 0.1 N HCl, hypoacidic stomach model and a transfer model simulating the transfer from the stomach to the intestine. All compacts released above 95% of drug after 10 min in 0.1 N HCl. In the hypoacidic model, the compacts with R 30 were superior compared to R 25, where they released >90% of drug after 10 min compared to 80% for R 25. After the transfer of the optimum compacts from Simulated gastric fluid fast (SGFfast) to fasted state simulated intestinal fluid, slight turbidity appeared after 30 min, and the amount of drug dissolved slightly decreased from 96.91% to 90.59%. However, after the transfer from SGFfast to fed state simulated intestinal fluid, no turbidity or precipitation occurred throughout time of the test (60 min). In vivo pharmacokinetic study in human volunteers proved the success of the in vitro models with enhancement of the oral bioavailability (121.20%) compared to the commercial product. PMID:24892630

  3. Effect of water soluble extract of nacre (Pinctada maxima) on alkaline phosphatase activity and Bcl-2 expression in primary cultured osteoblasts from neonatal rat calvaria.

    PubMed

    Moutahir-Belqasmi, F; Balmain, N; Lieberrher, M; Borzeix, S; Berland, S; Barthelemy, M; Peduzzi, J; Milet, C; Lopez, E

    2001-01-01

    The nacre (mother of pearl) layer of the oyster Pinctada maxima shell can initiate bone formation by human osteoblasts in vivo and in vitro and is a new biomaterial that induces osteogenesis. This activity of nacre could be due to its water-soluble matrix. We examined the action of a water-soluble extract of nacre on the osteoblast phenotype of cells isolated from rat neonatal calvaria by measuring alkaline phosphatase (ALP) activity and by localization of the anti-apoptotic protein Bcl-2 by immunocytochemistry. ALP activity was increased 7% (p<0.001) by 100 microg proteins/ml extract and 20% (p<0.001) by 50 microg proteins/ml extract, but a low concentration of extract decreased the ALP activity by 8%. Cells treated with a high aspartic acid content fraction of the extract had increased ALP activity (23%, p<0.0001). Nacre extract and the fraction have no effect on the proliferation of mature osteoblasts. Immunoreactive Bcl-2 was overproduced in the cytoplasm and nuclei of osteoblasts at all stages of culture. Bcl-2 was found over the whole chromatin in quiescent and mitotic cells at the end of mitosis in the two nuclei in one cell, before cytodieresis. Bcl-2 was also found over chromosomes. Thus, nacre extract stimulates Bcl-2 production in osteoblasts, that is correlated with the cell cycle. Bcl-2 was also abundant in the nucleoli of extract-treated cells. Thus, the concentration and subcellular distribution of Bcl-2 in osteoblasts in primary cultures is influenced by nacre extract, and related to the cell cycle and the regulation of gene expression. Hence, knowledge of how water-soluble extracts of Pinctada maxima nacre act on osteoblasts in vitro may reveal the mechanisms involved in its action in vivo on bone cells and bone regeneration. PMID:15348370

  4. Composition and topology of activity cliff clusters formed by bioactive compounds.

    PubMed

    Stumpfe, Dagmar; Dimova, Dilyana; Bajorath, Jürgen

    2014-02-24

    The assessment of activity cliffs has thus far mostly focused on compound pairs, although the majority of activity cliffs are not formed in isolation but in a coordinated manner involving multiple active compounds and cliffs. However, the composition of coordinated activity cliff configurations and their topologies are unknown. Therefore, we have identified all activity cliff configurations formed by currently available bioactive compounds and analyzed them in network representations where activity cliff configurations occur as clusters. The composition, topology, frequency of occurrence, and target distribution of activity cliff clusters have been determined. A limited number of large cliff clusters with unique topologies were identified that were centers of activity cliff formation. These clusters originated from a small number of target sets. However, most clusters were of small to moderate size. Three basic topologies were sufficient to describe recurrent activity cliff cluster motifs/topologies. For example, frequently occurring clusters with star topology determined the scale-free character of the global activity cliff network and represented a characteristic activity cliff configuration. Large clusters with complex topology were often found to contain different combinations of basic topologies. Our study provides a first view of activity cliff configurations formed by currently available bioactive compounds and of the recurrent topologies of activity cliff clusters. Activity cliff clusters of defined topology can be selected, and from compounds forming the clusters, SAR information can be obtained. The SAR information of activity cliff clusters sharing a/one specific activity and topology can be compared. PMID:24437577

  5. Effects of motor patterns on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue

    NASA Technical Reports Server (NTRS)

    Pevzner, L. Z.; Venkov, L.; Cheresharov, L.

