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Sample records for determining ligand potency

  1. The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering

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    Bing-Mae Chen

    2017-07-01

    Full Text Available T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11 in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

  2. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3

    DEFF Research Database (Denmark)

    Hudson, Brian D; Tikhonova, Irina G; Pandey, Sunil K

    2012-01-01

    Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C...

  3. Extracellular Ionic Locks Determine Variation in Constitutive Activity and Ligand Potency between Species Orthologs of the Free Fatty Acid Receptors FFA2 and FFA3*

    Science.gov (United States)

    Hudson, Brian D.; Tikhonova, Irina G.; Pandey, Sunil K.; Ulven, Trond; Milligan, Graeme

    2012-01-01

    Free fatty acid receptors 2 and 3 (FFA2 and FFA3) are G protein-coupled receptors for short chain free fatty acids (SCFAs). They respond to the same set of endogenous ligands but with distinct rank-order of potency such that acetate (C2) has been described as FFA2-selective, whereas propionate (C3) is non-selective. Although C2 was confirmed to be selective for human FFA2 over FFA3, this ligand was not selective between the mouse orthologs. Moreover, although C3 was indeed not selective between the human orthologs, it displayed clear selectivity for mouse FFA3 over mouse FFA2. This altered selectivity to C2 and C3 resulted from broad differences in SCFAs potency at the mouse orthologs. In studies to define the molecular basis for these observations, marked variation in ligand-independent constitutive activity was identified using a [35S]GTPγS assay. The orthologs with higher potency for the SCFAs, human FFA2 and mouse FFA3, displayed high constitutive activity in this assay, whereas the orthologs with lower potency for the agonist ligands, mouse FFA2 and human FFA3, did not. Sequence alignments of the second extracellular loop identified single negatively charged residues in FFA2 and FFA3 not conserved between species and predicted to form ionic lock interactions with arginine residues within the FFA2 or FFA3 agonist binding pocket to regulate constitutive activity and SCFA potency. Reciprocal mutation of these residues between species orthologs resulted in the induction (or repression) of constitutive activity and in most cases also yielded corresponding changes in SCFA potency. PMID:23066016

  4. MATHEMATICAL MODELING FOR ERYTHROMYCIN POTENCY DETERMINATION OF MASTIKER

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    Victorita Burghelea

    2016-12-01

    Full Text Available Mastiker E is an antibacterial suspension for intramammary infusion in cattle mastitis. The active drug is erythromycin, a macrolide antibiotic. The main characteristic of commercial product is erythromycin potency. The potency of erythromycin is estimated by comparing the inhibition of growth of sensitive micro-organisms produced by known concentrations of the antibiotic to be examined and a reference substance. The validation study aims to demonstrate the determination of the potency of erythromycin, it is an appropriate analytical method, reproducible and meets the quality requirements of Mastiker product. The paper establishes the performance characteristics of the method considered and identify the factors that influence these characteristics. The diameters of inhibition zones, directly proportional to the logarithm of the concentration of the antibiotic used for the assay, measured and calculated using statistical methods (Combistats Soft. The assay is designed in such a way that the mathematical model on which the potency equation is based can be proved to be valid. A parallel-line model is chosen. The two log dose response lines of the preparation under examination and the standard preparation are parallel; they are rectilinear over the range of doses used in the calculation. These conditions are verified by validity tests for a given probability (P = 0.05. The test is not valid unless the confidence limits (P = 0.95 are not less than 50% and not more than 200% of the estimated potency. The estimated potency is not less than 95% and not more than 105% of the stated potency. The stated potency is not less than 50.000 IU/g. The validation procedure includes details on protocol working to determine the potency of erythromycin, validation criteria, experimental results, mathematical modeling for determining the potency, inter laboratory comparisons.

  5. Influence of a threonine residue in the S2 ligand binding domain in determining agonist potency and deactivation rate of recombinant NR1a/NR2D NMDA receptors.

    Science.gov (United States)

    Chen, Philip E; Johnston, Alexander R; Mok, M H Selina; Schoepfer, Ralf; Wyllie, David J A

    2004-07-01

    NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose-response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.

  6. Potency determination of antidandruff shampoos in nystatin international unit equivalents

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    Anusha Hewage D.B.G

    2008-01-01

    Full Text Available A convenient standard microbiological potency determination test for the antidandruff shampoos was developed by adopting the pharmacopoeial microbiological assay procedure of the drug nystatin. A standard curve was drawn consisting of the inhibition zone diameters vs. logarithm of nystatin concentrations in international units using the fungus Saccharomyces cerevisiae (yeast strain National Collection of Type Culture (NCTC 1071606 as the test organism. From the standard curve the yeast inhibitory potencies of the shampoos in nystatin international unit equivalents were determined from the respective inhibition zones of the test samples of the shampoos. Under test conditions four shampoo samples showed remarkable fungal inhibitory potencies of 10227, 10731, 12396 and 18211 nystatin international unit equivalents/ml while two shampoo samples had extremely feeble inhibitory potencies 4.07 and 4.37 nystatin international unit equivalents/ml although the latter two products claimed antifungal activity. The potency determination method could be applied to any antidandruff shampoo with any one or a combination of active ingredients.

  7. Fluorescence polarization competition assay: the range of resolvable inhibitor potency is limited by the affinity of the fluorescent ligand.

    Science.gov (United States)

    Huang, Xinyi

    2003-02-01

    For the development of fluorescence polarization (FP) competition assays, there is a widespread belief that tight-binding fluorescent ligands should be avoided to identify inhibitors of low or intermediate potency in the screening of small-molecule compound libraries. It is demonstrated herein that this statement is a misconception; in fact, the higher the affinity of the fluorescent ligand, the wider the range of inhibitor potency that can be resolved. An approximate estimate for the low end of inhibitor K(i) values that can be resolved is the K(d) value of the fluorescent ligand. Because FP competition assays are typically conducted under nonstoichiometric titration conditions, it is suggested that a fluorescent ligand of highest affinity that also has an adequate quantum yield to satisfy such conditions be selected.

  8. A new ELISA for determination of potency in snake antivenoms.

    Science.gov (United States)

    Rial, A; Morais, V; Rossi, S; Massaldi, H

    2006-09-15

    A competitive ELISA for potency determination of bothropic equine antivenom was developed and compared to the conventional in vivo ED(50) assay, with the aim of partially substituting the in vivo assay in the monitoring of antivenom immunoglobulin levels. On this purpose, blood samples were taken at different times during and after the immunization protocol of the lot of horses used for production of snake antivenom at the Instituto de Higiene, Uruguay. Both the competitive ELISA and the ED(50) assay were performed on those samples. In addition, a group of five commercial pepsin-digested antivenoms were tested by both methods. A significant (P<0.001) correlation (Pearson's r=0.957) was found between the ELISA titres and the corresponding ED(50) values, indicating that the in vitro test can estimate the neutralizing antibody capacity of the sera as well as the in vivo assay. By means of this new ELISA, it was found that the immunized animals maintained good venom antibody titres, in the order of 20-50% of the maximum achieved, even 10 month after the end of the immunization schedule. The main advantage of our ELISA design is its ability to correctly estimate the neutralization capacity of crude hyperimmune plasma and antivenom sera independently of their antibody composition in terms of whole IgG or F(ab')(2) fragment.

  9. Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

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    Thompson, Andrew J; Alqazzaz, Mona; Price, Kerry L; Weston, David A; Lummis, Sarah C R

    2014-10-01

    The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

  10. Disturbances of ligand potency and enhanced degradation of the human glycine receptor at affected positions G160 and T162 originally identified in patients suffering from hyperekplexia

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    Sinem eAtak

    2015-12-01

    Full Text Available Ligand-binding of Cys-loop receptors is determined by N-terminal extracellular loop structures from the plus as well as from the minus side of two adjacent subunits in the pentameric receptor complex. An aromatic residue in loop B of the glycine receptor (GlyR undergoes direct interaction with the incoming ligand via cation-π interactions. Recently we showed that mutated residues in loop B identified from human patients suffering from hyperekplexia disturb ligand-binding. Here, we exchanged the affected human residues by amino acids found in related members of the Cys-loop receptor family to determine the effects of side chain volume for ion channel properties. GlyR variants were characterized in vitro following transfection into cell lines in order to analyze protein expression, trafficking, degradation and ion channel function. GlyR α1 G160 mutations significantly decrease glycine potency arguing for a positional effect on neighboring aromatic residues and consequently glycine-binding within the ligand-binding pocket. Disturbed glycinergic inhibition due to T162 α1 mutations is an additive effect of affected biogenesis and structural changes within the ligand-binding site. Protein trafficking from the ER towards ER-Golgi intermediate compartment, the secretory Golgi pathways and finally the cell surface is largely diminished, but still sufficient to deliver ion channels that are functional at least at high glycine concentrations. The majority of T162 mutant protein accumulates in the ER and is conducted to ER-associated proteasomal degradation. Hence, G160 is an important determinant during glycine binding. In contrast, T162 assigns primarily receptor biogenesis whereas exchanges in functionality are secondary effects thereof.

  11. MATHEMATICAL MODELING FOR BENZYLPENICILIN POTASSIUM AND STREPTOMYCIN SULPHATE POTENCY DETERMINATION OF ASCOMICIN

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    Viviana Ciuca

    2016-12-01

    Full Text Available Ascomicin is an antibacterial unguent for treatment of local infections of skin, eyes, outer ear, in cattle, sheep, pig, dog and cat. The product contains two active substances: benzylpenicillin potassium (Penicillin G potassium and streptomycin sulphate. The main characteristic of commercial product is benzylpenicillin potassium and streptomycin sulphate potency. The potency is estimated by comparing the inhibition of growth of sensitive micro-organisms produced by known concentrations of the antibiotic to be examined and a reference substance. The validation study aims to demonstrate the determination of the potency of benzylpenicillin potassium and streptomycin sulphate, it is an appropriate analytical method, reproducible and meets the quality requirements of Ascomicin product. The paper establishes the performance characteristics of the method considered and identify the factors that influence these characteristics. The diameters of inhibition zones, directly proportional to the logarithm of the concentration of the antibiotic used for the assay, measured and calculated using statistical methods (Combistats Soft. The assay is designed in such a way that the mathematical model on which the potency equation is based can be proved to be valid. A parallel-line model is chosen. The two log dose response lines of the preparation under examination and the standard preparation are parallel; they are rectilinear over the range of doses used in the calculation. These conditions are verified by validity tests for a given probability (P = 0.05. The test is not valid unless the confidence limits (P = 0.95 are not less than 50 per cent and not more than 200 per cent of the estimated potency. The estimated potency is not less than 95 per cent and not more than 105 per cent of the stated potency. The stated potency is not less than 19400 international units/g benzylpenicillin potassium and 13960 international units/g streptomycin sulphate. The validation

  12. Selection of appropriate analytical tools to determine the potency and bioactivity of antibiotics and antibiotic resistance$

    Institute of Scientific and Technical Information of China (English)

    Nishant A. Dafale n; Uttam P. Semwal; Rupak K. Rajput; G.N. Singh

    2016-01-01

    Antibiotics are the chemotherapeutic agents that kill or inhibit the pathogenic microorganisms. Re-sistance of microorganism to antibiotics is a growing problem around the world due to indiscriminate and irrational use of antibiotics. In order to overcome the resistance problem and to safely use antibiotics, the correct measurement of potency and bioactivity of antibiotics is essential. Microbiological assay and high performance liquid chromatography (HPLC) method are used to quantify the potency of antibiotics. HPLC method is commonly used for the quantification of potency of antibiotics, but unable to determine the bioactivity; whereas microbiological assay estimates both potency and bioactivity of antibiotics. Additionally, bioassay is used to estimate the effective dose against antibiotic resistant microbes. Simultaneously, microbiological assay addresses the several parameters such as minimal inhibitory concentration (MIC), minimum bactericidal concentration (MBC), mutation prevention concentration (MPC) and critical concentration (Ccr) which are used to describe the potency in a more informative way. Microbiological assay is a simple, sensitive, precise and cost effective method which gives reproducible results similar to HPLC. However, the HPLC cannot be a complete substitute for microbiological assay and both methods have their own significance to obtain more realistic and precise results.

  13. Determination of relative potencies for chemical inhibition of spontaneous neuronal activity using a four amplifier MEA system.

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    Potency determination is important to identify the most promising drug candidates as well as identification of and ranking of compound toxicity. In our laboratory, we have utilized MEA recording techniques to determine the relative potency of 11 insecticidal compounds and rank th...

  14. Estrogenic potencies of several environmental pollutants, as determined by vitellogenin induction in a carp hepatocyte assay.

    Science.gov (United States)

    Smeets, J M; van Holsteijn, I; Giesy, J P; Seinen, W; van den Berg, M

    1999-08-01

    Estrogenic potencies of several xenoestrogens were determined in vitro, using cultured hepatocytes from a genetically uniform male carp strain (Cyprinus carpio). Estrogenicity was measured as induction of the yolk protein precursor vitellogenin (Vtg), and compared to Vtg induction by 17beta-estradiol (E2). The order of estrogenic potency was: methoxychlor (MXCL) > o,p-DDT > chlordecone approximately/= bisphenol-A approximately/= 4-t-pentylphenol. Estrogenic potencies of these compounds varied from 1 x 10(-3) to 1 x 10(-4) relative to E2. The synthetic estrogen DES had a relative estrogenic potency of 0.5, whereas dieldrin, beta-endosulfan, o,p-DDE, and toxaphene (technical mixture) did not induce vitellogenesis at concentrations up to 100 microM. Experiments in which cells were simultaneously exposed to E2 and these xenoestrogens showed that the Vtg-inducing activities of E2 and 4-t-pentylphenol or bisphenol-A were (partially) additive, whereas E2 antagonized the estrogenic effects of MXCL and o,p-DDT. The effect of cytochrome P4501A (CYP1A)-induction on the estrogenicity of MXCL was studied by co-exposing cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). TCDD (10 pM) caused a greater than 50-fold induction of CYP1A, measured as ethoxyresorufin O-deethylase (EROD) activity, but Vtg induction by MXCL was not significantly affected. This indicates that CYP1A is not involved in the bioactivation of MXCL to more potent estrogenic metabolites in carp. The CARP-HEP (hepatocyte) assay can detect xenoestrogens with a potency > or = 2 x 10(-5) relative to E2. It allows simultaneous testing of more than 10 compounds for both estrogenic and antiestrogenic effects, which makes it a promising tool for the screening of suspected xenoestrogens.

  15. Titer on chip: new analytical tool for influenza vaccine potency determination.

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    Laura R Kuck

    Full Text Available Titer on Chip (Flu-ToC is a new technique for quantification of influenza hemagglutinin (HA concentration. In order to evaluate the potential of this new technique, a comparison of Flu-ToC to more conventional methods was conducted using recombinant HA produced in a baculovirus expression system as a test case. Samples from current vaccine strains were collected from four different steps in the manufacturing process. A total of 19 samples were analysed by Flu-ToC (blinded, single radial immunodiffusion (SRID, an enzyme-linked immunosorbent assay (ELISA, and the purity adjusted bicinchoninic acid assay (paBCA. The results indicated reasonable linear correlation between Flu-ToC and SRID, ELISA, and paBCA, with regression slopes of log-log plots being 0.91, 1.03, and 0.91, respectively. The average ratio for HA content measured by Flu-ToC relative to SRID, ELISA, and paBCA was 83%, 147%, and 81%, respectively; indicating nearly equivalent potency determination for Flu-ToC relative to SRID and paBCA. These results, combined with demonstrated multiplexed analysis of all components within a quadrivalent formulation and robust response to HA strains over a wide time period, support the conclusion that Flu-ToC can be used as a reliable and time-saving alternative potency assay for influenza vaccines.

  16. The Binding Mode Prediction and Similar Ligand Potency in the Active Site of Vitamin D Receptor with QM/MM Interaction, MESP, and MD Simulation.

    Science.gov (United States)

    Selvaraman, Nagamani; Selvam, Saravana Kumar; Muthusamy, Karthikeyan

    2016-08-01

    Non-secosteroidal ligands are well-known vitamin D receptor (VDR) agonists. In this study, we described a combined QM/MM to define the protein-ligand interaction energy a strong positive correlation in both QM-MM interaction energy and binding free energy against the biological activity. The molecular dynamics simulation study was performed, and specific interactions were extensively studied. The molecular docking results and surface analysis shed light on steric and electrostatic complementarities of these non-secosteroidal ligands to VDR. Finally, the drug likeness properties were also calculated and found within the acceptable range. The results show that bulky group substitutions in side chain decrease the VDR activity, whereas a small substitution increased it. Functional analyses of H393A and H301A mutations substantiate their roles in the VDR agonistic and antagonistic activities. Apart from the His393 and His301, two other amino acids in the hinge region viz. Ser233 and Arg270 acted as an electron donor/acceptor specific to the agonist in the distinct ligand potency. The results from this study disclose the binding mechanism of VDR agonists and structural modifications required to improve the selectivity.

  17. Procedure for Calculation of Potency and Efficacy for Ligands Acting on G(s)- and G (i)-Coupled Receptors

    DEFF Research Database (Denmark)

    Meier, Eddi; Schousboe, Arne; Belhage, Bo

    2012-01-01

    Structure activity relationship (SAR) analyses of pharmacological data of compounds constitute an important part of the discovery process in the design of new drug candidates with improved pharmacological properties. In particular G-Protein Coupled Receptors (GPCRs) associated with the cAMP second...... messenger systems G(s) and G(i) have constituted one of the most widely used basis for pharmacological in vitro assays for assessing functional receptor effects. Such assays are based on Radio Immuno Assay (RIA) analysis to measure the cellular cAMP concentration as readout of receptor activation....... It appears, however, to be a common practice to omit the use of cAMP standard curves to transform the measured signals (cpm or cps) into cAMP concentrations on which estimations of potencies (EC(50) values) and efficacies (E(MAX) values) in G(s) and G(i) coupled receptor stimulation are based. Such practice...

  18. Collaborative studies on the development of national reference standards for potency determination of H7N9 influenza vaccine

    Science.gov (United States)

    Li, Changgui; Xu, Kangwei; Hashem, Anwar; Shao, Ming; Liu, Shuzhen; Zou, Yong; Gao, Qiang; Zhang, Yongchao; Yuan, Liyong; Xu, Miao; Li, Xuguang; Wang, Junzhi

    2015-01-01

    The outbreak of human infections of a novel avian influenza virus A (H7N9) prompted the development of the vaccines against this virus. Like all types of influenza vaccines, H7N9 vaccine must be tested for its potency prior to being used in humans. However, the unavailability of international reference reagents for the potency determination of H7N9 vaccines substantially hinders the progress in vaccine development. To facilitate clinical development, we enlisted 5 participants in a collaborative study to develop critical reagents used in Single Radial Immunodiffusion (SRID), the currently acceptable assay for potency determination of influenza vaccine. Specifically, the hemagglutinin (HA) content of one vaccine bulk for influenza A (H7N9), herein designated as Primary Liquid Standard (PLS), was determined by SDS-PAGE. In addition, the freeze-dried antigen references derived from PLS were prepared to enhance the stability for long term storage. The final HA content of lyophilized antigen references were calibrated against PLS by SRID assay in a collaborative study. Importantly, application of these national reference standards to potency analyses greatly facilitated the development of H7N9 vaccines in China. PMID:25970793

  19. Selection of appropriate analytical tools to determine the potency and bioactivity of antibiotics and antibiotic resistance

    OpenAIRE

    Nishant A. Dafale; Uttam P. Semwal; Rupak K. Rajput; Singh, G. N.

    2016-01-01

    Antibiotics are the chemotherapeutic agents that kill or inhibit the pathogenic microorganisms. Resistance of microorganism to antibiotics is a growing problem around the world due to indiscriminate and irrational use of antibiotics. In order to overcome the resistance problem and to safely use antibiotics, the correct measurement of potency and bioactivity of antibiotics is essential. Microbiological assay and high performance liquid chromatography (HPLC) method are used to quantify the pote...

  20. Selection of appropriate analytical tools to determine the potency and bioactivity of antibiotics and antibiotic resistance

    Directory of Open Access Journals (Sweden)

    Nishant A. Dafale

    2016-08-01

    Simultaneously, microbiological assay addresses the several parameters such as minimal inhibitory concentration (MIC, minimum bactericidal concentration (MBC, mutation prevention concentration (MPC and critical concentration (Ccr which are used to describe the potency in a more informative way. Microbiological assay is a simple, sensitive, precise and cost effective method which gives reproducible results similar to HPLC. However, the HPLC cannot be a complete substitute for microbiological assay and both methods have their own significance to obtain more realistic and precise results.

  1. Development of a Time and Cost Benefit Antibody Binding Test-Based Method for Determination of Rabies Vaccine Potency.

    Science.gov (United States)

    Asgary, Vahid; Mojtabavi, Nazanin; Janani, Alireza; Mousavi, Tahereh; Hadjati, Jamshid; Khosravy, Mohammad Sadeq; Ahangari Cohan, Reza

    2017-01-24

    This study is an improvement on the antibody binding test, known as ABT method, to develop a simple and fast method in comparison with NIH for determination of rabies vaccine potency. In the current study, several commercial human and veterinary vaccines were tested using both modified ABT and NIH methods. The ED50 was calculated using the probit method and the relative potency of each vaccine was measured based on the reference vaccine. The test was repeated four times to calculate the reproducibility of the method. Statistical analysis indicated that there was no significant difference between the result obtained from NIH and modified ABT method for either human or veterinary vaccines (p > 0.05). In addition, the linearity of the method (R(2)) was calculated as 0.94 by serial dilution of a test vaccine. Coefficient variances were determined as less than and more than 10% for the human and veterinary rabies vaccines, respectively. In conclusion, the findings suggest that the modified method could be considered as an alternative approach for rabies vaccine potency determination in in-process quality control tests at industrial scale. It is a time and cost benefit method and accuracy may further be increased by employing monoclonal antibodies against trimeric form of G glycoprotein. However, the use of serum samples may be useful compared with an artificial mix of antibodies because other components from the serum samples could have a positive impact on cell sensitivity and mimic more the complexity of the immune response. Although the modified test has solved a fundamental problem, it is still not sensitive enough for veterinary vaccine assessment and needs further modifications to obtain the acceptability criteria.

  2. Evaluation of nanoparticle-ligand distributions to determine nanoparticle concentration.

    Science.gov (United States)

    Uddayasankar, Uvaraj; Shergill, Ravi T; Krull, Ulrich J

    2015-01-20

    The concentration of nanoparticles in solution is an important, yet challenging, parameter to quantify. In this work, a facile strategy for the determination of nanoparticle concentration is presented. The method relies on the quantitative analysis of the inherent distribution of nanoparticle-ligand conjugates that are generated when nanoparticles are functionalized with ligands. Validation of the method was accomplished by applying it to gold nanoparticles and semiconductor nanoparticles (CdSe/ZnS; core/shell). Poly(ethylene glycol) based ligands, with functional groups that quantitatively react with the nanoparticles, were incubated with the nanoparticles at varying equivalences. Agarose gel electrophoresis was subsequently used to separate and quantify the nanoparticle-ligand conjugates of varying valences. The distribution in the nanoparticle-ligand conjugates agreed well with that predicted by the Poisson model. A protocol was then developed, where a series of only eight different ligand amounts could provide an estimate of the nanoparticle concentration that spans 3 orders of magnitude (1 μM to 1 mM). For the gold nanoparticles and semiconductor nanoparticles, the measured concentrations were found to deviate by only 7% and 2%, respectively, from those determined by UV-vis spectroscopy. The precision of the assay was evaluated, resulting in a coefficient of variation of 5-7%. Finally, the protocol was used to determine the extinction coefficient of alloyed semiconductor nanoparticles (CdSxSe1-x/ZnS), for which a reliable estimate is currently unavailable, of three different emission wavelengths (525, 575, and 630 nm). The extinction coefficient of the nanoparticles of all emission wavelengths was similar and was found to be 2.1 × 10(5) M(-1)cm(-1).

  3. Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo

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    Hassan, Hatem A.F.M.; Smyth, Lesley; Rubio, Noelia; Ratnasothy, Kulachelvy; Wang, Julie T.-W.; Bansal, Sukhvinder S.; Summers, Huw D.; Diebold, Sandra S.; Lombardi, Giovanna; Al-Jamal, Khuloud T.

    2016-01-01

    Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~ 386 nm), bearing net positive charge (5.8 mV), or short MWNTs-OVA (~ 122 nm) of increasing negative charges (− 23.4, − 35.8 or − 39 mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system. PMID:26802552

  4. In vitro potency determination of botulinum neurotoxin B based on its receptor-binding and proteolytic characteristics.

    Science.gov (United States)

    Wild, Emina; Bonifas, Ursula; Klimek, Jolanta; Trösemeier, Jan-Hendrik; Krämer, Beate; Kegel, Birgit; Behrensdorf-Nicol, Heike A

    2016-08-01

    Botulinum neurotoxins (BoNTs) are the most potent toxins known. However, the paralytic effect caused by BoNT serotypes A and B is taken advantage of to treat different forms of dystonia and in cosmetic procedures. Due to the increasing areas of application, the demand for BoNTs A and B is rising steadily. Because of the high toxicity, it is mandatory to precisely determine the potency of every produced BoNT batch, which is usually accomplished by performing toxicity testing (LD50 test) in mice. Here we describe an alternative in vitro assay for the potency determination of the BoNT serotype B. In this assay, the toxin is first bound to its specific receptor molecules. After the proteolytic subunit of the toxin has been released and activated by chemical reduction, it is exposed to synaptobrevin, its substrate protein. Finally the proteolytic cleavage is quantified by an antibody-mediated detection of the neoepitope, reaching a detection limit below 0.1mouseLD50/ml. Thus, the assay, named BoNT/B binding and cleavage assay (BoNT/B BINACLE), takes into account the binding as well as the protease function of the toxin, thereby measuring its biological activity.

  5. Integration of in silico approaches to determination of endocrine-disrupting perfluorinated chemicals binding potency with steroidogenic acute regulatory protein.

    Science.gov (United States)

    Kranthi Kumar, K; Uma Devi, B; Neeraja, P

    2017-09-30

    A myriad of perfluorinated compounds (PFCs) have the ability to interfere with steroidogenic acute regulatory (StAR) protein. Consequently, PFCs breaches cholesterol biotransformation in mitochondria and cause fatal consequences in steroidogenesis, however, these were poorly characterized. In the present study, we have evaluated toxic potencies, nuclear mediated probabilities and interaction profiles with StAR of PFCs using computational system biology tools. Toxicity endpoints revealed that PFCs contain high carcinogenicity, developmental toxicity, skin sensitization effects with low mutagenic activity. Consensus qualitative nuclear receptor agonist models show higher probability rates towards ER and PPAR-γ receptor than AR and AhR models were observed. To poise the subtle fluctuations of actual predictions, balanced accuracy and MCC were computed, and they signify perfect correlation ranges in all models. Screening studies resulting protein-ligand interaction profiles showed that residues Asn148, Asn150, Glu169, Ala171, Arg182, Phe184, Arg188, Trp241, Thr263 and Phe267 were identified as novel hotspots, participated in halogen bonds, H-bonds, atomic π-stacking, π-cation interactions and salt-bridges formation. Thus, the additional bonds contribute conformer stability that holds the protein structure at flexible state, so that PFCs acts as a barrier to cholesterol binding. From docking outcomes, representation space was created, that specifies high and medium StAR binders were occupied in toxic endpoints space with active concern. PFCs restrain molecular features and mitochondrial membrane disruption functions were revealed by efficient toxicogenomics studies. These data indicate toxicity and StAR protein binding levels of PFCs, sorted pinpoints could be useful in a promising way to know the other environmental pollutants and health risks. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Determination of estrogen receptor {beta}-mediated estrogenic potencies of hydroxylated PCBS by a yeast two-hybrid assay

    Energy Technology Data Exchange (ETDEWEB)

    Kuroki, H.; Kumate, M.; Nakaoka, H.; Yonekura, S. [Daiichi Coll. of Pharmaceutical Sciences, Fukuoka (Japan); Nishikawa, J.; Nishihara, T. [Osaka Univ., Osaka (Japan)

    2004-09-15

    Several environmental phenolic chemicals such as Nonylphenol and Bisphenol A (BPA) have been previously shown to possess estrogenic properties. In the previous paper, we have investigated the estrogenic activity of a series of hydroxylated PCBs (OH-PCBs) by a yeast two-hybrid assay (estrogen receptor{alpha} (ER{alpha}) -TIF2), in which the expression of estrogenic activity is based on the interaction of chemicals with ER{alpha}, and demonstrated that 4'-OH-CB30 and 4'-OH-CB61 are more estrogenic than BPA, one of the environmental estrogens. We have showed that one chlorine substitution adjacent to 4-OH at 3- or 5-position significantly reduces the ER{alpha}-mediated estrogenic activity of 4-OH-PCBs. Thus, 4'-OH-CB25 and 4-OH-CB56 showed a very weak estrogenicity. We have also showed that 4-OH-PCBs with two chlorine substitutions adjacent to 4-OH at 3- and 5-position such as 4'-OH-CB79 (hydroxylated metabolite of CB77) and persistent 4-OH-PCBs retained in human blood (4-OH-CB107, 4-OH-CB146 and 4-OH-CB187) have no ER{alpha}-mediated estrogenic activity. ER is known to have two subtypes, namely ER{alpha} and ER{beta} and it is reported that ligand, some agonist and antagonist have a different binding affinity for ER{alpha} and ER{beta}. However, there is limited information on ER{beta}-mediated endocrine disrupting potency. In this study, we examined the ER{beta}-mediated estrogenic activity of a series of OH-PCBs, including environmentally relevant 4-OH-PCBs by a yeast two-hybrid assay (ER{beta}-TIF2).

  7. Evaluation of natural substances from Evolvulus alsinoides L. with the purpose of determining their antioxidant potency.

    Science.gov (United States)

    Cervenka, Frantisek; Koleckar, Vit; Rehakova, Zuzana; Jahodar, Ludek; Kunes, Jiri; Opletal, Lubomir; Hyspler, Radomir; Jun, Daniel; Kuca, Kamil

    2008-08-01

    In recent years, great attention has been given to the search for natural compounds or extracts with the purpose of medical use. Evolvulus alsinoides L. (Convolvulaceae) is a plant used in traditional medicine of East Asia in many indications and has known nootropic and anti-inflammatory activity. However, the bioactive constituents have been described poorly in the literature. Four substances isolated from the ethanol extract of E. alsinoides by means of polyamide and Silica-gel chromatography are reported here. Their molecular structures were determined using NMR analyses. There were identified as scopoletin, umbelliferone, scopolin and 2-methyl-1,2,3,4-butanetetrol. The quantity of these substances was determined using HPLC-UV and GC-FID detection. Antioxidant activity of the isolated substances was measured by DPPH assay using the SIA method. Antioxidant activity and total phenolic content of the prepared fractions are also described. The prepared fractions and isolated substances did not exhibit any significant activity in DPPH test.

  8. Role of ligand-dependent GR phosphorylation and half-life in determination of ligand-specific transcriptional activity.

    Science.gov (United States)

    Avenant, Chanel; Ronacher, Katharina; Stubsrud, Elisabeth; Louw, Ann; Hapgood, Janet P

    2010-10-07

    A central question in glucocorticoid mechanism of action via the glucocorticoid receptor (GR) is what determines ligand-selective transcriptional responses. Using a panel of 12 GR ligands, we show that the extent of GR phosphorylation at S226 and S211, GR half-life and transcriptional response, occur in a ligand-selective manner. While GR phosphorylation at S226 was shown to inhibit maximal transcription efficacy, phosphorylation at S211 is required for maximal transactivation, but not for transrepression efficacy. Both ligand-selective GR phosphorylation and half-life correlated with efficacy for transactivation and transrepression. For both expressed and endogenous GR, in two different cell lines, agonists resulted in the greatest extent of phosphorylation and the greatest extent of GR downregulation, suggesting a link between these functions. However, using phosphorylation-deficient GR mutants we established that phosphorylation of the GR at S226 or S211 does not determine the rank order of ligand-selective GR transactivation. These results are consistent with a model whereby ligand-selective GR phosphorylation and half-life are a consequence of upstream events, such as ligand-specific GR conformations, which are maintained in the phosphorylation mutants.

  9. Determination of in vitro relative potency (REP) values for mono-ortho polychlorinated biphenyls after purification with active charcoal

    NARCIS (Netherlands)

    Peters, A.K.; Leonards, P.E.G.; Zhao, B.; Bergman, A.; Denison, M.S.; Berg, van den M.

    2006-01-01

    The TEF system for dioxin-like compounds has included assignment of TEF values for mono-ortho polychlorinated biphenyls (MO-PCBs). Small traces of aryl hydrocarbon receptor (AhR)-active impurities could result in artifactually higher relative potency (REP) values. MO-PCBs -105, -118, -156, and -167

  10. pFe(3+) determination of multidentate ligands by a fluorescence assay.

    Science.gov (United States)

    Ma, Yongmin; Zhou, Tao; Hider, Robert C

    2015-05-21

    The fluorescence intensity of the iron-CP691 complex in the presence of a competing multidentate ligand is associated with pFe(3+) of the competing ligand and the relative fluorescence has a linear correlation with the pFe(3+) values. A correlation was also found to exist between the relative fluorescence and the ratio of a competing ligand to the probe CP691. Based on this assay, the pFe(3+) value of a range of hexadentate ligands, dendrimers and polymers can be determined when they fall in the range 24.5-30.5. Only small quantities of chelators are required for this assay.

  11. Ability of Bruton's Tyrosine Kinase Inhibitors to Sequester Y551 and Prevent Phosphorylation Determines Potency for Inhibition of Fc Receptor but not B-Cell Receptor Signaling.

    Science.gov (United States)

    Bender, Andrew T; Gardberg, Anna; Pereira, Albertina; Johnson, Theresa; Wu, Yin; Grenningloh, Roland; Head, Jared; Morandi, Federica; Haselmayer, Philipp; Liu-Bujalski, Lesley

    2017-03-01

    Bruton's tyrosine kinase (Btk) is expressed in a variety of hematopoietic cells. Btk has been demonstrated to regulate signaling downstream of the B-cell receptor (BCR), Fc receptors (FcRs), and toll-like receptors. It has become an attractive drug target because its inhibition may provide significant efficacy by simultaneously blocking multiple disease mechanisms. Consequently, a large number of Btk inhibitors have been developed. These compounds have diverse binding modes, and both reversible and irreversible inhibitors have been developed. Reported herein, we have tested nine Btk inhibitors and characterized on a molecular level how their interactions with Btk define their ability to block different signaling pathways. By solving the crystal structures of Btk inhibitors bound to the enzyme, we discovered that the compounds can be classified by their ability to trigger sequestration of Btk residue Y551. In cells, we found that sequestration of Y551 renders it inaccessible for phosphorylation. The ability to sequester Y551 was an important determinant of potency against FcεR signaling as Y551 sequestering compounds were more potent for inhibiting basophils and mast cells. This result was true for the inhibition of FcγR signaling as well. In contrast, Y551 sequestration was less a factor in determining potency against BCR signaling. We also found that Btk activity is regulated differentially in basophils and B cells. These results elucidate important determinants for Btk inhibitor potency against different signaling pathways and provide insight for designing new compounds with a broader inhibitory profile that will likely result in greater efficacy. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  12. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J;

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA...

  13. Rationale for Determining the Functional Potency of Mesenchymal Stem Cells in Preventing Regulated Cell Death for Therapeutic Use.

    Science.gov (United States)

    Naji, Abderrahim; Suganuma, Narufumi; Espagnolle, Nicolas; Yagyu, Ken-Ichi; Baba, Nobuyasu; Sensebé, Luc; Deschaseaux, Frédéric

    2016-10-11

    : Mesenchymal stem (stromal) cells (MSCs) are being investigated for treating degenerative and inflammatory disorders because of their reparative and immunomodulatory properties. Intricate mechanisms relate cell death processes with immune responses, which have implications for degenerative and inflammatory conditions. We review the therapeutic value of MSCs in terms of preventing regulated cell death (RCD). When cells identify an insult, specific intracellular pathways are elicited for execution of RCD processes, such as apoptosis, necroptosis, and pyroptosis. To some extent, exacerbated RCD can provoke an intense inflammatory response and vice versa. Emerging studies are focusing on the molecular mechanisms deployed by MSCs to ameliorate the survival, bioenergetics, and functions of unfit immune or nonimmune cells. Given these aspects, and in light of MSC actions in modulating cell death processes, we suggest the use of novel functional in vitro assays to ensure the potency of MSCs for preventing RCD. Such analyses should be associated with existing functional assays measuring the anti-inflammatory capabilities of MSCs in vitro. MSCs selected on the basis of two in vitro functional criteria (i.e., prevention of inflammation and RCD) could possess optimal therapeutic efficacy in vivo. In addition, we underline the implications of these perspectives in clinical studies of MSC therapy, with particular focus on acute respiratory distress syndrome. Most studies of mesenchymal stem (stromal) cells (MSCs) focus on their anti-inflammatory, trophic and differentiation abilities, but their ability to prevent regulated cell death (RCD) remains undefined. However, this last function could explain both the regenerative and anti-inflammatory therapeutic effect of MSCs observed in preclinical and clinical studies. The present report reviews the role of MSCs in preventing RCD, with implications for enhancing their therapeutic efficacy in the clinic. Development of in vitro

  14. Rationale for Determining the Functional Potency of Mesenchymal Stem Cells in Preventing Regulated Cell Death for Therapeutic Use.

    Science.gov (United States)

    Naji, Abderrahim; Suganuma, Narufumi; Espagnolle, Nicolas; Yagyu, Ken-Ichi; Baba, Nobuyasu; Sensebé, Luc; Deschaseaux, Frédéric

    2017-03-01

    Mesenchymal stem (stromal) cells (MSCs) are being investigated for treating degenerative and inflammatory disorders because of their reparative and immunomodulatory properties. Intricate mechanisms relate cell death processes with immune responses, which have implications for degenerative and inflammatory conditions. We review the therapeutic value of MSCs in terms of preventing regulated cell death (RCD). When cells identify an insult, specific intracellular pathways are elicited for execution of RCD processes, such as apoptosis, necroptosis, and pyroptosis. To some extent, exacerbated RCD can provoke an intense inflammatory response and vice versa. Emerging studies are focusing on the molecular mechanisms deployed by MSCs to ameliorate the survival, bioenergetics, and functions of unfit immune or nonimmune cells. Given these aspects, and in light of MSC actions in modulating cell death processes, we suggest the use of novel functional in vitro assays to ensure the potency of MSCs for preventing RCD. Such analyses should be associated with existing functional assays measuring the anti-inflammatory capabilities of MSCs in vitro. MSCs selected on the basis of two in vitro functional criteria (i.e., prevention of inflammation and RCD) could possess optimal therapeutic efficacy in vivo. In addition, we underline the implications of these perspectives in clinical studies of MSC therapy, with particular focus on acute respiratory distress syndrome. Stem Cells Translational Medicine 2017;6:713-719. © 2016 The Authors Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  15. Determination of protein-ligand interactions using accelerator mass spectrometry: modified crosslinking assay.

    Science.gov (United States)

    Hah, Sang Soo

    2009-05-01

    A highly sensitive detection method for the determination of protein-ligand interactions has been developed. Radiocarbon-labeled 17beta-estradiol was incubated with estrogen receptor-alpha; as a selective binding partner, and covalently attached using crosslinking agents, to form covalently linked protein-ligand complexes. After separation using a denaturing gel, the (14)C content in the sliced gels was identified by accelerator mass spectrometry. The obtained data demonstrated specific binding of the small molecule to its binding partner. In theory, this method can be applied to most protein-ligand interaction studies.

  16. Transfer of a two-tiered keratinocyte assay: IL-18 production by NCTC2544 to determine the skin sensitizing capacity and epidermal equivalent assay to determine sensitizer potency

    DEFF Research Database (Denmark)

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke

    2013-01-01

    . The two tiered approach may offer an unique opportunity to provide an alternative method to the Local Lymph Node Assay (LLNA). These assays are both based on the use of human keratinocytes, which have been shown over the last two decades, to play a key role in all phases of skin sensitization.......At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it is very important from ethical, safety and economic point of view to have biological markers to discriminate allergy and irritation events, and to be able to classify sensitizers...... sensitizers from non-sensitizers. This study describes two of these assays, which when used in a tiered strategy, may be able to identify contact sensitizers and also to quantify sensitizer potency. Tier 1 is the human keratinocyte NCTC2544 IL-18 assay and tier 2 is the Epidermal Equivalent potency assay...

  17. Determination of potency and paraspecific effects of Androctonus crassicauda (Olivier, 1807 antivenom against Mesobuthus gibbosus (Brullé, 1832 venom (Scorpiones: Buthidae

    Directory of Open Access Journals (Sweden)

    O. Ozkan

    2007-01-01

    Full Text Available Scorpion envenomation remains a real health problem in many countries. In scorpionism cases, it is often recommended that patients be treated with species-specific antivenom. Androctonus crassicauda venom has been used as antigen for antivenom production in Turkey, where this antivenom, called Turkish antivenom, has also been effective in the treatment of envenomation caused by species other than A. crassicauda. The present study aimed at determining the paraspecific effects and potency of the Turkish antivenom against Mesobuthus gibbosus (Brullé, 1832 venom. To assess the venom toxicity and the antivenom efficacy, we determined the Minimum Lethal Dose (MLD and the Minimum Effective Dose (MED instead of LD50 and ED50, respectively. Androctonus crassicauda antivenom was capable of neutralizing M. gibbosus venom (20 MLD. This was the first study indicating that A. crassicauda antivenom can be used for the treatment of Mesobuthus gibbosus stings, especially in Aegean Region, Turkey.

  18. Quantitative Determination of DNA-Ligand Binding Using Fluorescence Spectroscopy

    Science.gov (United States)

    Healy, Eamonn F.

    2007-01-01

    The effective use of fluorescence spectroscopy for determining the binding of the intercalcating agent crhidium bromide to DNA is being described. The analysis used simple measurement techniques and hence can be easily adopted by the students for a better understanding.

  19. Protein-ligand binding affinity determination by the waterLOGSY method: An optimised approach considering ligand rebinding

    Science.gov (United States)

    Huang, Renjie; Bonnichon, Arnaud; Claridge, Timothy D. W.; Leung, Ivanhoe K. H.

    2017-03-01

    WaterLOGSY is a popular ligand-observed NMR technique to screen for protein-ligand interactions, yet when applied to measure dissociation constants (KD) through ligand titration, the results were found to be strongly dependent on sample conditions. Herein, we show that accurate KDs can be obtained by waterLOGSY with optimised experimental setup.

  20. Direct determination of unbound lipophilic ligands in aqueous solutions

    Directory of Open Access Journals (Sweden)

    Bojesen Inge N.

    2004-01-01

    Full Text Available Due to their hydrophobic nature, lipophilic compounds are always bound to proteins when transported in the organism. The transfer of such compounds between their binding proteins and cells as well as intracellular trafficking is mediated by a very low water-phase concentration of monomers. The use of protein filled resealed red cell membranes (erythrocyte ghosts as semipermeable bags enables us to determine directly such water-phase concentrations in a biological system where the lipophilic compound is in equilibrium with the compound bound to its binding protein. Equilibrium dissociation constants (Kd’s and number of binding sites are determined by regression analyses of data. We describe the method with the hydrophobic anion arachidonate and the neutral N-arachidonoylethanolamide as examples.