    1980-01-01

    Albino rats were kept for a year under conditions of daily motor load or constant hypokinesia. An increase in motor activity results in a rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesia but does not change under the motor load pattern. Motor load causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

  6. 76 FR 69276 - Agency Information Collection Activities: Form N-336, Revision of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-11-08

    ... SECURITY Citizenship and Immigration Services Agency Information Collection Activities: Form N-336... Information Collection Under Review: Form N- 336, Request for Hearing on a Decision in Naturalization...: Form N-336. U.S. Citizenship and Immigration Services. (4) Affected public who will be asked...

  7. 76 FR 53144 - Agency Information Collection Activities: Form N-336; Revision of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-08-25

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-336... Information Collection Under Review: Form N- 336, Request for Hearing on a Decision in Naturalization...: Form N-336; U.S. Citizenship and Immigration Services (USCIS). (4) Affected public who will be asked...

  8. 75 FR 78264 - Agency Information Collection Activities: Form N-336, Revision to an Existing Information...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-12-15

    ... Register at 75 FR 51095 announcing the extension of the Form N-336. The 60-day notice announced that during... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-336... collection under review: Form N- 336, Request for Hearing on a Decision in Naturalization Proceedings...

  9. 76 FR 78674 - Agency Information Collection Activities: Form N-470, Revision of a Currently Approved...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-19

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-470... Information Collection Under Review: Form N- 470, Application To Preserve Residence for Naturalization; OMB..., and the applicable component of the Department of Homeland Security sponsoring the collection: Form...

  10. 75 FR 51096 - Agency Information Collection Activities: Form N-400; Extension of an Existing Information...

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  14. Dance as a Fitness Activity: The Impact of Teaching Style and Dance Form.

    ERIC Educational Resources Information Center

    Fromel, Karel; Vasendova, Jana; Stratton, Gareth; Pangrazi, Robert P.

    2002-01-01

    Analyzed the amount of activity, intensity of activity, and attitudes of participants in Czech high school physical education classes taught using different teaching styles and dance forms. Measurements of heart rate and dance intensity and student surveys indicated that teaching style and dance form significantly impacted the intensity and volume…

  15. Determination of water-soluble hexavalent chromium in clinker samples by wavelength-dispersive X-ray fluorescence spectrometry after concentration in activated layers.

    PubMed

    Marguí, Eva; Fontàs, Claudia; Toribio, Marta; Guillem, Manel; Hidalgo, Manuela; Queralt, Ignacio

    2010-05-01

    The determination of hexavalent chromium (Cr(VI)) in cement-related material extracts is frequently monitored in cement industries to comply with the European Directive (2003/53/EC) that limits the use of cements containing more than 2 mg kg(-1) of water-soluble Cr(VI). In the present work, a rapid and simple method for the determination of water-soluble Cr(VI) in clinker samples has been developed. The analytical methodology is based on the combined use of a low cost Cr(VI) isolation procedure using activated layers followed by their analysis using wavelength-dispersive X-ray fluorescence (WDXRF) spectrometry. WDXRF instrumentation is a common tool used for determining the chemical composition of all materials involved in cement production and also for the quality control of the products produced in cement and concrete factories. Therefore, the presented methodology does not imply the use of additional instrumentation in cement-industries laboratories and can be used as a comparative method to the spectrophotometric reference (EN 196-10:2006). The analytical parameters evaluated (selectivity, limit of detection, linearity, and precision) prove to be suitable for the intended purpose, and the methodology has successfully been applied to determine water-soluble Cr(VI) in several clinker samples. PMID:20482975

  16. Decrease in neuroimmune activation by HSV-mediated gene transfer of TNFα soluble receptor alleviates pain in rats with diabetic neuropathy.

    PubMed

    Ortmann, Kathryn L Maier; Chattopadhyay, Munmun

    2014-10-01

    The mechanisms of diabetic painful neuropathy are complicated and comprise of peripheral and central pathophysiological phenomena. A number of proinflammatory cytokines are involved in this process. Tumor necrosis factor α (TNF-α) is considered to be one of the major contributors of neuropathic pain. In order to explore the potential role of inflammation in the peripheral nervous system of Type 1 diabetic animals with painful neuropathy, we investigated whether TNF-α is a key inflammatory mediator to the diabetic neuropathic pain and whether continuous delivery of TNFα soluble receptor from damaged axons achieved by HSV vector mediated transduction of DRG would block or alter the pain perception in animals with diabetic neuropathy. Diabetic animals exhibited changes in threshold of mechanical and thermal pain perception compared to control rats and also demonstrated increases in TNFα in the DRG, spinal cord dorsal horn, sciatic nerve and in the foot skin, 6 weeks after the onset of diabetes. Therapeutic approaches by HSV mediated expression of p55 TNF soluble receptor significantly attenuated the diabetes-induced hyperalgesia and decreased the expression of TNFα with reduction in the phosphorylation of p38MAPK in the spinal cord dorsal horn and DRG. The overall outcome of this study suggests that neuroinflammatory activation in the peripheral nervous system may be involved in the pathogenesis of painful neuropathy in Type 1 diabetes which can be alleviated by local expression of HSV vector expressing p55 TNF soluble receptor. PMID:24880032

  17. The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

    PubMed Central

    Zon, L I; Moreau, J F; Koo, J W; Mathey-Prevot, B; D'Andrea, A D

    1992-01-01

    The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55. Images PMID:1320192

  18. Antifungal activity, experimental infections and nail permeation of an innovative ciclopirox nail lacquer based on a water-soluble biopolymer.

    PubMed

    Togni, Giuseppe; Mailland, Federico

    2010-05-01

    P-3051 is an innovative 8% ciclopirox nail lacquer, based on hydroxypropyl chitosan (HPCH) as a film-forming agent. The authors' aim was to investigate P-3051's in vitro antifungal activity, as well as its in vitro and in vivo nail permeation. The dilution susceptibility tests performed for Trichophyton rubrum (T. rubrum) and Candida parapsilosis (C. parapsilosis) showed that the minimum inhibitory concentrations (MICs) of P-3051, as percent of ciclopirox, was for both fungi < or = 0.0015% (equivalent to a concentration of 15.6 mg/ ml). In the biological assay of in vitro nail permeation and fungal inhibition, the authors observed that P-3051 permeated well through bovine hoof membranes and produced dose-dependent inhibitory effects on dermatophyte, yeast and mold strains. Moreover, the inhibition effects were higher than those obtained by equal amounts of the ciclopirox reference nail lacquer. P-3051 and the reference showed the same protective activity in experimental infections with strains of dermatophytes isolated from clinical samples. The amount of ciclopirox remained in cut fingernails washed six hours after in vivo application of P-3051 ranged between 18 and 35% of the applied dose. After in vitro application to cut human nails, 40-50% of the applied ciclopirox penetrated during the first six hours, independent of nails being infected or uninfected, intact or filed. In both experiments, the concentration of ciclopirox is largely higher (three to four orders of magnitude) than the MICs for nail pathogens. PMID:20480796