  1. Structural determinants of ligand migration in Mycobacterium tuberculosis truncated hemoglobin O.

    Science.gov (United States)

    Boechi, Leonardo; Martí, Marcelo A; Milani, Mario; Bolognesi, Martino; Luque, F Javier; Estrin, Darío A

    2008-11-01

    Mycobacterium tuberculosis is the causative agent of human tuberculosis, one of the most prevalent infectious diseases in the world. Its genome hosts the glbN and glbO genes coding for two proteins, truncated hemoglobin N (trHbN) and truncated hemoglobin O (trHbO), that belong to different groups (I and II, respectively) of the recently discovered trHb family of hemeproteins. The different expression pattern and kinetics rates constants for ligand association and NO oxidation rate suggest different functions for these proteins. Previous experimental and theoretical studies showed that, in trHbs, ligand migration along the internal tunnel cavity system is a key issue in determining the ligand-binding characteristics. The X-ray structure of trHbO has been solved and shows several internal cavities and secondary-docking sites. In this work, we present an extensive investigation of the tunnel/cavity system ofM. tuberculosis trHbO by means of computer-simulation techniques. We have computed the free-energy profiles for ligand migration along three found tunnels in the oxy and deoxy w.t. and mutant trHbO proteins. Our results show that multiple-ligand migration paths are possible and that several conserved residues such as TrpG8 play a key role in the ligand-migration regulation.

  2. Development, Optimization, and Validation of a Microplate Bioassay for Relative Potency Determination of Linezolid Using a Design Space Concept, and its Measurement Uncertainty.

    Science.gov (United States)

    Saviano, Alessandro Morais; Francisco, Fabiane Lacerda; Ostronoff, Celina Silva; Lourenço, Felipe Rebello

    2015-01-01

    The aim of this study was to develop, optimize, and validate a microplate bioassay for relative potency determination of linezolid in pharmaceutical samples using quality-by-design and design space approaches. In addition, a procedure is described for estimating relative potency uncertainty based on microbiological response variability. The influence of culture media composition was studied using a factorial design and a central composite design was adopted to study the influence of inoculum proportion and triphenyltetrazolium chloride in microbial growth. The microplate bioassay was optimized regarding the responses of low, medium, and high doses of linezolid, negative and positive controls, and the slope, intercept, and correlation coefficient of dose-response curves. According to optimization results, design space ranges were established using: (a) low (1.0 μg/mL), medium (2.0 μg/mL), and high (4.0 μg/mL) doses of pharmaceutical samples and linezolid chemical reference substance; (b) Staphylococcus aureus ATCC 653 in an inoculum proportion of 10%; (c) antibiotic No. 3 culture medium pH 7.0±0.1; (d) 6 h incubation at 37.0±0.1ºC; and (e) addition of 50 μL of 0.5% (w/v) triphenyltetrazolium chloride solution. The microplate bioassay was linear (r2=0.992), specific, precise (repeatability RSD=2.3% and intermediate precision RSD=4.3%), accurate (mean recovery=101.4%), and robust. The overall measurement uncertainty was reasonable considering the increased variability inherent in microbiological response. Final uncertainty was comparable with those obtained with other microbiological assays, as well as chemical methods.

  3. An ELISA Based Binding and Competition Method to Rapidly Determine Ligand-receptor Interactions.

    Science.gov (United States)

    Syedbasha, Mohameedyaseen; Linnik, Janina; Santer, Deanna; O'Shea, Daire; Barakat, Khaled; Joyce, Michael; Khanna, Nina; Tyrrell, D Lorne; Houghton, Michael; Egli, Adrian

    2016-01-01

    A comprehensive understanding of signaling pathways requires detailed knowledge regarding ligand-receptor interaction. This article describes two fast and reliable point-by-point protocols of enzyme-linked immunosorbent assays (ELISAs) for the investigation of ligand-receptor interactions: the direct ligand-receptor interaction assay (LRA) and the competition LRA. As a case study, the ELISA based analysis of the interaction between different lambda interferons (IFNLs) and the alpha subunit of their receptor (IL28RA) is presented: the direct LRA is used for the determination of dissociation constants (KD values) between receptor and IFN ligands, and the competition LRA for the determination of the inhibitory capacity of an oligopeptide, which was designed to compete with the IFNLs at their receptor binding site. Analytical steps to estimate KD and half maximal inhibitory concentration (IC50) values are described. Finally, the discussion highlights advantages and disadvantages of the presented method and how the results enable a better molecular understanding of ligand-receptor interactions.

  4. Two-tiered keratinocyte assay: IL-18 production by NCTC2544 cells to determine the skin sensitizing capacity and an epidermal equivalent assay to determine sensitizer potency

    DEFF Research Database (Denmark)

    Teunis, Marc; Corsini, Emanuela; Smits, Mieke

    2012-01-01

    method to the LLNA. Both assays are based on the use of human keratinocytes, which have been shown, over the last two decades, to play a key role in all phases of skin sensitization. First, 4 known chemicals were tested during a transferability study in which 6 laboratories participated. Three......At present, the identification of potentially sensitizing chemicals is carried out using animal models. However, it should be very important, both from ethical and economic point of view, to discriminate allergy and irritation events, and to classify sensitizers according to their potency, without...... the use of animals. The aim of the EU FP6 Integrated Project Sens-it-iv was to develop and optimize an integrated testing strategy consisting of in vitro, human cell based assays which will closely mimic sensitization mechanisms in vivo. These assays should be an alternative approach to the LLNA. The NCTC...

  5. Determination of colloidal gold nanoparticle surface areas, concentrations, and sizes through quantitative ligand adsorption.

    Science.gov (United States)

    Gadogbe, Manuel; Ansar, Siyam M; He, Guoliang; Collier, Willard E; Rodriguez, Jose; Liu, Dong; Chu, I-Wei; Zhang, Dongmao

    2013-01-01

    Determination of the true surface areas, concentrations, and particle sizes of gold nanoparticles (AuNPs) is a challenging issue due to the nanoparticle morphological irregularity, surface roughness, and size distributions. A ligand adsorption-based technique for determining AuNP surface areas in solution is reported. Using a water-soluble, stable, and highly UV-vis active organothiol, 2-mercaptobenzimidazole (MBI), as the probe ligand, we demonstrated that the amount of ligand adsorbed is proportional to the AuNP surface area. The equivalent spherical AuNP sizes and concentrations were determined by combining the MBI adsorption measurement with Au(3+) quantification of aqua regia-digested AuNPs. The experimental results from the MBI adsorption method for a series of commercial colloidal AuNPs with nominal diameters of 10, 30, 50, and 90 nm were compared with those determined using dynamic light scattering, transmission electron microscopy, and localized surface plasmonic resonance methods. The ligand adsorption-based technique is highly reproducible and simple to implement. It only requires a UV-vis spectrophotometer for characterization of in-house-prepared AuNPs.

  6. Evaluacion de la tecnica de contraimmunoelectroforesis para determinar la potencia antigena de las vacunas antirrabicas Evaluation ot the counterimmunoelectrophoresis technique to determine the antigenic potency of antirrabic vaccines

    Directory of Open Access Journals (Sweden)

    Graciela Miceli

    1993-12-01

    paper presents the results obtained when using a counterimmunoelectrophoresis (CIE techniques to determine the antigens content in samples of 84 and 40 samples of lots of suckling mouse rabies and tissue culture rabies vaccines, respectively. The evaluation of rabies vaccines under, at, near and up to 0.3 UI by the NIH and CIE tests showed that for practical use, a CIE titer of 1:4 would match the minimum of NIH potency test requirement. Low degree of CIE test variability within our laboratory was observed, when two working reference and 7 lots of rabies vaccines from different origin and activity were tested in five independent assays. All titres fell within one double dilution what is an indication of its reproducibility. Good sensitivity to detect antigen deterioration in thermal degradation test was observed when samples of 3 lots of liquid suckling mouse rabies vaccine were held stored at 4ºC or at 37ºC for 4 weeks and assays by CIE and NIH tests. Finally, CIE test could apply by manufactures to follow the concentration procedures to determine the final end point which could match a desired antigenic value in the NIH potency test.

  7. Ligand placement based on prior structures: the guided ligand-replacement method

    Energy Technology Data Exchange (ETDEWEB)

    Klei, Herbert E. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Moriarty, Nigel W., E-mail: nwmoriarty@lbl.gov; Echols, Nathaniel [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Terwilliger, Thomas C. [Los Alamos National Laboratory, Los Alamos, NM 87545-0001 (United States); Baldwin, Eric T. [Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Natural Discovery LLC, Princeton, NJ 08542-0096 (United States); Pokross, Matt; Posy, Shana [Bristol-Myers Squibb, Princeton, NJ 08543-4000 (United States); Adams, Paul D. [Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); University of California at Berkeley, Berkeley, CA 94720-1762 (United States)

    2014-01-01

    A new module, Guided Ligand Replacement (GLR), has been developed in Phenix to increase the ease and success rate of ligand placement when prior protein-ligand complexes are available. The process of iterative structure-based drug design involves the X-ray crystal structure determination of upwards of 100 ligands with the same general scaffold (i.e. chemotype) complexed with very similar, if not identical, protein targets. In conjunction with insights from computational models and assays, this collection of crystal structures is analyzed to improve potency, to achieve better selectivity and to reduce liabilities such as absorption, distribution, metabolism, excretion and toxicology. Current methods for modeling ligands into electron-density maps typically do not utilize information on how similar ligands bound in related structures. Even if the electron density is of sufficient quality and resolution to allow de novo placement, the process can take considerable time as the size, complexity and torsional degrees of freedom of the ligands increase. A new module, Guided Ligand Replacement (GLR), was developed in Phenix to increase the ease and success rate of ligand placement when prior protein–ligand complexes are available. At the heart of GLR is an algorithm based on graph theory that associates atoms in the target ligand with analogous atoms in the reference ligand. Based on this correspondence, a set of coordinates is generated for the target ligand. GLR is especially useful in two situations: (i) modeling a series of large, flexible, complicated or macrocyclic ligands in successive structures and (ii) modeling ligands as part of a refinement pipeline that can automatically select a reference structure. Even in those cases for which no reference structure is available, if there are multiple copies of the bound ligand per asymmetric unit GLR offers an efficient way to complete the model after the first ligand has been placed. In all of these applications, GLR

  8. Determination of copper speciation in highway stormwater runoff using competitive ligand exchange - Adsorptive cathodic stripping voltammetry.

    Science.gov (United States)

    Nason, Jeffrey A; Sprick, Matthew S; Bloomquist, Don J

    2012-11-01

    Low concentrations of dissolved copper have been shown to adversely affect the olfactory system of salmonid species, impairing their ability to avoid predators and likely increasing mortality. These studies have resulted in increased regulatory scrutiny of stormwater discharges to surface waters inhabited by threatened and endangered salmonid species. Because it is primarily the free ionic (Cu(2+)) and weakly complexed forms of copper that are bioavailable, it is critical to understand the speciation of copper in stormwater. This paper reports on the characterization of copper binding ligands and copper speciation in composite samples of highway stormwater runoff collected at four sites in Oregon, USA using competitive ligand exchange - adsorptive cathodic stripping voltammetry (CLE-ACSV). Although the concentration and strength of copper binding ligands in stormwater varied considerable between sites and storms, the vast majority (>99.9%) of the total dissolved copper in composite samples was complexed by organic ligands in stormwater. Although total dissolved copper concentrations range from 2 to 20 μg/L, the analytically determined free ionic copper concentrations did not exceed 10(-10) M (6.3 ng/L) in any of the fully characterized samples, suggesting that much of the copper in highway stormwater is not bioavailable. Analytically determined free ionic copper concentrations were compared with those predicted by a readily available chemical equilibrium models and found to be in reasonable agreement. Copyright © 2012 Elsevier Ltd. All rights reserved.

  9. Estrogen potency of oral contraceptive pills.

    Science.gov (United States)

    Chihal, H J; Peppler, R D; Dickey, R P

    1975-01-01

    The estrogen potencies of 9 oral contraceptive pills, Enovid-E, Enovid-5, Ovulen, Demulen, Norinyl+80, Norinyl+50, Ovral, Norlestrin 1 mg. and Norlestrin 2.5 mg., were determined by bioassay. Relative estrogen potency was determined by analysis of variance. Enovid-5, the most estrogenic compound, had a potency of 4.88 compared to ethinyl estradiol, 50 mcg. equal 1.00; Ovral, the least estrogenic compound, had a potency of 0.81, a sixfold difference. Estrogen potencies at a fractional dose of 0.00155 correlate with reports of the incidence of minor side effects and thromboembolic disease. The effect of progestins on estrogen potency was purely additive (norgestrel and norethynodrel), purely antagonistic, or additive at low concentrations and antagonistic at high concentrations (norethindrone, norethindrone acetate, and ethynodiol diacetate). These results suggest that pills with a greater margin of safety might be developed by utilizing greater ratios of progestin to estrogen. In addition, differences in relative estrogen potency of oral contraceptive pills may be used as a basis for better clinical selection.

  10. Determining the size-dependent structure of ligand-free gold-cluster ions.

    Science.gov (United States)

    Schooss, Detlef; Weis, Patrick; Hampe, Oliver; Kappes, Manfred M

    2010-03-28

    Ligand-free metal clusters can be prepared over a wide size range, but only in comparatively small amounts. Determining their size-dependent properties has therefore required the development of experimental methods that allow characterization of sample sizes comprising only a few thousand mass-selected particles under well-defined collision-free conditions. In this review, we describe the application of these methods to the geometric structural determination of Au(n)(+) and Au(n)(-) with n = 3-20. Geometries were assigned by comparing experimental data, primarily from ion-mobility spectrometry and trapped ion electron diffraction, to structural models from quantum chemical calculations.

  11. Potencies of oral contraceptives.

    Science.gov (United States)

    Edgren, R A; Sturtevant, F M

    1976-08-15

    Oral contraceptives are combinations of estrogens and progestogens or, in the case of the mini-pills, progestogens alone. With specific test procedures in laboratory animals or human subjects, it is possible to assign potency evaluations to the components relative to the progestational, estrogenic, or antiestrogenic activities of the progestogen or to the estrogenic potencies of the estrogenic component. It might even be possible to quantify the synergistic effects of the estrogen on the progestational agent. Unfortunately, however, it is impossible now to amalgamate such assay results into single estimates of the potencies of the combinations (either the combination products per se or the combination tablets of sequential products). For example, an over-all estrogenic potency of a combination preparation would involve the integration of contributions form the estrogen itself plus the estrogenic products of metabolism of the progestogen minus the antagonistic effect of the progestational agent, if any. These factors cannot now be quantified independently, much less merged into a single figure of clinical significance. Further, even if it were possible to produce such an estimate, it is unlikely that the evaluation would be meaningful in relation to any putative side effect or adverse reaction, i.e., the alleged thrombogenic effects of oral contraceptives cannot currently be related directly to any measure of potency that will allow prediction of these clinical conditions from laboratory models. Any evaluation of the potential of a given contraceptive to produce a specific side effect will depend upon data generated with specific regard to that adverse reaction and the individual product in question.

  12. Role of the T cell receptor ligand affinity in T cell activation by bacterial superantigens

    DEFF Research Database (Denmark)

    Andersen, P S; Geisler, C; Buus, S

    2001-01-01

    Similar to native peptide/MHC ligands, bacterial superantigens have been found to bind with low affinity to the T cell receptor (TCR). It has been hypothesized that low ligand affinity is required to allow optimal TCR signaling. To test this, we generated variants of Staphylococcus enterotoxin C3...... (SEC3) with up to a 150-fold increase in TCR affinity. By stimulating T cells with SEC3 molecules immobilized onto plastic surfaces, we demonstrate that increasing the affinity of the SEC3/TCR interaction caused a proportional increase in the ability of SEC3 to activate T cells. Thus, the potency...... correlation between ligand affinity and ligand potency indicating that it is the density of receptor-ligand complexes in the T cell contact area that determines TCR signaling strength....

  13. Role of the Sulfonium Center in Determining the Ligand Specificity of Human S-Adenosylmethionine Decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Bale, Shridhar; Brooks, Wesley; Hanes, Jeremiah W.; Mahesan, Arnold M.; Guida, Wayne C.; Ealick, Steven E.; (Moffitt); (Cornell)

    2009-08-13

    S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requirement for a positive charge at the position equivalent to the sulfonium of S-adenosylmethionine. We investigated the ligand specificity of AdoMetDC through crystallography, quantum chemical calculations, and stopped-flow experiments. We determined crystal structures of the enzyme cocrystallized with 5{prime}-deoxy-5{prime}-dimethylthioadenosine and 5{prime}-deoxy-5{prime}-(N-dimethyl)amino-8-methyladenosine. The crystal structures revealed a favorable cation-{pi} interaction between the ligand and the aromatic side chains of Phe7 and Phe223. The estimated stabilization from this interaction is 4.5 kcal/mol as determined by quantum chemical calculations. Stopped-flow kinetic experiments showed that the rate of the substrate binding to the enzyme greatly depends on Phe7 and Phe223, thus supporting the importance of the cation-{pi} interaction.

  14. Optimization of Electrospray Ionization by Statistical Design of Experiments and Response Surface Methodology: Protein-Ligand Equilibrium Dissociation Constant Determinations

    Science.gov (United States)

    Pedro, Liliana; Van Voorhis, Wesley C.; Quinn, Ronald J.

    2016-09-01

    Electrospray ionization mass spectrometry (ESI-MS) binding studies between proteins and ligands under native conditions require that instrumental ESI source conditions are optimized if relative solution-phase equilibrium concentrations between the protein-ligand complex and free protein are to be retained. Instrumental ESI source conditions that simultaneously maximize the relative ionization efficiency of the protein-ligand complex over free protein and minimize the protein-ligand complex dissociation during the ESI process and the transfer from atmospheric pressure to vacuum are generally specific for each protein-ligand system and should be established when an accurate equilibrium dissociation constant (KD) is to be determined via titration. In this paper, a straightforward and systematic approach for ESI source optimization is presented. The method uses statistical design of experiments (DOE) in conjunction with response surface methodology (RSM) and is demonstrated for the complexes between Plasmodium vivax guanylate kinase ( PvGK) and two ligands: 5'-guanosine monophosphate (GMP) and 5'-guanosine diphosphate (GDP). It was verified that even though the ligands are structurally similar, the most appropriate ESI conditions for KD determination by titration are different for each.

  15. Determination of multivalent protein-ligand binding kinetics by second-harmonic correlation spectroscopy.

    Science.gov (United States)

    Sly, Krystal L; Conboy, John C

    2014-11-18

    Binding kinetics of the multivalent proteins peanut agglutinin (PnA) and cholera toxin B subunit (CTB) to a GM1-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid bilayer were investigated by both second-harmonic correlation spectroscopy (SHCS) and a traditional equilibrium binding isotherm. Adsorption and desorption rates, as well as binding affinity and binding free energy, for three bulk protein concentrations were determined by SHCS. For PnA binding to GM1, the measured adsorption rate decreased with increasing bulk PnA concentration from (3.7 ± 0.3) × 10(6) M(-1)·s(-1) at 0.43 μM PnA to (1.1 ± 0.1) × 10(5) M(-1)·s(-1) at 12 μM PnA. CTB-GM1 exhibited a similar trend, decreasing from (1.0 ± 0.1) × 10(9) M(-1)·s(-1) at 0.5 nM CTB to (3.5 ± 0.2) × 10(6) M(-1)·s(-1) at 240 nM CTB. The measured desorption rates in both studies did not exhibit any dependence on initial protein concentration. As such, 0.43 μM PnA and 0.5 nM CTB had the strongest measured binding affinities, (3.7 ± 0.8) × 10(9) M(-1) and (2.8 ± 0.5) × 10(13) M(-1), respectively. Analysis of the binding isotherm data suggests there is electrostatic repulsion between protein molecules when PnA binds GM1, while CTB-GM1 demonstrates positive ligand-ligand cooperativity. This study provides additional insight into the complex interactions between multivalent proteins and their ligands and showcases SHCS for examining these complex yet technologically important protein-ligand complexes used in biosensors, immunoassays, and other biomedical diagnostics.

  16. Selectivity and affinity determinants for ligand binding to the aromatic amino acid hydroxylases.

    Science.gov (United States)

    Teigen, Knut; McKinney, Jeffrey Alan; Haavik, Jan; Martínez, Aurora

    2007-01-01

    Hydroxylation of the aromatic amino acids phenylalanine, tyrosine and tryptophan is carried out by a family of non-heme iron and tetrahydrobiopterin (BH4) dependent enzymes, i.e. the aromatic amino acid hydroxylases (AAHs). The reactions catalyzed by these enzymes are important for biomedicine and their mutant forms in humans are associated with phenylketonuria (phenylalanine hydroxylase), Parkinson's disease and DOPA-responsive dystonia (tyrosine hydroxylase), and possibly neuropsychiatric and gastrointestinal disorders (tryptophan hydroxylase 1 and 2). We attempt to rationalize current knowledge about substrate and inhibitor specificity based on the three-dimensional structures of the enzymes and their complexes with substrates, cofactors and inhibitors. In addition, further insights on the selectivity and affinity determinants for ligand binding in the AAHs were obtained from molecular interaction field (MIF) analysis. We applied this computational structural approach to a rational analysis of structural differences at the active sites of the enzymes, a strategy that can help in the design of novel selective ligands for each AAH.

  17. ANTIOXIDANT POTENCY OF WATER KEFIR

    Directory of Open Access Journals (Sweden)

    Muneer Alsayadi M.S.

    2013-06-01

    Full Text Available Reactive oxygen species (ROS have strong relationship with several diseases. Many fermented foods were reported to be important sources for antioxidant compounds. Antioxidant activity of water kefir never reported in the scientific literature. The objective of this study was to detect and investigate the antioxidant potency of water kefir. Water kefir was prepared by fermentation of sugar solution with kefir grains for 24h. Antioxidant activity of fresh water kefir drink and its extract with (0.125–5 mg/ml was evaluated using 2,2,-diphenyl-1-pricrylhydrozyl (DPPH scavenging method, and inhibition of ascorbate autoxidation and the reducing power of water kefir were determined, Butylated hydroxyanisole (BHA and ascorbic acid were used for comparison. Water kefir demonstrated great ability to DPPH scavenging ranged (9.88-63.17%. And inhibit ascorbate oxidation by (6.08-25.57% increased in consequent with concentration raising. These results prime to conclude that water kefir could be promisor source of natural antioxidants with good potency in health developing.

  18. Comparative assay of Vipera ammodytes antivenom potency.

    Science.gov (United States)

    Capitanescu, Cristian; Macovei Oprescu, Anca Monica; Supeanu, Alexandru; Coculescu, Bogdan Ioan; Strambu, Victor; Macovei, Radu Alexandru; Manole, Gheorghe

    2016-12-01

    The finding of the most appropriate way to assess precisely the antivenom efficacy represents one of the major issues for antivenom standardization and success increasing of antivenom therapy. The efficacy of experimental Vipera ammodytes antivenom raised in sheep was determined using in vivo mouse lethality test, respectively, L-aminoacid oxidase, total proteinase and phospholipase A2 antienzymatic effectiveness. The values gained for the antivenom potency depend on the method of measure. So, some of the most toxic venom proteins own phospholipase A2 activity and provide the highest antivenom potency (lowest effective dose) values by antienzymatic assay method. This value is similar with total antiproteolytic antivenom potency value, but almost three times higher than value obtained by L-aminoacid oxidase (low toxic viper venom protein) antienzymatic assay method.

  19. Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Jacob Lauwring, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Schrøder, Tenna Juul; Christensen, Søren [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Strandbygård, Dorthe [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Pallesen, Lone Tjener [Aarhus University, Ole Worms Allé 3, 8000 Aarhus C (Denmark); García-Alai, Maria Marta [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark); Lindberg, Samsa; Langgård, Morten; Eskildsen, Jørgen Calí; David, Laurent; Tagmose, Lena; Simonsen, Klaus Baek; Maltas, Philip James; Rønn, Lars Christian Biilmann; Jong, Inge E. M. de; Malik, Ibrahim John; Egebjerg, Jan; Karlsson, Jens-Jacob [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Uppalanchi, Srinivas; Sakumudi, Durga Rao; Eradi, Pradheep [GVK BioScience, Plot No. 28 A, IDA Nacharam, Hyderabad 500 076 (India); Watson, Steven P., E-mail: jla@mb.au.dk [H. Lundbeck A/S, Ottiliavej 9, 2500 Valby (Denmark); Thirup, Søren, E-mail: jla@mb.au.dk [Aarhus University, Gustav Wieds Vej 10C, 8000 Aarhus C (Denmark)

    2014-02-01

    The identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor–ligand complex are reported. Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin–AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Å resolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.

  20. Determination of the stability constants for cobalt, nickel and palladium homogeneous catalyst complexes containing triphenylphosphine ligands

    NARCIS (Netherlands)

    Djekic, T.; Zivkovic, Z.; van der Ham, A.G.J.; de Haan, A.B.

    2006-01-01

    Homogeneous catalysts are complex compounds that are always in equilibrium with their free metal, free ligand and other forms of complexes. The ratios between different species are defined by the stability constants, which are influenced by different parameters such as the type of metal, ligand, cou

  1. Comparison of Automated Continuous Flow Method With Shake- Flask Method in Determining Partition Coefficients of Bidentate Hydroxypyridinone Ligands

    Directory of Open Access Journals (Sweden)

    Lotfollah Saghaie

    2003-08-01

    Full Text Available The partition coefficients (Kpart , in octanol/water system of a range of bidentate ligands containing the 3-hydroxypyridin-4-one moiety were determined using shake flask and automated continuous flow methods (filter probe method. The shake flask method was used for extremely hydrophilic or hydrophobic compounds with a Kpart values greater than 100 and less than 0.01. For other ligands which possess moderate lipophilicity (Kpart values between 0.01-100 the filter probe method was used. Also the partition coefficient of four ligands with moderate lipophilicity was determined by shake flask method in order to check comparability of these two methods. While the shake flask method was able to determine either extremely hydrophilic or hydrophobic compounds efficiently, the filter probe method was unable to measure such Kpart values. Although, determination of the Kpart values of all compounds is possible with the classical shake-flask method, the procedure is time consuming. In contrast, the filter probe method offers many advantages over the traditional shake-flask method in terms of speed, efficiency of separation and degree of automation. The shake-flask method is the method of choice for determination of partition coefficients of extremely hydrophilic and hydrophobic ligands.

  2. Epitope distance to the target cell membrane and antigen size determine the potency of T cell-mediated lysis by BiTE antibodies specific for a large melanoma surface antigen.

    Science.gov (United States)

    Bluemel, Claudia; Hausmann, Susanne; Fluhr, Petra; Sriskandarajah, Mirnalini; Stallcup, William B; Baeuerle, Patrick A; Kufer, Peter

    2010-08-01

    Melanoma chondroitin sulfate proteoglycan (MCSP; also called CSPG4, NG2, HMW-MAA, MSK16, MCSPG, MEL-CSPG, or gp240) is a surface antigen frequently expressed on human melanoma cells, which is involved in cell adhesion, invasion and spreading, angiogenesis, complement inhibition, and signaling. MCSP has therefore been frequently selected as target antigen for development of antibody- and vaccine-based therapeutic approaches. We have here used a large panel of monoclonal antibodies against human MCSP for generation of single-chain MCSP/CD3-bispecific antibodies of the BiTE (for bispecific T cell engager) class. Despite similar binding affinity to MCSP, respective BiTE antibodies greatly differed in their potency of redirected lysis of CHO cells stably transfected with full-length human MCSP, or with various MCSP deletion mutants and fusion proteins. BiTE antibodies binding to the membrane proximal domain D3 of MCSP were more potent than those binding to more distal domains. This epitope distance effect was corroborated with EpCAM/CD3-bispecific BiTE antibody MT110 by testing various fusion proteins between MCSP and EpCAM as surface antigens. CHO cells expressing small surface target antigens were generally better lysed than those expressing larger target antigens, indicating that antigen size was also an important determinant for the potency of BiTE antibody. The present study for the first time relates the positioning of binding domains and size of surface antigens to the potency of target cell lysis by BiTE-redirected cytotoxic T cells. In case of the MCSP antigen, this provides the basis for selection of a maximally potent BiTE antibody candidate for development of a novel melanoma therapy.

  3. Determinants governing ligand specificity of the Vibrio harveyi LuxN quorum-sensing receptor.

    Science.gov (United States)

    Ke, Xiaobo; Miller, Laura C; Bassler, Bonnie L

    2015-01-01

    Quorum sensing is a process of bacterial cell-cell communication that relies on the production, release and receptor-driven detection of extracellular signal molecules called autoinducers. The quorum-sensing bacterium Vibrio harveyi exclusively detects the autoinducer N-((R)-3-hydroxybutanoyl)-L-homoserine lactone (3OH-C4 HSL) via the two-component receptor LuxN. To discover the principles underlying the exquisite selectivity LuxN has for its ligand, we identified LuxN mutants with altered specificity. LuxN uses three mechanisms to verify that the bound molecule is the correct ligand: in the context of the overall ligand-binding site, His210 validates the C3 modification, Leu166 surveys the chain-length and a strong steady-state kinase bias imposes an energetic hurdle for inappropriate ligands to elicit signal transduction. Affinities for the LuxN kinase on and kinase off states underpin whether a ligand will act as an antagonist or an agonist. Mutations that bias LuxN to the agonized, kinase off, state are clustered in a region adjacent to the ligand-binding site, suggesting that this region acts as the switch that triggers signal transduction. Together, our analyses illuminate how a histidine sensor kinase differentiates between ligands and exploits those differences to regulate its signaling activity.

  4. Determination of the folding of proteins as a function of denaturants, osmolytes or ligands using circular dichroism

    Science.gov (United States)

    Greenfield, Norma J.

    2009-01-01

    Circular dichroism (CD) is an excellent tool for examining the interactions and stability of proteins. This protocol covers methods to obtain and analyze circular dichroism spectra to measure changes in the folding of proteins as a function of denaturants, osmolytes or ligands. Applications include determination of the free energy of folding of a protein, the effects of mutations on protein stability and the estimation of binding constants for the interactions of proteins with other proteins, DNA or ligands, such as substrates or inhibitors. The experiments take 2-5 h. PMID:17406529

  5. Opioid analgesics: does potency matter?

    Science.gov (United States)

    Passik, Steven D; Webster, Lynn

    2014-01-01

    Prescription opioid analgesics with a wide range of potencies are currently used for the treatment of chronic pain. Yet understanding the clinical relevance and therapeutic consequences of opioid potency remains ill defined. Both patients and clinicians alike have misperceptions about opioid potency, expecting that less-potent opioids will be less effective or fearing that more-potent opioids are more dangerous or more likely to be abused. In this review, common myths about the potency of opioid analgesics will be discussed. Clinicians should understand that pharmacologic potency per se does not necessarily imply more effective analgesia or higher abuse liability. Published dose conversion tables may not accurately calculate the dose for effective and safe rotation from one opioid to another in patients receiving long-term opioid therapy because they are based on limited data that may not apply to chronic pain. Differences in pharmacologic potency are largely accounted for by the actual doses prescribed, according to individualized patient need. Factors for achieving effective analgesia and reducing the risks involved with opioid use include careful medication selection based on patient characteristics, appropriate dosing titration and opioid rotation practices, knowledge of product formulation characteristics (eg, extended release, immediate release, and tamper-resistant features), and an awareness of differences in opioid pharmacokinetics and metabolism. Clinicians should remain vigilant in monitoring patients on any opioid medication, regardless of classification along the opioid potency continuum.

  6. Determining PPARγ-ligand binding affinity using fluorescent assay with cis-parinaric acid as a probe

    Institute of Scientific and Technical Information of China (English)

    GAO Zhenting; LUO Haibin; CHEN Lili; SHEN Jianhua; CHEN Kaixian; JIANG Hualiang; SHEN Xu

    2005-01-01

    Upon the study of small-molecules binding to proteins, the traditional methods for calculating dissociation constants (Kd and Ki) have shortcomings in dealing with the single binding site models. In this paper, two equations have been derived to solve this problem. These two equations are independent of the total concentration or initial degree of saturation of receptor and the activity of the competitive molecule. Through nonlinear fitting against these two equations, Kd value of a probe can be obtained by binding assay, and Ki value of a ligand can be obtained by competitive assay. Moreover, only the total concentrations of receptor([R]t), ligand([L]t) and probe([P]t) are required for the data fitting. In this work, Ki values of some typical ligands of PPARγ were successfully determined by use of our equations, among which the Ki value of PPARγ-LY171883 was reported for the first time.

  7. Reverse depletion effects and the determination of ligand density on some spherical bioparticles

    Science.gov (United States)

    Wang, Chunxiang; Liu, Yanhui; Fan, Yangtao; Liu, Yijun; Li, Qiancheng; Xu, Houqiang

    2016-06-01

    In cell environments crowded with macromolecules, the depletion effects act and assist in the assembly of a wide range of cellular structures, from the cytoskeleton to the chromatin loop, which are well accepted. But a recent quantum dot experiment indicated that the dimensions of the receptor-ligand complex have strong effects on the size-dependent exclusion of proteins in cell environments. In this article, a continuum elastic model is constructed to resolve the competition between the dimension of the receptor-ligand complex and depletion effects in the endocytosis of a spherical virus-like bioparticle. Our results show that the depletion effects do not always assist endocytosis of a spherical virus-like bioparticle; while the dimension of the ligand-receptor complex is larger than the size of a small bioparticle in cell environments, the depletion effects do not work and reverse effects appear. The ligand density covered on the virus can be identified quantitatively.

  8. Structural determinants for the inhibitory ligands of orotidine-5′-monophosphate decarboxylase

    Energy Technology Data Exchange (ETDEWEB)

    Meza-Avina, Maria Elena; Wei, Lianhu; Liu, Yan; Poduch, Ewa; Bello, Angelica M.; Mishra, Ram K.; Pai, Emil F.; Kotra, Lakshmi P. (TGRI); (Toronto)

    2010-06-14

    In recent years, orotidine-5{prime}-monophosphate decarboxylase (ODCase) has gained renewed attention as a drug target. As a part of continuing efforts to design novel inhibitors of ODCase, we undertook a comprehensive study of potent, structurally diverse ligands of ODCase and analyzed their structural interactions in the active site of ODCase. These ligands comprise of pyrazole or pyrimidine nucleotides including the mononucleotide derivatives of pyrazofurin, barbiturate ribonucleoside, and 5-cyanouridine, as well as, in a computational approach, 1,4-dihydropyridine-based non-nucleoside inhibitors such as nifedipine and nimodipine. All these ligands bind in the active site of ODCase exhibiting distinct interactions paving the way to design novel inhibitors against this interesting enzyme. We propose an empirical model for the ligand structure for rational modifications in new drug design and potentially new lead structures.

  9. The crystal structure of the non-liganded 14-3-3σ protein: insights into determinants of isoform specific ligand binding and dimerization

    Institute of Scientific and Technical Information of China (English)

    Anne BENZINGER; Grzegorz M. POPOWICZ; Joma K. JOY; Sudipta MAJUMDAR; Tad A. HOLAK; Heiko HERMEKING

    2005-01-01

    Seven different, but highly conserved 14-3-3 proteins are involved in diverse signaling pathways in human cells. It is unclear how the 14-3-3σ isoform, a transcriptional target of p53, exerts its inhibitory effect on the cell cycle in the presence of other 14-3-3 isoforms, which are constitutively expressed at high levels. In order to identify structural differences between the 14-3-3 isoforms, we solved the crystal structure of the human 14-3-3σ protein at a resolution of 2.8 A and compared it to the known structures of 14-3-3ζ and 14-3-3τ. The global architecture of the 14-3-3σ fold is similar to the previously determined structures of 14-3-3ζ and 14-3-3τ: two 14-3-3σ molecules form a cup-shaped dimer. Significant differences between these 14-3-3 isoforms were detected adjacent to the amphipathic groove, which mediates the binding to phosphorylated consensus motifs in 14-3-3-1igands. Another specificity determining region is localized between amino-acids 203 to 215. These differences presumably select for the interaction with specific ligands,which may explain the different biological functions of the respective 14-3-3 isoforms. Furthermore, the two 14-3-3σ molecules forming a dimer differ by the spatial position of the ninth helix, which is shifted to the inside of the ligand interaction surface, thus indicating adaptability of this part of the molecule. In addition, 5 non-conserved residues are located at the interface between two 14-3-3σ proteins forming a dimer and represent candidate determinants of homoand hetero-dimerization specificity. The structural differences among the 14-3-3 isoforms described here presumably contribute to isoform-specific interactions and functions.

  10. The met axial ligand determines the redox potential in Cu-A sites

    DEFF Research Database (Denmark)

    Ledesma, G.N.; Murgida, D.H.; Ly, H.K.;

    2007-01-01

    The replacement of the axial methionine ligand in a native Cu-A protein rendered a series of stable mutants with spectroscopic features of a mixed valence center. The mutations resulted in minor perturbations of the electronic structure of this site but led to significant changes in the redox pot...

  11. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Arunima; Pasquel, Danielle [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Tyagi, Rakesh Kumar [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Mani, Sridhar, E-mail: sridhar.mani@einstein.yu.edu [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States)

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  12. The met axial ligand determines the redox potential in Cu-A sites

    DEFF Research Database (Denmark)

    Ledesma, G.N.; Murgida, D.H.; Ly, H.K.

    2007-01-01

    The replacement of the axial methionine ligand in a native Cu-A protein rendered a series of stable mutants with spectroscopic features of a mixed valence center. The mutations resulted in minor perturbations of the electronic structure of this site but led to significant changes in the redox pot...

  13. Ligand Migration in the Gaseous Insulin-CB7 Complex—A Cautionary Tale About the Use of ECD-MS for Ligand Binding Site Determination

    Science.gov (United States)

    Heath, Brittany L.; Jockusch, Rebecca A.

    2012-11-01

    Knowledge of the structure of protein-ligand complexes can aid in understanding their roles within complex biological processes. Here we use electrospray ionization (ESI) coupled to a Fourier transform ion cyclotron resonance mass spectrometer to investigate the noncovalent binding of the macrocycle cucurbit[7]uril (CB7) to bovine insulin. Recent condensed-phase experiments (Chinai et al., J. Am. Chem. Soc. 133:8810-8813, 2011) indicate that CB7 binds selectively to the N-terminal phenylalanine of the insulin B-chain. Competition experiments employing ESI mass spectrometry to assess complex formation between CB7 and wild type insulin B-chain vs. a mutant B-chain, confirm that the N-terminal phenylalanine plays in important role in solution-phase binding. However, analysis of fragment ions produced by electron capture dissociation (ECD) of CB7 complexed to intact insulin and to the insulin B-chain suggests a different picture. The apparent gas-phase binding site, as identified by the ECD, lies further along the insulin B-chain. Together, these studies thus indicate that the CB7 ligand migrates in the ESI mass spectrometry analysis. Migration is likely aided by the presence of additional interactions between CB7 and the insulin B-chain, which are not observed in the crystal structure. While this conformational difference may result simply from the removal of solvent and addition of excess protons by the ESI, we propose that the migration may be enhanced by charge reduction during the ECD process itself because ion-dipole interactions are key to CB7 binding. The results of this study caution against using ECD-MS as a stand-alone structural probe for the determination of solution-phase binding sites.

  14. Quantitative determination of ligand densities on nanomaterials by X-ray photoelectron spectroscopy.

    Science.gov (United States)

    Torelli, Marco D; Putans, Rebecca A; Tan, Yizheng; Lohse, Samuel E; Murphy, Catherine J; Hamers, Robert J

    2015-01-28

    X-ray photoelectron spectroscopy (XPS) is a nearly universal method for quantitative characterization of both organic and inorganic layers on surfaces. When applied to nanoparticles, the analysis is complicated by the strong curvature of the surface and by the fact that the electron attenuation length can be comparable to the diameter of the nanoparticles, making it necessary to explicitly include the shape of the nanoparticle to achieve quantitative analysis. We describe a combined experimental and computational analysis of XPS data for molecular ligands on gold nanoparticles. The analysis includes scattering in both Au core and organic shells and is valid even for nanoparticles having diameters comparable to the electron attenuation length (EAL). To test this model, we show experimentally how varying particle diameter from 1.3 to 6.3 nm leads to a change in the measured AC/AAu peak area ratio, changing by a factor of 15. By analyzing the data in a simple computational model, we demonstrate that ligand densities can be obtained, and, moreover, that the actual ligand densities for these nanoparticles are a constant value of 3.9 ± 0.2 molecules nm(-2). This model can be easily extended to a wide range of core-shell nanoparticles, providing a simple pathway to extend XPS quantitative analysis to a broader range of nanomaterials.

  15. Conformational changes in Bcl-2 pro-survival proteins determine their capacity to bind ligands.

    Science.gov (United States)

    Lee, Erinna F; Czabotar, Peter E; Yang, Hong; Sleebs, Brad E; Lessene, Guillaume; Colman, Peter M; Smith, Brian J; Fairlie, W Douglas

    2009-10-30

    Antagonists of anti-apoptotic Bcl-2 family members hold promise as cancer therapeutics. Apoptosis is triggered when a peptide containing a BH3 motif or a small molecule BH3 peptidomimetic, such as ABT 737, binds to the relevant Bcl-2 family members. ABT-737 is an antagonist of Bcl-2, Bcl-x(L), and Bcl-w but not of Mcl-1. Here we describe new structures of mutant BH3 peptides bound to Bcl-x(L) and Mcl-1. These structures suggested a rationale for the failure of ABT-737 to bind Mcl-1, but a designed variant of ABT-737 failed to acquire binding affinity for Mcl-1. Rather, it was selective for Bcl-x(L), a result attributable in part to significant backbone refolding and movements of helical segments in its ligand binding site. To date there are few reported crystal structures of organic ligands in complex with their pro-survival protein targets. Our structure of this new organic ligand provided insights into the structural transitions that occur within the BH3 binding groove, highlighting significant differences in the structural properties of members of the Bcl-2 pro-survival protein family. Such differences are likely to influence and be important in the quest for compounds capable of selectively antagonizing the different family members.

  16. Evaluation of Phytocannabinoids from High Potency Cannabis sativa using In Vitro Bioassays to Determine Structure-Activity Relationships for Cannabinoid Receptor 1 and Cannabinoid Receptor 2

    OpenAIRE

    2014-01-01

    Cannabis has been around for thousands of years and has been used recreationally, medicinally, and for fiber. Over 500 compounds have been isolated from Cannabis sativa with approximately 105 being cannabinoids. Of those 105 compounds, Δ9-tetrahydrocannabinol has been determined as the primary constituent, which is also responsible for the psychoactivity associated with Cannabis. Cannabinoid receptors belong to the large superfamily of G protein-coupled receptors. Targeting the cannabinoid re...

  17. The probability distribution of side-chain conformations in [Leu] and [Met]enkephalin determines the potency and selectivity to mu and delta opiate receptors

    DEFF Research Database (Denmark)

    Nielsen, Bjørn Gilbert; Jensen, Morten Østergaard; Bohr, Henrik

    2003-01-01

    The structure of enkephalin, a small neuropeptide with five amino acids, has been simulated on computers using molecular dynamics. Such simulations exhibit a few stable conformations, which also have been identified experimentally. The simulations provide the possibility to perform cluster analysis...... in the space defined by potentially pharmacophoric measures such as dihedral angles, side-chain orientation, etc. By analyzing the statistics of the resulting clusters, the probability distribution of the side-chain conformations may be determined. These probabilities allow us to predict the selectivity...

  18. Reliable Determinations of Protein-Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

    Science.gov (United States)

    Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.

    2012-03-01

    The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein-ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

  19. Structural and Biochemical Determinants of Ligand Binding by the c-di-GMP Riboswitch

    Energy Technology Data Exchange (ETDEWEB)

    Smith, K.; Lipchock, S; Livingston,; Shanahan, C; Strobel, S

    2010-01-01

    The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 {angstrom}) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3{prime}-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.