  19. In vitro activity of Rutaceae species against the trypomastigote form of Trypanosoma cruzi.

    PubMed

    Mafezoli, J; Vieira, P C; Fernandes, J B; da Silva, M F; de Albuquerque, S

    2000-11-01

    The activity of crude plant extracts of nine species of Rutaceae against the trypomastigote form of Trypanosoma cruzi was evaluated at 4 mg/ml. Thirty-two crude extracts were tested and eight of them showed significant activity (>80%). The most active extract was obtained from the stems of Pilocarpus spicatus (97.3%). Fractionation of the active crude extracts provided 25 fractions which were tested against the trypomastigote form of T. cruzi at 2 mg/ml. Of these six showed significant activity (>80%). The most active fractions (100%) were obtained from the leaves of Almeidea coerulea (butanol fraction) and Conchocarpus inopinatus (dichloromethane fraction). PMID:11025175

  20. ¹H, ¹³C and ¹⁵N resonance assignment of the soluble form of the lipid-modified Azurin from Neisseria gonorrhoeae.

    PubMed

    Nóbrega, Cláudia S; Matzapetakis, Manolis; Pauleta, Sofia R

    2013-10-01

    Lipid-modified azurin (Laz) from Neisseria gonorrhoeae is a type 1 copper protein proposed to be the electron donor to several enzymes involved in the resistance mechanism to reactive oxygen and nitrogen species. Here we report the backbone and side-chain resonance assignment of Laz in the reduced form, which has been complete at 97%. The predicted secondary structure indicates that this protein belongs to the azurin subfamily of type 1 copper proteins. PMID:23070845

  1. Free radical scavenging and anti-oxidative activities of an ethanol-soluble pigment extract prepared from fermented Zijuan Pu-erh tea.

    PubMed

    Fan, Jiang Ping; Fan, Chong; Dong, Wen Min; Gao, Bin; Yuan, Wei; Gong, Jia Shun

    2013-09-01

    An ethanol-soluble pigment extract was separated from fermented Zijuan Pu-erh tea. The compositions of the ethanol soluble pigment extract were analyzed by high-performance liquid chromatography-tandem mass spectroscopy (HPLC-MS/MS). The extract was prepared into a series of ethanol solutions and analyzed for free radical-scavenging activities (against two free radicals: 1,1-diphenyl-2-picrylhydrazyl (DPPH) and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)) and in vitro anti-oxidative properties. Electron spin resonance spectroscopy showed that the peaks of DPPH and TEMPO decreased with increasing extract concentration, suggesting that the extract had excellent free radical-scavenging activities. In vitro cell culture suggested that, at 50-200 mg/L, the extract had no measurable effect on the viability of vascular endothelial cells (ECV340) but produced significant protective effects for cells that underwent oxidative injuries due to hydrogen peroxide (H₂O₂) treatment. Compared with the H₂O₂ treatment alone cells group, 200 mg/L of the extract increased the activity of superoxide dismutase (SOD) in cells by 397.3%, and decreased the concentration of malondialdehyde (MDA) and the activity of lactate acid dehydrogenase (LDH) by 47.8% and 69.6%, respectively. These results suggest that the extract has excellent free radical scavenging and anti-oxidative properties. PMID:23831194

  2. [Determination of six C-Glycoside flavones and antitumor activity of water-soluble total flavonoids from Isodon lophanthoides var. gerardianus].

    PubMed

    Zhang, Yang; Tang, Hai-ming; Li, Ai; Xu, Lan-fang; Chen, Jian-nan; Huang, Song; He, Lian

    2015-04-01

    This research established an HPLC method for determination of six C-Glycoside flavones of warer-soluble total flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara, and studied the antitumor activity of the warer-soluble total flavonoids. The HPLC system consisted of Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) column and a solution system of methanol, acetonitrile and 0.5% formic acid gradient elution at a flow rate of 0. 8 mL x min(-1) and the wavelength of detector was at 334 nm. The column temperature was 25 degrees C. The antitumor activity of water-soluble flavonoids was assayed using HepG2 cell as the tested cell. The linear ranges of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabinosylapigenin were 0.25-2.53, 0.12-1.20, 0.37-3.69, 0.16-1.63, 0.19-1.92, 0.14-1.42 microg, respectively. The average recoveries (n = 6) were 99.6% (RSD 0.87%), 100.2% (RSD 2.0%), 99.6% (RSD 1.8%), 97.9% (RSD 1.5%), 98.8% (RSD 1.2%), 98.6% (RSD 1.2%), respectively. After exposure in 24, 48, 72 h, the total flavonoids showed inhibitory effect on the proliferation of HepG2 cells with IC50 as the evaluation index, the IC50 values of 1.89, 1.71, 1.51 g x L(-1), respectively. The method is quick, simple and accurate with good re- producibility, and can be used for determination of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabino- sylapigenin in the warer-soluble total flavonoids from L lophanthoides var. gerardianus. The warer-soluble total flavonoids from L lophanthoides have inhibitory effect on the proliferation of HepG2 cells. PMID:26281595