  20. Synthesis and Electronic Structure Determination of Uranium(VI) Ligand Radical Complexes

    OpenAIRE

    Herasymchuk, Khrystyna; Chiang, Linus; Hayes, Cassandra E.; Brown, Matthew L; Ovens, Jeffrey S.; Patrick, Brian; Leznoff, Daniel B.; Storr, Tim

    2016-01-01

       Pentagonal bipyramidal uranyl complexes of salen ligands, N,N’-bis(3-tert-butyl-(5R)-salicylidene)-1,2-phenylenediamine, in which R = tBu (1a), OMe (1b), and NMe2 (1c), were prepared and the electronic structure of the one-electron oxidized species [1a-c]+ were investigated in solution. The solid-state structures of 1a and 1b were solved by X-ray crystallography, and in the case of 1b an asymmetric UO22+ unit was found due to an intermolecular hydrogen bonding interaction. Ele...

  1. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists.

    Science.gov (United States)

    Charfi, Iness; Nagi, Karim; Mnie-Filali, Ouissame; Thibault, Dominic; Balboni, Gianfranco; Schiller, Peter W; Trudeau, Louis-Eric; Pineyro, Graciela

    2014-04-01

    Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

  2. Development and Validation of a Rapid Turbidimetric Assay to Determine the Potency of Cefuroxime Sodium in Powder for Dissolution for Injection

    Directory of Open Access Journals (Sweden)

    Daniela C. M. Vieira

    2014-07-01

    Full Text Available The cefuroxime sodium is a second generation cephalosporin indicated for infections caused by Gram-positive and Gram-negative microorganisms. Although this drug is highly studied and researched regarding the antimicrobial activity, pharmacokinetics and pharmacodynamics, there are few studies regarding the development of analytical methodology for this cephalosporin. Thus, research involving analytical methods is essential and highly relevant to optimize its analysis in the pharmaceutical industry and guarantee the quality of the product already sold. This study describes the development and validation of a microbiological assay applying the turbidimetric method for the determination of cefuroxime, using Micrococcus luteus ATCC 9341 as micro-organism test and 3x3 parallel line assay design, with nine tubes for each assay, as recommended by the Brazilian Pharmacopoeia. The developed and validated method showed excellent results of linearity, seletivity, precision and robustness, in the concentration range from 30.0 to 120.0 mg/mL, with 100.21% accuracy and content 99.97% to cefuroxime sodium in injectable pharmaceutical form.

  3. Classification of contact allergens according to potency: proposals.

    Science.gov (United States)

    Kimber, I; Basketter, D A; Butler, M; Gamer, A; Garrigue, J-L; Gerberick, G F; Newsome, C; Steiling, W; Vohr, H-W

    2003-12-01

    It is clear that contact allergens vary substantially with regard to the relative potency with which they are able to induce skin sensitisation. Considerations of potency will in the future become a significant factor in the classification of skin sensitising chemicals. It is therefore appropriate to establish what is known of potency and thresholds in the induction of skin sensitisation and the elicitation of allergic contact dermatitis, and to identify approaches that might be available for assessment of relative potency for the purposes of categorising chemical allergens. This paper was prepared by an ECETOC (European Centre for Ecotoxicology and Toxicology) Task Force that had the objective of recommending approaches for the measurement of potency and definition of thresholds for both the induction and elicitation of contact sensitisation. The deliberations recorded here build upon recommendations made previously by an ECETOC Task Force that considered the conduct of standard skin sensitisation test methods for the purposes of hazard identification and risk assessment (ECETOC, Monograph No. 29, Brussels, 2000). The emphasis in this present paper is also on standard and accepted methods for the assessment of skin sensitisation, and for which OECD guidelines are available: the local lymph node assay (LLNA), the guinea pig maximisation test and the occluded patch test of Buehler. For various reasons, discussed in detail herein, attention focused primarily upon consideration of categorisation of chemical allergens and the identification of thresholds with respect to the induction of skin sensitisation, rather than the elicitation of allergic contact dermatitis. It is concluded that although the LLNA is the method of choice for the determination of skin sensitisation potency for the purposes of categorisation, if data are already available from appropriate guinea pig tests then their judicious interpretation may provide information of value in determinations of

  4. Determination of Heparin Sodium Injection Potency by Chemicalreagent%亚甲基蓝测定微量肝素钠的研究

    Institute of Scientific and Technical Information of China (English)

    亓培培

    2014-01-01

    The Interaction between heparin sodium and basic methylene blue and the reaction mechanism were preliminary studied.The results showed that the dye was faded dramatically in the formation of the ion association product both heparin sodium and methylene blue basic dye in the HAc-NaAc buffer solution for 4.6.The absorbance was decreased significantly at the maximum absorption wavelength of 664 nm.In addition, the decline for absorbance was of a linear relationship with the content of heparin sodium.Thus the new meth⁃od was proposed and established for the determination of trace heparin sodium based on the results.The lin⁃ear range was 0~1.6 mg/L,the coefficient correlation 0.996 4,and the relatively standard deviation 0.8%,re⁃spectively.The method with high selectivity and sensitivity was simple and rapid.At the same time ,it was ap⁃plied to the detection of heparin sodium content with satisfactory.%研究肝素钠与碱性亚甲基蓝之间的相互作用,对反应机理进行初步的探讨.结果表明,在pH 4.6的HAc-NaAc缓冲溶液中,肝素钠与亚甲基蓝碱性染料形成离子缔合物时,染料发生明显褪色,并且在最大吸收波长664 nm处的吸光度显著下降,下降程度与肝素钠的含量呈线性关系,建立了测定痕量肝素钠含量的新方法.线性范围0~1.6 mg/L,相关系数为0.9964,相对标准偏差为0.8%.该法简便、快速,对样品中肝素钠的测定令人满意.

  5. Determination of the potency of a novel saw palmetto supercritical CO2 extract (SPSE for 5α-reductase isoform II inhibition using a cell-free in vitro test system

    Directory of Open Access Journals (Sweden)

    Pais P

    2016-04-01

    Full Text Available Pilar Pais, Agustí Villar, Santiago Rull Euromed, Barcelona, Spain Background: The nicotinamide adenine dinucleotide phosphate-dependent membrane protein 5α-reductase catalyses the conversion of testosterone to the most potent androgen – 5α-dihydrotestosterone. Two 5α-reductase isoenzymes are expressed in humans: type I and type II. The latter is found primarily in prostate tissue. Saw palmetto extract (SPE has been used extensively in the treatment of lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH. The pharmacological effects of SPE include the inhibition of 5α-reductase, as well as anti-inflammatory and antiproliferative effects. Clinical studies of SPE have been inconclusive – some have shown significant results, and others have not – possibly the result of varying bioactivities of the SPEs used in the studies. Purpose: To determine the in vitro potency in a cell-free test system of a novel SP supercritical CO2 extract (SPSE, an inhibitor of the 5α-reductase isoenzyme type II. Materials and methods: The inhibitory potency of SPSE was compared to that of finasteride, an approved 5α-reductase inhibitor, on the basis of the enzymatic conversion of the substrate androstenedione to the 5α-reduced product 5α-androstanedione. Results: By concentration-dependent inhibition of 5α-reductase type II in vitro (half-maximal inhibitory concentration 3.58±0.05 µg/mL, SPSE demonstrated competitive binding toward the active site of the enzyme. Finasteride, the approved 5α-reductase inhibitor tested as positive control, led to 63%–75% inhibition of 5α-reductase type II. Conclusion: SPSE effectively inhibits the enzyme that has been linked to BPH, and the amount of extract required for activity is comparatively low. It can be confirmed from the results of this study that SPSE has bioactivity that promotes prostate health at a level that is superior to that of many other phytotherapeutic extracts. The

  6. Programmed death-1 concentration at the immunological synapse is determined by ligand affinity and availability.

    Science.gov (United States)

    Pentcheva-Hoang, Tsvetelina; Chen, Lieping; Pardoll, Drew M; Allison, James P

    2007-11-06

    Despite the importance of programmed death-1 (PD-1) for T cell inhibition, little is known about its intracellular trafficking or requirements for localization to the immunological synapse. Here, we show that in activated T cells, PD-1 is present at the plasma membrane, near the Golgi and in the trans-Golgi network. Unlike CD28 and CTLA-4, PD-1 accumulation at the synapse is extensive only when T cells interact with dendritic cells (DCs) expressing high B7-DC levels. However, B7-H1 is also critically important, especially when the DCs have little B7-DC. Despite this preference, B7-H1(-/-) DCs elicit greater cytokine secretion than B7-DC(-/-) DCs during T cell restimulation, possibly because they also express less B7-DC. PD-1 and CD28 have similar kinetics of synaptic accumulation, suggesting that the process involves T cell receptor-triggered cytoskeletal reorganization followed by ligand binding.

  7. Molecular determinants of ligand selectivity for the human multidrug and toxin extruder proteins MATE1 and MATE2-K.

    Science.gov (United States)

    Astorga, Bethzaida; Ekins, Sean; Morales, Mark; Wright, Stephen H

    2012-06-01

    The present study compared the selectivity of two homologous transport proteins, multidrug and toxin extruders 1 and 2-K (MATE1 and MATE2-K), and developed three-dimensional pharmacophores for inhibitory ligand interaction with human MATE1 (hMATE1). The human orthologs of MATE1 and MATE2-K were stably expressed in Chinese hamster ovary cells, and transport function was determined by measuring uptake of the prototypic organic cation (OC) substrate 1-methyl-4-phenylpyridinium (MPP). Both MATEs had similar apparent affinities for MPP, with K(tapp) values of 4.4 and 3.7 μM for MATE1 and MATE2-K, respectively. Selectivity was assessed for both transporters from IC(50) values for 59 structurally diverse compounds. Whereas the two transporters discriminated markedly between a few of the test compounds, the IC(50) values for MATE1 and MATE2-K were within a factor of 3 for most of them. For hMATE1 there was little or no correlation between IC(50) values and the individual molecular descriptors LogP, total polar surface area, or pK(a). The IC(50) values were used to generate a common-features pharmacophore, quantitative pharmacophores for hMATE1, and a bayesian model suggesting molecular features favoring and not favoring the interaction of ligands with hMATE1. The models identified hydrophobic regions, hydrogen bond donor and hydrogen bond acceptor sites, and an ionizable (cationic) feature as key determinants for ligand binding to MATE1. In summary, using a combined in vitro and computational approach, MATE1 and MATE2-K were found to have markedly overlapping selectivities for a broad range of cationic compounds, including representatives from seven novel drug classes of Food and Drug Administration-approved drugs.

  8. Determination of Adenosine A1 Receptor Agonist and Antagonist Pharmacology Using Saccharomyces cerevisiae: Implications for Ligand Screening and Functional Selectivity

    Science.gov (United States)

    Stewart, Gregory D.; Valant, Celine; Dowell, Simon J.; Mijaljica, Dalibor; Devenish, Rodney J.; Scammells, Peter J.; Sexton, Patrick M.

    2009-01-01

    The budding yeast, Saccharomyces cerevisiae, is a convenient system for coupling heterologous G protein-coupled receptors (GPCRs) to the pheromone response pathway to facilitate empirical ligand screening and/or GPCR mutagenesis studies. However, few studies have applied this system to define GPCR-G protein-coupling preferences and furnish information on ligand affinities, efficacies, and functional selectivity. We thus used different S. cerevisiae strains, each expressing a specific human Gα/yeast Gpa1 protein chimera, and determined the pharmacology of various ligands of the coexpressed human adenosine A1 receptor. These assays, in conjunction with the application of quantitative models of agonism and antagonism, revealed that (−)-N6-(2-phenylisopropyl)adenosine was a high-efficacy agonist that selectively coupled to Gpa/1Gαo, Gpa1/Gαi1/2, and Gpa1/Gαi3, whereas the novel compound, 5′-deoxy-N6-(endo-norborn-2-yl)-5′-(2-fluorophenylthio)adenosine (VCP-189), was a lower-efficacy agonist that selectively coupled to Gpa1/Gαi proteins; the latter finding suggested that VCP-189 might be functionally selective. The affinity of the antagonist, 8-cyclopentyl-1,3-dipropylxanthine, was also determined at the various strains. Subsequent experiments performed in mammalian Chinese hamster ovary cells monitoring cAMP formation/inhibition, intracellular calcium mobilization, phosphorylation of extracellular signal-regulated kinase 1 and 2 or 35S-labeled guanosine 5′-(γ-thio)triphosphate binding, were in general agreement with the yeast data regarding agonist efficacy estimation and antagonist affinity estimation, but revealed that the apparent functional selectivity of VCP-189 could be explained by differences in stimulus-response coupling between yeast and mammalian cells. Our results suggest that this yeast system is a useful tool for quantifying ligand affinity and relative efficacy, but it may lack the sensitivity required to detect functional selectivity of

  9. Androgen receptor functional analyses by high throughput imaging: determination of ligand, cell cycle, and mutation-specific effects.

    Directory of Open Access Journals (Sweden)

    Adam T Szafran

    Full Text Available BACKGROUND: Understanding how androgen receptor (AR function is modulated by exposure to steroids, growth factors or small molecules can have important mechanistic implications for AR-related disease therapies (e.g., prostate cancer, androgen insensitivity syndrome, AIS, and in the analysis of environmental endocrine disruptors. METHODOLOGY/PRINCIPAL FINDINGS: We report the development of a high throughput (HT image-based assay that quantifies AR subcellular and subnuclear distribution, and transcriptional reporter gene activity on a cell-by-cell basis. Furthermore, simultaneous analysis of DNA content allowed determination of cell cycle position and permitted the analysis of cell cycle dependent changes in AR function in unsynchronized cell populations. Assay quality for EC50 coefficients of variation were 5-24%, with Z' values reaching 0.91. This was achieved by the selective analysis of cells expressing physiological levels of AR, important because minor over-expression resulted in elevated nuclear speckling and decreased transcriptional reporter gene activity. A small screen of AR-binding ligands, including known agonists, antagonists, and endocrine disruptors, demonstrated that nuclear translocation and nuclear "speckling" were linked with transcriptional output, and specific ligands were noted to differentially affect measurements for wild type versus mutant AR, suggesting differing mechanisms of action. HT imaging of patient-derived AIS mutations demonstrated a proof-of-principle personalized medicine approach to rapidly identify ligands capable of restoring multiple AR functions. CONCLUSIONS/SIGNIFICANCE: HT imaging-based multiplex screening will provide a rapid, systems-level analysis of compounds/RNAi that may differentially affect wild type AR or clinically relevant AR mutations.

  10. Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

    Science.gov (United States)

    Zhou, Han; Peng, Yan; Halikhedkar, Aneetha; Fan, Pusheng; Janero, David R; Thakur, Ganesh A; Mercier, Richard W; Sun, Xin; Ma, Xiaoyu; Makriyannis, Alexandros

    2017-06-21

    TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

  11. Recyclable non-ligand dual cloud point extraction method for determination of lead in food samples.

    Science.gov (United States)

    Wang, Yun; Han, Juan; Liu, Yingying; Wang, Lei; Ni, Liang; Tang, Xu

    2016-01-01

    A new pH-mediated non-ligand dual cloud point extraction (NL-DCPE) was first developed for extraction Pb(II) from food samples. The NL-DCPE method includes two cloud point extraction (CPE) steps and the recycling of the copolymer. The first procedure was based on the forming of lead hydroxide at pH 9.5 and subsequent lead hydroxide was entrapped in a thermoseparating triblock copolymer [(PEO)10(PPO)23(PEO)10] (L44) phase. At second stage, the copolymer-rich phase was treated with the acidic solution, and Pb(II) was back extracted into the aqueous phase. So the problem emerging from the high viscosity of the copolymer-rich phase can be well solved. Under the optimized conditions, the extraction efficiency of 97.20% and detection limit of 1.9 μg L(-1) were obtained. Moreover, the copolymer L44 was successfully recycled and reused for more than two times. This method was successfully used for analyzing Pb(II) in food samples with satisfactory recoveries in the range of 94.01-101.19%.

  12. Structural determinants for ligand capture by a class II preQ1 riboswitch.

    Science.gov (United States)

    Kang, Mijeong; Eichhorn, Catherine D; Feigon, Juli

    2014-02-11

    Prequeuosine (preQ1) riboswitches are RNA regulatory elements located in the 5' UTR of genes involved in the biosynthesis and transport of preQ1, a precursor of the modified base queuosine universally found in four tRNAs. The preQ1 class II (preQ1-II) riboswitch regulates preQ1 biosynthesis at the translational level. We present the solution NMR structure and conformational dynamics of the 59 nucleotide Streptococcus pneumoniae preQ1-II riboswitch bound to preQ1. Unlike in the preQ1 class I (preQ1-I) riboswitch, divalent cations are required for high-affinity binding. The solution structure is an unusual H-type pseudoknot featuring a P4 hairpin embedded in loop 3, which forms a three-way junction with the other two stems. (13)C relaxation and residual dipolar coupling experiments revealed interhelical flexibility of P4. We found that the P4 helix and flanking adenine residues play crucial and unexpected roles in controlling pseudoknot formation and, in turn, sequestering the Shine-Dalgarno sequence. Aided by divalent cations, P4 is poised to act as a "screw cap" on preQ1 recognition to block ligand exit and stabilize the binding pocket. Comparison of preQ1-I and preQ1-II riboswitch structures reveals that whereas both form H-type pseudoknots and recognize preQ1 using one A, C, or U nucleotide from each of three loops, these nucleotides interact with preQ1 differently, with preQ1 inserting into different grooves. Our studies show that the preQ1-II riboswitch uses an unusual mechanism to harness exquisite control over queuosine metabolism.

  13. Determination of Stability Constants of Triazole Ligand Carrying Naphthol Group with Heavy Metal Ions in Aqueous Solutions

    Directory of Open Access Journals (Sweden)

    Nurhan Gümrükçüoğlu

    2013-12-01

    Full Text Available Interaction of 4-(2-hydroxy-1-naphthylmethylamino-3-methyl-5-(4-tolyl-4H-1,2,4-triazole with heavy metal cations such as Cu2+, Co2+, Cd2+, Ni2+ and Pb2+ was investigated by using UV-visible spectrophotometric technique. The complex stability constants (Log β were determined in aqueous as well as in methanol: water (1:1 system at 25 ± 0.1°C by Buschmann’s method and Valeur’s methods, respectively. The ligand showed good sensitivity for Co2+ with a linear range of 210-6M to 310-5M.

  14. Quantitative structure - mesothelioma potency model ...

    Science.gov (United States)

    Cancer potencies of mineral and synthetic elongated particle (EP) mixtures, including asbestos fibers, are influenced by changes in fiber dose composition, bioavailability, and biodurability in combination with relevant cytotoxic dose-response relationships. A unique and comprehensive rat intra-pleural (IP) dose characterization data set with a wide variety of EP size, shape, crystallographic, chemical, and bio-durability properties facilitated extensive statistical analyses of 50 rat IP exposure test results for evaluation of alternative dose pleural mesothelioma response models. Utilizing logistic regression, maximum likelihood evaluations of thousands of alternative dose metrics based on hundreds of individual EP dimensional variations within each test sample, four major findings emerged: (1) data for simulations of short-term EP dose changes in vivo (mild acid leaching) provide superior predictions of tumor incidence compared to non-acid leached data; (2) sum of the EP surface areas (ÓSA) from these mildly acid-leached samples provides the optimum holistic dose response model; (3) progressive removal of dose associated with very short and/or thin EPs significantly degrades resultant ÓEP or ÓSA dose-based predictive model fits, as judged by Akaike’s Information Criterion (AIC); and (4) alternative, biologically plausible model adjustments provide evidence for reduced potency of EPs with length/width (aspect) ratios 80 µm. Regar

  15. Determination of Enantiomeric Compositions of Tryptophan by Chemometric Analysis of the Fluorescence Spectra of Bovine Serum Albumin Receptor-ligand Mixtures

    Institute of Scientific and Technical Information of China (English)

    Yun Xia WANG; Fang ZHANG; Jing LIANG; Hua LI; Ji Lie KONG

    2006-01-01

    In this work, a novel method was constructed to determine the enantiomeric composition of tryptophan (Trp) by bovine serum albumin (BSA) based on the fluorescence spectra of the receptor-ligand mixtures coupled with partial least squares (PLS-1) analysis. As a result the enantiomeric composition of Trp was accurately determined.

  16. General, Label-Free Method for Determining K(d) and Ligand Concentration Simultaneously.

    Science.gov (United States)

    Jalali-Yazdi, Farzad; Takahashi, Terry T; Roberts, Richard W

    2015-12-01

    Some of the most commonly used affinity reagents (e.g., antibodies) are often developed and used in conditions where their input concentrations ([L]0) and affinities (K(d)) are not known. Here, we have developed a general approach to determine both [L]0 and K(d) values simultaneously for affinity reagents (small molecules, proteins, and antibodies). To do this, we perform quantitative equilibrium exclusion immunoassays with two different concentrations of target and fit the data simultaneously to determine K(d) and [L]0. The results give accurate and reproducible measures of both values compared to established methods. By performing detailed error analysis, we demonstrate that our fitting gives unique solutions and indicates where K(d) and [L]0 measures are reliable. Furthermore, we found that a divalent model of antibody binding gives accurate K(d) and [L]0 values in both the forward (antibody immobilized) and the reverse (target immobilized) assays-addressing the long-term problem of obtaining quantitative data from reverse assays.

  17. Quantitative Determination and Comparison of the Surface Binding of Phosphonic Acid, Carboxylic Acid, and Catechol Ligands on TiO2 Nanoparticles.

    Science.gov (United States)

    Zeininger, Lukas; Portilla, Luis; Halik, Marcus; Hirsch, Andreas

    2016-09-12

    The adsorption, desorption, co-adsorption, and exchange behavior of phosphonic acid, carboxylic acid, and catechol derivatives on the surface of titanium oxide (anatase) nanoparticles are investigated. Thermogravimetric analysis provides a facile and fast-track quantitative determination of the wet-chemical monolayer adsorption constants and grafting densities of ten adsorbates, all under neutral pH conditions. This characterization protocol allows straightforward quantification of the relevant thermodynamic data of ligand adsorption and a comparison of ligand adsorption strengths. The reported procedure is proposed as a universal tool and it should be applicable to many other colloidal metal oxide materials. Moreover, the determined values for the adsorption constants and the monolayer grafting densities provide a toolbox for the assessment of the adsorbates' behavior in desorption, exchange, and co-adsorption equilibria. This versatile evaluation procedure will help to identify optimal monolayer-surface combinations and to evaluate critical parameters, such as monolayer robustness, ligand exchange rates, or targeted mixed assembly of functionalities.

  18. Magnetic interactions in oxide-bridged dichromium(III) complexes. Computational determination of the importance of non-bridging ligands

    DEFF Research Database (Denmark)

    Morsing, Thorbjørn Juul; Sauer, Stephan P. A.; Weihe, Høgni

    2013-01-01

    The magnetic susceptibility of the dinuclear chromium(III) complex [(CH3CN)5CrOCr(NCCH3)5](BF4)4 · 2 CH3CN has been measured and analyzed. With a fitted value of the triplet energy J = 650 cm-1, the antiferromagnetic coupling is the strongest hitherto determined for an unsupported linear oxide-br...... relative errors typically of less than 10 % ranging from the strongest coupled systems to systems with moderately strong couplings. A significant influence (>20%) of the chemical nature of the peripheral, non-bridging ligands on the exchange coupling was found and rationalized.......The magnetic susceptibility of the dinuclear chromium(III) complex [(CH3CN)5CrOCr(NCCH3)5](BF4)4 · 2 CH3CN has been measured and analyzed. With a fitted value of the triplet energy J = 650 cm-1, the antiferromagnetic coupling is the strongest hitherto determined for an unsupported linear oxide...

  19. Vasoconstriction Potency Induced by Aminoamide Local Anesthetics Correlates with Lipid Solubility

    Directory of Open Access Journals (Sweden)

    Hui-Jin Sung

    2012-01-01

    Full Text Available Aminoamide local anesthetics induce vasoconstriction in vivo and in vitro. The goals of this in vitro study were to investigate the potency of local anesthetic-induced vasoconstriction and to identify the physicochemical property (octanol/buffer partition coefficient, pKa, molecular weight, or potency of local anesthetics that determines their potency in inducing isolated rat aortic ring contraction. Cumulative concentration-response curves to local anesthetics (levobupivacaine, ropivacaine, lidocaine, and mepivacaine were obtained from isolated rat aorta. Regression analyses were performed to determine the relationship between the reported physicochemical properties of local anesthetics and the local anesthetic concentration that produced 50% (ED50 of the local anesthetic-induced maximum vasoconstriction. We determined the order of potency (ED50 of vasoconstriction among local anesthetics to be levobupivacaine > ropivacaine > lidocaine > mepivacaine. The relative importance of the independent variables that affect the vasoconstriction potency is octanol/buffer partition coefficient > potency > pKa > molecular weight. The ED50 in endothelium-denuded aorta negatively correlated with the octanol/buffer partition coefficient of local anesthetics (r2=0.9563; P<0.001. The potency of the vasoconstriction in the endothelium-denuded aorta induced by local anesthetics is determined primarily by lipid solubility and, in part, by other physicochemical properties including potency and pKa.

  20. Structural Determinants for the Interaction of Formyl Peptide Receptor 2 with Peptide Ligands*

    Science.gov (United States)

    He, Hui-Qiong; Troksa, Erica L.; Caltabiano, Gianluigi; Pardo, Leonardo; Ye, Richard D.

    2014-01-01

    Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-2817.32 is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-2817.32 might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D2817.32G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides. PMID:24285541

  1. Structural determinants for the interaction of formyl peptide receptor 2 with peptide ligands.

    Science.gov (United States)

    He, Hui-Qiong; Troksa, Erica L; Caltabiano, Gianluigi; Pardo, Leonardo; Ye, Richard D

    2014-01-24

    Unlike formyl peptide receptor 1 (FPR1), FPR2/ALX (FPR2) interacts with peptides of diverse sequences but has low affinity for the Escherichia coli-derived chemotactic peptide fMet-Leu-Phe (fMLF). Using computer modeling and site-directed mutagenesis, we investigated the structural requirements for FPR2 to interact with formyl peptides of different length and composition. In calcium flux assay, the N-formyl group of these peptides is necessary for activation of both FPR2 and FPR1, whereas the composition of the C-terminal amino acids appears more important for FPR2 than FPR1. FPR2 interacts better with pentapeptides (fMLFII, fMLFIK) than tetrapeptides (fMLFK, fMLFW) and tripeptide (fMLF) but only weakly with peptides carrying negative charges at the C terminus (e.g. fMLFE). In contrast, FPR1 is less sensitive to negative charges at the C terminus. A CXCR4-based homology model of FPR1 and FPR2 suggested that Asp-281(7.32) is crucial for the interaction of FPR2 with certain formyl peptides as its negative charge may be repulsive with the terminal COO- group of fMLF and negatively charged Glu in fMLFE. Asp-281(7.32) might also form a stable interaction with the positively charged Lys in fMLFK. Site-directed mutagenesis was performed to remove the negative charge at position 281 in FPR2. The D281(7.32)G mutant showed improved affinity for fMLFE and fMLF and reduced affinity for fMLFK compared with wild type FPR2. These results indicate that different structural determinants are used by FPR1 and FPR2 to interact with formyl peptides.

  2. LIBSA--a method for the determination of ligand-binding preference to allosteric sites on receptor ensembles.

    Science.gov (United States)

    Hocker, Harrison J; Rambahal, Nandini; Gorfe, Alemayehu A

    2014-02-24

    Incorporation of receptor flexibility into computational drug discovery through the relaxed complex scheme is well suited for screening against a single binding site. In the absence of a known pocket or if there are multiple potential binding sites, it may be necessary to do docking against the entire surface of the target (global docking). However no suitable and easy-to-use tool is currently available to rank global docking results based on the preference of a ligand for a given binding site. We have developed a protocol, termed LIBSA for LIgand Binding Specificity Analysis, that analyzes multiple docked poses against a single or ensemble of receptor conformations and returns a metric for the relative binding to a specific region of interest. By using novel filtering algorithms and the signal-to-noise ratio (SNR), the relative ligand-binding frequency at different pockets can be calculated and compared quantitatively. Ligands can then be triaged by their tendency to bind to a site instead of ranking by affinity alone. The method thus facilitates screening libraries of ligand cores against a large library of receptor conformations without prior knowledge of specific pockets, which is especially useful to search for hits that selectively target a particular site. We demonstrate the utility of LIBSA by showing that it correctly identifies known ligand binding sites and predicts the relative preference of a set of related ligands for different pockets on the same receptor.

  3. Synthesis, characterization and structural determination of some nickel(II) complexes containing imido Schiff bases and substituted phosphine ligands.

    Science.gov (United States)

    Kianfar, Ali Hossein; Ebrahimi, Mostafa

    2013-11-01

    Some new tridentate ONN Schiff base complexes of [NiL(PR3)] (where L=Salicylidene2-amino4-nitrobenzene (L(1)), 5-BrSalicylidene2-amino4-nitrobenzene (L(2)), 5-NO2Salicylidene2-amino4-nitrobenzene (L(3)), 5-MeOSalicylidene2-amino4-nitrobenzene (L(4)) and 3-MeOSalicylidene2-amino4-nitrobenzene (L(5)), R=Bu and Ph (with L(1))) were synthesised and characterized by IR, UV-Vis, (1)H NMR spectroscopy and elemental analysis. The geometry of [NiL(1)(PPh3)] was determined by X-ray crystallography. It indicated that the complex had a planar structure and four coordinates in the solid state. The thermogravimetry (TG) and differential thermoanalysis (DTA) of the synthesized complexes were carried out in the range of 20-600°C, leading to the decomposition of L(1)-L(3) type in three stages and of L(4)-L(5) and [NiL(1)(PPh3)] type in four stages. Thermal decomposition of the complexes was closely the dependent upon the nature of the Schiff base ligands and proceeded via the first order kinetics.

  4. Ligand Replacement Approach to Raman-Responded Molecularly Imprinted Monolayer for Rapid Determination of Penicilloic Acid in Penicillin.

    Science.gov (United States)

    Zhang, Liying; Jin, Yang; Huang, Xiaoyan; Zhou, Yujie; Du, Shuhu; Zhang, Zhongping

    2015-12-01

    Penicilloic acid (PA) is a degraded byproduct of penicillin and often causes fatal allergies to humans, but its rapid detection in penicillin drugs remains a challenge due to its similarity to the mother structure of penicillin. Here, we reported a ligand-replaced molecularly imprinted monolayer strategy on a surface-enhanced Raman scattering (SERS) substrate for the specific recognition and rapid detection of Raman-inactive PA in penicillin. The bis(phenylenediamine)-Cu(2+)-PA complex was first synthesized and stabilized onto the surface of silver nanoparticle film that was fabricated by a bromide ion-added silver mirror reaction. A molecularly imprinted monolayer was formed by the further modification of alkanethiol around the stabilized complex on the Ag film substrate, and the imprinted recognition site was then created by the replacement of the complex template with Raman-active probe molecule p-aminothiophenol. When PA rebound into the imprinted site in the alkanethiol monolayer, the SERS signal of p-aminothiophenol exhibited remarkable enhancement with a detection limit of 0.10 nM. The imprinted monolayer can efficiently exclude the interference of penicillin and thus provides a selective determination of 0.10‰ (w/w) PA in penicillin, which is about 1 order of magnitude lower than the prescribed residual amount of 1.0‰. The strategy reported here is simple, rapid and inexpensive compared to the traditional chromatography-based methods.

  5. Ligand size is a major determinant of specificity in periplasmic oxyanion-binding proteins: the 1.2 A resolution crystal structure of Azotobacter vinelandii ModA.

    Science.gov (United States)

    Lawson, D M; Williams, C E; Mitchenall, L A; Pau, R N

    1998-12-15

    . Periplasmic receptors constitute a diverse class of binding proteins that differ widely in size, sequence and ligand specificity. Nevertheless, almost all of them display a common beta/alpha folding motif and have similar tertiary structures consisting of two globular domains. The ligand is bound at the bottom of a deep cleft, which lies at the interface between these two domains. The oxyanion-binding proteins are notable in that they can discriminate between very similar ligands. . Azotobacter vinelandii is unusual in that it possesses two periplasmic molybdate-binding proteins. The crystal structure of one of these with bound ligand has been determined at 1.2 A resolution. It superficially resembles the structure of sulphate-binding protein (SBP) from Salmonella typhimurium and uses a similar constellation of hydrogen-bonding interactions to bind its ligand. However, the detailed interactions are distinct from those of SBP and the more closely related molybdate-binding protein of Escherichia coli. . Despite differences in the residues involved in binding, the volumes of the binding pockets in the A. vinelandii and E. coli molybdate-binding proteins are similar and are significantly larger than that of SBP. We conclude that the discrimination between molybdate and sulphate shown by these binding proteins is largely dependent upon small differences in the sizes of these two oxyanions.

  6. Mathematical analysis of the pharmacokinetic-pharmacodynamic (PKPD) behaviour of monoclonal antibodies: predicting in vivo potency.

    Science.gov (United States)

    Aston, Philip J; Derks, Gianne; Raji, Adewale; Agoram, Balaji M; van der Graaf, Piet H

    2011-07-21

    We consider the relationship between the target affinity of a monoclonal antibody and its in vivo potency. The dynamics of the system is described mathematically by a target-mediated drug disposition model. As a measure of potency, we consider the minimum level of the free receptor following a single bolus injection of the ligand into the plasma compartment. From the differential equations, we derive two expressions for this minimum level in terms of the parameters of the problem, one of which is valid over the full range of values of the equilibrium dissociation constant K(D) and the other which is valid only for a large drug dose or for a small value of K(D). Both of these formulae show that the potency achieved by increasing the association constant k(on) can be very different from the potency achieved by decreasing the dissociation constant k(off). In particular, there is a saturation effect when decreasing k(off) where the increase in potency that can be achieved is limited, whereas there is no such effect when increasing k(on). Thus, for certain monoclonal antibodies, an increase in potency may be better achieved by increasing k(on) than by decreasing k(off).

  7. An Accurate Method for Determination of Receptor-Ligand and Enzyme-Inhibitor Dissociation Constants from Displacement Curves

    Science.gov (United States)

    Horovitz, Amnon; Levitzki, Alexander

    1987-10-01

    Receptor-ligand dissociation constants are usually calculated from the displacement curve of a radioactively labeled ligand bound to the receptor. The formula used is restricted to cases in which the concentration of receptor is negligible compared to the concentration of both the displacing ligand and the radioactive ligand used. In this study, we rigorously derive a simple equation that can be used for calculating receptor-ligand dissociation constants for any set of experimental conditions. A linearized form of this equation provides a convenient plot from which the dissociation constant of the displacing ligand can be directly obtained. The plot is also a test for the competitive mode of binding. This exact equation now allows us to estimate the error incurred by the conventionally used equations. Similarly, we show that for competitive inhibition in enzymology, one can derive the analogous formula. Our new formula is free of the usual restrictions--namely, that the enzyme concentration is very small compared to the concentration of both the substrate and the inhibitor. It may therefore be applied to any set of experimental conditions.

  8. Mutagenic Potency of Food-Derived Heterocyclic Amines

    Energy Technology Data Exchange (ETDEWEB)

    Felton, J S; Knize, M G; Wu, R W; Colvin, M E; Hatch, F T; Malfatti, M A

    2006-10-26

    The understanding of mutagenic potency has been primarily approached using ''quantitative structure activity relationships'' (QSAR). Often this method allows the prediction of mutagenic potency of the compound based on its structure. But it does not give the underlying reason why the mutagenic activities differ. We have taken a set of heterocyclic amine structures and used molecular dynamic calculations to dock these molecules into the active site of a computational model of the cytochrome P-450 1A1 enzyme. The calculated binding strength using Boltzman distribution constants was then compared to the QSAR value (HF/6-31G* optimized structures) and the Ames/Salmonella mutagenic potency. Further understanding will only come from knowing the complete set of mutagenic determinants. These include the nitrenium ion half-life, DNA adduct half-life, efficiency of repair of the adduct, and ultimately fixation of the mutation through cellular processes. For two isomers, PhIP and 3-Me-PhIP, we showed that for the 100-fold difference in the mutagenic potency a 5-fold difference can be accounted for by differences in the P450 oxidation. The other factor of 20 is not clearly understood but is downstream from the oxidation step. The application of QSAR (chemical characteristics) to biological principles related to mutagenesis is explored in this report.

  9. Determination of formation constants of uranyl(VI complexes with a hydrophilic SO3-Ph-BTP ligand, using liquid-liquid extraction

    Directory of Open Access Journals (Sweden)

    Steczek Lukasz

    2015-12-01

    Full Text Available Complex formation between uranyl ion, UO22+, and a hydrophilic anionic form of SO3-Ph-BTP4- ligand, L4-, in water was studied by liquid-liquid extraction experiments performed over a range of the ligand and HNO3 concentrations in the aqueous phase, at a constant concentration of nitrate anions at 25°C . The competition for UO22+ ions between the lipophilic TODGA extractant and the hydrophilic L4- ligand leads to the decrease in the uranyl distribution ratios, D, with an increasing L4- concentration. The model of the solvent extraction process used accounts - apart from uranyl complexation by TODGA and SO3-Ph-BTP4- - also for uranyl complexation by nitrates and for the decrease in the concentration of the free L4- ligand in the aqueous phase, due to its protonation, bonding in the uranyl complex and the distribution between the two liquid phases. The unusually strong dependence of the D values on the acidity, found in the experiment, could hardly be explained as due to L4- protonation merely. Three hypotheses were experimentally tested, striving to interpret the data in terms of additional extraction to the organic phase of ion associates of protonated TODGA cation with either partly protonated anionic L4- ligands or anionic UO22+ complexes with NO3 - or L4-. None of them has been confirmed. The analysis of the results, based on the formal correction of free ligand concentrations, points to the formation of 1 : 1 and 1 : 2 uranyl - SO3-Ph-BTP complexes in the aqueous phase. The conditional formation constant of the 1:1 complex has been determined, logßL,1 = 2.95 ± 0.15.

  10. High-affinity multivalent wheat germ agglutinin ligands by one-pot click reaction

    Directory of Open Access Journals (Sweden)

    Henning S. G. Beckmann

    2012-06-01

    Full Text Available A series of six mono-, di-, and trivalent N,N’-diacetylchitobiose derivatives was conveniently prepared by employing a one-pot procedure for Cu(II-catalyzed diazo transfer and Cu(I-catalyzed azide–alkyne cycloaddition (CuAAC starting from commercially available amines. These glycoclusters were probed for their binding potencies to the plant lectin wheat germ agglutinin (WGA from Triticum vulgaris by an enzyme-linked lectin assay (ELLA employing covalently immobilized N-acetylglucosamine (GlcNAc as a reference ligand. IC50 values were in the low micromolar/high nanomolar range, depending on the linker between the two disaccharides. Binding enhancements β up to 1000 for the divalent ligands and 2800 for a trivalent WGA ligand, compared to N,N’-diacetylchitobiose as the corresponding monovalent ligand, were observed. Molecular modeling studies, in which the chitobiose moieties were fitted into crystallographically determined binding sites of WGA, correlate the binding enhancements of the multivalent ligands with their ability to bind to the protein in a chelating mode. The best WGA ligand is a trivalent cluster with an IC50 value of 220 nM. Calculated per mol of contained chitobiose, this is the best WGA ligand known so far.

  11. Antibacterial potency screening of Capparis zeylanica Linn

    Institute of Scientific and Technical Information of China (English)

    Rezaul Haque; Wahedul Islam; Selina Parween

    2016-01-01

    Objective: To conduct the antibacterial potency and minimum inhibitory concentration of extracts (n-hexane, acetone, chloroform and methanol) obtained from the root, leaf and stem of Capparis zeylanica. Methods: The powdered leaf, root and stem samples were Soxhlet extracted sequentially in n-hexane, acetone, chloroform and methanol. Antibacterial potency was evaluated by following the agar diffusion method and amoxicillin disc was used as a control. Results: In vitro antibacterial activity against 12 bacteria was performed with crude extracts. Among them, all the bacteria showed the moderate activity but chloroform and methanolic extracts showed promising antibacterial potency against Staphylococcus aureus, Sarcina lutea, Bacillus megaterium, Bacillus subtilis, Salmonella typhi and Shigella dysenteriae (leaf > root > stem). This activity was evaluated using disc diffusion method with a standard antibiotic, 30 µg/disc of amoxicillin. Conclusions: Strong antibacterial potency of chloroform and methanolic extracts provides new antibacterial compounds.

  12. Development of electrochemical sensors for nano scale Tb(III) ion determination based on pendant macrocyclic ligands.

    Science.gov (United States)

    Singh, Ashok K; Singh, Prerna; Banerjee, Shibdas; Mehtab, Sameena

    2009-02-02

    The two macrocyclic pendant ligands 3,4,5:12,13,14-dipyridine-2,6,11,15-tetramethyl-1,7,10,16-tetramethylacrylate-1,4,7,10,13,16-hexaazacyclooctadeca-3,13-di ene (L(1)) and 3,4,5:12,13,14-dipyridine-2,6,11,15-tetramethyl-1,7,10,16-tetra(2-cyano ethane)-1,4,7,10,13,16-hexaazacyclooctadeca-3,13-diene (L(2)) have been synthesized and explored as neutral ionophores for preparing poly(vinylchloride) (PVC) based membrane sensors selective to Tb(III) ions. Effects of various plasticizers and anion excluders were studied in detail and improved performance was observed. The best performance was obtained for the membrane sensor having a composition of L(1): PVC:1-CN:NaTPB in the ratio of 6: 32: 58: 4 (w/w; mg). The performance of the membrane based on L(1) was compared with polymeric membrane electrode (PME) as well as with coated graphite electrode (CGE). The electrodes exhibit Nernstian slope for Tb(3+) ions with limits of detection of 3.4 x 10(-8)mol L(-1) for PME and 5.7 x 10(-9)mol L(-1) for CGE. The response time for PME and CGE was found to be 10s and 8s, respectively. The potentiometric responses are independent of the pH of the test solution in the pH range 3.0-7.5 for PME and 2.0-8.5 for CGE. The CGE has found to work satisfactorily in partially non-aqueous media upto 30% (v/v) content of methanol, ethanol and 20% (v/v) content of acetonitrile and could be used for a period of 5 months. The CGE was used as indicator electrode in the potentiometric titration of Tb(3+) ions with EDTA and in determination of fluoride ions in various samples. It can also be used in direct determination of Tb(3+) ions in tap water and various binary mixtures with quantitative results.

  13. The XChemExplorer graphical workflow tool for routine or large-scale protein-ligand structure determination.

    Science.gov (United States)

    Krojer, Tobias; Talon, Romain; Pearce, Nicholas; Collins, Patrick; Douangamath, Alice; Brandao-Neto, Jose; Dias, Alexandre; Marsden, Brian; von Delft, Frank

    2017-03-01

    XChemExplorer (XCE) is a data-management and workflow tool to support large-scale simultaneous analysis of protein-ligand complexes during structure-based ligand discovery (SBLD). The user interfaces of established crystallographic software packages such as CCP4 [Winn et al. (2011), Acta Cryst. D67, 235-242] or PHENIX [Adams et al. (2010), Acta Cryst. D66, 213-221] have entrenched the paradigm that a `project' is concerned with solving one structure. This does not hold for SBLD, where many almost identical structures need to be solved and analysed quickly in one batch of work. Functionality to track progress and annotate structures is essential. XCE provides an intuitive graphical user interface which guides the user from data processing, initial map calculation, ligand identification and refinement up until data dissemination. It provides multiple entry points depending on the need of each project, enables batch processing of multiple data sets and records metadata, progress and annotations in an SQLite database. XCE is freely available and works on any Linux and Mac OS X system, and the only dependency is to have the latest version of CCP4 installed. The design and usage of this tool are described here, and its usefulness is demonstrated in the context of fragment-screening campaigns at the Diamond Light Source. It is routinely used to analyse projects comprising 1000 data sets or more, and therefore scales well to even very large ligand-design projects.

  14. Insights into the structural determinants required for high-affinity binding of chiral cyclopropane-containing ligands to α4β2-nicotinic acetylcholine receptors: an integrated approach to behaviorally active nicotinic ligands.

    Science.gov (United States)

    Zhang, Han-Kun; Eaton, J Brek; Yu, Li-Fang; Nys, Mieke; Mazzolari, Angelica; van Elk, René; Smit, August B; Alexandrov, Vadim; Hanania, Taleen; Sabath, Emily; Fedolak, Allison; Brunner, Daniela; Lukas, Ronald J; Vistoli, Giulio; Ulens, Chris; Kozikowski, Alan P

    2012-09-27

    Structure-based drug design can potentially accelerate the development of new therapeutics. In this study, a cocrystal structure of the acetylcholine binding protein (AChBP) from Capitella teleta (Ct) in complex with a cyclopropane-containing selective α4β2-nicotinic acetylcholine receptor (nAChR) partial agonist (compound 5) was acquired. The structural determinants required for ligand binding obtained from this AChBP X-ray structure were used to refine a previous model of the human α4β2-nAChR, thus possibly providing a better understanding of the structure of the human receptor. To validate the potential application of the structure of the Ct-AChBP in the engineering of new α4β2-nAChR ligands, homology modeling methods, combined with in silico ADME calculations, were used to design analogues of compound 5. The most promising compound, 12, exhibited an improved metabolic stability in comparison to the parent compound 5 while retaining favorable pharmacological parameters together with appropriate behavioral end points in the rodent studies.

  15. Relative developmental toxicity potencies of retinoids in the embryonic stem cell test compared with their relative potencies in in vivo and two other in vitro assays for developmental toxicity

    NARCIS (Netherlands)

    Louisse, J.; Gönen, S.; Rietjens, I.M.C.M.; Verwei, M.

    2011-01-01

    The present study determines the relative developmental toxicity potencies of retinoids in the embryonic stem (ES)-D3 cell differentiation assay of the embryonic stem cell test, and compares the outcomes with their relative potencies in in vivo and two other in vitro assays for developmental toxicit

  16. Different Concentrations of FGF Ligands, FGF2 or FGF8 Determine Distinct States of WNT-Induced Presomitic Mesoderm.

    Science.gov (United States)

    Sudheer, Smita; Liu, Jinhua; Marks, Matthias; Koch, Frederic; Anurin, Anna; Scholze, Manuela; Senft, Anna Dorothea; Wittler, Lars; Macura, Karol; Grote, Phillip; Herrmann, Bernhard G

    2016-07-01

    Presomitic mesoderm (PSM) cells are the precursors of the somites, which flank both sides of the neural tube and give rise to the musculo-skeletal system shaping the vertebrate body. WNT and FGF signaling control the formation of both the PSM and the somites and show a graded distribution with highest levels in the posterior PSM. We have used reporters for the mesoderm/PSM control genes T, Tbx6, and Msgn1 to investigate the differentiation of mouse ESCs from the naïve state via EpiSCs to PSM cells. Here we show that the activation of WNT signaling by CHIR99021 (CH) in combination with FGF ligand induces embryo-like PSM at high efficiency. By varying the FGF ligand concentration, the state of PSM cells formed can be altered. High FGF concentration supports posterior PSM formation, whereas low FGF generates anterior/differentiating PSM, in line with in vivo data. Furthermore, the level of Msgn1 expression depends on the FGF ligand concentration. We also show that Activin/Nodal signaling inhibits CH-mediated PSM induction in EpiSCs, without affecting T-expression. Inversely, Activin/Nodal inhibition enhances PSM induction by WNT/high FGF signaling. The ability to generate PSM cells of either posterior or anterior PSM identity with high efficiency in vitro will promote the investigation of the gene regulatory networks controlling the formation of nascent PSM cells and their switch to differentiating/somitic paraxial mesoderm. Stem Cells 2016;34:1790-1800.

  17. Mononuclear mercury(II) complexes containing bipyridine derivatives and thiocyanate ligands: Synthesis, characterization, crystal structure determination, and luminescent properties

    Science.gov (United States)

    Amani, Vahid; Alizadeh, Robabeh; Alavije, Hanieh Soleimani; Heydari, Samira Fadaei; Abafat, Marzieh

    2017-08-01

    A series of mercury(II) complexes, [Hg(Nsbnd N)(SCN)2] (Nsbnd N is 4,4‧-dimethyl-2,2‧-bipyridine in 1, 5,5‧-dimethyl-2,2‧-bipyridine in 2, 6,6‧-dimethyl-2,2‧-bipyridine in 3 and 6-methyl-2,2‧-bipyridine in 4), were prepared from the reactions of Hg(SCN)2 with mentioned ligands in methanol. Suitable crystals of these complexes were obtained for X-ray diffraction measurement by methanol diffusion into a DMSO solution. The four complexes were thoroughly characterized by spectral methods (IR, UV-Vis, 13C{1H}NMR, 1H NMR and luminescence), elemental analysis (CHNS) and single crystal X-ray diffraction. The X-ray structural analysis indicated that in the structures of these complexes, the mercury(II) cation is four-coordinated in a distorted tetrahedral configuration by two S atoms from two thiocyanate anions and two N atoms from one chelating 2,2‧-bipyridine derivative ligand. Also, in these complexes intermolecular interactions, for example Csbnd H⋯N hydrogen bonds (in 1-4), Csbnd H⋯S hydrogen bonds (in 1, 2 and 4), π … π interactions (in 2-4), Hg⋯N interactions (in 2) and S⋯S interactions (in 4), are effective in the stabilization of the crystal structures and the formation of the 3D supramolecular complexes. Furthermore, the luminescence spectra of the title complexes show that the intensity of their emission bands are stronger than the emission bands for the free bipyridine derivative ligands.

  18. Plant-determined variation in the cardenolide content, thin-layer chromatography profiles, and emetic potency of monarch butterflies,Danaus plexippus L. Reared on milkweed plants in California: 2.Asclepias speciosa.

    Science.gov (United States)

    Brower, L P; Seiber, J N; Nelson, C J; Lynch, S P; Holland, M M

    1984-04-01

    The pattern of variation in gross cardenolide concentration of 111Asclepias speciosa plants collected in six different areas of California is a positively skewed distribution which ranges from 19 to 344 μg of cardenolide per 0.1 g dry weight with a mean of 90 μg per 0.1 g. Butterflies reared individually on these plants in their native habitats ranged from 41 to 547 μg of cardenolide per 0.1 g dry weight with a mean of 179 μg. Total cardenolide per butterfly ranged from 54 to 1279 μg with a mean of 319 μg. Differences in concentrations and total cardenolide contents in the butterflies from the six geographic areas appeared minor, and there were no differences between the males and the females, although the males did weigh significantly more than females. The uptake of cardenolide by the butterflies was found to be a logarithmic function of the plant concentration. This results in regulation: larvae which feed on low-concentration plants produce butterflies with increased cardenolide concentrations relative to those of the plants, and those which feed on high-concentration plants produce butterflies with decreased concentrations. No evidence was adduced that high concentrations of cardenolides in the plants affected the fitness of the butterflies. The mean emetic potencies of the powdered plant and butterfly material were 5.62 and 5.25 blue jay emetic dose fifty units per milligram of cardenolide and the number of ED50 units per butterfly ranged from 0.28 to 6.7 with a mean of 1.67. Monarchs reared onA. speciosa, on average, are only about one tenth as emetic as those reared onA. eriocarpa. UnlikeA. eriocarpa which is limited to California,A. speciosa ranges from California to the Great Plains and is replaced eastwards byA. syriaca L. These two latter milkweed species appear to have a similar array of chemically identical cardenolides, and therefore both must produce butterflies of relatively low emetic potency to birds, with important ecological implications

  19. DATA MINING POTENCY ESTIMATORS FROM TOXICOLOGICAL DATABASES

    OpenAIRE

    Piegorsch, Walter W.; Simmons, Susan J.; Zeiger, Errol

    2004-01-01

    We discuss use of data mining techniques to study estimators of toxic potency (activity) in toxicological databases. The methods are slight variations on the standard data mining motif, but fit fully within the larger context of knowledge discovery in databases. An example illustrates the general theme of the approach, using results from a U.S. National Toxicology Program Salmonella mutagenicity database.

  20. Kinetic microplate bioassays for relative potency of antibiotics improved by partial Least Square (PLS) regression.

    Science.gov (United States)

    Francisco, Fabiane Lacerda; Saviano, Alessandro Morais; Almeida, Túlia de Souza Botelho; Lourenço, Felipe Rebello

    2016-05-01

    Microbiological assays are widely used to estimate the relative potencies of antibiotics in order to guarantee the efficacy, safety, and quality of drug products. Despite of the advantages of turbidimetric bioassays when compared to other methods, it has limitations concerning the linearity and range of the dose-response curve determination. Here, we proposed to use partial least squares (PLS) regression to solve these limitations and to improve the prediction of relative potencies of antibiotics. Kinetic-reading microplate turbidimetric bioassays for apramacyin and vancomycin were performed using Escherichia coli (ATCC 8739) and Bacillus subtilis (ATCC 6633), respectively. Microbial growths were measured as absorbance up to 180 and 300min for apramycin and vancomycin turbidimetric bioassays, respectively. Conventional dose-response curves (absorbances or area under the microbial growth curve vs. log of antibiotic concentration) showed significant regression, however there were significant deviation of linearity. Thus, they could not be used for relative potency estimations. PLS regression allowed us to construct a predictive model for estimating the relative potencies of apramycin and vancomycin without over-fitting and it improved the linear range of turbidimetric bioassay. In addition, PLS regression provided predictions of relative potencies equivalent to those obtained from agar diffusion official methods. Therefore, we conclude that PLS regression may be used to estimate the relative potencies of antibiotics with significant advantages when compared to conventional dose-response curve determination.

  1. Analysis of macromolecules, ligands and macromolecule-ligand complexes

    Science.gov (United States)

    Von Dreele, Robert B [Los Alamos, NM

    2008-12-23

    A method for determining atomic level structures of macromolecule-ligand complexes through high-resolution powder diffraction analysis and a method for providing suitable microcrystalline powder for diffraction analysis are provided. In one embodiment, powder diffraction data is collected from samples of polycrystalline macromolecule and macromolecule-ligand complex and the refined structure of the macromolecule is used as an approximate model for a combined Rietveld and stereochemical restraint refinement of the macromolecule-ligand complex. A difference Fourier map is calculated and the ligand position and points of interaction between the atoms of the macromolecule and the atoms of the ligand can be deduced and visualized. A suitable polycrystalline sample of macromolecule-ligand complex can be produced by physically agitating a mixture of lyophilized macromolecule, ligand and a solvent.

  2. Direct simultaneous determination of Co, Cu, Fe, Ni and V in pore waters by means of adsorptive cathodic stripping voltammetry with mixed ligands.

    Science.gov (United States)

    Santos-Echeandía, Juan

    2011-07-15

    An analytical procedure is proposed for the direct simultaneous determination in a single scan of Co, Cu, Fe, Ni and V in sediment pore waters by means of adsorptive cathodic stripping voltammetry (ACSV) with mixed ligands (DMG and catechol). Optimum conditions for the determination of these five elements were studied. Detection limits of the technique depended upon the reproducibility of the procedure blank, and were found to be 0.04 nM Co, 0.09 nM Cu, 1.29 nM Fe, 0.46 nM Ni and 2,52 nMV making the method suitable for the direct simultaneous determination of these five metals in pore waters, estuarine waters and probably coastal waters. Copyright © 2011 Elsevier B.V. All rights reserved.

  3. Structure-guided optimization of estrogen receptor binding affinity and antagonist potency of pyrazolopyrimidines with basic side chains.

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, H.; Sheng, S.; Compton, D.; Kim, Y.; Joachimiak, A.; Sharma, S.; Carlson, K.; Katzenellenbogen, B.; Nettles, K.; Greene, G.; Katzenellenbogen, J.; Biosciences Division; Univ. of Illinois; Univ. of Chicago; The Scripps Research Inst.

    2007-01-01

    2,3-Diarylpyrazolo[1,5-a]pyrimidines are estrogen receptor (ER) antagonists of modest potency that we have described previously. Guided by the crystal structure of an ER-ligand complex that we have obtained with one of these compounds, we prepared analogs that contain a basic side chain at the 2- or 3-aryl group and quickly found one that, according to the structure-based prediction, shows an increase in binding affinity and antagonist potency and a loss of residual agonist activity.

  4. Simultaneous spectrophotometric determination of copper, cobalt, nickel and iron in foodstuffs and vegetables with a new bis thiosemicarbazone ligand using chemometric approaches.

    Science.gov (United States)

    Rohani Moghadam, Masoud; Poorakbarian Jahromi, Sayedeh Maria; Darehkordi, Ali

    2016-02-01

    A newly synthesized bis thiosemicarbazone ligand, (2Z,2'Z)-2,2'-((4S,5R)-4,5,6-trihydroxyhexane-1,2-diylidene)bis(N-phenylhydrazinecarbothioamide), was used to make a complex with Cu(2+), Ni(2+), Co(2+) and Fe(3+) for their simultaneous spectrophotometric determination using chemometric methods. By Job's method, the ratio of metal to ligand in Ni(2+) was found to be 1:2, whereas it was 1:4 for the others. The effect of pH on the sensitivity and selectivity of the formed complexes was studied according to the net analyte signal (NAS). Under optimum conditions, the calibration graphs were linear in the ranges of 0.10-3.83, 0.20-3.83, 0.23-5.23 and 0.32-8.12 mg L(-1) with the detection limits of 2, 3, 4 and 10 μg L(-1) for Cu(2+), Co(2+), Ni(2+) and Fe(3+) respectively. The OSC-PLS1 for Cu(2+) and Ni(2+), the PLS1 for Co(2+) and the PC-FFANN for Fe(3+) were selected as the best models. The selected models were successfully applied for the simultaneous determination of elements in some foodstuffs and vegetables.

  5. LigandRNA: computational predictor of RNA-ligand interactions.

    Science.gov (United States)

    Philips, Anna; Milanowska, Kaja; Lach, Grzegorz; Bujnicki, Janusz M

    2013-12-01

    RNA molecules have recently become attractive as potential drug targets due to the increased awareness of their importance in key biological processes. The increase of the number of experimentally determined RNA 3D structures enabled structure-based searches for small molecules that can specifically bind to defined sites in RNA molecules, thereby blocking or otherwise modulating their function. However, as of yet, computational methods for structure-based docking of small molecule ligands to RNA molecules are not as well established as analogous methods for protein-ligand docking. This motivated us to create LigandRNA, a scoring function for the prediction of RNA-small molecule interactions. Our method employs a grid-based algorithm and a knowledge-based potential derived from ligand-binding sites in the experimentally solved RNA-ligand complexes. As an input, LigandRNA takes an RNA receptor file and a file with ligand poses. As an output, it returns a ranking of the poses according to their score. The predictive power of LigandRNA favorably compares to five other publicly available methods. We found that the combination of LigandRNA and Dock6 into a "meta-predictor" leads to further improvement in the identification of near-native ligand poses. The LigandRNA program is available free of charge as a web server at http://ligandrna.genesilico.pl.

  6. A two-step strategy to enhance activity of low potency peptides.

    Directory of Open Access Journals (Sweden)

    Jamie R Doyle

    Full Text Available Novel strategies are needed to expedite the generation and optimization of peptide probes targeting G protein-coupled receptors (GPCRs. We have previously shown that membrane tethered ligands (MTLs, recombinant proteins comprised of a membrane anchor, an extracellular linker, and a peptide ligand can be used to identify targeted receptor modulators. Although MTLs provide a useful tool to identify and/or modify functionally active peptides, a major limitation of this strategy is the reliance on recombinant protein expression. We now report the generation and pharmacological characterization of prototype peptide-linker-lipid conjugates, synthetic membrane anchored ligands (SMALs, which are designed as mimics of corresponding MTLs. In this study, we systematically compare the activity of selected peptides as MTLs versus SMALs. As prototypes, we focused on the precursor proteins of mature Substance P (SubP and Cholecystokinin 4 (CCK4, specifically non-amidated SubP (SubP-COOH and glycine extended CCK4 (CCK4-Gly-COOH. As low affinity soluble peptides these ligands each presented a challenging test case for assessment of MTL/SMAL technology. For each ligand, MTLs and corresponding SMALs showed agonist activity and comparable subtype selectivity. In addition, our results illustrate that membrane anchoring increases ligand potency. Furthermore, both MTL and SMAL induced signaling can be blocked by specific non-peptide antagonists suggesting that the anchored constructs may be orthosteric agonists. In conclusion, MTLs offer a streamlined approach for identifying low activity peptides which can be readily converted to higher potency SMALs. The ability to recapitulate MTL activity with SMALs extends the utility of anchored peptides as probes of GPCR function.

  7. GABA potency at GABAA receptors found in synaptic and extrasynaptic zones

    Directory of Open Access Journals (Sweden)

    Martin eMortensen

    2012-01-01

    Full Text Available The potency of GABA is vitally important for its primary role in activating GABAA receptors and acting as an inhibitory neurotransmitter. Although numerous laboratories have presented information, directly or indirectly, on GABA potency, it is often difficult to compare across such studies given the inevitable variations in the methods used, the cell types studied, whether native or recombinant receptors are examined, and their relevance to native synaptic and extrasynaptic GABAA receptors. In this review, we list the most relevant isoforms of synaptic and extrasynaptic GABAA receptors that are thought to assemble in surface membranes of neurons in the central nervous system. Using consistent methodology in one cell type, the potencies of the endogenous neurotransmitter GABA are compared across a spectrum of GABAA receptors. The highest potency for GABA is measured when activating extrasynaptic-type α6 subunit-containing receptors, whereas synaptic-type α2β3γ2 and α3β3γ2 receptors exhibited the lowest potency, and other GABAA receptor subtypes that are found both in synaptic and extrasynaptic compartments, showed intermediate sensitivities to GABA. The relatively simple potency relationship between GABA and its target receptors is important as it serves as one of the major determinants of GABAA receptor activation, with consequences for the development of inhibition, either by tonic or phasic mechanisms.

  8. 21 CFR 640.56 - Quality control test for potency.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Quality control test for potency. 640.56 Section...) BIOLOGICS ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Cryoprecipitate § 640.56 Quality control test for potency. (a) Quality control tests for potency of antihemophilic factor shall be...

  9. 9 CFR 113.9 - New potency test.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false New potency test. 113.9 Section 113.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES... New potency test. A potency test written into the filed Outline of Production for a product shall be...

  10. Incipient cytotoxicity: A time-independent measure of cytotoxic potency in vitro.

    Science.gov (United States)

    Gülden, Michael; Kähler, Daria; Seibert, Hasso

    2015-09-01

    Time is an important determinant of toxicity but largely ignored in in vitro toxicity assays where exposure times chosen are rather arbitrary. To investigate the impact of time on the cytotoxic potency of chemicals in vitro, the concentration dependent cytotoxic action of selected chemicals (surfactants, metals, oxidative stressors, a mitochondrial poison) was determined after various exposure times (1-72 h) in cultures of Balb/c 3T3 cells. Time affected the cytotoxic potency as well as the cytotoxic efficacy. The median cytotoxic concentrations, EC50, decreased and in most cases approached an "incipient" value, EC50,∞, within 72 h. Cytotoxicity due to mitochondrial insult occurred after a threshold time which was dependent on the medium glucose concentration. Within the chemicals studied the extent of potency change with time ranged from 3- to >1000-fold and the "time to incipient cytotoxicity", tic, from 4 to >72 h. Hence, also the relative cytotoxic potencies depend on exposure time. Ignoring this may lead to severe bias in toxicological hazard and risk assessment. Therefore it is recommended to determine the incipient cytotoxic potency of chemical compounds, represented by, e.g., the incipient median effect (EC50,∞), no effect (NEC∞) or lowest effect concentrations (LEC∞) instead of measures obtained after arbitrary exposure times. If this is not possible, the 72 h-potency measurements appear to be useful surrogates. These time-independent incipient potency values can be reasonably compared between substances, endpoints, cells and biological test systems and may serve to define points of departure for quantitative in vitro-in vivo extrapolations.

  11. High-speed gas chromatography in doping control: fast-GC and fast-GC/MS determination of beta-adrenoceptor ligands and diuretics.

    Science.gov (United States)

    Brunelli, Claudio; Bicchi, Carlo; Di Stilo, Antonella; Salomone, Alberto; Vincenti, Marco

    2006-12-01

    In official doping controls, about 300 drugs and metabolites have to be screened for each sample. Moreover, the number of determinations to be routinely processed increases continuously as the number of both samples and potential illicit drugs keeps growing. As a consequence, increasingly specific, sensitive, and, above all, fast methods for doping controls are needed. The present study presents an efficient fast-GC/MS approach to the routine screening of two different classes of doping agents, namely beta-adrenoceptor ligands and diuretics (belonging to the S3, P2, and S5 groups of the WADA list of prohibited substances). Narrow bore columns (100 mm id) of different lengths and coated with apolar stationary phases were successfully used to separate the derivatized analytes; preliminary experiments (results not shown) showed better performances with OV-1701 for the separation of beta-adrenoceptor ligands. On the same stationary phase some diuretics required too high a temperature or a long isothermal time for elution, in which case a DB1-MS column was preferred. Two methods of sample preparation, derivatization, and analysis were used on aqueous standard mixtures of, respectively, (i) eight beta-adrenoceptor ligands, including five beta-antagonists (acebutolol, alprenolol, atenolol, metoprolol, pindolol) and three beta2-agonists (salbutamol, clenbuterol, terbutaline) and (ii) seventeen diuretic drugs (acetazolamide, althiazide, bendroflumethiazide, bumethanide, canrenone, chlorothiazide, chlortalidone, clopamide, ethacrinic acid, furosemide, hydrochlorothiazide, hydroflumethiazide, indapamide, indomethacine, spironolactone, triamterene, trichloromethiazide) and one masking agent (probenecid). The mixture of beta-adrenoceptor ligand derivatives was efficiently separated in about 5.6 min, while the one of 18 diuretics and masking agents required less than 5 min for analysis. Limits of detection were from 1 microg/L for pindolol, ethacrinic acid, furosemide

  12. A QSAR for the Mutagenic Potencies of Twelve 2-Amino-trimethylimidazopyridine Isomers: Structural, Quantum Chemical,and Hydropathic Factors

    Energy Technology Data Exchange (ETDEWEB)

    Knize, M G; Hatch, F T; Tanga, M J; Lau, E V; Colvin, M E

    2005-04-23

    An isomeric series of heterocyclic amines related to one found in heated muscle meats was investigated for properties that predict their measured mutagenic potency. Eleven of the 12 possible 2-amino-trimethylimidazopyridine (TMIP) isomers were tested for mutagenic potency in the Ames/Salmonella test with bacterial strain TA98, and resulted in a 600-fold range in potency. Structural, quantum chemical and hydropathic data were calculated on the parent molecules and the corresponding nitrenium ions of all of the tested isomers to establish models for predicting the potency of the unknown isomer. The regression model accounting for the largest fraction of the total variance in mutagenic potency contains four predictor variables: dipole moment, a measure of the gap between amine LUMO and HOMO energies, percent hydrophilic surface, and energy of amine LUMO. The most important determinants of high mutagenic potency in these amines are: (1) a small dipole moment, (2) the combination of b-face ring fusion and N3-methyl group, and (3) a lower calculated energy of the {pi} electron system. Based on predicted potency from the average of five models, the isomer not yet synthesized and tested is expected to have a mutagenic potency of 0.84 revertants/{micro}g in test strain TA98.

  13. Geohydrology, geochemistry, geothermal potency of Rianiate Toba Lake North Sumatera

    Science.gov (United States)

    Nainggolan, Juliper; Sitepu, Cristin; Pardede, Sanggam; Diantoro, Markus

    2017-09-01

    This research was performed to determine the potency of Rianiate’s geothermal an alternative of energy source and determine the types of geothermal that was begun with the position’s measurement by using GPS (Global Position System), and then the direct observation of chemical and physical properties such as pH, surface’s temperature, color etc. The following steps were taking sample in four different springs indicated by spring 1, spring 2, spring 3, and spring 4. The chemical nature was measured by titrimetry method by using AAS, XRD, and gas Chromathology. The calculating of temperature of subsurface has done by using geothermometer and sequentially followed by geothermal potency’s calculation. The position of four springs are located about N: 02° 31,852’ and E: 098° 44. 021’ where were average height from sea’s level is 958 m. The highest surface’s temperature is 80 °C and the temperature under soil is about 130.5 °C described the average of geothermal. The calculation of content of chloride, sulfate, bicarbonations revealed that the water can be categorized as chloride type. The trilateral diagram Na/1000 – K/100 - √Mg of hot water is in the regime of immature water. From the calculation of Indonesia’s Standardized Geotherm, it was obtained that the estimated reservoir potency of Rianiate geotherm is 2,68 MWe.

  14. Temperature controlled ionic liquid-based dispersive micro-extraction using two ligands, for determination of aluminium in scalp hair samples of Alzheimer's patients: A multivariate study

    Science.gov (United States)

    Arain, Mariam S.; Arain, Salma A.; Kazi, Tasneem G.; Afridi, Hassan I.; Ali, Jamshaid; Naeemulllah; Arain, Sadaf S.; Brahman, Kapil Dev; Mughal, Moina Akhtar

    2015-02-01

    A green and sensitive temperature controlled dispersive liquid-liquid microextraction (TIL-DLLME) methodology based on the application of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [C4mim][PF6], as an extractant solvent was proposed for the preconcentration of trace levels of aluminium (Al3+) in scalp hair samples of Alzheimer's (AD) patients, prior to analyzing by flame atomic absorption spectrometry (FAAS). The Al3+ was complexed with 8-hydrooxyquinoline (oxine) (L1) and 3,5,7,2‧-4‧ pentahydroxy flavone (morin) (L2) separately and then extracted by IL at temperature (50 ± 2.0 °C). Some effective factors that influence the TIL-DLLME efficiency such as pH, ligands concentrations, volume of IL, ionic strength, and incubation time were investigated and optimized by multivariate analysis. In the optimum experimental conditions, the limit of detection (3 s) and enhancement factor were 0.56 μg L-1, 0.64 μg L-1 and 85, 73 for both ligands, respectively. The relative standard deviation (RSD) for six replicate determinations of 100 μg L-1 Al3+ complexed with oxine and morin were found to be 3.88% and 4.74%, respectively. The developed method was validated by the analysis of certified reference material of human hair (NCSZC81002).and applied satisfactorily to the determination of Al3+ in acid digested scalp hair samples of AD patients and healthy controls. The resulted data shows significant higher level in scalp hair samples of AD male patients with related to referents of same age and socioeconomic status.

  15. Temperature controlled ionic liquid-based dispersive micro-extraction using two ligands, for determination of aluminium in scalp hair samples of Alzheimer's patients: a multivariate study.

    Science.gov (United States)

    Arain, Mariam S; Arain, Salma A; Kazi, Tasneem G; Afridi, Hassan I; Ali, Jamshaid; Naeemulllah; Arain, Sadaf S; Brahman, Kapil Dev; Mughal, Moina Akhtar

    2015-02-25

    A green and sensitive temperature controlled dispersive liquid-liquid microextraction (TIL-DLLME) methodology based on the application of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate, [C4mim][PF6], as an extractant solvent was proposed for the preconcentration of trace levels of aluminium (Al(3+)) in scalp hair samples of Alzheimer's (AD) patients, prior to analyzing by flame atomic absorption spectrometry (FAAS). The Al(3+) was complexed with 8-hydrooxyquinoline (oxine) (L1) and 3,5,7,2'-4' pentahydroxy flavone (morin) (L2) separately and then extracted by IL at temperature (50±2.0°C). Some effective factors that influence the TIL-DLLME efficiency such as pH, ligands concentrations, volume of IL, ionic strength, and incubation time were investigated and optimized by multivariate analysis. In the optimum experimental conditions, the limit of detection (3s) and enhancement factor were 0.56 μg L(-1), 0.64 μg L(-1) and 85, 73 for both ligands, respectively. The relative standard deviation (RSD) for six replicate determinations of 100 μg L(-1) Al(3+) complexed with oxine and morin were found to be 3.88% and 4.74%, respectively. The developed method was validated by the analysis of certified reference material of human hair (NCSZC81002).and applied satisfactorily to the determination of Al(3+) in acid digested scalp hair samples of AD patients and healthy controls. The resulted data shows significant higher level in scalp hair samples of AD male patients with related to referents of same age and socioeconomic status.

  16. Potency of Animal Models in KANSEI Engineering

    Science.gov (United States)

    Ozaki, Shigeru; Hisano, Setsuji; Iwamoto, Yoshiki

    Various species of animals have been used as animal models for neuroscience and provided critical information about the brain functions. Although it seems difficult to elucidate a highly advanced function of the human brain, animal models have potency to clarify the fundamental mechanisms of emotion, decision-making and social behavior. In this review, we will pick up common animal models and point to both the merits and demerits caused by the characteristics. We will also mention that wide-ranging approaches from animal models are advantageous to understand KANSEI as well as mind in humans.

  17. Patient Controlled Analgesia Used to Assess the Efficacy and Potency of a New Opioid

    Directory of Open Access Journals (Sweden)

    Brian Ginsberg

    1996-01-01

    Full Text Available Patient controlled analgesia (PCA is widely used for the management of postoperative pain. PCA also permits a comparison to be made among analgesics in the clinical setting because it limits the variability introduced by third parties. Use of PCA to establish efficacy and potency data for an investigational drug, pentamorphone, compared with morphine is reported. Pentamorphone was found to be more efficacious than morphine in the first hour after surgery because significantly more patients were able to achieve a visual analogue scale of less than 30 mm with pentamorphone. Thereafter pentamorphone and morphine were found to be equally efficacious. Initially pentamorphone may be more potent than morphine based on the greater volume of morphine used in the first hour of therapy. However, a potency ratio could not be determined because this result was under conditions of unequal analgesia. The potency ratio determined at 24 h of therapy under equianalgesic conditions (252:1 is similar to previously reported potency data from laboratory studies (200:1. This study supports the use of PCA as a model to investigate and compare new drugs to establish their efficacy and potency.

  18. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins

    Directory of Open Access Journals (Sweden)

    Osnat Rosen

    2016-09-01

    Full Text Available Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV < 13%. Most importantly, the antitoxin potency measured by the in vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p < 0.0001. Thus, this new in vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

  19. Anticonvulsant properties of histamine H3 receptor ligands belonging to N-substituted carbamates of imidazopropanol.

    Science.gov (United States)

    Sadek, Bassem; Shehab, Safa; Więcek, Małgorzata; Subramanian, Dhanasekaran; Shafiullah, Mohamed; Kieć-Kononowicz, Katarzyna; Adem, Abdu

    2013-09-01

    Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. Therefore, the previously described and structurally strongly related imidazole-based derivatives belonging to carbamate class with high H3R in vitro affinity, in-vivo antagonist potency, and H3R selectivity profile were investigated on their anticonvulsant activity in maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-kindled seizure models in Wistar rats. The effects of systemic injection of H3R ligands 1-13 on MES-induced and PTZ-kindled seizures were screened and evaluated against the reference antiepileptic drug (AED) Phenytoin (PHT) and the standard histamine H3R inverse agonist/antagonist Thioperamide (THP) to determine their potential as new antiepileptic drugs. Following administration of the H3R ligands 1-13 (5, 10 and 15 mg/kg, ip) there was a significant dose dependent reduction in MES-induced seizure duration. The protective action observed for the pentenyl carbamate derivative 4, the most protective H3R ligand among 1-13, was significantly higher (P histamine (RAMH) (10mg/kg), or with the CNS penetrant H1R antagonist Pyrilamine (PYR) (10mg/kg). In addition, subeffective dose of H3R ligand 4 (5mg/kg, ip) significantly potentiated the protective action in rats pretreated with PHT (5mg/kg, ip), a dose without appreciable protective effect when given alone. In contrast, pretreatment with H3R ligand 4 (10mg/kg ip) failed to modify PTZ-kindled convulsion, whereas the reference drug PHT was found to fully protect PTZ-induced seizure. These results indicate that some of the investigated imidazole-based H3R ligands 1-13 may be of future therapeutic value in epilepsy.

  20. Solid phase extraction of ultra traces silver(I) using octadecyl silica membrane disks modified by 1,3-bis(2-cyanobenzene) triazene (CBT) ligand prior to determination by flame atomic absorption

    Energy Technology Data Exchange (ETDEWEB)

    Rofouei, Mohammad Kazem, E-mail: rofouei@tmu.ac.ir [Faculty of Chemistry, Tarbiat Moalem University, Tehran (Iran, Islamic Republic of); Payehghadr, Mahmood [Department of Chemistry, Payame Noor University (PNU) (Iran, Islamic Republic of); Shamsipur, Mojtaba [Department of Chemistry, Razi University, Kermanshah (Iran, Islamic Republic of); Ahmadalinezhad, Asieh [Department of Chemistry, Lakehead University, Thunder Bay, Ontario P7B 5E1 (Canada)

    2009-09-15

    A simple, reliable and rapid method for preconcentration and determination of the ultra trace amount of silver using octadecyl silica membrane disk modified by a recently synthesized triazene ligand, 1,3-bis(2-cyanobenzene)triazene (CBT), and flame atomic absorption spectrometry is presented. Various parameters including pH of aqueous solution, flow rates, the amount of ligand and the type of stripping solvents were optimized. The breakthrough volume was greater than 1800 ml with an enrichment factor of more than 360 and 6.0 ng l{sup -1} detection limit. The capacity of the membrane disks modified by 5 mg of the ligand was found to be 1070 {mu}g of silver. The effects of various cationic interferences on the percent recovery of silver ion were studied. The method was successfully applied to the determination of silver ion in different samples, especially determination of ultra trace amount of silver in the presence of large amount of lead.

  1. Conjugation of mono and di-GalNAc sugars enhances the potency of antisense oligonucleotides via ASGR mediated delivery to hepatocytes.

    Science.gov (United States)

    Kinberger, Garth A; Prakash, Thazha P; Yu, Jinghua; Vasquez, Guillermo; Low, Audrey; Chappell, Alfred; Schmidt, Karsten; Murray, Heather M; Gaus, Hans; Swayze, Eric E; Seth, Punit P

    2016-08-01

    Antisense oligonucleotides (ASOs) conjugated to trivalent GalNAc ligands show 10-fold enhanced potency for suppressing gene targets expressed in hepatocytes. Trivalent GalNAc is a high affinity ligand for the asialoglycoprotein receptor (ASGR)-a C-type lectin expressed almost exclusively on hepatocytes in the liver. In this communication, we show that conjugation of two and even one GalNAc sugar to single stranded chemically modified ASOs can enhance potency 5-10 fold in mice. Evaluation of the mono- and di-GalNAc ASO conjugates in an ASGR binding assay suggested that chemical features of the ASO enhance binding to the receptor and provide a rationale for the enhanced potency.

  2. Ultra-trace determination of silver in water samples by electrothermal atomic absorption spectrometry after preconcentration with a ligand-less cloud point extraction methodology

    Energy Technology Data Exchange (ETDEWEB)

    Manzoori, Jamshid L. [Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz (Iran, Islamic Republic of)]. E-mail: manzoori@tabrizu.ac.ir; Abdolmohammad-Zadeh, Hossein [Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz (Iran, Islamic Republic of); Amjadi, Mohammad [Department of Analytical Chemistry, Faculty of Chemistry, University of Tabriz, Tabriz (Iran, Islamic Republic of)

    2007-06-01

    A very simple and ligand-less cloud point extraction (CPE) methodology for the preconcentration of ultra-trace amounts of silver as a prior step to its determination by electrothermal atomic absorption spectrometry (ETAAS) has been developed. The method is based on the extraction of silver at pH 9 by using non-ionic surfactant polyethyleneglycolmono-p-nonylphenylether (PONPE 7.5) without adding any chelating agent. Several important variables that affect the CPE efficiency and ETAAS signal were investigated and optimized. The preconcentration of 15 ml sample solution allowed us to achieve an enhancement factor of 60. The calibration graph using the preconcentration system was linear in the range of 5-100 ng l{sup -1} with a correlation coefficient of 0.9991. The lower limit of detection (3s) obtained in the optimal conditions was 1.2 ng l{sup -1}. The relative standard deviation (R.S.D.) for eight replicate determinations at 30 ng l{sup -1} Ag level was 4.2%. The proposed method was successfully applied to the ultra-trace determination of silver in water samples.

  3. Simultaneous determination of trace uranium(VI) and zinc(II) by adsorptive cathodic stripping voltammetry with aluminon ligand.

    Science.gov (United States)

    Cha, K W; Park, C I; Park, S H

    2000-09-05

    Uranium(VI) complexed with aluminon (3-[bis(3-carboxy-4-hydroxy-phenyl)methylene]-6-oxo-1,4-cyclohexadiene-1-carboxylic acid triammonium salt) was determined by adsorptive cathodic stripping voltammetry (ACSV) using a hanging mercury drop electrode. Trace uranium(VI) and zinc(II) can be simultaneously determined in a single scan in the presence of aluminon and urea. Optimal conditions were found to be: accumulation time; 180-200 s, accumulation potential; 50 mV versus Ag/AgCl, scan rate; 40 mV s(-1), supporting electrolyte; 0.1 M sodium acetate buffer at pH 6.5-7.0, and concentration of aluminon; 1x10(-6) M. The linear range of uranium(VI) and zinc(II) were observed over the concentration range 2-33 and 30-120 ng ml(-1), respectively. The detection limit (S/N=3) are 0.2 ng ml(-1) (uranium) and 30 ng ml(-1) (zinc). A good reproducibility shows RSDs of 2.5-4.0% (n=10). The procedure offers high selectivity, with the presence of urea masking some metal ions.

  4. Plant-determined variation in the cardenolide content, thin-layer chromatography profiles, and emetic potency of monarch butterflies,Danaus plexippus reared on the milkweed,Asclepias eriocarpa in California.

    Science.gov (United States)

    Brower, L P; Seiber, J N; Nelson, C J; Lynch, S P; Tuskes, P M

    1982-03-01

    This paper is the first in a series on cardenolide fingerprinting of the monarch butterfly. New methodologies are presented which allow both qualitative and quantitative descriptions of the constituent cardenolides which these insects derive in the wild from specificAsclepias foodplants. Analyses of thin-layer Chromatographic profiles ofAsclepias eriocarpa cardenolides in 85 individual plant-butterfly pairs collected at six widely separate localities in California indicate a relatively invariant pattern of 16-20 distinct cardenolides which we here define as theAsclepias eriocarpa cardenolide fingerprint profile. Cardenolide concentrations vary widely in the plant samples, but monarchs appear able to regulate total storage by increasing their concentrations relative to their larval host plant when reared on plants containing low concentrations, and vice versa. Forced-feeding of blue jays with powdered butterfly and plant material and with one of the constituent plant cardenolides, labriformin, established that theA. eriocarpa cardenolides are extremely emetic, and that monarchs which have fed on this plant contain an average of 16 emetic-dose fifty (ED50) units. The relatively nonpolar labriformin and labriformidin in the plant are not stored by the monarch but are metabolized in vivo to desglucosyrioside which the butterfly does store. This is chemically analogous to the way in which monarchs and grasshoppers metabolize another series of milkweed cardenolides, those found inA. curassavica. It appears that the sugar moiety through functionality at C-3' determines which cardenolides are metabolized and which are stored. The monarch also appears able to store several lowR f cardenolides fromA. eriocarpa without altering them. Differences in the sequestering process in monarchs and milkweed bugs (Oncopeltus) may be less than emphasized in the literature. The monarch is seen as a central organism involved in a coevolutionary triad simultaneously affecting and affected

  5. Determining the magnitude and direction of photoinduced ligand field switching in photochromic metal-organic complexes: molybdenum-tetracarbonyl spirooxazine complexes.

    Science.gov (United States)

    Paquette, Michelle M; Patrick, Brian O; Frank, Natia L

    2011-07-06

    The ability to optically switch or tune the intrinsic properties of transition metals (e.g., redox potentials, emission/absorption energies, and spin states) with photochromic metal-ligand complexes is an important strategy for developing "smart" materials. We have described a methodology for using metal-carbonyl complexes as spectroscopic probes of ligand field changes associated with light-induced isomerization of photochromic ligands. Changes in ligand field between the ring-closed spirooxazine (SO) and ring-opened photomerocyanine (PMC) forms of photochromic azahomoadamantyl and indolyl phenanthroline-spirooxazine ligands are demonstrated through FT-IR, (13)C NMR, and computational studies of their molybdenum-tetracarbonyl complexes. The frontier molecular orbitals (MOs) of the SO and PMC forms differ considerably in both electron density distributions and energies. Of the multiple π* MOs in the SO and PMC forms of the ligands, the LUMO+1, a pseudo-b(1)-symmetry phenanthroline-based MO, mixes primarily with the Mo(CO)(4) fragment and provides the major pathway for Mo(d)→phen(π*) backbonding. The LUMO+1 is found to be 0.2-0.3 eV lower in energy in the SO form relative to the PMC form, suggesting that the SO form is a better π-acceptor. Light-induced isomerization of the photochromic ligands was therefore found to lead to changes in the energies of their frontier MOs, which in turn leads to changes in π-acceptor ability and ligand field strength. Ligand field changes associated with photoisomerizable ligands allow tuning of excited-state and ground-state energies that dictate energy/electron transfer, optical/electrical properties, and spin states of a metal center upon photoisomerization, positioning photochromic ligand-metal complexes as promising targets for smart materials.

  6. Simultaneous determination of copper, mercury and zinc in water with a tailored fluorescent bipyridine ligand entrapped in silica sol-gel

    Energy Technology Data Exchange (ETDEWEB)

    Lopes Pinheiro, Silvia C. [University of Campinas, Institute of Chemistry, P.O. Box 6154, Campinas (Brazil); Universidad Complutense de Madrid, Optical Chemosensors and Applied Photochemistry Group (GSOLFA), Faculty of Chemistry, Madrid (Spain); Raimundo, Ivo M. [University of Campinas, Institute of Chemistry, P.O. Box 6154, Campinas (Brazil); Moreno-Bondi, Maria C.; Orellana, Guillermo [Universidad Complutense de Madrid, Optical Chemosensors and Applied Photochemistry Group (GSOLFA), Faculty of Chemistry, Madrid (Spain)

    2010-12-15

    A novel fluorescent ligand, (4-[(E)-2-(4{sup '}-methyl-2,2{sup '}-bipyridin-4-yl)vinyl]phenol) (abbreviated BSOH), has been designed and prepared for simultaneous determination of heavy metals in water. Its photophysical and photochemical properties in the absence and in the presence of Cd(II), Cu(II), Hg(II), Ni(II) and Zn(II) were determined, and the respective complexation constants (7.4 x 10{sup 3}-2.8 x 10{sup 8} l mol{sup -1}) and stoichiometries were extracted thereof. The Stern-Volmer emission intensity and lifetime plots indicate an efficient static quenching of the indicator dye with the heavy metals. The BSOH fluorescent reagent has been successfully immobilised in a silica sol-gel matrix for automation of the analytical method, and the sensing phase demonstrated a reversible response to Cu(II), Hg(II) and Zn(II) but not to Cd(II) and Ni(II). Characterisation of the sensor showed that its response to those heavy metals is linear in the 2.5 to 50 {mu}mol l{sup -1} range, with a response time (t{sub 90}) on the order of 100 min, providing detection limits of 9.0 x 10{sup -7}, 4.7 x 10{sup -7} and 2.9 x 10{sup -7} mol l{sup -1} for Zn(II), Cu(II) and Hg(II), respectively. Due to the stability of the immobilised ligand, which presented no leaching from the sol-gel matrix, the simultaneous determination of the three cations in water was feasible by employing multivariate calibration techniques coupled to fluorescence quenching measurements. The sensor was validated with recovery tests by addition of Cu(II) and Hg(II) ions to spring waters, providing results with standard errors lower than 4.1 {mu}mol l{sup -1}. (orig.)