  3. Soluble peptide-MHC monomers cause activation of CD8+ T cells through transfer of the peptide to T cell MHC molecules

    NASA Astrophysics Data System (ADS)

    Ge, Qing; Stone, Jennifer D.; Thompson, M. Todd; Cochran, Jennifer R.; Rushe, Mia; Eisen, Herman N.; Chen, Jianzhu; Stern, Lawrence J.

    2002-10-01

    T cell receptor (TCR)-mediated activation of CD4+ T cells is known to require multivalent engagement of the TCR by, for example, oligomeric peptide-MHC complexes. In contrast, for CD8+ T cells, there is evidence for TCR-mediated activation by univalent engagement of the TCR. We have here compared oligomeric and monomeric Ld and Kb peptide-MHC complexes and free peptide as stimulators of CD8+ T cells expressing the 2C TCR. We found that the monomers are indeed effective in activating naïve and effector CD8+ T cells, but through an unexpected mechanism that involves transfer of peptide from soluble monomers to T cell endogenous MHC (Kb) molecules. The result is that T cells, acting as antigen-presenting cells, are able to activate other naïve T cells.

  4. Soluble vs. insoluble fiber

    MedlinePlus

    ... soluble and insoluble. Both are important for health, digestion, and preventing diseases. Soluble fiber attracts water and turns to gel during digestion. This slows digestion. Soluble fiber is found in ...

  5. Evaluation of the activity and molecular form of bi in cu smelting slags: Part I. ternary silicate slags

    NASA Astrophysics Data System (ADS)

    Marschman, S. C.; Lynch, D. C.

    1988-08-01

    The thermodynamic behavior of bismuth in the chemical systems associated with copper processing is not well understood. This study was designed to further the understanding of the physical chemistry of bismuth in slags that have similar compositions to those found in copper extractive metallurgical processing. The silicate system investigated was the FeO-Fe2O3-SiO2 ternary system in which bismuth was dissolved using an isopiestic experimental technique. Bismuth vapor pressures of 1 • 10-5 atm and 7.5 • 10-4 atm were used, and the silicates were equilibrated with this vapor at temperatures of 1458 K and 1523 K. In these experiments, the slag composition was varied such that P O 2 ranged from 10-12 to 10-8 atm. Bismuth was found to enter the silicate slag in both neutral and oxidic molecular forms. The oxidic form identified was that of BiO. The data suggest that the activity coefficient of neutral bismuth, γBi, is dependent on the solubility of that species in slag, even at the low concentrations observed in this study. It has been hypothesized, based on the large diameter of neutral Bi, that only a limited number of sites are available to accommodate neutral Bi, and that as the limit is approached γBi increases significantly. That hypothesis is shown to be consistent with the experimental results obtained in the present work as well as the results obtained by other investigators.