  7. Quantitative autoradiographic determination of binding sites for a peripheral benzodiazepine ligand ((/sup 3/H)PK 11195) in human iris

    Energy Technology Data Exchange (ETDEWEB)

    Valtier, D.; Malgouris, C.; Uzan, A.

    1987-01-01

    Specific binding sites of peripheral-type benzodiazepines were investigated in human iris/ciliary body (8 eyes). Examination of color-coded prints and densitometric quantification of autoradiograms were performed on slides (20 ..mu..m) labelled with (/sup 3/H)PK 11195 (1 nM) at 25 deg C. Nonspecific binding was determined with PK 11211 (5 ..mu..M) or Ro 5-4864 (5 ..mu..M). Binding sites were present on all the slides, with equivalent density in the 3 regions of the preparation (ciliary body, iris and pupil margin). The numbers of binding sites in ciliary body, iris, and pupil margin, respectively were: 42.7 +- 0.2, 30.1 +- 0.5 and 37.4 +- 0.4 femtomol/mg protein. Labelling on the pupil margin seemed to coincide with the iris sphincter muscle. The presence of peripheral benzodiazepine binding sites in iris muscular tissue, and particularly in the pupil margin, suggests that the iris preparation may be a valuable tool to detect putative physiological effects of peripheral benzodiazepines on muscular motility.

  8. Relative potency of ketamine and S(+)-ketamine in dogs.

    Science.gov (United States)

    Duque, J C; Oleskovicz, N; Guirro, E C B P; Valadão, C A A; Soares, V E

    2008-08-01

    The aim of this study was to determine the relative potency of racemic ketamine and S(+)-ketamine for the hypnotic effect and to evaluate the clinical anesthesia produced by equianesthetic doses of these two substances in dogs. One hundred and eight dogs were allocated in groups R2, R2.5, R3, R6, R9, R12, S2, S2.5, S3, S6, S9, and S12, to receive by intravenous route 2, 2.5, 3, 6, 9, and 12 mg/kg of ketamine or S(+)-ketamine, respectively. A dose-effect curve was drawn with the dose logarithm and the percentage of dogs that presented hypnosis in each group. The curve was used to obtain a linear regression, to determine the effective doses 100 and the potency relationship. In another experimental phase, eight groups of five dogs received 3, 6, 9 and 12 mg/kg of ketamine or S(+)-ketamine to evaluate the periods of latency, hypnosis, and total recovery. The times in which the dogs reached the sternal position, attempted to stand up for the first time, recovered the standing position, and started to walk were also recorded. The hypnotic dose for ketamine was 9.82 +/- 3.02 (6.86-16.5) mg/kg and for S(+)-ketamine was 7.76 +/- 2.17 (5.86-11.5) mg/kg. The time of hypnosis was longer in R3 and the first attempt to stand up occurred early in R6 when compared with S3 and S6 respectively. When R9 (100% of hypnosis with ketamine) and S6 [100% of hypnosis with S(+)-ketamine] were compared (1:1.5 ratio), the time to sternal position (12 +/- 2.5 and 20.2 +/- 5.6 min respectively) and the total recovery time (45 +/- 5.5 and 60.2 +/- 5.2 min respectively) were significantly shorter with S(+)-ketamine. It was concluded that the potency ratio between ketamine and S(+)-ketamine in dogs is smaller than the one reported in other species, and that the dose obtained after a reduction of 50%, as usually performed in humans, would not be enough to obtain equianesthetic effects in dogs.

  9. Relative Contribution of Endocrine-disrupting Chemicals to the Estrogenic Potency of Marine Sediments of Osaka Bay, Japan

    OpenAIRE

    KOYAMA, Jiro; KITOH, Asami; NAKAI, Makoto; KOHNO, Kumiko; Tanaka, Hiroyuki; Uno, Seiichi; コヤマ, ジロウ; キトウ, アサミ; ナカイ, マコト; コウノ, クミコ; タナカ, ヒロユキ; ウノ, セイイチ; 小山, 次朗; 中井, 誠; 河野, 久美子

    2013-01-01

    Although concentrations of endocrine-disrupting chemicals (EDCs) in seawaters of Japan are relatively low, vitellogenin and ovotestis inductions are still being observed in some males of mullet and flounder collected in coastal areas. These fish species are benthic and could be affected by EDCs in marine sediments. Therefore, the concentrations of EDCs in marine sediments of Osaka Bay were determined by LC-MS/MS. In addition, the estrogen receptor binding potencies as estrogenic potencies of ...

  10. Antibacterial potency of methanol extracts of lower plants

    Institute of Scientific and Technical Information of China (English)

    OJO O.O.; AJAYI A.O.; ANIBIJUWON I.I.

    2007-01-01

    Antibacterial potency of methanol extracts of three green lower plants, Pneumatopteris afra, Platycerium bifurcatum and Nephrolepsis bisserata was determined using agar dilution method on clinical strains of Escherichia coli, Staphylococcus aureus, Klebsiella spp. and Salmomelia typhi. Antibacterial activities were observed at concentrations of 12.5, 25.0, 50.0 and 100.0 μg/ml. Their minimum inhibitory concentrations ranged from 12.5~100 μg/ml. Extracts ofP. afra and P. bifurcatum were most active. Antibacterial activities observed with N. bisserata were less pronounced with no detectable activity at extract concentrations of 12.5 and 25.0 μg/ml. E. coli, together with S. aureus appeared to be the most susceptible of the test bacteria while Klebsiella spp. was least sensitive. The significance of our findings is discussed.

  11. Enantiomers of a nonylphenol isomer: absolute configurations and estrogenic potencies.

    Science.gov (United States)

    Zhang, Haifeng; Oppel, Iris M; Spiteller, Michael; Guenther, Klaus; Boehmler, Gabriele; Zuehlke, Sebastian

    2009-02-01

    Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay.

  12. Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

    Science.gov (United States)

    Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

    2017-07-01

    The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

  13. Relative potencies and combination effects of steroidal estrogens in fish.

    Science.gov (United States)

    Thorpe, Karen L; Cummings, Rob I; Hutchinson, Thomas H; Scholze, Martin; Brighty, Geoff; Sumpter, John P; Tyler, Charles R

    2003-03-15

    The natural steroids estradiol-17beta (E2) and estrone (E1) and the synthetic steroid ethynylestradiol-17alpha (EE2) have frequently been measured in waters receiving domestic effluents. All of these steroids bind to the estrogen receptor(s) and have been shown to elicit a range of estrogenic responses in fish at environmentally relevant concentrations. At present, however, no relative potency estimates have been derived for either the individual steroidal estrogens or their mixtures in vivo. In this study the estrogenic activity of E2, E1, and EE2, and the combination effects of a mixture of E2 and EE2 (equi-potent fixed-ratio mixture), were assessed using vitellogenin induction in a 14-day in vivo juvenile rainbow trout screening assay. Median effective concentrations, relative to E2, for induction of vitellogenin were determined from the concentration-response curves and the relative estrogenic potencies of each of the test chemicals calculated. Median effective concentrations were between 19 and 26 ng L(-1) for E2, 60 ng L(-1) for E1, and between 0.95 and 1.8 ng L(-1) for EE2, implying that EE2 was approximately 11 to 27 times more potent than E2, while E2 was 2.3 to 3.2 times more potent than E1. The median effective concentration, relative to E2, for the binary mixture of E2 and EE2 was 15 ng L(-1) (comprising 14.4 ng L(-1) E2 and 0.6 ng L(-1) EE2). Using the model of concentration addition it was shown that this activity of the binary mixture could be predicted from the activity of the individual chemicals. The ability of each individual steroid to contribute to the overall effect of a mixture, even at individual no-effect concentrations, combined with the high estrogenic potency of the steroids, particularly the synthetic steroid EE2, emphasizes the need to consider the total estrogenic load of these chemicals in our waterways.

  14. Expression and purification of a natural N-terminal pre-ligand assembly domain of tumor necrosis factor receptor 1 (TNFR1 PLAD) and preliminary activity determination.

    Science.gov (United States)

    Cao, Jin; Meng, Fang; Gao, Xiangdong; Dong, Hongxia; Yao, Wenbing

    2011-04-01

    A domain at the NH(2) terminal (N-terminal) of tumor necrosis factor receptor (TNFR) termed the pre-ligand binding assembly domain (PLAD). The finding that PLAD can mediate a selective TNFR assembly in previously researches provides a novel target to the prevention of TNFR signaling in immune-mediated inflammatory diseases (IMID). In this study, a natural N-terminal TNFR1 PLAD was obtained for the first time through the methods of GST-tag fusion protein expression and enterokinase cleavage. After purification with a Q Sepharose Fast Flow column, a natural N-terminal TNFR1 PLAD which purity was up to 95%, was obtained and was identified using Nano LC-ECI-MS/MS. Secondary structure analysis of PLAD was carried out using circular dichroism spectra (CD). After that, the TNFR1 PLAD in vitro anti-TNFα activity and the specific TNFR1 affinity were determined. The results proved that the natural N-terminal TNFR1 PLAD can selectively inhibit TNFα bioactivity mainly through TNFR1. It infers an effective and safe strategy for treating variety of IMID with a low risk of side effects in future.

  15. Optimizing Ligand Efficiency of Selective Androgen Receptor Modulators (SARMs).

    Science.gov (United States)

    Handlon, Anthony L; Schaller, Lee T; Leesnitzer, Lisa M; Merrihew, Raymond V; Poole, Chuck; Ulrich, John C; Wilson, Joseph W; Cadilla, Rodolfo; Turnbull, Philip

    2016-01-14

    A series of selective androgen receptor modulators (SARMs) containing the 1-(trifluoromethyl)benzyl alcohol core have been optimized for androgen receptor (AR) potency and drug-like properties. We have taken advantage of the lipophilic ligand efficiency (LLE) parameter as a guide to interpret the effect of structural changes on AR activity. Over the course of optimization efforts the LLE increased over 3 log units leading to a SARM 43 with nanomolar potency, good aqueous kinetic solubility (>700 μM), and high oral bioavailability in rats (83%).

  16. In vitro screening of the endocrine disrupting potency of brominated flame retardants and their metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Hamers, T.; Kamstra, J.H. [Inst. for Environmental Studies (IVM), Amsterdam (Netherlands); Sonneveld, E. [BioDetection Systems (BDS), Amsterdam (Netherlands); Murk, A.J. [Wageningen Univ., Toxicology Group, Wageningen (Netherlands); Zegers, B.N.; Boon, J.P. [Royal Netherlands Inst. for Sea Research (NIOZ), Den Burg (Netherlands); Brouwer, A. [Umea Univ., Umea (Sweden)

    2004-09-15

    Substantial evidence is recently becoming available that brominated flame retardants (BFRs) are potential endocrine disruptors. The toxicological profile of BFRs, however, is too incomplete and insufficient to perform human and ecological risk assessment. To fill these gaps, the EU funded research program FIRE was started in December 2002. This program aims at the identification and toxicological characterization of the most potent and environmentally relevant BFRs and their possible risk for human and wildlife health. As part of a hazard identification approach, twenty seven BFRs have been selected within the framework of FIRE for pre-screening their endocrinedisrupting potencies. Selection of test compounds was based on a maximal variation in physicochemical characteristics of BFRs within the test set, allowing the establishment of quantitative structure-activity relationships (QSARs). In addition, environmental relevance (e.g. high production volumes and persistence) and availability for testing were used as selection criteria. BFRs were tested in seven different in vitro bioassays for their potency to interfere via estrogenic, thyroidal, androgenic, progestagenic, and Ah-receptor mediated pathways. Metabolisation rates of BFRs were determined using phenobarbital-induced rat liver microsomes. Finally, the endocrine disrupting potency of the metabolites was determined in the same in vitro bio-assays and compared to the potency of the parent compounds.

  17. Development of an Innovative in Vitro Potency Assay for Anti-Botulinum Antitoxins.

    Science.gov (United States)

    Rosen, Osnat; Ozeri, Eyal; Barnea, Ada; David, Alon Ben; Zichel, Ran

    2016-09-24

    Botulinum neurotoxins are bacterial proteins that cause botulism, a life-threatening disease. Therapy relies mostly on post-intoxication antibody treatment. The only accepted method to measure the potency of, and to approve, antitoxin preparations is the mouse lethality neutralization bioassay. However, this assay is time-consuming, labor-intensive, costly, and raises ethical issues related to the large numbers of laboratory animals needed. Until now, all efforts to develop an alternative in vitro assay have not provided a valid replacement to the mouse potency assay. In the present study, we report the development of an innovative in vitro assay for determining botulinum antitoxin potency, using botulinum type B as a model. The concept of the assay is to mimic two fundamental steps in botulinum intoxication: receptor binding and catalytic activity. By simulating these steps in vitro we were able to accurately determine the potency of antitoxin preparations. The reproducibility of the assay was high with a CV vitro assay highly correlated with that measured by the standard in vivo mouse assay (r = 0.9842, p vitro assay has the potential to be considered, after validation, as a replacement to the mouse assay for quantitating neutralizing antibody concentrations in pharmaceutical botulinum antitoxin preparations. Future adoption of this in vitro assay would minimize the use of laboratory animals, speed up the time, and reduce the cost of botulinum antitoxin approval.

  18. Prion potency in stem cells biology.

    Science.gov (United States)

    Lopes, Marilene H; Santos, Tiago G

    2012-01-01

    Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.

  19. Inhibitory Potency of 4-Carbon Alkanes and Alkenes toward CYP2E1 Activity

    OpenAIRE

    2014-01-01

    CYP2E1 has been implicated in the bioactivation of many small molecules into reactive metabolites which form adducts with proteins and DNA, and thus a better understanding of the molecular determinants of its selectivity are critical for accurate toxicological predictions. In this study, we determined the potency of inhibition of human CYP2E1 for various 4-carbon alkanes, alkenes and alcohols. In addition, known CYP2E1 substrates and inhibitors including 4-methylpyrazole, aniline, and dimethy...

  20. Novel method demonstrates differential ligand activation and phosphatase-mediated deactivation of insulin receptor tyrosine-specific phosphorylation.

    Science.gov (United States)

    Cieniewicz, Anne M; Cooper, Philip R; McGehee, Jennifer; Lingham, Russell B; Kihm, Anthony J

    2016-08-01

    Insulin receptor signaling is a complex cascade leading to a multitude of intracellular functional responses. Three natural ligands, insulin, IGF1 and IGF2, are each capable of binding with different affinities to the insulin receptor, and result in variable biological responses. However, it is likely these affinity differences alone cannot completely explain the myriad of diverse cellular outcomes. Ligand binding initiates activation of a signaling cascade resulting in phosphorylation of the IR itself and other intracellular proteins. The direct catalytic activity along with the temporally coordinated assembly of signaling proteins is critical for insulin receptor signaling. We hypothesized that determining differential phosphorylation among individual tyrosine sites activated by ligand binding or dephosphorylation by phosphatases could provide valuable insight into insulin receptor signaling. Here, we present a sensitive, novel immunoassay adapted from Meso Scale Discovery technology to quantitatively measure changes in site-specific phosphorylation levels on endogenous insulin receptors from HuH7 cells. We identified insulin receptor phosphorylation patterns generated upon differential ligand activation and phosphatase-mediated deactivation. The data demonstrate that insulin, IGF1 and IGF2 elicit different insulin receptor phosphorylation kinetics and potencies that translate to downstream signaling. Furthermore, we show that insulin receptor deactivation, regulated by tyrosine phosphatases, occurs distinctively across specific tyrosine residues. In summary, we present a novel, quantitative and high-throughput assay that has uncovered differential ligand activation and site-specific deactivation of the insulin receptor. These results may help elucidate some of the insulin signaling mechanisms, discriminate ligand activity and contribute to a better understanding of insulin receptor signaling. We propose this methodology as a powerful approach to characterize

  1. Piperidine-Based Nocaine/Modafinil Hybrid Ligands as Highly Potent Monoamine Transporter Inhibitors: Efficient Drug Discovery by Rational Lead Hybridization

    Science.gov (United States)

    Zhou, Jia; He, Rong; Johnson, Kenneth M.; Ye, Yanping; Kozikowski, Alan P.

    2005-01-01

    Some piperidine-based nocaine/modafinil hybrid ligands have been designed, synthesized, and found to display an improved potency at all three monoamine transporters and particularly for DAT and/or NET. Some highly active and selective monoamine transporter inhibitors with low nanomolar to subnanomolar potency were identified. Ligands of this type may find important applications as positron emission tomography imaging tools and in the treatment of central nervous system disorders such as depression and sleep apnea. PMID:15537337

  2. Synthesis, CMC determination, and intercalative binding interaction with nucleic acid of a surfactant-copper(II) complex with modified phenanthroline ligand (dpq).

    Science.gov (United States)

    Nagaraj, Karuppiah; Ambika, Subramanian; Arunachalam, Sankaralingam

    2015-01-01

    A surfactant-copper(II) complex, [Cu(dpq)2DA](ClO4)2 (dpq = dipyrido[3,2-d:2'-3'-f]quinoxaline; DA-dodecylamine), was synthesized and characterized on the basis of elemental analyses, UV-vis, IR, and EPR spectra. The critical micelle concentration (CMC) value of this surfactant-copper(II) complex in aqueous solution was found out from conductance measurements. Specific conductivity data at different temperature served for the evaluation of the temperature-dependent CMC and the thermodynamics of micellization (ΔG°(m), ΔH°(m) and ΔS°(m)). In addition, the complex has been examined by its ability to bind to nucleic acids (DNA and RNA) in tris-HCl buffer by UV-vis absorption, emission spectroscopy techniques, and viscosity measurements. The complex has been found to bind strongly to nucleic acids with apparent binding constants at DNA and RNA is 4.3 × 10(5), 9.0 × 10(5) M(-1), respectively. UV-vis studies of the interaction of the complex with DNA/RNA have revealed that the complex can bind to both DNA and RNA by the intercalative binding mode via ligand dpq into the base pairs of DNA and RNA which has been verified by viscosity measurements. The presence of long aliphatic chain in the surfactant complex increases this hydrophobic interaction. The binding constants have been calculated. The cytotoxic activity of this complex on human liver carcinoma cancer cells was determined by adopting 3-(4, 5-dimethylthiazol-2-yl)-2, 5- diphenyl tetrazolium bromide assay and specific staining techniques. The antimicrobial and antifungal screening tests of this complex have shown good results.

  3. Biodistribution and radiation dosimetry of the A{sub 1} adenosine receptor ligand {sup 18}F-CPFPX determined from human whole-body PET

    Energy Technology Data Exchange (ETDEWEB)

    Herzog, Hans; Elmenhorst, David; Winz, Oliver [Forschungszentrum Juelich GmbH, Institute of Neuroscience and Biophysics - Medicine, Juelich (Germany); Bauer, Andreas [Forschungszentrum Juelich GmbH, Institute of Neuroscience and Biophysics - Medicine, Juelich (Germany); University Hospital Duesseldorf, Department of Neurology, Duesseldorf (Germany)

    2008-08-15

    {sup 18}F-8-cyclopentyl-3-(3-fluoropropyl)-1-propylxanthine ({sup 18}F-CPFPX) is a potent radioligand to study human cerebral A{sub 1} adenosine receptors and their neuromodulatory and neuroprotective functions with positron emission tomography (PET). The purpose of this study was to determine the biodistribution and the radiation dose of {sup 18}F-CPFPX by whole-body scans in humans. Six normal volunteers were examined with 12 whole-body PET scans from 1.5 min to 4.5 h after injection. Volumes of interest were defined over all visually identifiable organs, i.e. liver, gallbladder, kidneys, small intestines, heart, and brain to obtain the organs' volumes and time-activity curves (TACs). TACs were fitted with exponential functions, extrapolated, multiplied with the physical decay and normalized to injected activities so that the residence times could be computed as area under the curve. Radiation doses were calculated using the OLINDA/EXM software for internal dose assessment in nuclear medicine. The liver uptake shows peak values (decay-corrected) of up to 35% of the injected radioactivity. About 30% is eliminated by bladder voiding. The highest radiation dose is received by the gallbladder (136.2 {+-} 66.1 {mu}Sv/MBq), followed by the liver (84.4 {+-} 10.6 {mu}Sv/MBq) and the urinary bladder (78.3 {+-} 7.1 {mu}Sv/MBq). The effective dose was 17.6 {+-} 0.5 {mu}Sv/MBq. With 300 MBq of injected {sup 18}F-CPFPX a subject receives an effective dose (ICRP 60) of 5.3 mSv. Thus the effective dose of an {sup 18}F-CPFPX study is comparable to that of other {sup 18}F-labelled neuroreceptor ligands. (orig.)

  4. Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A receptor determine its activation and membrane-driven oligomerization properties.

    Directory of Open Access Journals (Sweden)

    Jufang Shan

    Full Text Available From computational simulations of a serotonin 2A receptor (5-HT(2AR model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011, we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i-the involvement of cholesterol in the activation of the 5-HT(2AR, and (ii-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.

  5. Multiplex Assay for Protein Profiling and Potency Measurement of German Cockroach Allergen Extracts.

    Directory of Open Access Journals (Sweden)

    Taruna Khurana

    Full Text Available German cockroach (GCr allergens induce IgE responses and may cause asthma. Commercial GCr allergen extracts are variable and existing assays may not be appropriate for determining extract composition and potency.Our aim was to develop a multiplex antibody/bead-based assay for assessment of GCr allergen extracts.Single chain fragment variable (scFv antibodies against GCr were obtained by screening libraries derived from naïve human lymphocytes and hyperimmunized chicken splenocytes and bone marrow. Selected clones were sequenced and characterized by immunoblotting. Eighteen scFv antibodies (17 chicken, 1 human coupled to polystyrene beads were used in this suspension assay; binding of targeted GCr allergens to antibody-coated beads was detected using rabbit antisera against GCr, and against specific allergens rBla g 1, rBla g 2, and rBla g 4. The assay was tested for specificity, accuracy, and precision. Extracts were also compared by IgE competition ELISA.Chicken scFv's generated eight different binding patterns to GCr proteins from 14 to 150 kDa molecular weight. Human scFv's recognized a 100 kDa GCr protein. The multiplex assay was found to be specific and reproducible with intra-assay coefficient of variation (CV of 2.64% and inter-assay CV of 10.0%. Overall potencies of various GCr extracts were calculated using mean logEC50s for eight selected scFvs. Overall potency measures were also analyzed by assessing the contributions to potency of each target.An scFv antibody-based multiplex assay has been developed capable of simultaneously measuring different proteins in a complex mixture, and to determine the potencies and compositions of allergen extracts.

  6. Potency determination of follitropin, lutropin And thyrotropin: a comparison between the quantification by reversed-phase high-performance liquid chromatography and in vivo bioassay; Determinacao de potencia de diferentes preparacoes de foliculotrofina, luteotrofina e tireotrofina: comparacao entre a quantificacao por cromatografia liquida em fase reversa e por bioensaio in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Almeida, Beatriz Elane de

    2013-07-01

    With the intention of setting up physico-chemical methods as an alternative to in vivo bioassay for determining biological activity, the hFSH, hTSH and hLH content of native and recombinant preparations was determined by reversed-phase high-performance liquid chromatography (RP-HPLC) and compared with the data obtained by the classical mouse or rat in vivo bioassays (BA). A linear relationship between the two methods was found for these hormones: hFSH BA{sub U=} 0.9925 RP-HPLC{sub U–} 1.3165, r = 0.9371, p < 0.001, n = 24; hTSH BAμg = 0.9790 RP-HPLCμg - 0.052, r = 0.8725, p < 0.001, n = 14; hLH BA{sub IU} = 0.8771 RP-HPLC{sub IU} + 12.41, r = 0.9786, p < 0.01, n = 5. For nine other hFSH and eleven hTSH preparations, the mean difference ( ) between the bioactivity predicted from RP-HPLC data via these equations and the mean of the bioactivities obtained with the two methods was as follows. For hLH this difference could not be estimated due to lack of different samples. In the case of hFSH, ± SD = -2.11 ± 3.49% with a precision of 1.16% and in the case of hTSH, ± SD = -2.01 ± 5.56 %, with precision of 1.68%. Partly-degraded hFSH, hTSH and hLH samples presented different activity degrees that could be predicted by RP-HPLC, with an acceptable agreement with the in vivo bioassays. These results demonstrate that the employment of a non-animal physico-chemical assay, such as RP-HPLC, is a viable alternative to the use of an in vivo bioassay for hFSH and hTSH potency determination, thus reducing the number of animals currently used for assuring quality and efficacy of a pharmaceutical product. (author)

  7. Potency of Traditional Insecticide Materials against Stored Bean ...

    African Journals Online (AJOL)

    Potency of Traditional Insecticide Materials against Stored Bean Weevil, ... AFRICAN JOURNALS ONLINE (AJOL) · Journals · Advanced Search · USING AJOL ... apply traditional insecticide materials in the protection of bean from insect pests.

  8. Effect of sugar positions in ginsenosides and their inhibitory potency on Na+/K+-ATPase activity

    Institute of Scientific and Technical Information of China (English)

    Ronald JY CHEN; Tse-yu CHUNG; Feng-yin LI; Nan-hei LIN; Jason TC TZEN

    2009-01-01

    Aim: To determine whether ginsenosides with various sugar attachments may act as active components responsible for the cardiac therapeutic effects of ginseng and sanqi (the roots of Panax ginseng and Panax notoginseng) via the same molecular mechanism triggered by cardiac glycosides, such as ouabain and digoxin. Methods: The structural similarity between ginsenosides and ouabain was analyzed. The inhibitory potency of ginseno-sides and ouabain on Na+/K+-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of ginsenosides to Na+/K+-ATPase. Results: Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, and possessed inhibitory potency on Na+/K+-ATPase activity. However, their inhibi-tory potency was significantly reduced or completely abolished when a monosaccharide was linked to the C-6 or C-20 posi-tion of the steroid-like structure; replacement of the monosaccharide with a disaccharide molecule at either of these posi-tions caused the disappearance of the inhibitory potency. Molecular modeling and docking confirmed that the difference in Na+/K+-ATPase inhibitory potency among ginsenosides was due to the steric hindrance of sugar attachment at the C-6 and C-20 positions of the steroid-like structure. Conclusion: The cardiac therapeutic effects of ginseng and sanqi should be at least partly attributed to the effective inhi-bition of Na+/K+-ATPase by their metabolized ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure.

  9. Imidazoline receptors ligands

    Directory of Open Access Journals (Sweden)

    Agbaba Danica

    2012-01-01

    Full Text Available Extensive biochemical and pharmacological studies have determined three different subtypes of imidazoline receptors: I1-imidazoline receptors (I1-IR involved in central inhibition of sympathicus that produce hypotensive effect; I2-imidazoline receptors (I2-IR modulate monoamine oxidase B activity (MAO-B; I3-imidazoline receptors (I3-IR regulate insulin secretion from pancreatic β-cells. Therefore, the I1/I2/I3 imidazoline receptors are selected as new, interesting targets for drug design and discovery. Novel selective I1/I2/I3 agonists and antagonists have been recently developed. In the present review, we provide a brief update to the field of imidazoline research, highlighting some of the chemical diversity and progress made in the 2D-QSAR, 3D-QSAR and quantitative pharmacophore development studies of I1-IR and I2-IR imidazoline receptor ligands. Theoretical studies of I3-IR ligands are not yet performed because of insufficient number of synthesized I3-IR ligands.

  10. RGS proteins destroy spare receptors: Effects of GPCR-interacting proteins and signal deamplification on measurements of GPCR agonist potency.

    Science.gov (United States)

    Chidiac, Peter

    2016-01-01

    Many GPCRs are able to activate multiple distinct signaling pathways, and these may include biochemical cascades activated via either heterotrimeric G proteins or by β-arrestins. The relative potencies and/or efficacies among a series of agonists that act on a common receptor can vary depending upon which signaling pathway is being activated. This phenomenon is known as biased signaling or functional selectivity, and is presumed to reflect underlying differences in ligand binding affinities for alternate conformational states of the receptor. The first part of this review discusses how various cellular GPCR interacting proteins (GIPs) can influence receptor conformation and thereby affect ligand-receptor interactions and contribute to signaling bias. Upon activation, receptors trigger biochemical cascades that lead to altered cellular function, and measuring points along the cascade (e.g., second messenger production) conveys information about receptor activity. As a signal continues along its way, the observed concentration dependence of a GPCR ligand may change due to amplification and saturation of biochemical steps. The second part of this review considers additional cellular factors that affect signal processing, focusing mainly on structural elements and deamplification mechanisms, and discusses the relevance of these to measurements of potency and functional selectivity.

  11. Effect of high-potency cannabis on corpus callosum microstructure.

    Science.gov (United States)

    Rigucci, S; Marques, T R; Di Forti, M; Taylor, H; Dell'Acqua, F; Mondelli, V; Bonaccorso, S; Simmons, A; David, A S; Girardi, P; Pariante, C M; Murray, R M; Dazzan, P

    2016-03-01

    The use of cannabis with higher Δ9-tetrahydrocannabinol content has been associated with greater risk, and earlier onset, of psychosis. However, the effect of cannabis potency on brain morphology has never been explored. Here, we investigated whether cannabis potency and pattern of use are associated with changes in corpus callosum (CC) microstructural organization, in patients with first-episode psychosis (FEP) and individuals without psychosis, cannabis users and non-users. The CC of 56 FEP (37 cannabis users) and 43 individuals without psychosis (22 cannabis users) was virtually dissected and segmented using diffusion tensor imaging tractography. The diffusion index of fractional anisotropy, mean diffusivity (MD), axial diffusivity (AD) and radial diffusivity was calculated for each segment. Across the whole sample, users of high-potency cannabis had higher total CC MD and higher total CC AD than both low-potency users and those who never used (p = 0.005 and p = 0.004, respectively). Daily users also had higher total CC MD and higher total CC AD than both occasional users and those who never used (p = 0.001 and p cannabis is associated with disturbed callosal microstructural organization in individuals with and without psychosis. Since high-potency preparations are now replacing traditional herbal drugs in many European countries, raising awareness about the risks of high-potency cannabis is crucial.

  12. Antigenotoxic potencies of a lichen species, Evernia prunastri.

    Science.gov (United States)

    Alpsoy, Lokman; Orhan, Furkan; Nardemir, Gokce; Agar, Guleray; Gulluce, Medine; Aslan, Ali

    2015-02-01

    In this article, the genotoxic and antigenotoxic effects of methanol extract of Evernia prunastri (Huds.) Willd. (MEP) were studied using WP2, Ames (TA1535 and TA1537) and sister chromatid exchange (SCE) test systems. The results obtained from bacterial test systems demonstrated that MEP has strong antimutagenic potencies on TA1537 and WP2 strains. The highest inhibition rates for MEP on TA1537 and WP2 strains were 37.70% and 69.70%, respectively. According to the SCE test system, MEP reduced the genotoxic effects of aflatoxin. In order to clarify the mechanism underlying the antigenotoxic effects of MEP, the antioxidants were determined. Cotreatments of 5, 10 and 20 µg/mL concentrations of MEP with aflatoxin B1 decreased the frequencies of SCE and the malondialdehyde level and increased amount of superoxide dismutase, glutathione and glutathione peroxidase which were decreased by aflatoxin. The data obtained from this work have clearly shown that MEP has significant antigenotoxic effects which are thought to be partly due to the antioxidant activities and antioxidant inducing capability of MEP. This is the first report indicating the antigenotoxic activities of MEP against several mutagen agents such as N-methyl-N'-nitro-N-nitrosoguanidine, acridin and aflatoxin. © The Author(s) 2012.

  13. Inhibitory Potency of 4-Carbon Alkanes and Alkenes toward CYP2E1 Activity

    Science.gov (United States)

    Hartman, Jessica H.; Miller, Grover P.; Boysen, Gunnar

    2016-01-01

    CYP2E1 has been implicated in the bioactivation of many small molecules into reactive metabolites which form adducts with proteins and DNA, and thus a better understanding of the molecular determinants of its selectivity are critical for accurate toxicological predictions. In this study, we determined the potency of inhibition of human CYP2E1 for various 4-carbon alkanes, alkenes and alcohols. In addition, known CYP2E1 substrates and inhibitors including 4-methylpyrazole, aniline, and dimethylnitrosamine were included to determine their relative potencies. Of the 1,3-butadiene-derived metabolites studied, 3,4-epoxy-1-butene was the strongest inhibitor with an IC50 of 110 μM compared to 1700 μM and 6600 μM for 1,2-butenediol and 1,2:3,4-diepoxybutane, respectively. Compared to known inhibitors, inhibitory potency of 3,4-epoxy-1-butene is between 4-methylpyrazole (IC50 = 1.8 μM) and dimethylnitrosamine (IC50 = 230 μM). All three butadiene metabolites inhibit CYP2E1 activity through a simple competitive mechanism. Among the 4-carbon compounds studied, the presence and location of polar groups seems to influence inhibitory potency. To further examine this notion, the investigation was extended to include structurally and chemically similar analogs, including propylene oxide and various butane alcohols. Those results demonstrated preferential recognition of CYP2E1 toward the type and location of polar and hydrophobic structural elements. Taken together, CYP2E1 metabolism may be modified in vivo by exposure to 4-carbon compounds, such as drugs, and nutritional constituents, a finding that highlights the complexity of exposure to mixtures. PMID:24561005

  14. Phototoxicity of strained Ru(ii) complexes: is it the metal complex or the dissociating ligand?

    Science.gov (United States)

    Azar, Daniel F; Audi, Hassib; Farhat, Stephanie; El-Sibai, Mirvat; Abi-Habib, Ralph J; Khnayzer, Rony S

    2017-07-27

    A photochemically dissociating ligand in Ru(bpy)2(dmphen)Cl2 [bpy = 2,2'-bipyridine; dmphen = 2,9-dimethyl-1,10-phenanthroline] was found to be more cytotoxic on the ML-2 Acute Myeloid Leukemia cell line than Ru(bpy)2(H2O)2(2+) and prototypical cisplatin. Our findings illustrate the potential potency of diimine ligands in photoactivatable Ru(ii) complexes.

  15. Potency Evaluation of Recombinant Human Erythropoietin in Brazil: Assessment of Reproducibility Using a Practical Approach

    Directory of Open Access Journals (Sweden)

    Michele Cardoso do Nascimento

    2015-08-01

    Full Text Available In this study, we compared the results of potency determination of recombinant human erythropoietin (rhEPO obtained between 2010 and 2012 by the National Institute of Quality Control in Health (INCQS/Fiocruz, i.e., the National Control Laboratory (NCL, and by a manufacturer of rhEPO. In total, 47 different batches of commercially prepared rhEPO (alpha isoform were analyzed. All results, including those of the control and warning limits, remained within the limits recommended by European Pharmacopoeia (Ph. Eur.. All relative error (RE values were less than ± 30%, wh ereas most were approximately ± 20%. Applying the Bland-Altman plot, only two of 47 values remained outside the limits of agreement (LA. In addition, agreement of potency determination between INCQS and the manufacturer coefficient of variation of reproducibility (% CVR was considered satisfactory. Taken together, our results demonstrate (i. the potency assay of rhEPO performed at INCQS, is standardized and controlled, (ii. the comparison of our results with those of the manufacturer, revealed an adequate inter-laboratory variation, and (iii. the critical appraisal proposed here appears to be a feasible tool to assess the reproducibility of biological activity, providing additional information regarding monitoring and production consistency to manufacturers and NCLs.

  16. Identification of mangiferin as a potential Glucokinase activator by structure-based virtual ligand screening.

    Science.gov (United States)

    Min, Qiuxia; Cai, Xinpei; Sun, Weiguang; Gao, Fei; Li, Zhimei; Zhang, Qian; Wan, Luo-Sheng; Li, Hua; Chen, Jiachun

    2017-03-20

    The natural product mangiferin (compound 7) has been identified as a potential glucokinase activator by structure-based virtual ligand screening. It was proved by enzyme activation experiment and cell-based assays in vitro, with potency in micromolar range. Meanwhile, this compound showed good antihyperglycemic activity in db/db mice without obvious side effects such as excessive hypoglycaemia.

  17. Estrogenic potency of food-packaging-associated plasticizers and antioxidants as detected in ERa and ERb reporter gene cell lines

    NARCIS (Netherlands)

    Veld, ter M.G.R.; Schouten, B.; Louisse, J.; Es, van D.S.; Saag, van der P.T.; Rietjens, I.M.C.M.; Murk, A.J.

    2006-01-01

    This study presents the estrogenic potency of 21 food-packaging-associated compounds determined for the first time, using two transfected U2-OS (human osteoblasts devoid of endogenous estrogen receptors) estrogen receptor (ER) alpha and beta cell lines. Six plasticizers and three antioxidants were s

  18. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality : A possible complementation to the Pertussis Serological Potency test

    NARCIS (Netherlands)

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-01-01

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunolog

  19. Stimulative influence of germination and growth of maize seedlings originating from aged seeds by 2,4-D potencies.

    Science.gov (United States)

    Dragicevic, Vesna; Spasic, Mihajlo; Simic, Milena; Dumanovic, Zoran; Nikolic, Bogdan

    2013-07-01

    The 2,4-D (2,4-dichlorophenoxyacetic acid) is using as a growth regulator in tissue culture media. Maize seeds have poor ability to maintain germination rate in the long term. To examine the possible restorative effect of homeopathic 2,4-D potencies on maize seedlings originating from seeds damaged by accelerated aging. Seeds of four maize lines were subjected to accelerated aging stress treatment. Seed samples were treated with distilled water (control) and a range of potencies of 2,4-D: 3C, 3.75C, 4.5C, 5.25C and 6C. The germination capacity, fresh substance (FS) and length of root and shoot were determined. Hydrolysis and biosynthesis, GSH/GSSG ratio and redox capacity were calculated. Induced seed aging decreased germination rate and growth of seedlings. 2,4-D potencies did not have a statistically significant effect on germination. However, there were statistically significant effects on FS production, root and shoot length and redox capacity. The 3C potency had the largest effect on the FS accumulation, 4.5C increased root and shoot length, compared to control (statistically significant). The GSH/GSSG ratio and the redox capacity were decreased by aging. The 3C and 4.5C potencies tended to reverse the GSH/GSSG ratio (statistically significant) in the root and shoot, (i.e., shifted the redox balance to the reduced state). Homeopathic potencies of 2,4-D appear to have a beneficial effect on artificially aged maize seeds: they stimulate growth through better substance conversion from seed rest, and shift the redox capacity towards a reduced environment. Further work is required to determine if this is an useful means of improving maize seed germination and growth. Copyright © 2013 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

  20. Use of biopartitioning micellar chromatography and RP-HPLC for the determination of blood-brain barrier penetration of α-adrenergic/imidazoline receptor ligands, and QSPR analysis.

    Science.gov (United States)

    Vucicevic, J; Popovic, M; Nikolic, K; Filipic, S; Obradovic, D; Agbaba, D

    2017-03-01

    For this study, 31 compounds, including 16 imidazoline/α-adrenergic receptor (IRs/α-ARs) ligands and 15 central nervous system (CNS) drugs, were characterized in terms of the retention factors (k) obtained using biopartitioning micellar and classical reversed phase chromatography (log kBMC and log kwRP, respectively). Based on the retention factor (log kwRP) and slope of the linear curve (S) the isocratic parameter (φ0) was calculated. Obtained retention factors were correlated with experimental log BB values for the group of examined compounds. High correlations were obtained between logarithm of biopartitioning micellar chromatography (BMC) retention factor and effective permeability (r(log kBMC/log BB): 0.77), while for RP-HPLC system the correlations were lower (r(log kwRP/log BB): 0.58; r(S/log BB): -0.50; r(φ0/Pe): 0.61). Based on the log kBMC retention data and calculated molecular parameters of the examined compounds, quantitative structure-permeability relationship (QSPR) models were developed using partial least squares, stepwise multiple linear regression, support vector machine and artificial neural network methodologies. A high degree of structural diversity of the analysed IRs/α-ARs ligands and CNS drugs provides wide applicability domain of the QSPR models for estimation of blood-brain barrier penetration of the related compounds.

  1. Complexation efficiency of differently fixed 8-hydroxyquinoline and salicylic acid ligand groups for labile aluminium species determination in soils--comparison of two methods.

    Science.gov (United States)

    Matús, Peter; Kubová, Jana

    2006-07-28

    Two methods utilizing the complexation of labile Al species by 8-hydroxyquinoline (HQN) and salicylic acid (SA) ligand groups were developed for aluminium operationally defined fractionation in acid soils. First, the solid phase extraction (SPE) procedure by a short-term ion-exchange batch reaction with chelating resins Iontosorb Oxin and Iontosorb Salicyl containing both ligand groups was used previously. Second, the 8-hydroxyquinoline, salicylic acid and ammonium salicylate agents with different concentrations by a single extraction protocol were applied in this paper. The flame atomic absorption spectrometry (FAAS) and optical emission spectrometry with inductively coupled plasma were used for aluminium quantification. The comparison of results from both methods show the possibility to supersede the first laborious method for the second simpler one in Al environmental risk assessment. The use of 1% 8-hydroxyquinoline in 2% acetic acid and 0.2% salicylic acid by a single extraction protocol without a need of sample filtration can supersede the SPE procedure in the Al pollution soil monitoring. Finally, the new scheme usable in a laboratory and moreover, directly in a field was proposed for Al fractionation in solid and liquid environmental samples. The labile Al species in soils and sediments are separated after their single leaching by 8-hydroxyquinoline or salicylic acid without a need of sample filtration. The labile Al species in soil solutions and natural waters are separated after their ultrafiltration followed by the SPE procedure with Iontosorb Oxin or Iontosorb Salicyl.

  2. Complexation efficiency of differently fixed 8-hydroxyquinoline and salicylic acid ligand groups for labile aluminium species determination in soils-comparison of two methods

    Energy Technology Data Exchange (ETDEWEB)

    Matus, Peter [Comenius University in Bratislava, Faculty of Natural Sciences, Mlynska dolina, 842 15 Bratislava (Slovakia)]. E-mail: matus@fns.uniba.sk; Kubova, Jana [Comenius University in Bratislava, Faculty of Natural Sciences, Mlynska dolina, 842 15 Bratislava (Slovakia)

    2006-07-28

    Two methods utilizing the complexation of labile Al species by 8-hydroxyquinoline (HQN) and salicylic acid (SA) ligand groups were developed for aluminium operationally defined fractionation in acid soils. First, the solid phase extraction (SPE) procedure by a short-term ion-exchange batch reaction with chelating resins Iontosorb Oxin and Iontosorb Salicyl containing both ligand groups was used previously. Second, the 8-hydroxyquinoline, salicylic acid and ammonium salicylate agents with different concentrations by a single extraction protocol were applied in this paper. The flame atomic absorption spectrometry (FAAS) and optical emission spectrometry with inductively coupled plasma were used for aluminium quantification. The comparison of results from both methods show the possibility to supersede the first laborious method for the second simpler one in Al environmental risk assessment. The use of 1% 8-hydroxyquinoline in 2% acetic acid and 0.2% salicylic acid by a single extraction protocol without a need of sample filtration can supersede the SPE procedure in the Al pollution soil monitoring. Finally, the new scheme usable in a laboratory and moreover, directly in a field was proposed for Al fractionation in solid and liquid environmental samples. The labile Al species in soils and sediments are separated after their single leaching by 8-hydroxyquinoline or salicylic acid without a need of sample filtration. The labile Al species in soil solutions and natural waters are separated after their ultrafiltration followed by the SPE procedure with Iontosorb Oxin or Iontosorb Salicyl.

  3. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells.

    Science.gov (United States)

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A; Ackerman, Janet M; Yaswen, Paul; Vulpe, Chris D; Leitman, Dale C

    2016-05-01

    Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM, Yaswen P, Vulpe CD, Leitman DC. 2016. Parabens and human epidermal

  4. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells

    Science.gov (United States)

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A.; Ackerman, Janet M.; Yaswen, Paul; Vulpe, Chris D.; Leitman, Dale C.

    2015-01-01

    Background: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. Objectives: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). Methods: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Results: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Conclusion: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Citation: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM

  5. A study of cannabis potency in France over a 25 years period (1992-2016).

    Science.gov (United States)

    Dujourdy, Laurence; Besacier, Fabrice

    2017-03-01

    Cannabis contains a unique class of compounds known as the cannabinoids. Pharmacologically, the principal psychoactive constituent is Δ(9)-tetrahydrocannabinol (THC). The amount of THC in conjunction with selected additional cannabinoid compounds (cannabidiol/CBD, cannabinol/CBN), determines the strength or potency of the cannabis product. Recently, reports have speculated over the change in the quality of cannabis products, from nearly a decade, specifically concerning the increase in cannabinoid content. This article exploits the analytical data of cannabis samples analyzed in the five French forensic police laboratories over 25 years. The increase potency of both herbal and resin cannabis in France is proved through the monitoring of THC content. For cannabis resin, it has slowly risen from 1992 to 2009, before a considerable increase in the last four years (mean THC content in mid-2016 is 23% compared to 10% in 2009). For herbal cannabis, it has known three main stages of growth (mean THC content is 13% in 2015 and mid-2016 compared to 7% in 2009 and 2% in 1995). The calculation of THC/CBD ratios in both herbal and resin samples confirms the recent change in chemotypes in favor of high potency categories. Finally, the CBN/THC ratios in marijuana samples were measured in order to evaluate the freshness of French seized hemp.

  6. Potency values from the local lymph node assay: application to classification, labelling and risk assessment.

    Science.gov (United States)

    Loveless, S E; Api, A-M; Crevel, R W R; Debruyne, E; Gamer, A; Jowsey, I R; Kern, P; Kimber, I; Lea, L; Lloyd, P; Mehmood, Z; Steiling, W; Veenstra, G; Woolhiser, M; Hennes, C

    2010-02-01

    Hundreds of chemicals are contact allergens but there remains a need to identify and characterise accurately skin sensitising hazards. The purpose of this review was fourfold. First, when using the local lymph node assay (LLNA), consider whether an exposure concentration (EC3 value) lower than 100% can be defined and used as a threshold criterion for classification and labelling. Second, is there any reason to revise the recommendation of a previous ECETOC Task Force regarding specific EC3 values used for sub-categorisation of substances based upon potency? Third, what recommendations can be made regarding classification and labelling of preparations under GHS? Finally, consider how to integrate LLNA data into risk assessment and provide a rationale for using concentration responses and corresponding no-effect concentrations. Although skin sensitising chemicals having high EC3 values may represent only relatively low risks to humans, it is not possible currently to define an EC3 value below 100% that would serve as an appropriate threshold for classification and labelling. The conclusion drawn from reviewing the use of distinct categories for characterising contact allergens was that the most appropriate, science-based classification of contact allergens according to potency is one in which four sub-categories are identified: 'extreme', 'strong', 'moderate' and 'weak'. Since draining lymph node cell proliferation is related causally and quantitatively to potency, LLNA EC3 values are recommended for determination of a no expected sensitisation induction level that represents the first step in quantitative risk assessment.

  7. Bioluminescence inhibition of bacterial luciferase by aliphatic alcohol, amine and carboxylic acid: inhibition potency and mechanism.

    Science.gov (United States)

    Yamasaki, Shinya; Yamada, Shuto; Takehara, Kô

    2013-01-01

    The inhibitory effects of hydrophobic molecules on the bacterial luciferase, BL, luminescence reaction were analyzed using an electrochemically-controlled BL luminescence system. The inhibition potency of alkyl amines, C(n)NH(2), and fatty acids, C(m)COOH (m = n - 1), on the BL reaction increased with an increase in the alkyl chain-length of these aliphatic compounds. C(m)COOH showed lower inhibition potency than C(n)NH(2) and alkyl alcohols, C(n)OH, data for which have been previously reported. To make clear the inhibition mechanisms of the aliphatic compounds on the BL reaction, the initial rate of the BL reaction was measured and analyzed using the Dixon plot and Cornish-Bowden plot. The C(12)OH inhibited the BL reaction in competition with the substrate C(11)CHO, while C(12)NH(2) and C(11)COOH inhibited in an uncompetitive manner with the C(11)CHO. These results suggest that the alkyl chain-length and the terminal unit of the aliphatic compound determine the inhibition potency and the inhibition mechanism, respectively.

  8. Factors Affecting the Inclusion Potency for Acicular Ferrite Nucleation in High-Strength Steel Welds

    Science.gov (United States)

    Kang, Yongjoon; Jeong, Seonghoon; Kang, Joo-Hee; Lee, Changhee

    2016-06-01

    Factors affecting the inclusion potency for acicular ferrite nucleation in high-strength weld metals were investigated and the contribution of each factor was qualitatively evaluated. Two kinds of weld metals with different hardenabilities were prepared, in both, MnTi2O4-rich spinel formed as the predominant inclusion phase. To evaluate the factors determining the inclusion potency, the inclusion characteristics of size, phase distribution in the multiphase inclusion, orientation relationship with ferrite, and Mn distribution near the inclusion were analyzed. Three factors affecting the ferrite nucleation potency of inclusions were evaluated: the Baker-Nutting (B-N) orientation relationship between ferrite and the inclusion; the formation of an Mn-depleted zone (MDZ) near the inclusion; and the strain energy around the inclusion. Among these, the first two factors were found to be the most important. In addition, it was concluded that the increased chemical driving force brought about by the formation of an MDZ contributed more to the formation of acicular ferrite in higher-strength weld metals, because the B-N orientation relationship between ferrite and the inclusion was less likely to form as the transformation temperature decreased.

  9. Establishment of animal model for potency evaluation of inactivated SARS virus experimental vaccine

    Institute of Scientific and Technical Information of China (English)

    GUANMUDONG; QIAN; YANKONG; WENXUELIU; LIHINGYANG; JUNZHIWANG; YONGXINYU; YAOLONGSHU; ZHENGWANG; WEIDONGYIN; QINGYUZHU; HAIFAZHENG

    2005-01-01

    The purpose of this study was to test the effectiveness of source virus strain for the manufacture of the inactivated SARS virus vaccine, and establish an experimental method and preliminary standard for potency evaluation. Mice were divided into groups for being immunized with corresponding serially diluted experimental SARS virus inactivated vaccine. And the rabbits were immunized with undiluted vaccine. Challenge assay was conducted with a heterologous SARS virus. And the neutralization antibody was determined with plaque reduction neutralization test(PRNT), to which the neutralization antibody in the convalescent serum of SARS patients was compared. The experimental vaccine viral strains were proved to be suitable for manufacturing the vaccine. Mice immunized by vaccines of serial dilutions were able to elicit neutralizing antibody. The antibody titer from mice immunized with the undiluted vaccine could reach up to 1:495.2, while those of rabbits immunized with the undiluted vaccine could reach a GMT of 55.0-79.9. The capability of the antibody to neutralize the virus from Guangdong is more efficient than that from Beijing. The GMT of neutralizing antibody in SARS convalescents living in south and north China ranged from 50.12 to 54.95, and the titers of convalescents from north China were higher than those from south China. Mice and rabbits used as the model for evaluation of potency are of sensitivity, and the test is of reproducibility. The candidate challenge viral strains showed a relatively consistent effect on evaluating antibodies produced by various batches and different vaccine-source strains,hence they can be used to evaluate potency of the vaccine. The method for testing the vaccine potency and the evaluation standard was established preliminarily.

  10. Phytoestrogens and sterols in waters with cyanobacterial blooms - Analytical methods and estrogenic potencies.

    Science.gov (United States)

    Procházková, Tereza; Sychrová, Eliška; Javůrková, Barbora; Večerková, Jaroslava; Kohoutek, Jiří; Lepšová-Skácelová, Olga; Bláha, Luděk; Hilscherová, Klára

    2017-03-01

    Compounds with estrogenic potencies and their adverse effects in surface waters have received much attention. Both anthropogenic and natural compounds contribute to overall estrogenic activity in freshwaters. Recently, estrogenic potencies were also found to be associated with cyanobacteria and their blooms in surface waters. The present study developed and compared the solid phase extraction and LC-MS/MS analytical approaches for determination of phytoestrogens (8 flavonoids - biochanin A, coumestrol, daidzein, equol, formononetin, genistein, naringenin, apigenin - and 5 sterols - ergosterol, β-sitosterol, stigmasterol, campesterol, brassicasterol) and cholesterol in water. The method was used for analyses of samples collected in stagnant water bodies dominated by different cyanobacterial species. Concentrations of individual flavonoids ranged from below the limit of detection to 3.58 ng/L. Sterols were present in higher amounts up to 2.25 μg/L. Biological potencies of these phytoestrogens in vitro were characterized using the hERα-HeLa-9903 cell line. The relative estrogenic potencies (compared to model estrogen - 17β-estradiol) of flavonoids ranged from 2.25E-05 to 1.26E-03 with coumestrol being the most potent. None of the sterols elicited estrogenic response in the used bioassay. Estrogenic activity was detected in collected field water samples (maximum effect corresponding to 2.07 ng/L of 17β-estradiol equivalents, transcriptional assay). At maximum phytoestrogens accounted for only 1.56 pg/L of 17β-estradiol equivalents, contributing maximally 8.5% of the total estrogenicity of the water samples. Other compounds therefore, most likely of anthropogenic origin such as steroid estrogens, are probably the major drivers of total estrogenic effects in these surface waters.

  11. Molecular Recognition and Ligand Association

    Science.gov (United States)

    Baron, Riccardo; McCammon, J. Andrew

    2013-04-01

    We review recent developments in our understanding of molecular recognition and ligand association, focusing on two major viewpoints: (a) studies that highlight new physical insight into the molecular recognition process and the driving forces determining thermodynamic signatures of binding and (b) recent methodological advances in applications to protein-ligand binding. In particular, we highlight the challenges posed by compensating enthalpic and entropic terms, competing solute and solvent contributions, and the relevance of complex configurational ensembles comprising multiple protein, ligand, and solvent intermediate states. As more complete physics is taken into account, computational approaches increase their ability to complement experimental measurements, by providing a microscopic, dynamic view of ensemble-averaged experimental observables. Physics-based approaches are increasingly expanding their power in pharmacology applications.

  12. Why mercury prefers soft ligands

    Energy Technology Data Exchange (ETDEWEB)

    Riccardi, Demian M [ORNL; Guo, Hao-Bo [ORNL; Gu, Baohua [ORNL; Parks, Jerry M [ORNL; Summers, Anne [University of Georgia, Athens, GA; Miller, S [University of California, San Francisco; Liang, Liyuan [ORNL; Smith, Jeremy C [ORNL

    2013-01-01

    Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg2+, is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg2+. Comparison of Hg2+ ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.

  13. Overexpression of KIR inhibitory ligands (HLA-I) determines that immunosurveillance of myeloma depends on diverse and strong NK cell licensing.

    Science.gov (United States)

    Martínez-Sánchez, María V; Periago, Adela; Legaz, Isabel; Gimeno, Lourdes; Mrowiec, Anna; Montes-Barqueros, Natividad R; Campillo, José A; Bolarin, José M; Bernardo, María V; López-Álvarez, María R; González, Consuelo; García-Garay, María C; Muro, Manuel; Cabañas-Perianes, Valentin; Fuster, Jose L; García-Alonso, Ana M; Moraleda, José M; Álvarez-Lopez, María R; Minguela, Alfredo

    2016-04-01

    Missing self recognition makes cancer sensitive to natural killer cell (NKc) reactivity. However, this model disregards the NKc licensing effect, which highly increases NKc reactivity through interactions of inhibitory killer cell immunoglobulin-like receptors (iKIR) with their cognate HLA-I ligands. The influence of iKIR/HLA-ligand (HLA-C1/C2) licensing interactions on the susceptibility to and progression of plasma cell (PC) dyscrasias was evaluated in 164 Caucasian patients and 286 controls. Compared to controls, myeloma accumulates KIR2DL1(-)L2(+)L3(-) genotypes (2.8% vs. 13.2%, p < 0.01, OR = 5.29) and less diverse peripheral repertoires of NKc clones. Less diverse and weaker-affinity repertoires of iKIR2D/HLA-C licensing interactions increased myeloma susceptibility. Thus, the complete absence of conventional iKIR2D/HLA-C licensing interactions (KIR2DL1(-)L2(+)L3(-)/C2C2, 2.56% vs. 0.35%; p < 0.05; OR = 15.014), single-KIR2DL3(+)/C1(+) (20.51% vs. 10.84%; p < 0.05; OR = 2.795) and single-KIR2DL2(+)/C1(+) (12.82% vs. 4.9%; p < 0.01; OR = 5.18) interactions were over-represented in myeloma, compared to controls. Additionally, KIR2DL1(-)L2(+)L3(-) (20% vs. 83%, p < 0.00001) as well as KIR3DL1(-) (23% vs. 82%, p < 0.00001) genotypes had a dramatic negative impact on the 3-y progression-free survival (PFS), particularly in patients with low-tumor burden. Remarkably, myeloma-PCs, compared to K562 and other hematological cancers, showed substantial over-expression of HLA-I ("increasing-self" instead of missing-self), including HLA-C, and mild expression of ligands for NKc activating receptors (aRec) CD112, CD155, ULBP-1 and MICA/B, which apparently renders myeloma-PCs susceptible to lysis mainly by licensed NKc. KIR2DL1(-)L2(+)L3(-)/C2C2 patients (with no conventional iKIR2D/HLA-C licensing interactions) lyse K562 but barely lyse myeloma-PCs (4% vs. 15%; p < 0.05, compared to controls). These results support a model where immunosurveillance of no

  14. Capping of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle Number Concentration and Surface Ligand Density.

    Science.gov (United States)

    Garcia-Cortes, Marta; Sotelo González, Emma; Fernández-Argüelles, María T; Encinar, Jorge Ruiz; Costa-Fernández, José M; Sanz-Medel, Alfredo

    2017-06-27

    Colloidal Mn(2+)-doped ZnS quantum dots (QDs) were synthesized, surface modified, and thoroughly characterized using a pool of complementary techniques. Cap exchange of the native l-cysteine coating of the QDs with dihydrolipoic acid (DHLA) ligands is proposed as a strategy to produce nanocrystals with a strong phosphorescent-type emission and improved aqueous stability. Moreover, such a stable DHLA coating can facilitate further bioconjugation of these QDs to biomolecules using established reagents such as cross-linker molecules. First, a structural and morphological characterization of the l-cysteine QD core was performed by resorting to complementary techniques, including X-ray powder diffraction (XRD) and microscopy tools. XRD patterns provided information about the local structure of ions within the nanocrystal structure and the number of metal atoms constituting the core of a QD. The judicious combination of the data obtained from these complementary characterization tools with the analysis of the QDs using inductively coupled plasma-mass spectrometry (ICP-MS) allowed us to assess the number concentration of nanoparticles in an aqueous sample, a key parameter when such materials are going to be used in bioanalytical or toxicological studies. Asymmetric flow field-flow fractionation (AF4) coupled online to ICP-MS detection proved to be an invaluable tool to compute the number of DHLA molecules attached to the surface of a single QD, a key feature that is difficult to estimate in nanoparticles and that critically affects the behavior of nanoparticles when entering the biological media (e.g., cellular uptake, biodistribution, or protein corona formation). This hybrid technique also allowed us to demonstrate that the elemental composition of the nanoparticle core remains unaffected after the ligand exchange process. Finally, the photostability and robustness of the DHLA-capped QDs, critical parameters for bioanalytical applications, were assessed by molecular

  15. A novel strategy for the determination of enantiomeric compositions of chiral compounds by chemometric analysis of the UV-vis spectra of bovine serum albumin receptor-ligand mixtures

    Science.gov (United States)

    Wang, Yunxia; Zhang, Fang; Liang, Jing; Li, Hua; Kong, Jilie

    2007-10-01

    In this work, a novel strategy was constructed to determine the enantiomeric composition of chiral substances discriminated by bovine serum albumin (BSA) based on the UV-vis spectra of the receptor-ligand mixtures coupled with partial least squares (PLS-1) analysis. Taking tryptophan (Trp) enantiomer as an example, when 20 μM BSA was used, the enantiomeric composition was accurately determined with concentration of only 100 nM and the corresponding enantiomeric excess as high as 98% (or -98%), which is relatively more sensitive than in literature. Furthermore, the BSA-based approach was also used to predict the enantiomeric composition of other chiral compounds, such as phenylalanine (Phe), tyrosine (Tyr), alanine (Ala), cysteine (Cys), DOPA and propranolol (Prop). The results fully demonstrate that BSA is effective in determination of enantiomeric composition of some chiral compounds.

  16. The Parathyroid Hormone Family of Ligands and Receptors

    Directory of Open Access Journals (Sweden)

    Damian G. D'Souza

    2015-07-01

    Full Text Available The PTH family of ligands and receptors have a wide range of vital functions from calcium homeostasis to tissue and bone development from the embryo to adult. This family has undergone whole genome duplication events predating vertebrate evolution, indicating more primitive and ancient functions other than skeletal development. The N-terminal region of the ligands, have been widely studied by biophysical and functional analysis, resulting in the discovery of key characteristics essential for ligand-receptor activation being elucidated. Multi-substituted amino acid analogs with differential binding affinities and either antagonistic or agonistic signalling potencies have been created based on these findings allowing for improvement on potential therapies affected by the PTH system in skeletal and embryonic development. The PTH family has diversely evolved to cover a wide range of pivotal pathways crucial to growth and development throughout all animal life.

  17. Multivalent drug design and inhibition of cholera toxin by specific and transient protein-ligand interactions.

    Science.gov (United States)

    Liu, Jiyun; Begley, Darren; Mitchell, Daniel D; Verlinde, Christophe L M J; Varani, Gabriele; Fan, Erkang

    2008-05-01

    Multivalent inhibitors of the cholera toxin B pentamer are potential therapeutic drugs for treating cholera and serve as models for demonstrating multivalent ligand effects through a structure-based approach. A crucial yet often overlooked aspect of multivalent drug design is the length, rigidity and chemical composition of the linker used to connect multiple binding moieties. To specifically study the role of chemical linkers in multivalent ligand design, we have synthesized a series of compounds with one and two binding motifs connected by several different linkers. These compounds have affinity for and potency against the cholera toxin B pentamer despite the fact that none can simultaneously bind two toxin receptor sites. Results from saturation transfer difference NMR reveal transient, non-specific interactions between the cholera toxin and linker groups contribute significantly to overall binding affinity of monovalent compounds. However, the same random protein-ligand interactions do not appear to affect binding of bivalent molecules. Moreover, the binding affinities and potencies of these 'non-spanning' bivalent ligands appear to be wholly independent of linker length. Our detailed analysis identifies multiple effects that account for the improved inhibitory potencies of bivalent ligands and suggest approaches to further improve the activity of this class of compounds.

  18. DNA interaction and cytotoxic activities of square planar platinum(II) complexes with N, S-donor ligands

    Science.gov (United States)

    Patel, Mohan N.; Patel, Chintan R.; Joshi, Hardik N.; Thakor, Khyati P.

    2014-06-01

    The platinum(II) complexes with N, S-donor ligands have been synthesized and characterized by physicochemical methods viz. elemental, electronic, FT-IR, 1H NMR and LC-MS spectra. The binding mode and potency of the complexes with HS DNA (Herring Sperm) have been examined by absorption titration and viscosity measurement studies. The results revealed that complexes bind to HS DNA via covalent mode with the intrinsic binding constant (Kb) in the range 1.37-7.76 × 105 M-1. Decrease in the relative viscosity of HS DNA also supports the covalent mode of binding. The DNA cleavage activity of synthesized complexes has been carried out by gel electrophoresis experiment using supercoiled form of pUC19 DNA; showing the unwinding of the negatively charged supercoiled DNA. Brine shrimp (Artemia Cysts) lethality bioassay technique has been applied for the determination of toxic property of synthesized complexes in terms of μM.

  19. Principles for identification of High Potency Category Chemicals for which the Dermal Sensitisation Threshold (DST) approach should not be applied.

    Science.gov (United States)

    Roberts, David W; Api, Anne Marie; Safford, Robert J; Lalko, Jon F

    2015-08-01

    An essential step in ensuring the toxicological safety of chemicals used in consumer products is the evaluation of their skin sensitising potential. The sensitising potency, coupled with information on exposure levels, can be used in a Quantitative Risk Assessment (QRA) to determine an acceptable level of a given chemical in a given product. Where consumer skin exposure is low, a risk assessment can be conducted using the Dermal Sensitisation Threshold (DST) approach, avoiding the need to determine potency experimentally. Since skin sensitisation involves chemical reaction with skin proteins, the first step in the DST approach is to assess, on the basis of the chemical structure, whether the chemical is expected to be reactive or not. Our accompanying publication describes the probabilistic derivation of a DST of 64 μg/cm(2) for chemicals assessed as reactive. This would protect against 95% of chemicals assessed as reactive, but the remaining 5% would include chemicals with very high potency. Here we discuss the chemical properties and structural features of high potency sensitisers, and derive an approach whereby they can be identified and consequently excluded from application of the DST.

  20. Synthesis and Characterization of a New Bivalent Ligand Combining Caffeine and Docosahexaenoic Acid.

    Science.gov (United States)

    Fernández-Dueñas, Víctor; Azuaje, Jhonny; Morató, Xavier; Cordobilla, Begoña; Domingo, Joan Carles; Sotelo, Eddy; Ciruela, Francisco

    2017-02-27

    Caffeine is a promising drug for the management of neurodegenerative diseases such as Parkinson's disease (PD), demonstrating neuroprotective properties that have been attributed to its interaction with the basal ganglia adenosine A2A receptor (A2AR). However, the doses needed to exert these neuroprotective effects may be too high. Thus, it is important to design novel approaches that selectively deliver this natural compound to the desired target. Docosahexaenoic acid (DHA) is the major omega-3 fatty acid in the brain and can act as a specific carrier of caffeine. Furthermore, DHA displays properties that may lead to its use as a neuroprotective agent. In the present study, we constructed a novel bivalent ligand covalently linking caffeine and DHA and assessed its pharmacological activity and safety profile in a simple cellular model. Interestingly, the new bivalent ligand presented higher potency as an A2AR inverse agonist than caffeine alone. We also determined the range of concentrations inducing toxicity both in a heterologous system and in primary striatal cultures. The novel strategy presented here of attaching DHA to caffeine may enable increased effects of the drug at desired sites, which could be of interest for the treatment of PD.

  1. Statin therapy in patients with acute coronary syndrome: low-density lipoprotein cholesterol goal attainment and effect of statin potency

    Directory of Open Access Journals (Sweden)

    Chinwong D

    2015-01-01

    Full Text Available Dujrudee Chinwong,1,2 Jayanton Patumanond,3 Surarong Chinwong,1 Khanchai Siriwattana,4 Siriluck Gunaparn,5 John Joseph Hall,6 Arintaya Phrommintikul5 1Department of Pharmaceutical Care, Faculty of Pharmacy, Chiang Mai University, Chiang Mai, Thailand; 2Clinical Epidemiology Program, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 3Center of Excellence in Applied Epidemiology, Faculty of Medicine, Thammasat University, Pathum Thani, Thailand; 4Division of Medicine, Nakornping Hospital, Chiang Mai, Thailand; 5Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand; 6Centre for Clinical Epidemiology and Biostatistics, School of Medicine and Public Health, Faculty of Health, University of Newcastle, Callaghan, NSW, Australia Background: Elevated low-density lipoprotein cholesterol (LDL-C is associated with an increased risk of coronary artery disease. Current guidelines recommend an LDL-C target of <70 mg/dL (<1.8 mmol/L for acute coronary syndrome (ACS patients, and the first-line treatment to lower lipids is statin therapy. Despite current guidelines and the efficacious lipid-lowering agents available, about half of patients at very high risk, including ACS patients, fail to achieve their LDL-C goal. This study assessed LDL-C goal attainment according to use of high and low potency statins in routine practice in Thailand.Methods: A retrospective cohort study was performed by retrieving data from medical records and the electronic hospital database for a tertiary care hospital in Thailand between 2009 and 2011. Included were ACS patients treated with statins at baseline and with follow-up of LDL-C levels. Patients were divided into high or low potency statin users, and the proportion reaching the LDL-C goal of <70 mg/dL was determined. A Cox proportional hazard model was applied to determine the relationship between statin potency and LDL-C goal attainment. Propensity score adjustment

  2. Solvolysis mechanisms of RNA phosphodiester analogues promoted by mononuclear zinc(II) complexes: mechanisic determination upon solvent medium and ligand effects.

    Science.gov (United States)

    Zhang, Xuepeng; Zhu, Yajie; Gao, Hui; Zhao, Cunyuan

    2014-11-17

    The solvolysis mechanisms of RNA phosphodiester model 2-(hydroxypropyl)-4-nitrophenyl phosphate (HpPNP) catalyzed by mononuclear zinc(II) complexes are investigated in the paper via a theoretical approach. The general-base-catalyzed (GBC) and specific-base-catalyzed (SBC) mechanisms are thoroughly discussed in the paper, and the calculations indicate a SBC mechanism (also named as the direct nucleophilic attack mechanism) when the cyclization of HpPNP is promoted by the Zn:[12]aneN3 complex ([12]aneN3 = 1,5,9-triazacyclododecane). The ligand effect is considered by involving two different catalysts, and the results show that the increasing size catalyst provides a lower energy barrier and a significant mechanistic preference to the SBC mechanism. The solvent medium effect is also explored, and reduced polarity/dielectric constant solvents, such as light alcohols methanol and ethanol, are more favorable. Ethanol is proven to be a good solvent medium because of its low dielectric constant. The computational results are indicative of concerted pathways. Our theoretical results are consistent with and well interpret the experimental observations and, more importantly, provide practical suggestions on the catalyst design and selection of reaction conditions.

  3. Comparative sensitizing potencies of fragrances, preservatives, and hair dyes

    DEFF Research Database (Denmark)

    Lidén, Carola; Yazar, Kerem; Johansen, Jeanne Duus

    2016-01-01

    the sensitizing potencies of fragrance substances, preservatives, and hair dye substances, which are skin sensitizers that frequently come into contact with the skin of consumers and workers, LLNA results and EC3 values for 72 fragrance substances, 25 preservatives and 107 hair dye substances were obtained from...... two published compilations of LLNA data and opinions by the Scientific Committee on Consumer Safety and its predecessors. The median EC3 values of fragrances (n = 61), preservatives (n = 19) and hair dyes (n = 59) were 5.9%, 0.9%, and 1.3%, respectively. The majority of sensitizing preservatives...... and hair dyes are thus strong or extreme sensitizers (EC3 value of ≤2%), and fragrances are mostly moderate sensitizers. Although fragrances are typically moderate sensitizers, they are among the most frequent causes of contact allergy. This indicates that factors other than potency need to be addressed...

  4. Can biochemistry drive drug discovery beyond simple potency measurements?

    Science.gov (United States)

    Chène, Patrick

    2012-04-01

    Among the fields of expertise required to develop drugs successfully, biochemistry holds a key position in drug discovery at the interface between chemistry, structural biology and cell biology. However, taking the example of protein kinases, it appears that biochemical assays are mostly used in the pharmaceutical industry to measure compound potency and/or selectivity. This limited use of biochemistry is surprising, given that detailed biochemical analyses are commonly used in academia to unravel molecular recognition processes. In this article, I show that biochemistry can provide invaluable information on the dynamics and energetics of compound-target interactions that cannot be obtained on the basis of potency measurements and structural data. Therefore, an extensive use of biochemistry in drug discovery could facilitate the identification and/or development of new drugs.

  5. Alternative splicing in nicotinic acetylcholine receptor subunits from Locusta migratoria and its influence on acetylcholine potencies.

    Science.gov (United States)

    Zhang, Yixi; Liu, Yang; Bao, Haibo; Sun, Huahua; Liu, Zewen

    2017-01-18

    Due to the great abundance within insect central nervous system (CNS), nicotinic acetylcholine receptors (nAChRs) play key roles in insect CNS, which makes it to be the targets of several classes of insecticides, such as neonicotinoids. Insect nAChRs are pentameric complexes consisting of five subunits, and a dozen subunits in one insect species can theoretically comprise diverse nAChRs. The alternative splicing in insect nAChR subunits may increase the diversity of insect nAChRs. In the oriental migratory locust (Locusta migratoria manilensis Meyen), a model insect species with agricultural importance, the alternative splicing was found in six α subunits among nine α and two β subunits, such as missing conserved residues in Loop D from Locα1, Locα6 and Locα9, a 34-residue insertion in Locα8 cytoplasmic loop, and truncated transcripts for Locα4, Locα7 and Locα9. Hybrid nAChRs were successfully constructed in Xenopus oocytes through co-expression with rat β2 and one α subunit from L. migratoria, which included Locα1, Locα2, Locα3, Locα4, Locα5, Locα8 and Locα9. Influences of alternative splicing in Locα1, Locα8 and Locα9 on acetylcholine potency were tested on hybrid nAChRs. The alternative splicing in Locα1 and Locα9 could increase acetylcholine sensitivities on recombinant receptors, while the splicing in Locα8 showed significant influences on the current amplitudes of oocytes. The results revealed that the alternative splicing at or close to the ligand-binding sites, as well as at cytoplasmic regions away from the ligand-binding sites, in insect nAChR subunits would change the agonist potencies on the receptors, which consequently increased nAChR diversity in functional and pharmacological properties.

  6. Incorporating potency into EU classification for carcinogenicity and reproductive toxicity.

    Science.gov (United States)

    Hennes, C; Batke, M; Bomann, W; Duhayon, S; Kosemund, K; Politano, V; Stinchcombe, S; Doe, J

    2014-11-01

    Although risk assessment, assessing the potential harm of each particular exposure of a substance, is desirable, it is not feasible in many situations. Risk assessment uses a process of hazard identification, hazard characterisation, and exposure assessment as its components. In the absence of risk assessment, the purpose of classification is to give broad guidance (through the label) on the suitability of a chemical in a range of use situations. Hazard classification in the EU is a process involving identification of the hazards of a substance, followed by comparison of those hazards (including degree of hazard) with defined criteria. Classification should therefore give guidance on degree of hazard as well as hazard identification. Potency is the most important indicator of degree of hazard and should therefore be included in classification. This is done for acute lethality and general toxicity by classifying on dose required to cause the effect. The classification in the EU for carcinogenicity and reproductive toxicity does not discriminate across the wide range of potencies seen (6 orders of magnitude) for carcinogenicity and for developmental toxicity and fertility. Therefore potency should be included in the classification process. The methodology in the EU guidelines for classification for deriving specific concentration limits is a rigorous process for assigning substances which cause tumours or developmental toxicity and infertility in experimental animals to high, medium or low degree of hazard categories by incorporating potency. Methods are suggested on how the degree of hazard so derived could be used in the EU classification process to improve hazard communication and in downstream risk management.

  7. A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

    Science.gov (United States)

    Aggarwal, Anshu; Tandon, Simran; Singla, Surinder Kumar; Tandon, Chanderdeep

    2012-08-01

    Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

  8. Epoxy resin monomers with reduced skin sensitizing potency.

    Science.gov (United States)

    O'Boyle, Niamh M; Niklasson, Ida B; Tehrani-Bagha, Ali R; Delaine, Tamara; Holmberg, Krister; Luthman, Kristina; Karlberg, Ann-Therese

    2014-06-16

    Epoxy resin monomers (ERMs), especially diglycidyl ethers of bisphenol A and F (DGEBA and DGEBF), are extensively used as building blocks for thermosetting polymers. However, they are known to commonly cause skin allergy. This research describes a number of alternative ERMs, designed with the aim of reducing the skin sensitizing potency while maintaining the ability to form thermosetting polymers. The compounds were designed, synthesized, and assessed for sensitizing potency using the in vivo murine local lymph node assay (LLNA). All six epoxy resin monomers had decreased sensitizing potencies compared to those of DGEBA and DGEBF. With respect to the LLNA EC3 value, the best of the alternative monomers had a value approximately 2.5 times higher than those of DGEBA and DGEBF. The diepoxides were reacted with triethylenetetramine, and the polymers formed were tested for technical applicability using thermogravimetric analysis and differential scanning calorimetry. Four out of the six alternative ERMs gave polymers with a thermal stability comparable to that obtained with DGEBA and DGEBF. The use of improved epoxy resin monomers with less skin sensitizing effects is a direct way to tackle the problem of contact allergy to epoxy resin systems, particularly in occupational settings, resulting in a reduction in the incidence of allergic contact dermatitis.

  9. Comparative anticonvulsant potency and pharmacokinetics of (+)-and (-)-enantiomers of stiripentol.

    Science.gov (United States)

    Shen, D D; Levy, R H; Savitch, J L; Boddy, A V; Tombret, F; Lepage, F

    1992-06-01

    The anticonvulsant potency and pharmacokinetics of the enantiomers of stiripentol were compared using the intravenous pentylenetetrazol infusion seizure model in the rat. Enantioselectivity was observed with respect to both the anticonvulsant activity and elimination kinetics of this compound. (+)-Stiripentol was 2.4 times more potent than its antipode against pentylenetetrazol-induced clonic seizure (brain EC50 of 15.2 micrograms/ml versus 36.1 micrograms/ml). The (+)-enantiomer was eliminated more rapidly than the (-)-enantiomer, as reflected in a higher plasma clearance (1.64 l/h/kg versus 0.557 l/h/kg) and a shorter half-life (2.83 h versus 6.50 h). Parallel studies with the racemate of stiripentol indicated that the anticonvulsant potency of the racemate was between the potency of the two enantiomers, suggesting that the combined activity reflects the additive action of (+)- and (-)-stiripentol. However, a marked metabolic interaction between enantiomers was evident after racemate administration. These results point to the need for information on the differential pharmacokinetics of stiripentol enantiomers following racemic drug administration.

  10. Potency of marbofloxacin for pig pneumonia pathogens Actinobacillus pleuropneumoniae and Pasteurella multocida: Comparison of growth media.

    Science.gov (United States)

    Dorey, L; Hobson, S; Lees, P

    2017-04-01

    Pharmacodynamic properties of marbofloxacin were established for six isolates each of the pig respiratory tract pathogens, Actinobacillus pleuropneumoniae and Pasteurella multocida. Three in vitro indices of potency were determined; Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and Mutant Prevention Concentration (MPC). For MIC determination Clinical Laboratory Standards Institute guidelines were modified in three respects: (1) comparison was made between two growth media, an artificial broth and pig serum; (2) a high inoculum count was used to simulate heavy clinical bacteriological loads; and (3) five overlapping sets of two-fold dilutions were used to improve accuracy of determinations. Similar methods were used for MBC and MPC estimations. MIC and MPC serum:broth ratios for A. pleuropneumoniae were 0.79:1 and 0.99:1, respectively, and corresponding values for P. multocida were 1.12:1 and 1.32:1. Serum protein binding of marbofloxacin was 49%, so that fraction unbound (fu) serum MIC values were significantly lower than those predicted by correction for protein binding; fu serum:broth MIC ratios were 0.40:1 (A. pleuropneumoniae) and 0.50:1 (P. multocida). For broth, MPC:MIC ratios were 13.7:1 (A. pleuropneumoniae) and 14.2:1 (P. multocida). Corresponding ratios for serum were similar, 17.2:1 and 18.8:1, respectively. It is suggested that, for dose prediction purposes, serum data might be preferable to potency indices measured in broths. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Targeting Selectins and Their Ligands in Cancer

    Directory of Open Access Journals (Sweden)

    Alessandro eNatoni

    2016-04-01

    Full Text Available Aberrant glycosylation is a hallmark of cancer cells with increased evidence pointing to a role in tumor progression. In particular, aberrant sialylation of glycoproteins and glycolipids have been linked to increased immune cell evasion, drug evasion, drug resistance, tumor invasiveness, and vascular dissemination leading to metastases. Hypersialylation of cancer cells is largely the result of overexpression of sialyltransferases. Humans differentially express twenty different sialyltransferases in a tissue-specific manner, each of which catalyze the attachment of sialic acids via different glycosidic linkages (2-3; 2-6 or 2-8 to the underlying glycan chain. One important mechanism whereby overexpression of sialyltransferases contributes to an enhanced metastatic phenotype is via the generation of selectin ligands. Selectin ligand function requires the expression of sialyl-Lewis X and its structural-isomer sialyl-Lewis A, which are synthesized by the combined action of alpha 1-3-fucosyltransferases, 2-3-sialyltransferases, 1-4-galactosyltranferases, and N-acetyl--glucosaminyltransferases. The α2-3-sialyltransferases ST3Gal4 and ST3Gal6 are critical to the generation of functional E- and P-selectin ligands and overexpression of these sialyltransferases have been linked to increased risk of metastatic disease in solid tumors and poor outcome in multiple myeloma. Thus, targeting selectins and their ligands as well as the enzymes involved in their generation, in particular sialyltransferases, could be beneficial to many cancer patients. Potential strategies include sialyltransferase inhibition and the use of selectin antagonists, such as glycomimetic drugs and antibodies. Here, we review ongoing efforts to optimize the potency and selectivity of sialyltransferase inhibitors, including the potential for targeted delivery approaches, as well as evaluate the potential utility of selectin inhibitors, which are now in early clinical

  12. Standardization and validation of Vero cell assay for potency estimation of diphtheria antitoxin serum.

    Science.gov (United States)

    Kumar, Sunil; Kanwar, Sarika; Bansal, Vivek; Sehgal, Rakesh

    2009-10-01

    Diphtheria toxin has the capacity to block protein synthesis in cultured mammalian cells, and thus causing cell death. This capacity of diphtheria toxin was utilized for in-vitro neutralization test to determine antibody titer, using Vero cells, which have been found to be susceptible to diphtheria toxin. In the present study, a Vero cell assay was standardized and validated for potency estimation of diphtheria antitoxin serum (DATS). The results obtained by Vero cell assay were compared with in-vivo biological assay. High degree of correlation (+0.98) was found between in-vivo biological assay and in-vitro Vero cell assay. The assay has also been found to be effective in determining the rising antibody titer in the equines inducted in DATS production. The present study indicated that although biological assays hold the key for final potency estimations till date but in the future scenario in-vitro Vero cell assay may be a good alternative to in-vivo biological assay.

  13. Ultra-performance liquid chromatography tandem mass spectrometry method for the determination of AZ66, a sigma receptor ligand, in rat plasma and its application to in vivo pharmacokinetics.

    Science.gov (United States)

    Jamalapuram, Seshulatha; Vuppala, Pradeep Kumar; Abdelazeem, Ahmed H; McCurdy, Christopher R; Avery, Bonnie A

    2013-08-01

    Methamphetamine abuse continues as a major problem in the USA owing to its powerful psychological addictive properties. AZ66, 3-[4-(4-cyclohexylpiperazine-1-yl)pentyl]-6-fluorobenzo[d]thiazole-2(3H)-one, an optimized sigma receptor ligand, is a promising therapeutic agent against methamphetamine. To study the in vivo pharmacokinetics of this novel sigma receptor ligand in rats, a sensitive ultra-performance liquid chromatography/tandem mass spectrometry (UPLC/MS/MS) method was developed in rat plasma and validated. The developed method requires a small volume of plasma (100 μL) and a simple liquid-liquid extraction. The chromatographic separations were achieved in 3.3 min using an Acquity UPLC BEH Shield RP18 column. The mass spectrophotometric detection was carried out using a Waters Micromass Quattro MicroTM triple-quadrupole system. Multiple reaction monitoring was used for the quantitation with transitions m/z 406 → m/z 181 for AZ66 and m/z 448 → m/z 285 for aripiprazole. The method was validated over a concentration range of 1-3500 ng/mL and the lower limit of quantitation was determined to be 1 ng/mL. Validation of the assay demonstrated that the developed UPLC/MS/MS method was sensitive, accurate and selective for the determination of AZ66 in rat plasma. The present method has been successfully applied to an i.v. pharmacokinetic study in Sprague-Dawley rats.

  14. Identification of mouse cathepsin K structural elements that regulate the potency of odanacatib.

    Science.gov (United States)

    Law, Simon; Andrault, Pierre-Marie; Aguda, Adeleke H; Nguyen, Nham T; Kruglyak, Natasha; Brayer, Gary D; Brömme, Dieter

    2017-02-20

    Cathepsin K (CatK) is the predominant mammalian bone-degrading protease and thus an ideal target for antiosteoporotic drug development. Rodent models of osteoporosis are preferred due to their close reflection of the human disease and their ease of handling, genetic manipulation and economic affordability. However, large differences in the potency of CatK inhibitors for the mouse/rat vs. the human protease orthologs have made it impossible to use rodent models. This is even more of a problem considering that the most advanced CatK inhibitors, including odanacatib (ODN) and balicatib, failed in human clinical trials due to side effects and rodent models are not available to investigate the mechanism of these failures. Here, we elucidated the structural elements of the potency differences between mouse and human CatK (hCatK) using ODN. We determined and compared the structures of inhibitor-free mouse CatK (mCatK), hCatK and ODN bound to hCatK. Two structural differences were identified and investigated by mutational analysis. Humanizing subsite 2 in mCatK led to a 5-fold improvement of ODN binding, whereas the replacement of Tyr61 in mCatK with Asp resulted in an hCatK with comparable ODN potency. Combining both sites further improved the inhibition of the mCatK variant. Similar results were obtained for balicatib. These findings will allow the generation of transgenic CatK mice that will facilitate the evaluation of CatK inhibitor adverse effects and to explore routes to avoid them.

  15. Pancuronium, gallamine, and d-tubocurarine compared: is speed of onset inversely related to drug potency?

    Science.gov (United States)

    Kopman, A F

    1989-06-01

    The relative potency and speed of onset of action of pancuronium, gallamine, and d-tubocurarine was studied in 55 adult female patients receiving nitrous oxide/oxygen-narcotic anesthesia. The integrated electromyogram of the adductor pollicis muscle was monitored using a cumulative dose-response technique; train-of-four stimuli were administered at 0.05 Hz. The measured ED95 values for pancuronium and gallamine were 0.069 and 2.38 mg/kg, respectively. In three separate groups, pancuronium 0.07 mg/kg, gallamine 2.4 mg/kg, or d-tubocurarine 0.45 mg/kg were given as a single bolus and the speed of onset and time to maximum effect determined. Peak twitch depression was essentially identical in all groups (92.7 +/- 1.4 [SE] vs. 93.3 +/- 1.1 vs. 93.7 +/- 1.1%, respectively). The rate of onset of neuromuscular blockade in these three groups was, however, quite different. After administration of pancuronium (n = 10) the times to 5%, 20%, 50%, and 80% twitch depression were 68 +/- 5, 97 +/- 6, 141 +/- 8, and 222 +/- 18 s. The comparable times following gallamine (n = 10) were 29 +/- 2, 42 +/- 3, 66 +/- 5, and 136 +/- 14 s; d-tubocurarine (n = 10) was intermediate in speed with onset times of 40 +/- 4, 63 +/- 6, 99 +/- 11, and 178 +/- 25 s. It appears that the onset times of different nondepolarizing blocking agents (even when given in equipotent doses) may vary by clinically appreciable amounts. The results of this study support the hypothesis that nondepolarizing neuromuscular blocking agents of low potency may have a more rapid onset of action than that seen with agents of high potency.