  6. Drug resinates an attractive approach of solubility enhancement of atorvastatin calcium.

    PubMed

    Kulthe, V V; Chaudhari, P D

    2013-09-01

    A substantial number of new chemical entities and marketed drugs show poor solubility characteristics and amorphisation is one of the favorable approaches to enhance solubility characteristics of such poorly soluble drugs. Formulation efforts in the present study were devoted to investigate amorphisation of a model poorly soluble drug, atorvastatin calcium by molecular complexation with anion exchange resin, Duolite(®)AP 143/1093 and hence enhancement in its solubility characteristics. Drug resinates in 1:1, 1:2, and 1:4 weight ratios were prepared by simple batch operation and subsequently studied for drug content, residual solvent content, molecular interactions, solid state characterisation and solubility characteristics. During initial characterisation, all the proportions of drug resinates, except 1:1 proportion showed partial amorphisation of the drug, whereas 1:1 proportion showed complete amorphisation of the drug. This proportion reported distinctly enhanced solubility characteristics over pure drug and other proportions. Such amorphisation and solubility enhancement could be attributed to the binding of individual drug molecules to the functional sites of the resin molecules, either partially or completely, resulting in reduction of crystal lattice energy, a main barrier to dissolution. Hydrophilic nature of ion exchange resin matrices also assisted in enhancing dissolution of the drug from the resinates. During accelerated stability study, there was an insignificant decrease in solubility characteristics of the drug and its amorphous form was also found to be stable in 1:1 proportion. Atorvastatin resinates formed in 1:1 weight ratio were in stoichiometric proportion and such drug resinates in stoichiometric proportion showed to have tremendous potential in conversion of crystalline form of drug substances to its amorphous form and subsequent stabilisation. It hence proved to be a very effective, yet simple approach for improving solubility

  7. Phylogenetic survey of soluble saxitoxin-binding activity in pursuit of the function and molecular evolution of saxiphilin, a relative of transferrin.

    PubMed Central

    Llewellyn, L E; Bell, P M; Moczydlowski, E G

    1997-01-01

    Saxiphilin is a soluble protein of unknown function which binds the neurotoxin, saxitoxin (STX), with high affinity. Molecular characterization of saxiphilin from the North American bullfrog, Rana catesbeiana, has previously shown that it is a member of the transferrin family. In this study we surveyed various animal species to investigate the phylogenetic distribution of saxiphilin, as detected by the presence of soluble [3H]STX binding activity in plasma, haemolymph or tissue extracts. We found that saxiphilin activity is readily detectable in a wide variety of arthropods, fish, amphibians, and reptiles. The pharmacological characteristics of [3H]STX binding activity in phylogenetically diverse species indicates that a protein homologous to bullfrog saxiphilin is likely to be constitutively expressed in many ectothermic animals. The results suggest that the saxiphilin gene is evolutionarily as old as an ancestral gene encoding bilobed transferrin, an Fe(2+)-binding and transport protein which has been identified in several arthropods and all the vertebrates which have been studied. PMID:9225480

  8. Influence of iron solubility and charged surface-active compounds on lipid oxidation in fatty acid ethyl esters containing association colloids.

    PubMed

    Homma, Rika; Johnson, David R; McClements, D Julian; Decker, Eric A

    2016-05-15

    The impact of iron compounds with different solubilities on lipid oxidation was studied in the presence and absence of association colloids. Iron (III) sulfate only accelerated lipid oxidation in the presence of association colloids while iron (III) oleate accelerated oxidation in the presence and absence of association colloids. Further, iron (III) oxide retarded lipid oxidation both with and without association colloids. The impact of charged association colloids on lipid oxidation in ethyl oleate was also investigated. Association colloids consisting of the anionic surface-active compound dodecyl sulphosuccinate sodium salt (AOT), cationic surface-active compound hexadecyltrimethylammonium bromide (CTAB), and nonionic surface-active compound 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol (Triton X-100) retarded, promoted, and had no effect on lipid oxidation rates, respectively. These results indicate that the polarity of metal compounds and the charge of association colloids play a big role in lipid oxidation. PMID:26776045

  9. Antioxidant activities of distiller dried grains with solubles as protein films containing tea extracts and their application in the packaging of pork meat.