  16. Potency and characterization of estrogen-receptor agonists in United Kingdom estuarine sediments.

    Science.gov (United States)

    Thomas, Kevin V; Balaam, Jan; Hurst, Mark; Nedyalkova, Zoya; Mekenyan, Ovanes

    2004-02-01

    The activity of estrogen-receptor (ER) agonists in sediments collected from the United Kingdom (UK) estuaries was assessed using the in vitro recombinant yeast estrogen screen (YES assay). The YES assay was successfully used to determine the in vitro ER agonist potency of pore waters and solvent extracts of sediments collected from UK estuaries. Estrogen-receptor agonists were detected in 66% of the pore water samples and in 91% of the sediment solvent extracts tested. The pore waters tested had ER agonist potencies from less than 2 to 68 ng 17beta-estradiol (E2) L(-1), whereas sediment extracts had potencies from less than 0.2 to 13 microg E2 kg(-1). A toxicity identification evaluation approach using bioassay-directed fractionation was used in an attempt to identify the ER agonists in extracts of sediments collected from the Tyne and Tees estuaries (UK). Gas chromatography-mass spectrometry was used to provide lists of compounds in the fractions obtained that were evaluated for known ER agonist activity using published data and an ER quantitative structure-activity relationship model. Toxicity identification evaluation characterization failed to identify any ER agonists in pore water extracts; however, three compounds in sediment solvent extracts were identified as ER agonists. Nonylphenol, cinnarizine, and cholesta-4,6-dien-3-one were identified in the sample collected from the Tyne estuary. Important ER agonist substances that contaminate marine sediments remain unidentified. The present study as well as previous work on effluents point toward the involvement of natural products in the estrogenic burdens of marine sediments. Further work is required to establish the relative contribution of natural products and anthropogenic chemicals to current environmental impacts in the context of the Oslo and Paris Commission strategy to eliminate hazardous substances by 2020.

  17. SnapShot: GPCR-Ligand Interactions.

    Science.gov (United States)

    Ghosh, Eshan; Nidhi, Kumari; Shukla, Arun K

    2014-12-18

    G-protein-coupled receptors enable cells to recognize numerous external stimuli and to transmit corresponding signals across the plasma membrane to trigger appropriate cellular responses. Crystal structures of a number of these receptors have now been determined in inactive and active conformations bound to chemically and functionally distinct ligands. These crystal structures illustrate overall receptor organization and atomic details of ligand-receptor interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Online size-exclusion high-performance liquid chromatography light scattering and differential refractometry methods to determine degree of polymer conjugation to proteins and protein-protein or protein-ligand association states.

    Science.gov (United States)

    Kendrick, B S; Kerwin, B A; Chang, B S; Philo, J S

    2001-12-15

    Characterizing the solution structure of protein-polymer conjugates and protein-ligand interactions is important in fields such as biotechnology and biochemistry. Size-exclusion high-performance liquid chromatography with online classical light scattering (LS), refractive index (RI), and UV detection offers a powerful tool in such characterization. Novel methods are presented utilizing LS, RI, and UV signals to rapidly determine the degree of conjugation and the molecular mass of the protein conjugate. Baseline resolution of the chromatographic peaks is not required; peaks need only be sufficiently separated to represent relatively pure fractions. An improved technique for determining the polypeptide-only mass of protein conjugates is also described. These techniques are applied to determining the degree of erythropoietin glycosylation, the degree of polyethylene glycol conjugation to RNase A and brain-derived neurotrophic factor, and the solution association states of these molecules. Calibration methods for the RI, UV, and LS detectors will also be addressed, as well as online methods to determine protein extinction coefficients and dn/dc values both unconjugated and conjugated protein molecules. (c)2001 Elsevier Science.

  19. Reversed-phase ion-pair liquid chromatographic method for determination of reaction equilibria involving ionic species: exemplification of the method using ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions.

    Science.gov (United States)

    Sato, Emiko; Miya, Seiko; Saitoh, Kazunori; Saito, Shingo; Shibukawa, Masami

    2011-02-18

    A reversed-phase ion-pair liquid chromatographic method is presented for the determination of reaction equilibria involving ionic species of the same charge sign as reactant and product compounds. It has been demonstrated that ion-exchange chromatography or reversed-phase ion-pair chromatography is a useful tool for the determination of equilibrium constants of chemical reactions involving ionic species such as metal complexation reactions. Previous work with these methods has been based on the assumption that the limiting retention factors of the reactant and product species are constant independent of concentration of the chemical species (X) in the mobile phase, which reacts with the analyte compound. However, when all the reactant and product species are ions of the same charge sign as that of the species X, it is virtually impossible to apply these methods to the equilibrium constant determination because the retention factors of both the reactant and product species may depend on the concentration of X. In this study, an alternative approach was developed that estimates the limiting retention factors of ionic species from the dependence of the retention factor on the ionic strength of the mobile phase. Ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions were used as model reactions to test this method. The equilibrium constants determined by this method are in good agreement with those obtained by a UV-visible spectrophotometric method.

  20. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B.P. [Pacific Northwest National Lab., Richland, WA (United States)

    1997-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used in the cost-effective removal of specific radionuclides from nuclear waste streams. Organic ligands with metal ion specificity are critical components in the development of solvent extraction and ion exchange processes that are highly selective for targeted radionuclides. The traditional approach to the development of such ligands involves lengthy programs of organic synthesis and testing, which in the absence of reliable methods for screening compounds before synthesis, results in wasted research effort. The author`s approach breaks down and simplifies this costly process with the aid of computer-based molecular modeling techniques. Commercial software for organic molecular modeling is being configured to examine the interactions between organic ligands and metal ions, yielding an inexpensive, commercially or readily available computational tool that can be used to predict the structures and energies of ligand-metal complexes. Users will be able to correlate the large body of existing experimental data on structure, solution binding affinity, and metal ion selectivity to develop structural design criteria. These criteria will provide a basis for selecting ligands that can be implemented in separations technologies through collaboration with other DOE national laboratories and private industry. The initial focus will be to select ether-based ligands that can be applied to the recovery and concentration of the alkali and alkaline earth metal ions including cesium, strontium, and radium.

  1. Contribution of the R8 substituent to the in vitro antibacterial potency of besifloxacin and comparator ophthalmic fluoroquinolones

    Directory of Open Access Journals (Sweden)

    Haas W

    2013-05-01

    Full Text Available Wolfgang Haas, Christine M Sanfilippo, Christine K Hesje, Timothy W Morris Department of Microbiology and Sterilization Sciences, Bausch + Lomb, Inc, Rochester, NY, USA Introduction: Previous work has shown that besifloxacin, an 8-chloro-fluoroquinolone, has more potent activity against gram-positive pathogens than moxifloxacin and gatifloxacin, which carry an 8-methoxy group. This study was conducted to determine the contribution of the R7 and R8 substituent to fluoroquinolone antibacterial activity. Materials and methods: Besifloxacin, moxifloxacin, gatifloxacin, their R8 structural analogs, and ciprofloxacin were tested against representative isolates of various gram-positive and gram-negative species and previously characterized fluoroquinolone-resistant mutants of Staphylococcus aureus. Minimum inhibitory and minimum bactericidal concentrations were determined according to Clinical and Laboratory Standards Institute (CLSI guidelines. Reserpine was used to determine the effect of efflux pumps on antibacterial activity. Results: In general, exchanging the R8 residue in besifloxacin slightly reduced the molecule's potency, while introducing an 8-chloro group in moxifloxacin increased its potency. A similar change in gatifloxacin had little to no effect. Substituting the R8 residues did not increase the susceptibility to the efflux pump inhibitor reserpine or result in a loss of bactericidal activity. In contrast, the positive control, ciprofloxacin, was shown to be a substrate for reserpine and lost bactericidal activity against some fluoroquinolone-resistant isolates of S. aureus. Conclusion: The data presented here show that, depending on the R7 substituent, replacing an 8-methoxy group with an 8-chloro substituent can improve potency or can have little-to-no effect. These findings highlight the importance of the interplay between the R7 and R8 substituents in determining antibacterial potency. Keywords: moxifloxacin, besifloxacin

  2. Potency of Microalgae as Biodiesel Source in Indonesia

    Directory of Open Access Journals (Sweden)

    H Hadiyanto

    2012-02-01

    Full Text Available Within 20 years, Indonesia should find another energy alternative to substitute current fossil oil. Current use of renewable energy is only 5% and need to be improved up to 17% of our energy mix program. Even though, most of the area in Indonesia is covered by sea, however the utilization of microalgae as biofuel production is still limited. The biodiesel from current sources (Jatropha, palm oil, and sorghum is still not able to cover all the needs if the fossil oil cannot be explored anymore. In this paper, the potency of microalgae in Indonesia was analysed as the new potential of energy (biodiesel sources.

  3. Potency of Microalgae as Biodiesel Source in Indonesia

    Directory of Open Access Journals (Sweden)

    H Hadiyanto

    2012-04-01

    Full Text Available Within 20 years, Indonesia should find another energy alternative to substitutecurrent fossil oil. Current use of renewable energy is only 5% and need to be improved up to 17%of our energy mix program. Even though, most of the area in Indonesia is covered by sea, howeverthe utilization of microalgae as biofuel production is still limited. The biodiesel from currentsources (Jatropha, palm oil, and sorghum is still not able to cover all the needs if the fossil oilcannot be explored anymore. In this paper, the potency of microalgae in Indonesia was analysed asthe new potential of energy (biodiesel sources.

  4. The Potency of Krokot (Portulaca oleracea) as Functional Food Ingredients

    OpenAIRE

    Irawan, Daisy; Hariyadi, Purwiyatno; Wijaya, Hanny

    2014-01-01

    Indonesia has many auxiliary plants that may have nutritional and or environmental benefits, so that it may increase the yield of the main crops. Krokot (Portulaca oleracea), one of the auxiliary plants, was traditionally consumed in many parts of the world for its delicacy and medicinal benefits. Our research indicated that krokot has high potency to improve the health status of the community. It has 5.4 mg/100 g of b-carotene, 22.2 mg/100 g of vitamin C, and significant amount of folic acid...

  5. Cannabis-induced psychosis associated with high potency "wax dabs".

    Science.gov (United States)

    Pierre, Joseph M; Gandal, Michael; Son, Maya

    2016-04-01

    With mounting evidence that the risk of cannabis-induced psychosis may be related to both dose and potency of tetrahydrocannbinol (THC), increasing reports of psychosis associated with cannabinoids containing greater amounts of THC are anticipated. We report two cases of emergent psychosis after using a concentrated THC extract known as cannabis "wax," "oil," or "dabs" raising serious concerns about its psychotic liability. Although "dabbing" with cannabis wax is becoming increasingly popular in the US for both recreational and "medicinal" intentions, our cases raise serious concerns about its psychotic liability and highlight the importance of understanding this risk by physicians recommending cannabinoids for purported medicinal purposes. Published by Elsevier B.V.

  6. A step forward in the quality control testing of inactivated rabies vaccines - extensive evaluation of European vaccines by using alternative methods to the in vivo potency tests.

    Science.gov (United States)

    Servat, Alexandre; Kempff, Sébastien; Brogat, Valère; Litaize, Estelle; Schereffer, Jean-Luc; Cliquet, Florence

    2015-03-01

    The mouse challenge test still remains the reference method for the potency determination of human and animal inactivated rabies vaccines, and it is still widely used throughout the world. This test suffers from many disadvantages - it is expensive and time consuming, uses a large number of mice, causes significant animal distress, and suffers from high variability. Recently, the European Pharmacopoeia has recognised the use of a serological potency assay (SPA) as an alternative method to the challenge test. This new test is based on the determination of rabies neutralising antibody titres in vaccinated mice, by using the modified Rapid Fluorescent Focus Inhibition Test (mRFFIT). With the objective of adopting this new method for the batch release of inactivated rabies vaccines, we evaluated its performance on a large collection of rabies vaccines currently assessed in our laboratory. The Fluorescent Antibody Virus Neutralisation test (FAVNt) was used in parallel with the mRFFIT, and the results were compared to the mouse challenge test. Our results demonstrate that the SPA is capable of estimating the potency of vaccines formulated with a potency margin well above the minimum of 1IU/dose. For low potency vaccines, this new method demonstrated some limitations, due to the recurrent invalidation of the assay. We have also demonstrated the superior sensitivity of the FAVNt when compared to the mRFFIT, and the importance of minimising the risk of detecting non-responders in vaccinated mice. 2015 FRAME.

  7. Determination of Lead Ion by a Modified Carbon Paste Electrode Based on Multi-Walled Carbon Nanotubes (MWCNTs and Ligand (N-(4-Hydroxyphenyl Ethanamide

    Directory of Open Access Journals (Sweden)

    Marzieh Bagheri

    2015-06-01

    Full Text Available The preparation of a new modified carbon paste electrode (CPE to measure lead ion has been reported in this study. Lead is a highly toxic element which can have a negative impact on the environment. Therefore, measurement of lead in aquatic environments is very important. Although several methods have been developed for determination of lead ion in aquatic environments, there is no a cheap, simple, accurate and rapid method to measure this ion. Aim of this study is to develop a new method to measure the lead based on using multi walls carbon nanotubes (MWCNTs and Paracetamol as an ionophore for modification of a CPE. The optimum composition of modified CPE was determined as 64% of graphite powder, 20% of paraffin oil, 12% of nanotube and 4% of ionophore. This optimum composition was shown high selectivity, with appropriate Nernestian slope (-29.73 mV/decade, linear range (from 1.0×10-1to 1.0×10-8M, low lead concentration detection limit (7.5×10-9M and good response time (equal of 25 sec.The results of this study to introduce a cheap, accurate and simple method for determination of lead ion in aquatic environments.

  8. Determination of Lead Ion by a Modified Carbon Paste Electrode Based on Multi-Walled Carbon Nanotubes (MWCNTs andLigand (N-(4-Hydroxyphenyl Ethanamide

    Directory of Open Access Journals (Sweden)

    Marzieh Bagheri

    2015-06-01

    Full Text Available The preparation of a new modified carbon paste electrode (CPEto measure lead ion has been reported in this study. Lead is a highly toxic element which can have a negative impact on the environment. Therefore, measurement of lead in aquatic environments is very important. Although several methods have been developed for determination of lead ion in aquatic environments, there is no a cheap, simple, accurate and rapid method to measure this ion. Aim of this study is to develop a new method to measure the lead based on using multi walls carbon nanotubes (MWCNTs and Paracetamol as an ionophore for modificationof a CPE.The optimum composition of modified CPE was determined as 64% of graphite powder, 20% of paraffin oil, 12% of nanotube and 4% of ionophore.This optimum composition was shown high selectivity, with appropriate Nernestian slope (-29.73 mV/decade, linear range (from 1.0×10-1to 1.0×10-8M, low lead concentration detection limit (7.5×10-9M and good response time (equal of 25 sec.The results of this study to introduce a cheap, accurate and simple method for determination of lead ion in aquatic environments.

  9. Determination of Trace Titanium by Solid Substrate-Room Temperature Phosphorimetry with 4,5-Dibromophenylfluorone as Luminescent Ligand and Triton X-100 as Sensitizer

    Institute of Scientific and Technical Information of China (English)

    LIU Jia-ming; YANG Tian-long; YAN Hai-gang; SHEN Li-shuang; LI Long-di

    2003-01-01

    A new highly sensitive solid substrate room temperature phosphorimetry for the determination of trace titanium is proposed based on the sensitization of Triton X-100 to the SS-RTP intensity of 4,5-dibromophenylfluorone-titanium complex adsorbed on the filter paper substrate modified by gelatin. When Triton X-100 was added into the luminescence system, the RTP intensity was raised 3 times stronger than that of the system without Triton X-100. The linear dynamic range of the new method is 0.64~3.2 fg/spot(0.4 μL) with a detection limit of 12.8 ag/spot, and the regression equation of the working curve is ΔIp =482.0+119.5mTi(Ⅳ)(fg/spot), the correlation coefficient r=0.9992, n=6. The phosphorescence lifetime(τ=0.85 ms) was also determined. The recoveries(and RSD) for the determinations of titanium in human hair and tea samples were 101.0%(3.0%) and 99.97%(4.2%), respectively.

  10. Ligand modeling and design

    Energy Technology Data Exchange (ETDEWEB)

    Hay, B. [Pacific Northwest Lab., Richland, WA (United States)

    1996-10-01

    The purpose of this work is to develop and implement a molecular design basis for selecting organic ligands that would be used tin applications for the cost-effective removal of specific radionuclides from nuclear waste streams.

  11. Sahara honey shows higher potency against Pseudomonas aeruginosa compared to north Algerian types of honey.

    Science.gov (United States)

    Boukraa, Laid; Niar, Abdellatif

    2007-12-01

    Six varieties of honey from different regions in Algeria were used to determine their potency against Pseudomonas aeruginosa. Four varieties originated from northern Algeria, and two from the Sahara. Three types of media were used. On nutrient agar the minimum inhibitory concentration (MIC) of the four northern varieties ranged between 30% (vol/vol) and 31% (vol/vol), while the MIC of the Sahara varieties was 11% (vol/vol) and 14% (vol/vol). On King A agar the MIC of the four northern varieties ranged from 25% (vol/vol) to 31% (vol/vol), whereas the MIC of the two varieties of Sahara honey was 12% (vol/vol) and 15% (vol/vol). On nutrient broth the MIC of the northern varieties ranged from 10% (vol/vol) to 21% (vol/vol), whereas the MIC of the two varieties of Sahara honey was 9% (vol/vol). The botanic flora of Sahara is known in Algeria for its medicinal uses, and thus the higher potency of the Sahara honey is most probably due to antibacterial substances in its plant derivates. These findings suggest that Sahara honey could be used for managing wounds and burns, which are mostly infected by P. aeruginosa.

  12. The effectivenes of science domain-based science learning integrated with local potency

    Science.gov (United States)

    Kurniawati, Arifah Putri; Prasetyo, Zuhdan Kun; Wilujeng, Insih; Suryadarma, I. Gusti Putu

    2017-08-01

    This research aimed to determine the significant effect of science domain-based science learning integrated with local potency toward science process skills. The research method used was a quasi-experimental design with nonequivalent control group design. The population of this research was all students of class VII SMP Negeri 1 Muntilan. The sample of this research was selected through cluster random sampling, namely class VII B as an experiment class (24 students) and class VII C as a control class (24 students). This research used a test instrument that was adapted from Agus Dwianto's research. The aspect of science process skills in this research was observation, classification, interpretation and communication. The analysis of data used the one factor anova at 0,05 significance level and normalized gain score. The significance level result of science process skills with one factor anova is 0,000. It shows that the significance level science domain-based science learning integrated with local potency toward science learning process skills. The results of analysis show that the normalized gain score are 0,29 (low category) in control class and 0,67 (medium category) in experiment class.

  13. Assessment of the sensitizing potency of preservatives with chance of skin contact by the loose-fit coculture-based sensitization assay (LCSA).

    Science.gov (United States)

    Sonnenburg, Anna; Schreiner, Maximilian; Stahlmann, Ralf

    2015-12-01

    Parabens, methylisothiazolinone (MI) and its derivative methylchloroisothiazolinone (MCI), are commonly used as preservatives in personal care products. They can cause hypersensitivity reactions of the human skin. We have tested a set of nine parabens, MI alone and in combination with MCI in the loose-fit coculture-based sensitization assay (LCSA). The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) in this assay emulates the in vivo situation of the human skin. Sensitization potency of the test substances was assessed by flow cytometric analysis of the DC-rc maturation marker CD86. Determination of the concentration required to cause a half-maximal increase in CD86-expression (EC50sens) allowed a quantitative evaluation. The cytotoxicity of test substances as indicator for irritative potency was measured by 7-AAD (7-amino-actinomycin D) staining. Parabens exhibited weak (methyl-, ethyl-, propyl- and isopropylparaben) or strong (butyl-, isobutyl-, pentyl- and benzylparaben) effects, whereas phenylparaben was found to be a moderate sensitizer. Sensitization potencies of parabens correlated with side chain length. Due to a pronounced cytotoxicity, we could not estimate an EC50sens value for MI, whereas MI/MCI was classified as sensitizer and also showed cytotoxic effects. Parabens showed no (methyl- and ethylparaben) or weak irritative potencies (propyl-, isopropyl-, butyl-, isobutyl-, phenyl- and benzylparaben), only pentylparaben was rated to be irritative. Overall, we were able to demonstrate and compare the sensitizing potencies of parabens in this in vitro test. Furthermore, we showed an irritative potency for most of the preservatives. The data further support the usefulness of the LCSA for comparison of the sensitizing potencies of xenobiotics.

  14. Automated design of ligands to polypharmacological profiles

    Science.gov (United States)

    Besnard, Jérémy; Ruda, Gian Filippo; Setola, Vincent; Abecassis, Keren; Rodriguiz, Ramona M.; Huang, Xi-Ping; Norval, Suzanne; Sassano, Maria F.; Shin, Antony I.; Webster, Lauren A.; Simeons, Frederick R.C.; Stojanovski, Laste; Prat, Annik; Seidah, Nabil G.; Constam, Daniel B.; Bickerton, G. Richard; Read, Kevin D.; Wetsel, William C.; Gilbert, Ian H.; Roth, Bryan L.; Hopkins, Andrew L.

    2012-01-01

    The clinical efficacy and safety of a drug is determined by its activity profile across multiple proteins in the proteome. However, designing drugs with a specific multi-target profile is both complex and difficult. Therefore methods to rationally design drugs a priori against profiles of multiple proteins would have immense value in drug discovery. We describe a new approach for the automated design of ligands against profiles of multiple drug targets. The method is demonstrated by the evolution of an approved acetylcholinesterase inhibitor drug into brain penetrable ligands with either specific polypharmacology or exquisite selectivity profiles for G-protein coupled receptors. Overall, 800 ligand-target predictions of prospectively designed ligands were tested experimentally, of which 75% were confirmed correct. We also demonstrate target engagement in vivo. The approach can be a useful source of drug leads where multi-target profiles are required to achieve either selectivity over other drug targets or a desired polypharmacology. PMID:23235874

  15. Ligand-free gold nanoparticles as colorimetric probes for the non-destructive determination of total dithiocarbamate pesticides after solid phase extraction.

    Science.gov (United States)

    Giannoulis, Kyriakos M; Giokas, Dimosthenis L; Tsogas, George Z; Vlessidis, Athanasios G

    2014-02-01

    In this work, we describe a simple and sensitive non-destructive method for the determination of the total concentration of dithiocarbamate fungicides (DTCs) in real samples. The proposed method combines for the first time the benefits of an extraction method for sample clean-up and preconcentration with a sensitive colorimetric assay based on gold nanoparticle probes. In this two-step procedure, the target DTCs are isolated from the matrix and preconcentrated by solid phase extraction onto commercially available C18 sorbents. Following elution, the extract containing the target dithiocarbamates, free from most interferences and matrix components, is delivered into an aqueous dispersion of plain citrate-capped gold nanoparticles (AuNPs) which aggregate in response to DTCs coordination on AuNPs surface through multiple gold thiolate bonds. This aggregation is evidenced by changes in the spectral properties of the solution involving a decrease in the original absorbance of Au nanoparticles at 522 nm and the appearance of a new absorption band above 700 nm. An ensuing chromatic shift of the solution from wine-red to purple-blue is observed which is visual by naked eye at concentrations as low as 50 μg L(-1). Further improvement in the detection limits can be accomplished by scaling-down the method to micro-volume conditions alleviating the need to preconcentrate larger sample volumes. Overall, by combining sample clean-up and preconcentration with the strong affinity of DTC thiol group for the gold surface, the total concentration of dithiocarbamate pesticides was successfully determined in various water samples at the low and ultra-low μg L(-1) levels without resorting to destructive techniques, sophisticated instrumentation or post-synthetic modification of gold nanoparticles. Method application in real samples showed good analytical features in terms of recoveries (81.0-94.0%), precision (5.6-8.9%) and reproducibility (~9%) rendering the method as an attractive

  16. PROBIOTIC POTENCY OF LACTOBACILLUS SPP. ISOLATED FROM SUMBAWA MARE MILK

    Directory of Open Access Journals (Sweden)

    I Nengah Sujaya

    2008-03-01

    Full Text Available This research was deigned to elucidate the potency of Lactobacillus spp. isolated from sumbawa mare milk to be developed as a probiotic. Sixteen lacobacilli were screened based on their resitancy to a model of gastric juice at pH 2, 3, and 4, then followed by their resistncy to small intestional fluid model containing deoxycholic. Three lactobacilli i.e. Lactobacillus sp. SKA13, Lactobacillus rhamnosus SKG34 and Lactobacillus rhamnosus SKG49 were found to be resistentent to gastric juice at pH 3 and 4. However, there were no lactobacilli resisted to pH 2. Lactobacillus rhamnosus SKG34 and Lactobacillus rhamnosus SKG49 were able to reach the colon even after being expossed to a model of intestinal fluid containing 0,4 mM deoxycholate and pancreatine. Therefore, these isolates have a potency to be developed as probiotic lactobacilli. Nevertherless, these lactobcailli could probably transform cholic acid into secondary bile acids, which were not expected to be found in the probiotic, and this capability is not appropriate for probiotic. This character is worthly to be studied since it has never been reported in lactobacilli.

  17. Alanine Mutagenesis in the Complementarity Determining Region 3 of the MTB and HIV-1 Peptide-Bispecific T Cell Receptor Beta Chain Affects Ligand Recognition

    Directory of Open Access Journals (Sweden)

    Chao-Ying Zhou

    2017-08-01

    Full Text Available Mycobacterium tuberculosis/human immunodeficiency virus (MTB/HIV coinfection presents a special challenge to the prevention and treatment of tuberculosis and HIV/AIDS. Adoptive transfer of high-affinity T cell receptor (TCR gene-modified T cells against MTB and HIV antigens is a promising approach to treating MTB/HIV coinfected patients whose cellular immunity is obviously disordered. We have previously successfully identified that a bispecific TCR screened out from peripheral blood mononuclear cells of a HLA-A*0201+ healthy individual using the complementarity determining region 3 (CDR3 spectratype analysis recognizes both MTB Ag85B199–207 and HIV-1 Env120–128 peptide. However, it has not been known how residues on CDR3 loops, which have been shown to play a leading role in antigen binding and specificity contribute to the bispecific TCR contact with the peptide–major histocompatibility complex (MHC complexes. In this study, we provided an extensive investigation of residues in the predicted CDR3 of the bispecific TCR beta (β chain using alanine scanning mutagenesis. Our data showed that three of the five substituted residues (G115A, T116A, A117G in CDR3β of the bispecific TCR caused a significantly diminished T cell response to antigen, whereas the remaining two substituted residues (D114A, S118A resulted in completely eliminated response, thus identifying the two residues that were particularly critical for the recognition of peptide–MHC in the bispecific TCR. These findings will provide an imperative foundation for generating an improved high-affinity bispecific TCR for use in T cell adoptive immunotherapy for MTB/HIV coinfected individuals.

  18. Cloud point extraction for determination of lead in blood samples of children, using different ligands prior to analysis by flame atomic absorption spectrometry: A multivariate study

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Faheem, E-mail: shah_ceac@yahoo.com [National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan); Kazi, Tasneem Gul, E-mail: tgkazi@yahoo.com [National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan); Afridi, Hassan Imran, E-mail: hassanimranafridi@yahoo.com [National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan); Naeemullah, E-mail: khannaeemullah@ymail.com [National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan); Arain, Muhammad Balal, E-mail: bilal_ku2004@yahoo.com [Department of Chemistry, University of Science and Technology, Bannu, KPK (Pakistan); Baig, Jameel Ahmed, E-mail: jab_mughal@yahoo.com [National Center of Excellence in Analytical Chemistry, University of Sindh, Jamshoro 76080 (Pakistan)

    2011-09-15

    Highlights: {yields} Trace levels of lead in blood samples of healthy children and with different kidney disorders {yields} Pre-concentration of Pb{sup +2} in acid digested blood samples after chelating with two complexing reagents. {yields} Multivariate technique was used for screening of significant factors that influence the CPE of Pb{sup +2} {yields} The level of Pb{sup +2} in diseased children was significantly higher than referents of same age group. - Abstract: The phase-separation phenomenon of non-ionic surfactants occurring in aqueous solution was used for the extraction of lead (Pb{sup 2+}) from digested blood samples after simultaneous complexation with ammonium pyrrolidinedithiocarbamate (APDC) and diethyldithiocarbamate (DDTC) separately. The complexed analyte was quantitatively extracted with octylphenoxypolyethoxyethanol (Triton X-114). The multivariate strategy was applied to estimate the optimum values of experimental factors. Acidic ethanol was added to the surfactant-rich phase prior to its analysis by flame atomic absorption spectrometer (FAAS). The detection limit value of Pb{sup 2+} for the preconcentration of 10 mL of acid digested blood sample was 1.14 {mu}g L{sup -1}. The accuracy of the proposed methods was assessed by analyzing certified reference material (whole blood). Under the optimized conditions of both CPE methods, 10 mL of Pb{sup 2+} standards (10 {mu}g L{sup -1}) complexed with APDC and DDTC, permitted the enhancement factors of 56 and 42, respectively. The proposed method was used for determination of Pb{sup 2+} in blood samples of children with kidney disorders and healthy controls.

  19. POTENSI NETRALISASI IMUNOGLOBULIN Y ANTITETANUS YANG DIISOLASI DARI TELUR AYAM (THE POTENCY NETRALIZATION OF ANTI TETANUS IMMUNOGLOBULIN Y THAT WERE ISOLATED FROM CHICKEN EGGS

    Directory of Open Access Journals (Sweden)

    I Nyoman Suartha

    2007-06-01

    Full Text Available The porpuse of study was to explore the potential use of? anti tetanus IgY from eggs yolk as a substitute for anti tetanus serum raised in ?horses. The eggs were collected from chickens which have previously been immunized with tetanus toxoid. Neutralization potency test of anti tetanus IgY determined by ?Spearman-Karber method.? The highest mean titer of anti tetanus of egg yolk was 80.16 ? 33.55 IU/ml and the lowest was 1.69 ? 0.63 IU/ml. The concentration? of purified IgY was 1.644 ? 0.424 mg/ml. Spearman-Karber value of potency of anti tetanus IgY are 35 IU/ml. ?This research concluded that Chickens was capable of produced of anti tetanus in eggs yolk with value of potency are 35 IU/ml.

  20. LM potencies: one of the hidden treasures of the sixth edition of the Organon.

    Science.gov (United States)

    De Schepper, L

    1999-07-01

    50 millesimal (LM) potencies have great advantages for all patients, not just those who are hypersensitive because of their great power to heal without major aggravations. Before discussing their advantages this paper describes what LM potencies are, and how they are administered, then addresses two questions: why do we want to avoid aggravations if most homeopaths look for aggravation to know if the remedy is working? And if LM potencies are indeed superior, why are they still relatively unknown and unused?

  1. Increasing the potency of a cytotoxin with an arginine graft.

    Science.gov (United States)

    Fuchs, Stephen M; Rutkoski, Thomas J; Kung, Vanessa M; Groeschl, Ryan T; Raines, Ronald T

    2007-10-01

    Variants and homologs of bovine pancreatic ribonuclease (RNase A) can exhibit cytotoxic activity. This toxicity relies on cellular internalization of the enzyme. Residues Glu49 and Asp53 form an anionic patch on the surface of RNase A. We find that replacing these two residues with arginine does not affect catalytic activity or affinity for the cytosolic ribonuclease inhibitor (RI) protein. This 'arginine graft' does, however, increase toxicity towards human cancer cells. Appending a nonaarginine domain to this cationic variant results in an additional increase in cytotoxicity, providing one of the most cytotoxic known variants of RNase A. These findings correlate the potency of a ribonuclease with its deliverance of ribonucleolytic activity to the cytosol, and indicate a rational means to enhance the efficacy of ribonucleases and other cytotoxic proteins.

  2. Development of a reliable analytical method for extraction spectrophotometric determination of ruthenium(III) from catalyst and fissium alloy using o-methylphenyl thiourea as a chromogenic chelating ligand

    Science.gov (United States)

    Kuchekar, Shashikant R.; Shelar, Yogesh S.; Aher, Haribhau R.; Han, Sung H.

    2013-04-01

    A simple and selective method is developed for the extraction spectrophotometric determination of ruthenium(III) using o-methylphenyl thiourea (OMPT) as a chromogenic chelating ligand. The basis of the proposed method is ruthenium(III)-OMPT complex formation in aqueous hydrochloric acid media (3.0 mol L-1) after 5.0 min heating on a boiling water bath and the complex formed is extracted into chloroform. The absorbance of green colored ruthenium(III)-OMPT complex is measured at 590 nm against the reagent blank. Beer's law was obeyed up to 42.5 μg mL-1 of ruthenium(III) and the optimum concentration range is 7.56-39.81 μg mL-1 of ruthenium(III) as evaluated by Ringbom's plot. Molar absorptivity and Sandell's sensitivity of ruthenium(III)-OMPT complex in chloroform are 2.34 × 103 L mol-1 cm-1 and 0.043 μg cm-2 respectively. The composition of ruthenium(III):OMPT complex (1:2) was established from slope ratio method, mole ratio method and Job's continuous variation method. Complex was stable for more than 48 h. The interfering effect of various foreign ions was studied and suitable masking agents are used wherever necessary to enhance the selectivity of the method. Proposed method is successfully applied for determination of ruthenium(III) from binary and ternary synthetic mixtures, synthetic mixtures corresponding to fissium alloy and ruthenium catalyst. Repetition of the method was checked by finding relative standard deviation (R.S.D) for 10 determinations which was 0.23%. A scheme for sequential separation of palladium(II), ruthenium(III), rhodium(III) and platinum(IV) has been developed.

  3. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines-part 1.

    Science.gov (United States)

    Winsnes, R; Sesardic, D; Daas, A; Behr-Gross, M-E

    2004-01-01

    A collaborative study on the evaluation of an alternative functional assay, the Vero cell method, to the Ph. Eur. in vivo challenge procedures for potency determination of diphtheria toxoid in 6 different combined vaccines was initiated in January 2001. The study was an extension of a previous study for the validation of serological methods for potency testing of tetanus toxoid vaccines for human use. To allow interim evaluation of test results and to monitor study progress, the project was divided into three consecutive phases. The results of Phase I and II studies are presented in this report. Pre-validation (Phase I) study, performed in two laboratories, indicated that comparable diphtheria potency estimates were obtained in the Ph. Eur. direct intradermal challenge assay in guinea pigs, in Vero cell assay and in indirect ELISA for five vaccines of different potencies (range of estimates: ca. 20-200 IU/ml). The correlation coefficients between the challenge assay and the Vero cell assay corresponded to those between the challenge assay and ELISA, confirming that the antibodies play an important role in protection and that predominantly protective/neutralising antibodies are present in guinea pigs, at the time point investigated. It was observed, for Vero cell assays, that about 16-35 (9-28 in Phase II study) fold lower titre of individual serum samples were obtained when using equine, rather than guinea pig reference serum. The study also provided preliminary information that sera from the same guinea pigs may be used for potency determination of both diphtheria and tetanus toxoid components of vaccines. In Phase II, another five laboratories analysed a subset of the vaccines included in Phase I study plus an additional vaccine. Four laboratories performed the lethal challenge assay and one laboratory carried out the intradermal challenge assay. All laboratories also performed the Vero cell assay and both ELISA for diphtheria antitoxin and ELISA for tetanus

  4. Tubulin acetylation promoting potency and absorption efficacy of deacetylase inhibitors

    Science.gov (United States)

    Mangas-Sanjuan, V; Oláh, J; Gonzalez-Alvarez, I; Lehotzky, A; Tőkési, N; Bermejo, M; Ovádi, J

    2015-01-01

    Background and Purpose Histone deacetylase 6 (HDAC6) and silent information regulator 2 (SIRT2) control the dynamics of the microtubule network via their deacetylase activities. Tubulin polymerization promoting protein (TPPP/p25) enhances microtubule acetylation by its direct binding to HDAC6. Our objective was to characterize the multiple interactions of the deacetylases and to establish the inhibitory potency and the pharmacokinetic features of the deacetylase inhibitors, trichostatin A (TSA) and AGK2. Experimental Approach The interactions of deacetylases with tubulin and TPPP/p25 were quantified by elisa using human recombinant proteins. The effect of inhibitors on the tubulin acetylation was established in HeLa cells transfected with pTPPP and CG-4 cells expressing TPPP/p25 endogenously by celisa (elisa on cells), Western blot and immunofluorescence microscopy. The pharmacokinetic features of the inhibitors were evaluated by in situ kinetic modelling of their intestinal transport in rats. Key Results Deacetylases interact with both tubulin and TPPP/p25, notwithstanding piggy-back binding of HDAC6 or SIRT2 to the TPPP/p25-associated tubulin was established. Much higher inhibitory potency for TSA than for AGK2 was detected in both HeLa and CG-4 cells. Pioneer pharmacokinetic studies revealed passive diffusion and diffusion coupled with secretion for TSA and AGK2 respectively. Both inhibitors exhibited greater permeability than some other well-established drugs. Conclusions and Implications TPPP/p25-directed deacetylase inhibition provides mechanisms for the fine control of the dynamics and stability of the microtubule network. Deacetylase inhibitors with chemical structures similar to TSA and AGK2 appear to be excellent candidates for oral drug absorption. PMID:25257800

  5. Potency of Mahakam Delta in East Kalimantan, Indonesia

    Directory of Open Access Journals (Sweden)

    Zairin Zain

    2014-04-01

    Full Text Available Normal 0 false false false IN X-NONE X-NONE Mahakam Delta is not only utilized for fishery activities but also for several industrial purposes such as coal and oil mining, shipping and timber. The objective of this paper is to provide information in term of the potency of Mahakam Delta located in East Kalimantan province, Indonesia. The study was conducted in Mahakam Delta, East Kalimantan Province, Indonesia. Participatory rural appraisal is a group of methods to collect information in a participatory fashion from rural communities. The method employed in this research was descriptive analysis describing the environmental potency found in Mahakam Delta. Massive conversion of mangrove forest particularly Nypa (Nypa fruticans area into pond has driven conflict among related stakeholders on the utilization of natural resource and land which subsequently impacts on the fish and shrimp source regeneration, loss of ecological functions of mangrove forest for feeding, nursery and spawning ground of fish and other organisms. Besides of fisheries, oil and gas activity also exists in Mahakam Delta. Mahakam Delta is regarded as important area due to the largest producer of oil and gas mining. Thus, oil and gas industry is the most reliable sector that contributes to the economic development of Kutai Kartanegara district. As a resource provider, Mahakam Delta ecosystem provides various resources for livelihoods. As a life-support service provider, Mahakam Delta ecosystem provides habitat and ideal environment to support variety of living kinds. As a convenience provider, Mahakam Delta ecosystem provides unique and interesting recreation site. As a protector from natural disaster, Mahakam Delta ecosystem is able to protect human kinds from natural disaster threatening coastal area.

  6. Naturally-Occurring Glucosinolates, Glucoraphanin and Glucoerucin, are Antagonists to Aryl Hydrocarbon Receptor as Their Chemopreventive Potency.

    Science.gov (United States)

    Abdull Razis, Ahmad Faizal; Noor, Noramaliza Mohd

    2015-01-01

    As a cytosolic transcription factor, the aryl hydrocarbon (Ah) receptor is involved in several patho- physiological events leading to immunosuppression and cancer; hence antagonists of the Ah receptor may possess chemoprevention properties. It is known to modulate carcinogen-metabolising enzymes, for instance the CYP1 family of cytochromes P450 and quinone reductase, both important in the biotransformation of many chemical carcinogens via regulating phase I and phase II enzyme systems. Utilising chemically-activated luciferase expression (CALUX) assay it was revealed that intact glucosinolates, glucoraphanin and glucoerucin, isolated from Brassica oleracea L. var. acephala sabellica and Eruca sativa ripe seeds, respectively, are such antagonists. Both glucosinolates were poor ligands for the Ah receptor; however, they effectively antagonised activation of the receptor by the avid ligand benzo[a]pyrene. Indeed, intact glucosinolate glucoraphanin was a more potent antagonist to the receptor than glucoerucin. It can be concluded that both glucosinolates effectively act as antagonists for the Ah receptor, and this may contribute to their established chemoprevention potency.

  7. Honing a harder-hitting hammerhead improves broadly neutralizing antibody breadth and potency.

    Science.gov (United States)

    Lewis, George K

    2015-06-01

    While current HIV-1 therapies have greatly improved the quality and duration of life for infected individuals, a vaccine to prevent transmission of the virus is lacking. Broadly neutralizing monoclonal antibodies (bnmAbs) with the capacity to neutralize multiple HIV-1 variants have been isolated from HIV-1-infected individuals, and there has been a great effort to investigate how these bnmAbs arise, due their potential for HIV-1 vaccination. In this issue of the JCI, Willis and colleagues apply a computational approach to design variants of the bnmAb PG9 in an attempt to enhance potency and neutralization breadth. One of these variants was able to target multiple PG9-resistant strains, as the result of stabilization of the long heavy chain complementarity determining region 3 (HCDR3). The results of this study provide important insight and a unique approach to optimizing HIV-1 bnmABs.

  8. Establishment of the relative antiinflammatory potency of deflazacort and prednisone in polymyalgia rheumatica

    DEFF Research Database (Denmark)

    Lund, B; Egsmose, C; Jørgensen, S;

    1987-01-01

    Conventional glucocorticoids exert a negative influence on calcium balance, and long-term treatment with these agents leads to osteopenia. Deflazacort is an oxazoline derivative of prednisolone with documented calcium-sparing properties when compared to prednisone on a weight basis. The purpose...... of the present study was to determine the relative antiinflammatory potency of deflazacort and prednisone. In a randomized, cross-over, double-blind trial, 11 patients, all suffering from polymyalgia rheumatica, and all on a stable maintenance dose of prednisone, were treated with equimolar doses of prednisone...... following prednisone treatment. Subsequently, in a similar regimen, prednisone was compared with deflazacort at a weight ratio of 1:1.2 in 10 patients, 1:1.5 in another 10 patients, and 1:1.8 in still another 10 patients for purposes of dose titration. Again, significant rises were seen in ESR, plasma...

  9. Discovery and computer aided potency optimization of a novel class of small molecule CXCR4 antagonists.

    Directory of Open Access Journals (Sweden)

    Victoria Vinader

    Full Text Available Amongst the chemokine signalling axes involved in cancer, chemokine CXCL12 acting on chemokine receptor CXCR4 is particularly significant since it orchestrates migration of cancer cells in a tissue-specific metastatic process. High CXCR4 tumour expression is associated with poor prognosis of lung, brain, CNS, blood and breast cancers. We have identified a new class of small molecule CXCR4 antagonists based on the use of computational modelling studies in concert with experimental determination of in vitro activity against CXCL12-induced intracellular calcium mobilisation, proliferation and chemotaxis. Molecular modelling proved to be a useful tool in rationalising our observed potencies, as well as informing the direction of the synthetic efforts aimed at producing more potent compounds.

  10. Antimicrobial Peptide Potency is Facilitated by Greater Conformational Flexibility when Binding to Gram-negative Bacterial Inner Membranes

    Science.gov (United States)

    Amos, Sarah-Beth T. A.; Vermeer, Louic S.; Ferguson, Philip M.; Kozlowska, Justyna; Davy, Matthew; Bui, Tam T.; Drake, Alex F.; Lorenz, Christian D.; Mason, A. James

    2016-11-01

    The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic α-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100 ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently α-helical cationic AMPs for enhanced antibacterial potency.