    PubMed

    Yang, Hyun-Ju; Lee, Ji-Hyeon; Won, Misun; Song, Kyung Bin

    2016-04-01

    Distiller dried grains with solubles (DDGS) as protein (DP) films were prepared. Additionally, to prepare anti-oxidant films, green tea extract (GTE), oolong tea extract (OTE), and black tea extract (BTE) were incorporated into the DP films. Consequently, the incorporation of the tea extracts did not alter the physical properties of the films much, whereas the antioxidant activities, such as ABTS and DPPH radical scavenging activities were observed. To apply the DP films containing tea extracts to food packaging, pork meat was wrapped with the films and stored at 4 °C for 10 d. During storage, the pork meat wrapped with the DP films containing GTE, OTE, and BTE had less lipid oxidation than did the control. Among the tea extracts, the DP film containing GTE had the greatest antioxidant activity. These results indicate that the DP films containing green tea extracts can be utilized as an anti-oxidative packaging material for pork meat. PMID:26593480

  10. Synthesis, spectroscopic characterization, DFT studies, and antibacterial and antitumor activities of a novel water soluble Pd(II) complex with L-alliin

    NASA Astrophysics Data System (ADS)

    Abbehausen, Camilla; Sucena, Suelen F.; Lancellotti, Marcelo; Heinrich, Tassiele A.; Abrão, Emiliana P.; Costa-Neto, Claudio M.; Formiga, André L. B.; Corbi, Pedro P.

    2013-03-01

    A new water soluble Pd(II) complex with L-alliin (S-allyl-L-cysteine sulfoxide) was obtained and characterized by a set of chemical and spectroscopic measurements. Elemental and mass spectrometric data are consistent with the formula [Pd(C6H10NO3S)2]. The 1H and 13C nuclear magnetic resonance (NMR) data, [1H-15N] two dimensional (2D) NMR and infrared spectroscopic measurements indicate coordination of the ligand to Pd(II) through N and O atoms. DFT studies showed that the trans isomer is the most stable and preferred geometry for the complex. The complex is soluble in water and dimethylsulfoxide. An antibiogram assay revealed that the complex possess antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus bacterial strains in the range 125-500 μg mL-1. Antitumor assays revealed that the complex presents cytotoxic activity over HeLa cells with an estimated IC50 of 20 μmol L-1.

  11. Solid inclusion complexes of oleanolic acid with amino-appended β-cyclodextrins (ACDs): Preparation, characterization, water solubility and anticancer activity.

    PubMed

    Ren, Yufeng; Liu, Ying; Yang, Zhikuan; Niu, Raomei; Gao, Kai; Yang, Bo; Liao, Xiali; Zhang, Jihong

    2016-12-01

    Oleanolic acid (OA) is a pentacyclic triterpenoid acid of natural abundance in plants which possesses important biological activities. However, its medicinal applications were severely impeded by the poor water solubility and resultant low bioavailability and potency. In this work, studies on solid inclusion complexes of OA with a series of amino-appended β-cyclodextrins (ACDs) were conducted in order to address this issue. These complexes were prepared by suspension method and were well characterized by NMR, SEM, XRD, TG, DSC and Zeta potential measurement. The 2:1 inclusion mode of ACDs/OA complexes was elucidated by elaborate 2D NMR (ROESY). Besides, water solubility of OA was dramatically promoted by inclusion complexation with ACDs. Moreover, in vitro anticancer activities of OA against human cancer cell lines HepG2, HT29 and HCT116 were significantly enhanced after formation of inclusion complexes, while the apoptotic response results indicated their induction of apoptosis of cancer cells. This could provide a novel approach to development of novel pharmaceutical formulations of OA. PMID:27612690

  12. Enhanced solubility and antibacterial activity of lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazoles by complexation with β-cyclodextrins.

    PubMed

    Trapani, A; De Laurentis, N; Armenise, D; Carrieri, A; Defrenza, I; Rosato, A; Mandracchia, D; Tripodo, G; Salomone, A; Capriati, V; Franchini, C; Corbo, F

    2016-01-30

    Some lipophilic fluoro-substituted N-benzoyl-2-aminobenzothiazole antibacterial agents have been evaluated for their activity in the presence of cyclodextrins (CDs) containing aqueous solutions where CDs are adopted as solubilizing excipients for improving the poor water solubility of these compounds. For such purpose both the natural β-CD and one of FDA/EMA approved CDs for parenteral use (i.e. HP-β-CD) have been employed. The solubility rank order observed was accounted for by thermal analysis (Differential Scanning Calorimetry) and FT-IR spectroscopy. The most promising compound was subjected to further NMR spectroscopic studies and molecular modelling simulations to verify the interactions between the guest molecule and the CD cavity. The assessment of the antibacterial activity of such compounds against selected Gram positive and Gram negative bacterial strains clearly showed that their antimicrobial effectiveness may, quite in all instances, be positively affected by complexation with β-CD and HP-β-CD. These results, which are in some ways in contrast with those already reported in the literature, are herein discussed on the basis of plausible mechanisms. Moreover, this investigation also reveals that the described methodology of complexing both lipophilic and hydrophilic antimicrobial agents with CDs may be an useful approach to enhance their effectiveness as well as a promising strategy to overcome even the microbial resistance problem. PMID:26611670

  13. 75 FR 30098 - Reports, Forms and RecordKeeping Requirements; Agency Information Collection Activity Under OMB...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-05-28

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  14. 78 FR 26766 - Commission Information Collection Activities (FERC Form 580); Comment Request; Revision

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-05-08

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  16. Mechanisms of relaxant activity of the nitric oxide-independent soluble guanylyl cyclase stimulator BAY 41-2272 in rat tracheal smooth muscle.

    PubMed

    Toque, Haroldo A; Mónica, Fabíola Z T; Morganti, Rafael P; De Nucci, Gilberto; Antunes, Edson

    2010-10-25

    The soluble guanylyl cyclase is expressed in airway smooth muscle, and agents that stimulate this enzyme activity cause airway smooth muscle relaxation and bronchodilation. The compound 5-Cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine (BAY 41-2272) is a potent nitric oxide (NO)-independent soluble guanylyl cyclase stimulator, but little is known about its effects in airway smooth muscle. Therefore, this study aimed to investigate the mechanisms underlying the relaxations of rat tracheal smooth muscle induced by BAY 41-2272. Tracheal rings were mounted in 10-ml organ baths for isometric force recording. BAY 41-2272 concentration-dependently relaxed carbachol-precontracted tracheal rings (pEC(50)=6.68+/-0.14). Prior incubation with the NO synthesis inhibitor l-NAME (100 microM) or the soluble guanylyl cyclase inhibitor ODQ (10 microM) caused significant rightward shifts in the concentration-response curves to BAY 41-2272. Sodium nitroprusside caused concentration-dependent relaxations, which were greatly potentiated by BAY 41-2272 and completely inhibited by ODQ. In addition, BAY 41-2272 shifted to the right the tracheal contractile responses to either carbachol (0.01-1 microM) or electrical field stimulation (EFS, 1-32 Hz). BAY 41-2272 (1 microM) also caused a marked rightward shift and decreased the maximal contractile responses to extracellular CaCl2, and such effect was not modified by pretreatment with ODQ. In addition, BAY 41-2272 (up to 1 microM) significantly increased the cGMP levels, and that was abolished by ODQ. Our results indicate that BAY 41-2272 causes cGMP-dependent rat tracheal smooth muscle relaxations in a synergistic fashion with exogenous NO. BAY 41-2272 has also an additional mechanism independently of soluble guanylyl cyclase activation possibly involving Ca(2+) entry blockade. PMID:20670622

  17. 76 FR 70747 - Agency Information Collection Activities: Form I-90; Revision of a Currently Approved Information...

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    2011-11-15

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  19. 77 FR 16047 - Agency Information Collection Activities: Form I-589; Extension of an Existing Information...

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    2012-03-19

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  20. 76 FR 52961 - Agency Information Collection Activities: Form N-300; Revision of an Existing Information...

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    2011-08-24

    ... SECURITY U.S. Citizenship and Immigration Services Agency Information Collection Activities: Form N-300.... The Department Homeland Security, U.S. Citizenship and Immigration Services (USCIS) will be submitting... collection: Form N-300; U.S. Citizenship and Immigration Services (USCIS). (4) Affected public who will...