  11. A low-density DNA microchip for the detection of (anti-)estrogenic compounds and their relative potencies.

    Science.gov (United States)

    Wang, Si; Rijk, Jeroen C W; Pen, Marieke J; Aarts, Jac M M J G; Peijnenburg, Ad A C M; Rietjens, Ivonne M C M; Bovee, Toine F H

    2013-04-01

    In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17β-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography-tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen).

  12. Validation and application of a rapid in vitro assay for assessing the estrogenic potency of halogenated phenolic chemicals.

    Science.gov (United States)

    Körner, W; Hanf, V; Schuller, W; Bartsch, H; Zwirner, M; Hagenmaier, H

    1998-01-01

    The E-Screen assay serves as an in vitro tool for the detection of estrogenic activity of chemicals and extracts of environmental samples. Based on the induction of proliferation in human estrogen receptor-positive MCF-7 breast cancer cells we could substantially simplify the assay. As one important step of validation we applied the modified assay for testing nine known xenoestrogens. We could confirm the results of other groups assuring the reproducibility of the E-Screen assay. The results provide evidence that the E-Screen assay is suitable for determination of estradiol equivalency factors (EEFs) for environmental estrogens to rank their estrogenic potency relative to the natural estrogen 17 beta-estradiol. Further, we used the optimized proliferation test to screen nine halogenated phenolic compounds for their possible estrogenic potency. Three widely applied chemicals expressed a weak receptor-mediated estrogenic activity: the flame retardant Tetrabromo-Bisphenol-A, the disinfectant 4-chloro-3-methylphenol, and the herbicide educt 4-chloro-2-methylphenol. Their estrogenic potencies were five to six orders of magnitude lower than that of 17 beta-estradiol.

  13. Determination of formal redox potentials in aqueous solution of copper(II) complexes with ligands having nitrogen and oxygen donor atoms and comparison with their EPR and UV-Vis spectral features.

    Science.gov (United States)

    Tabbì, Giovanni; Giuffrida, Alessandro; Bonomo, Raffaele P

    2013-11-01

    Formal redox potentials in aqueous solution were determined for copper(II) complexes with ligands having oxygen and nitrogen as donor atoms. All the chosen copper(II) complexes have well-known stereochemistries (pseudo-octahedral, square planar, square-based pyramidal, trigonal bipyramidal or tetrahedral) as witnessed by their reported spectroscopic, EPR and UV-visible (UV-Vis) features, so that a rough correlation between the measured redox potential and the typical geometrical arrangement of the copper(II) complex could be established. Negative values have been obtained for copper(II) complexes in tetragonally elongated pseudo-octahedral geometries, when measured against Ag/AgCl reference electrode. Copper(II) complexes in tetrahedral environments (or flattened tetrahedral geometries) show positive redox potential values. There is a region, always in the field of negative redox potentials which groups the copper(II) complexes exhibiting square-based pyramidal arrangements. Therefore, it is suggested that a measurement of the formal redox potential could be of great help, when some ambiguities might appear in the interpretation of spectroscopic (EPR and UV-Vis) data. Unfortunately, when the comparison is made between copper(II) complexes in square-based pyramidal geometries and those in square planar environments (or a pseudo-octahedral) a little perturbed by an equatorial tetrahedral distortion, their redox potentials could fall in the same intermediate region. In this case spectroscopic data have to be handled with great care in order to have an answer about a copper complex geometrical characteristics.

  14. Discovery of pyrrolidine-based β-secretase inhibitors: lead advancement through conformational design for maintenance of ligand binding efficiency.

    Science.gov (United States)

    Stachel, Shawn J; Steele, Thomas G; Petrocchi, Alessia; Haugabook, Sharie J; McGaughey, Georgia; Katharine Holloway, M; Allison, Timothy; Munshi, Sanjeev; Zuck, Paul; Colussi, Dennis; Tugasheva, Katherine; Wolfe, Abigail; Graham, Samuel L; Vacca, Joseph P

    2012-01-01

    We have developed a novel series of pyrrolidine derived BACE-1 inhibitors. The potency of the weak initial lead structure was enhanced using library-based SAR methods. The series was then further advanced by rational design while maintaining a minimal ligand binding efficiency threshold. Ultimately, the co-crystal structure was obtained revealing that these inhibitors interacted with the enzyme in a unique fashion. In all, the potency of the series was enhanced by 4 orders of magnitude from the HTS lead with concomitant increases in physical properties needed for series advancement. The progression of these developments in a systematic fashion is described.

  15. Interferon-α Subtypes in an Ex Vivo Model of Acute HIV-1 Infection: Expression, Potency and Effector Mechanisms.

    Directory of Open Access Journals (Sweden)

    Michael S Harper

    Full Text Available HIV-1 is transmitted primarily across mucosal surfaces and rapidly spreads within the intestinal mucosa during acute infection. The type I interferons (IFNs likely serve as a first line of defense, but the relative expression and antiviral properties of the 12 IFNα subtypes against HIV-1 infection of mucosal tissues remain unknown. Here, we evaluated the expression of all IFNα subtypes in HIV-1-exposed plasmacytoid dendritic cells by next-generation sequencing. We then determined the relative antiviral potency of each IFNα subtype ex vivo using the human intestinal Lamina Propria Aggregate Culture model. IFNα subtype transcripts from the centromeric half of the IFNA gene complex were highly expressed in pDCs following HIV-1 exposure. There was an inverse relationship between IFNA subtype expression and potency. IFNα8, IFNα6 and IFNα14 were the most potent in restricting HIV-1 infection. IFNα2, the clinically-approved subtype, and IFNα1 were both highly expressed but exhibited relatively weak antiviral activity. The relative potencies correlated with binding affinity to the type I IFN receptor and the induction levels of HIV-1 restriction factors Mx2 and Tetherin/BST-2 but not APOBEC3G, F and D. However, despite the lack of APOBEC3 transcriptional induction, the higher relative potency of IFNα8 and IFNα14 correlated with stronger inhibition of virion infectivity, which is linked to deaminase-independent APOBEC3 restriction activity. By contrast, both potent (IFNα8 and weak (IFNα1 subtypes significantly induced HIV-1 GG-to-AG hypermutation. The results unravel non-redundant functions of the IFNα subtypes against HIV-1 infection, with strong implications for HIV-1 mucosal immunity, viral evolution and IFNα-based functional cure strategies.

  16. Several synthetic progestins with different potencies adversely affect reproduction of fish.

    Science.gov (United States)

    Runnalls, Tamsin J; Beresford, Nicola; Losty, Erin; Scott, Alexander P; Sumpter, John P

    2013-02-19

    Synthetic progestins are widely used as a component in both contraceptives and in hormone replacement therapy (HRT), both on their own and in combination with EE2. Their presence in the environment is now established in wastewater effluent and river water and this has led to concerns regarding their potential effects on aquatic organisms living in these waters. We carried out in vivo experiments to determine the potencies of four different synthetic progestins on the reproductive capabilities of the fathead minnow (Pimephales promelas). We then performed a series of in vitro assays to try and determine the reason for the effects seen in the in vivo experiments. In the first experiment, fathead minnow exposed to a single concentration of 100 ng/L of either Levonorgestrel or Gestodene stopped spawning almost completely. The same nominal concentration of Desogestrel and Drospirenone did not affect reproduction (21 d NOECs of 100 ng/L). The second experiment investigated two progestins of different potency: Gestodene at 1, 10, and 100 ng/L and Desogestrel at 100 ng/L, 1 μg/L, and 10 μg/L. Gestodene concentrations as low as 1 ng/L had significant effects on reproduction over 21 d, whereas concentrations of Desogestrel at or above 1 μg/L were required to significantly reduce egg production. The synthetic progestins also masculinized the female fish in a concentration-dependent manner. Results from yeast-based in vitro assays demonstrated that the progestins are all strongly androgenic, thereby explaining the masculinization effects. The results strongly suggest that synthetic progestins merit serious consideration as environmental pollutants.

  17. Bioactive potency of epidermal mucus extracts from greasy grouper,Epinephelus tauvina (Forsskal, 1775)

    Institute of Scientific and Technical Information of China (English)

    Ganesh Manikantan; Somasundarannair Lyla; Syed Ajmal Khan; Packiaraj Vijayanand; George Edward Gnana Jothi

    2016-01-01

    Objective: To study the bio-potency of epidermal mucus fromEpinephelus tauvina. Methods: Mucus was extracted with acidic, organic and aqueous solvents. Protein, carbohydrate, lipid, amino acid and fatty acid content of mucus extracts were quantified by UV-spectrophotometer, high performance liquid chromatography and gas chromatography-mass spectrometer, respectively. Antimicrobial activity was tested against five human and fish pathogens by using agar well diffusion method. The molecular weight of peptides was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The haemolytic activity of extracts was tested against chick, goat, cow and human red blood cell. Results:Protein contributed with maximum of 26.25% in crude mucus. Arginine was recorded maximum of (133.9 nmol/mL) in crude mucus. 2,4,6-Decatrienoic acid and bis (a-chloroethyl) sulfone were confirmed in organic extract. The antimicrobial activity of acidic extract was significant. Among the human pathogens, maximum zone of inhibition [(26.0 ± 0.3) mm] was observed againstProteus mirabilis. Whereas, among fish pathogens maximum zone of inhibition [(25.0 ± 0.1) mm] was observed againstVibrio parahemolyticus. The activity of other two extracts was not remarkable. The molecular weight of peptides ranged from 115.5–37.1 kDa in acidic extract was determined. Chicken and goat blood were found to be highly vulnerable to the lysis. Conclusions: The whole mucus could be a promising source with numerous bioactive- potency. Consequently, this preliminary information suggested that mucus is a source of novel antimicrobial agents for fish and human health related applications.

  18. POTENCY OF HONEY AS ANTIBACTERIAL AGENT AGAINST MULTIPLE ANTIBIOTIC RESISTANT PATHOGENS EVALUATED BY DIFFERENT METHODS

    Directory of Open Access Journals (Sweden)

    Mohamed Mustafa Aween

    2014-01-01

    Full Text Available Honey is rich with complex natural components which could be useful as antibacterial agents or as preservative. Honey contains high concentration of sugars, low amount of water, high osmolality and often dark colour which influence its antibacterial activity. Disc diffusion, well method, micro dilution assay are methods commonly used to determine the antibacterial activity of honey. In this study, microtiter and microbial plate count were included to ascertain the potency of honey as antibacterial agent against multiple antibiotic resistant pathogenic bacteria (Staphylococcus aureus, Salmonella Typhimurium, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa with concentration of 0.2 g mL-1. Results found that well diffusion method tends to give higher inhibitory zone than disc diffusion method but there was no correlation among the bacteria was observed except for S. Typhimurium, E. coli (R = 0.310, 0.505 and 0.316, respectively. Nan photometer assay and microtiter plates assay showed comparable results with moderately strong correlation (R2 = 0.681 and 0.767, respectively for S. aureus and S. typhimurium, but poor correlation was found for E. coli, B. subtilis and P. aeruginosa (R2 = 0.441, 0.308 and 0.383, respectively. Determining the number of survivors by plating on agar after nanophotometer assay or microtiter plate assay had confirmed the effectiveness of honey as antimicrobial agent against target bacteria; which confirmed that honey has the potency to inhibit pathogens even at low concentration.

  19. Sexual Function and the Use of Medical Devices or Drugs to Optimize Potency After Prostate Brachytherapy

    Energy Technology Data Exchange (ETDEWEB)

    Whaley, J. Taylor; Levy, Lawrence B. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX (United States); Swanson, David A. [Department of Urology, University of Texas MD Anderson Cancer Center, Houston, TX (United States); Pugh, Thomas J. [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX (United States); Kudchadker, Rajat J.; Bruno, Teresa L. [Department of Radiation Physics, University of Texas MD Anderson Cancer Center, Houston, TX (United States); Frank, Steven J., E-mail: sjfrank@mdnaderson.org [Department of Radiation Oncology, University of Texas MD Anderson Cancer Center, Houston, TX (United States)

    2012-04-01

    Purpose: Prospective evaluation of sexual outcomes after prostate brachytherapy with iodine-125 seeds as monotherapy at a tertiary cancer care center. Methods and Materials: Subjects were 129 men with prostate cancer with I-125 seed implants (prescribed dose, 145 Gy) without supplemental hormonal or external beam radiation therapy. Sexual function, potency, and bother were prospectively assessed at baseline and at 1, 4, 8, and 12 months using validated quality-of-life self-assessment surveys. Postimplant dosimetry values, including dose to 10% of the penile bulb (D10), D20, D33, D50, D75, D90, and penile volume receiving 100% of the prescribed dose (V100) were calculated. Results: At baseline, 56% of patients recorded having optimal erections; at 1 year, 62% of patients with baseline erectile function maintained optimal potency, 58% of whom with medically prescribed sexual aids or drugs. Variables associated with pretreatment-to-posttreatment decline in potency were time after implant (p = 0.04) and age (p = 0.01). Decline in urinary function may have been related to decline in potency. At 1 year, 69% of potent patients younger than 70 years maintained optimal potency, whereas 31% of patients older than 70 maintained optimal potency (p = 0.02). Diabetes was related to a decline in potency (p = 0.05), but neither smoking nor hypertension were. For patients with optimal potency at baseline, mean sexual bother scores had declined significantly at 1 year (p < 0.01). Sexual potency, sexual function, and sexual bother scores failed to correlate with any dosimetric variable tested. Conclusions: Erections firm enough for intercourse can be achieved at 1 year after treatment, but most men will require medical aids to optimize potency. Although younger men were better able to maintain erections firm enough for intercourse than older men, there was no correlation between potency, sexual function, or sexual bother and penile bulb dosimetry.

  20. Robust Array-Based Coregulator Binding Assay Predicting ERa-Agonist Potency and Generating Binding Profiles Reflecting Ligand Structure

    NARCIS (Netherlands)

    Aarts, J.M.M.J.G.; Wang, S.; Houtman, R.; Beuningen, van R.M.G.J.; Westerink, W.M.A.; Waart, van de B.J.; Rietjens, I.M.C.M.; Bovee, T.F.H.

    2013-01-01

    Testing chemicals for their endocrine-disrupting potential, including interference with estrogen receptor (ER) signaling, is an important aspect of chemical safety testing. Because of the practical drawbacks of animal testing, the development of in vitro alternatives for the uterotrophic assay and o

  1. Ligand fitting with CCP4

    Science.gov (United States)

    2017-01-01

    Crystal structures of protein–ligand complexes are often used to infer biology and inform structure-based drug discovery. Hence, it is important to build accurate, reliable models of ligands that give confidence in the interpretation of the respective protein–ligand complex. This paper discusses key stages in the ligand-fitting process, including ligand binding-site identification, ligand description and conformer generation, ligand fitting, refinement and subsequent validation. The CCP4 suite contains a number of software tools that facilitate this task: AceDRG for the creation of ligand descriptions and conformers, Lidia and JLigand for two-dimensional and three-dimensional ligand editing and visual analysis, Coot for density interpretation, ligand fitting, analysis and validation, and REFMAC5 for macromolecular refinement. In addition to recent advancements in automatic carbohydrate building in Coot (LO/Carb) and ligand-validation tools (FLEV), the release of the CCP4i2 GUI provides an integrated solution that streamlines the ligand-fitting workflow, seamlessly passing results from one program to the next. The ligand-fitting process is illustrated using instructive practical examples, including problematic cases such as post-translational modifications, highlighting the need for careful analysis and rigorous validation. PMID:28177312

  2. Predicting Nanocrystal Shape through Consideration of Surface-Ligand Interactions

    KAUST Repository

    Bealing, Clive R.

    2012-03-27

    Density functional calculations for the binding energy of oleic acid-based ligands on Pb-rich {100} and {111} facets of PbSe nanocrystals determine the surface energies as a function of ligand coverage. Oleic acid is expected to bind to the nanocrystal surface in the form of lead oleate. The Wulff construction predicts the thermodynamic equilibrium shape of the PbSe nanocrystals. The equilibrium shape is a function of the ligand surface coverage, which can be controlled by changing the concentration of oleic acid during synthesis. The different binding energy of the ligand on the {100} and {111} facets results in different equilibrium ligand coverages on the facets, and a transition in the equilibrium shape from octahedral to cubic is predicted when increasing the ligand concentration during synthesis. © 2012 American Chemical Society.

  3. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity

    NARCIS (Netherlands)

    Teunis, M.A.T.; Spiekstra, S.W.; Smits, M.; Adriaens, E.; Eltze, T.; Galbiati, V.; Krul, C.A.M.; Landsiedel, R.; Pieters, R.; Reinders, J.; Roggen, E.; Corsini, E.; Gibbs, S.

    2014-01-01

    This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50%

  4. 76 FR 9028 - Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products; Availability

    Science.gov (United States)

    2011-02-16

    ... Industry: Potency Tests for Cellular and Gene Therapy Products'' dated January 2011. The guidance document provides manufacturers of cellular and gene therapy (CGT) products with recommendations for developing... document entitled ``Guidance for Industry: Potency Tests for Cellular and Gene Therapy Products''...

  5. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive capacity

    NARCIS (Netherlands)

    Teunis, M.A.T.; Spiekstra, S.W.; Smits, M.; Adriaens, E.; Eltze, T.; Galbiati, V.; Krul, C.; Landsiedel, R.; Pieters, R.; Reinders, J.; Roggen, E.; Corsini, E.; Gibbs, S.

    2014-01-01

    This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50%

  6. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive capacity

    NARCIS (Netherlands)

    Teunis, M.A.T.; Spiekstra, S.W.; Smits, M.; Adriaens, E.; Eltze, T.; Galbiati, V.; Krul, C.; Landsiedel, R.; Pieters, R.; Reinders, J.; Roggen, E.; Corsini, E.; Gibbs, S.

    2014-01-01

    This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50%

  7. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants

    NARCIS (Netherlands)

    Hamers, T.H.M.; Kamstra, J.H.; Sonneveld, E.; Murk, A.J.; Kester, M.H.A.; Andersson, P.L.; Legler, J.; Brouwer, A.

    2006-01-01

    Over the last years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action

  8. Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products

    DEFF Research Database (Denmark)

    Hansen, Martin; Krogh, Kristine A; Brandt, Asbjørn

    2009-01-01

    )). The potency of mixtures of two of the compounds, narasin and nicarbazin, was synergistic (more than additive) with 10-fold greater antibacterial potency of the mixture than can be explained by their individual EC(50)-values. The influence of pH, temperature, oxygen concentration and light...

  9. In vitro profiling of the endocrine-disrupting potency of brominated flame retardants

    NARCIS (Netherlands)

    Hamers, T.H.M.; Kamstra, J.H.; Sonneveld, E.; Murk, A.J.; Kester, M.H.A.; Andersson, P.L.; Legler, J.; Brouwer, A.

    2006-01-01

    Over the last years, increasing evidence has become available that some brominated flame retardants (BFRs) may have endocrine disrupting (ED) potencies. The goal of the current study was to perform a systematic in vitro screening of the ED potencies of BFRs (1) to elucidate possible modes of action

  10. International ring trial of the epidermal equivalent sensitizer potency assay: reproducibility and predictive-capacity

    NARCIS (Netherlands)

    Teunis, M.A.T.; Spiekstra, S.W.; Smits, M.; Adriaens, E.; Eltze, T.; Galbiati, V.; Krul, C.A.M.; Landsiedel, R.; Pieters, R.; Reinders, J.; Roggen, E.; Corsini, E.; Gibbs, S.

    2014-01-01

    This study describes the international ring trial of the epidermal-equivalent (EE) sensitizer potency assay. This assay does not distinguish a sensitizer from a non-sensitizer, but may classify known skin sensitizers according to their potency. It assesses the chemical concentration resulting in 50%

  11. Analysis of cannabis seizures in NSW, Australia: cannabis potency and cannabinoid profile.

    Directory of Open Access Journals (Sweden)

    Wendy Swift

    Full Text Available Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ(9-tetrahydrocannabinol (THC and low levels of other phytocannabinoids, particularly cannabidiol (CBD. Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales "Cannabis Cautioning" scheme. A further 26 "Known Provenance" samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG, and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC, cannabinol (CBN and tetrahydrocannabivarin (THC-V. The "Cannabis Cautioning" samples showed high mean THC content (THC+THC-A = 14.88% and low mean CBD content (CBD+CBD-A = 0.14%. A modest level of CBG was detected (CBG+CBG-A = 1.18% and very low levels of CBC, CBN and THC-V (<0.1%. "Known Provenance" samples showed no significant differences in THC content between those seized from indoor versus outdoor cultivation sites. The present analysis echoes trends reported in other countries towards the use of high potency cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed.

  12. Comparison of the disposition of diethylstilbestrol and estradiol in the fetal rat. Correlation with teratogenic potency.

    Science.gov (United States)

    Henry, E C; Miller, R K

    1986-06-15

    The dispositions of radiolabeled diethylstilbestrol (DES) and estradiol (E2) in the fetal rat were compared to determine whether kinetic differences accounted for their differences in teratogenic potency. 14C (from DES) was concentrated in fetal tissues relative to plasma, whereas 3H (from E2) was largely retained protein-bound in fetal plasma. Both compounds were rapidly metabolized in the fetus (and mother) to less or non-estrogenic products. Fetal levels of E2 declined faster than those of DES (E1 was the primary circulating estrogen within 1-3 hr of E2 injection) so that exposure to unchanged DES was of longer duration than to E2. The unchanged compounds were retained longer and at higher concentrations in the target genital tissue compared to other tissues. Although these differences were consistent with the potencies, the concentration of the unchanged estrogen in fetal genital tract was lower after a teratogenic dose of DES than after a threshold teratogenic dose of E2. However, the 3H in fetal plasma and genital tract cytosol at 1 hr after injection of [3H]E2 at 2 ng or 10 micrograms/fetus was found to be highly protein-bound. DES competed poorly for these binding sites. It is suggested that the concentration of E2 which is "free" in the cell (as DES is), rather than the total content, correlates with its teratogenicity. Thus, in the rat, rapid metabolism and extensive protein-binding, both extra- and intracellularly, reduce the teratogenicity of the natural estrogen compared to the synthetic estrogen.

  13. Building-related symptoms and inflammatory potency of dust from office buildings

    DEFF Research Database (Denmark)

    Allermann, Leila; Pejtersen, Jan; Gunnarsen, Lars Bo

    2007-01-01

    Abstract The aim was to investigate the association between building-related symptoms (BRS) in office buildings and the inflammatory potency of dust (PD). Furthermore, the association between dust potency and various building characteristics was investigated. Occupants of 22 office buildings...... received a retrospective questionnaire about BRS (2301 respondents). Dust was collected from groups of offices and building characteristics were recorded. The potency of a dust sample to induce interleukin-8 (IL-8) secretion from the lung epithelial cell line A549 was measured as the slope of the initial...... linear part of the concentration- response curve. Symptoms of the central nervous system (CNS) were associated with the potency of surface dust (OR ¼ 1.4). This association may be due to an association between an index of CNS symptoms and dust potency in offices of 1-6 occupants (OR ¼ 1.5). No single...

  14. Building-related symptoms and inflammatory potency of dust from office buildings

    DEFF Research Database (Denmark)

    Allermann, L; Pejtersen, J; Gunnarsen, L

    2007-01-01

    UNLABELLED: The aim was to investigate the association between building-related symptoms (BRS) in office buildings and the inflammatory potency of dust (PD). Furthermore, the association between dust potency and various building characteristics was investigated. Occupants of 22 office buildings...... received a retrospective questionnaire about BRS (2301 respondents). Dust was collected from groups of offices and building characteristics were recorded. The potency of a dust sample to induce interleukin-8 (IL-8) secretion from the lung epithelial cell line A549 was measured as the slope of the initial...... linear part of the concentration-response curve. Symptoms of the central nervous system (CNS) were associated with the potency of surface dust (OR = 1.4). This association may be due to an association between an index of CNS symptoms and dust potency in offices of 1-6 occupants (OR = 1.5). No single...

  15. Building-related symptoms and inflammatory potency of dust from office buildings

    DEFF Research Database (Denmark)

    Allermann, Leila; Pejtersen, Jan; Gunnarsen, Lars Bo

    2007-01-01

    Abstract The aim was to investigate the association between building-related symptoms (BRS) in office buildings and the inflammatory potency of dust (PD). Furthermore, the association between dust potency and various building characteristics was investigated. Occupants of 22 office buildings...... linear part of the concentration- response curve. Symptoms of the central nervous system (CNS) were associated with the potency of surface dust (OR ¼ 1.4). This association may be due to an association between an index of CNS symptoms and dust potency in offices of 1-6 occupants (OR ¼ 1.5). No single...... symptoms correlated with the potency of surface dust. The PD was not related to single building factors. The inflammatory PD may be used as an integrated proxy measure of biologically active compounds in dust, reflecting health relevant properties of the dust....

  16. Reducing animal experimentation in foot-and-mouth disease vaccine potency tests.

    Science.gov (United States)

    Reeve, Richard; Cox, Sarah; Smitsaart, Eliana; Beascoechea, Claudia Perez; Haas, Bernd; Maradei, Eduardo; Haydon, Daniel T; Barnett, Paul

    2011-07-26

    The World Organisation for Animal Health (OIE) Terrestrial Manual and the European Pharmacopoeia (EP) still prescribe live challenge experiments for foot-and-mouth disease virus (FMDV) immunogenicity and vaccine potency tests. However, the EP allows for other validated tests for the latter, and specifically in vitro tests if a "satisfactory pass level" has been determined; serological replacements are also currently in use in South America. Much research has therefore focused on validating both ex vivo and in vitro tests to replace live challenge. However, insufficient attention has been given to the sensitivity and specificity of the "gold standard"in vivo test being replaced, despite this information being critical to determining what should be required of its replacement. This paper aims to redress this imbalance by examining the current live challenge tests and their associated statistics and determining the confidence that we can have in them, thereby setting a standard for candidate replacements. It determines that the statistics associated with the current EP PD(50) test are inappropriate given our domain knowledge, but that the OIE test statistics are satisfactory. However, it has also identified a new set of live animal challenge test regimes that provide similar sensitivity and specificity to all of the currently used OIE tests using fewer animals (16 including controls), and can also provide further savings in live animal experiments in exchange for small reductions in sensitivity and specificity.

  17. GABA receptor subunit composition relative to insecticide potency and selectivity.

    Science.gov (United States)

    Ratra, G S; Casida, J E

    2001-07-01

    Three observations on the 4-[(3)H]propyl-4'-ethynylbicycloorthobenzoate ([(3)H]EBOB) binding site in the gamma-aminobutyric acid (GABA) receptor indicate the specific target for insecticide action in human brain and a possible mechanism for selectivity. First, from published data, alpha-endosulfan, lindane and fipronil compete for the [(3)H]EBOB binding site with affinities of 0.3--7 nM in both human recombinant homooligomeric beta 3 receptors and housefly head membranes. Second, from structure-activity studies, including new data, GABAergic insecticide binding potency on the pentameric receptor formed from the beta 3 subunit correlates well with that on the housefly receptor (r=0.88, n=20). This conserved inhibitor specificity is consistent with known sequence homologies in the housefly GABA receptor and the human GABA(A) receptor beta 3 subunit. Third, as mostly new findings, various combinations of alpha 1, alpha 6, and gamma 2 subunits coexpressed with a beta 1 or beta 3 subunit confer differential insecticide binding sensitivity, particularly to fipronil, indicating that subunit composition is a major factor in insecticide selectivity.

  18. Differential potencies of effector genes in adult Drosophila.

    Science.gov (United States)

    Thum, Andreas S; Knapek, Stephan; Rister, Jens; Dierichs-Schmitt, Eva; Heisenberg, Martin; Tanimoto, Hiromu

    2006-09-10

    The GAL4/UAS gene expression system in Drosophila has been crucial in revealing the behavioral significance of neural circuits. Transgene products that block neurotransmitter release and induce cell death have been proved to inhibit neural function powerfully. Here we compare the action of the five effector genes shibire(ts1), Tetanus toxin light chain (TNT), reaper, Diphtheria toxin A-chain (DTA), and inwardly rectifying potassium channel (Kir2.1) and show differences in their efficiency depending on the target cells and the timing of induction. Specifically, effectors blocking neuronal transmission or excitability led to adult-induced paralysis more efficiently than those causing cell ablation. We contrasted these differential potencies in adult to their actions during development. Furthermore, we induced TNT expression in the adult mushroom bodies. In contrast to the successful impairment in short-term olfactory memory by shibire(ts1), adult TNT expression in the same set of cells did not lead to any obvious impairment. Altogether, the efficiency of effector genes depends on properties of the targeted neurons. Thus, we conclude that the selection of the appropriate effector gene is critical for evaluating the function of neural circuits.

  19. Potency Of Bacteriocin For Animal Health And Food Safety

    Directory of Open Access Journals (Sweden)

    Siti Chotiah

    2013-06-01

    Full Text Available The emergence of antibiotic resistance in many bacteria related to animal and public health stresses the importance of decreasing the use of antibiotics in animal production. The reduction of antibiotic application in livestock can only be achieved if alternative antimicrobial strategies are available. A number of strategies have been explored to control microbial pathogens and to improve growth and feed efficiency in livestock without the use of antibiotics. Bacteriocins have been more extensively studied and proposed as potential alternatives to conventional antibiotics in animal husbandry. Bacteriocins are antimicrobial peptides ribosomally synthesized by many species of Bacteria and some strains of Archaea. In general, bacteriocins just exhibited bactericidal or bacteriostatic activity against other bacteria that are closely related to the producing strain. The main mechanisms of bacteriocin activity vary from pore formation in cytoplasmic membranes to the inhibition of cell wall biosynthesis and enzyme activities (RNAse or DNAse in target cells. The use of bacteriocins in probiotic applications, as preservatives, and most excitingly as alternatives to conventional antibiotics is being broadly explored and studied. This review will describe the bacteriocins potency for animal health and food safety, as well as the results of bacteriocin study that had been conducted in Indonesia.

  20. Radioiodination of chicken luteinizing hormone without affecting receptor binding potency

    Energy Technology Data Exchange (ETDEWEB)

    Kikuchi, M.; Ishii, S. (Waseda Univ., Tokyo (Japan))

    1989-12-01

    By improving the currently used lactoperoxidase method, we were able to obtain radioiodinated chicken luteinizing hormone (LH) that shows high specific binding and low nonspecific binding to a crude plasma membrane fraction of testicular cells of the domestic fowl and the Japanese quail, and to the ovarian granulosa cells of the Japanese quail. The change we made from the original method consisted of (1) using chicken LH for radioiodination that was not only highly purified but also retained a high receptor binding potency; (2) controlling the level of incorporation of radioiodine into chicken LH molecules by employing a short reaction time and low temperature; and (3) fractionating radioiodinated chicken LH further by gel filtration using high-performance liquid chromatography. Specific radioactivity of the final {sup 125}I-labeled chicken LH preparation was 14 microCi/micrograms. When specific binding was 12-16%, nonspecific binding was as low as 2-4% in the gonadal receptors. {sup 125}I-Labeled chicken LH was displaced by chicken LH and ovine LH but not by chicken follicle-stimulating hormone. The equilibrium association constant of quail testicular receptor was 3.6 x 10(9) M-1. We concluded that chicken LH radioiodinated by the present method is useful for studies of avian LH receptors.

  1. Potency of Fish Collagen as a Scaffold for Regenerative Medicine

    Directory of Open Access Journals (Sweden)

    Shizuka Yamada

    2014-01-01

    Full Text Available Cells, growth factors, and scaffold are the crucial factors for tissue engineering. Recently, scaffolds consisting of natural polymers, such as collagen and gelatin, bioabsorbable synthetic polymers, such as polylactic acid and polyglycolic acid, and inorganic materials, such as hydroxyapatite, as well as composite materials have been rapidly developed. In particular, collagen is the most promising material for tissue engineering due to its biocompatibility and biodegradability. Collagen contains specific cell adhesion domains, including the arginine-glycine-aspartic acid (RGD motif. After the integrin receptor on the cell surface binds to the RGD motif on the collagen molecule, cell adhesion is actively induced. This interaction contributes to the promotion of cell growth and differentiation and the regulation of various cell functions. However, it is difficult to use a pure collagen scaffold as a tissue engineering material due to its low mechanical strength. In order to make up for this disadvantage, collagen scaffolds are often modified using a cross-linker, such as gamma irradiation and carbodiimide. Taking into account the possibility of zoonosis, a variety of recent reports have been documented using fish collagen scaffolds. We herein review the potency of fish collagen scaffolds as well as associated problems to be addressed for use in regenerative medicine.

  2. Ligand-Receptor Interactions

    CERN Document Server

    Bongrand, Pierre

    2008-01-01

    The formation and dissociation of specific noncovalent interactions between a variety of macromolecules play a crucial role in the function of biological systems. During the last few years, three main lines of research led to a dramatic improvement of our understanding of these important phenomena. First, combination of genetic engineering and X ray cristallography made available a simultaneous knowledg of the precise structure and affinity of series or related ligand-receptor systems differing by a few well-defined atoms. Second, improvement of computer power and simulation techniques allowed extended exploration of the interaction of realistic macromolecules. Third, simultaneous development of a variety of techniques based on atomic force microscopy, hydrodynamic flow, biomembrane probes, optical tweezers, magnetic fields or flexible transducers yielded direct experimental information of the behavior of single ligand receptor bonds. At the same time, investigation of well defined cellular models raised the ...

  3. Identification and pharmacological characterization of a series of new 1H-4-substituted-imidazoyl histamine H3 receptor ligands.

    Science.gov (United States)

    Yates, S L; Phillips, J G; Gregory, R; Pawlowski, G P; Fadnis, L; Khan, M A; Ali, S M; Tedford, C E

    1999-05-01

    A new series of 1H-4-substituted imidazole compounds were synthesized and identified as potent and selective histamine (HA) H3 receptor ligands. These ligands establish that HA H3 antagonists exhibit stereoselective and conformational preferences in their binding to the HA H3 receptor. Structure-activity relationships were determined in vitro by HA H3 receptor-binding affinities using [3H]Nalpha-methylhistamine and rat cerebral cortical tissue homogenates. Several derivatives containing olefin, amide, and acetylene functional groups were identified as potent HA H3 receptor ligands. In the olefin series, GT-2227 (4-(6-cyclohexylhex-cis-3-enyl)imidazole) was identified as a potent HA H3 receptor ligand with a Ki of 4.2 +/- 0.6 nM, while the trans isomer (GT-2228) displayed a reduced potency (Ki = 15.2 +/- 2.4 nM). GT-2227 was also found to have excellent central nervous system penetration in an ex vivo binding paradigm (ED50 = 0.7 mg/kg i.p.). In the acetylene series, GT-2260 and GT-2286 both exhibited high affinity (Ki = 2.9 +/- 0.2 and 0.95 +/- 0.3 nM) and excellent central nervous system penetration profiles (ED50 = 0.43 and 0.48 mg/kg i.p., respectively). As a prototype for the series, GT-2227 showed high affinity for the human HA H3 receptor (3.2 nM) and minimal affinity for the human HA H1 (Ki = 13,407 +/- 540 nM) and H2 (Ki = 4,469 +/- 564 nM) receptor subtypes. GT-2227 also showed good selectivity for the HA H3 receptor over a broad spectrum of other neurotransmitter receptors (IC50 >/= 1 microM). Furthermore, GT-2227 improved acquisition in a cognitive paradigm without behavioral excitation or effect on spontaneous locomotor activity. In summary, the present studies demonstrate the development of novel HA H3-selective ligands, and lend support for the use of such agents in the treatment of disorders associated with cognitive or attentional deficits.

  4. Therapeutic androgen receptor ligands

    OpenAIRE

    Allan, George F.; Sui, Zhihua

    2003-01-01

    In the past several years, the concept of tissue-selective nuclear receptor ligands has emerged. This concept has come to fruition with estrogens, with the successful marketing of drugs such as raloxifene. The discovery of raloxifene and other selective estrogen receptor modulators (SERMs) has raised the possibility of generating selective compounds for other pathways, including androgens (that is, selective androgen receptor modulators, or SARMs).

  5. Superior serum half life of albumin tagged TNF ligands

    Energy Technology Data Exchange (ETDEWEB)

    Mueller, Nicole [Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg (Germany); Schneider, Britta; Pfizenmaier, Klaus [Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart (Germany); Wajant, Harald, E-mail: harald.wajant@mail.uni-wuerzburg.de [Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg, Roentgenring 11, 97070 Wuerzburg (Germany)

    2010-06-11

    Due to their immune stimulating and apoptosis inducing properties, ligands of the TNF family attract increasing interest as therapeutic proteins. A general limitation of in vivo applications of recombinant soluble TNF ligands is their notoriously rapid clearance from circulation. To improve the serum half life of the TNF family members TNF, TWEAK and TRAIL, we genetically fused soluble variants of these molecules to human serum albumin (HSA). The serum albumin-TNF ligand fusion proteins were found to be of similar bioactivity as the corresponding HSA-less counterparts. Upon intravenous injection (i.v.), serum half life of HSA-TNF ligand fusion proteins, as determined by ELISA, was around 15 h as compared to approximately 1 h for all of the recombinant control TNF ligands without HSA domain. Moreover, serum samples collected 6 or 24 h after i.v. injection still contained high TNF ligand bioactivity, demonstrating that there is only limited degradation/inactivation of circulating HSA-TNF ligand fusion proteins in vivo. In a xenotransplantation model, significantly less of the HSA-TRAIL fusion protein compared to the respective control TRAIL protein was required to achieve inhibition of tumor growth indicating that the increased half life of HSA-TNF ligand fusion proteins translates into better therapeutic action in vivo. In conclusion, our data suggest that genetic fusion to serum albumin is a powerful and generally applicable mean to improve bioavailability and in vivo activity of TNF ligands.

  6. Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity

    Directory of Open Access Journals (Sweden)

    Douglas Bledsoe

    2017-05-01

    Full Text Available N-methyl D-aspartate receptors (NMDAR play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A–D and two GluN3 (A–B subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD and ligand binding domain (LBD, transmembrane domain (TMD and an intracellular C-terminal domain (CTD. Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.

  7. First systematic evaluation of the potency of Cannabis sativa plants grown in Albania.

    Science.gov (United States)

    Bruci, Zana; Papoutsis, Ioannis; Athanaselis, Sotirios; Nikolaou, Panagiota; Pazari, Ermira; Spiliopoulou, Chara; Vyshka, Gentian

    2012-10-10

    Cannabis products (marijuana, hashish, cannabis oil) are the most frequently abused illegal substances worldwide. Delta-9-tetrahydrocannabinol (THC) is the main psychoactive component of Cannabis sativa plant, whereas cannabidiol (CBD) and cannabinol (CBN) are other major but no psychoactive constituents. Many studies have already been carried out on these compounds and chemical research was encouraged due to the legal implications concerning the misuse of marijuana. The aim of this study was to determine THC, CBD and CBN in a significant number of cannabis samples of Albanian origin, where cannabis is the most frequently used drug of abuse, in order to evaluate and classify them according to their cannabinoid composition. A GC-MS method was used, in order to assay cannabinoid content of hemp samples harvested at different maturation degree levels during the summer months and grown in different areas of Albania. This method can also be used for the determination of plant phenotype, the evaluation of psychoactive potency and the control of material quality. The highest cannabinoid concentrations were found in the flowers of cannabis. The THC concentrations in different locations of Albania ranged from 1.07 to 12.13%. The influence of environmental conditions on cannabinoid content is discussed. The cannabinoid content of cannabis plants were used for their profiling, and it was used for their classification, according to their geographical origin. The determined concentrations justify the fact that Albania is an area where cannabis is extensively cultivated for illegal purposes.

  8. Part 2: Potencies and interactions of polybrominated aromatic hydrocarbons in rainbow trout early life stage mortality

    Energy Technology Data Exchange (ETDEWEB)

    Hornung, M.W.; Zabel, E.W.; Peterson, R.E. [Univ. of Wisconsin, Madison, WI (United States); Bergman, A. [Stockholm Univ. (Sweden); Safe, S. [Texas A and M Univ., College Station, TX (United States)

    1994-12-31

    Polybrominated biphenyls (PBBs), dibenzo-p-dioxins (PBDDs), dibenzofurans (PBDFs), and diphenylethers (PBDPES) in aquatic environments may pose a risk to fish early life stage survival. Following rainbow trout egg microinjection, the potencies of these polybrominated aromatic hydrocarbons were determined using fish specific toxic equivalency factors (TEFs). TEFs are defined as the ratio of TCDD LD{sub 50} to brominated congener LD{sub 50}. Sac fry stage specific TCDD like toxicity included yolk sac edema, pericardial edema, multifocal hemorrhages and craniofacial malformations. TEFs of active congeners were: 2,3,7,8-TBDF = 0.23; 2,3,4,7,8-PBDF = 0.069; 1,2,3,4,7,8-HxBDD = 0.009. No signs of toxicity with 2,2{prime},4,4{prime}-TBDPE, 2,2{prime},3,4,4{prime}-PBDPE, or 2,2{prime},4,4{prime},5-PBDPE occurred at egg concentrations up to 126,37 {mu}g/g. Since these congeners occur as complex mixtures, the potential for additive, antagonistic, or synergistic interactions must also be determined for accurate risk assessment. Graded doses of 2,3,7,8-TBDD or 1,2,3,7,8-PBDD alone, or graded doses of fixed ratios of the two congeners were injected into newly fertilized rainbow trout eggs. Separate dose response curves were determined for each ratio and each individual congener. Isobolographic analysis supports the hypothesis that these congeners act additively.

  9. High affinity ligands for 'diazepam-insensitive' benzodiazepine receptors.

    Science.gov (United States)

    Wong, G; Skolnick, P

    1992-01-14

    Structurally diverse compounds have been shown to possess high affinities for benzodiazepine receptors in their 'diazepam-sensitive' (DS) conformations. In contrast, only the imidazobenzodiazepinone Ro 15-4513 has been shown to exhibit a high affinity for the 'diazepam-insensitive' (DI) conformation of benzodiazepine receptors. We examined a series of 1,4-diazepines containing one or more annelated ring systems for their affinities at DI and DS benzodiazepine receptors, several 1,4-diazepinone carboxylates including Ro 19-4603, Ro 16-6028 and Ro 15-3505 were found to possess high affinities (Ki approximately 2.6-20 nM) for DI. Nonetheless, among the ligands examined, Ro 15-4513 was the only substance with a DI/DS potency ratio approximately 1; other substances had ratios ranging from 13 to greater than 1000. Ligands with high to moderate affinities at DI were previously classified as partial agonists, antagonists, or partial inverse agonists at DS benzodiazepine receptors, but behaved as 'GABA neutral' (antagonist) substances at DI. The identification of several additional high affinity ligands at DI benzodiazepine receptors may be helpful in elucidating the pharmacological and physiological importance of these sites.

  10. Emergency planning and the acute toxic potency of inhaled ammonia.

    Science.gov (United States)

    Michaels, R A

    1999-08-01

    Ammonia is present in agriculture and commerce in many if not most communities. This report evaluates the toxic potency of ammonia, based on three types of data: anecdotal data, in some cases predating World War 1, reconstructions of contemporary industrial accidents, and animal bioassays. Standards and guidelines for human exposure have been driven largely by the anecdotal data, suggesting that ammonia at 5,000-10,000 parts per million, volume/volume (ppm-v), might be lethal within 5-10 min. However, contemporary accident reconstructions suggest that ammonia lethality requires higher concentrations. For example, 33,737 ppm-v was a 5-min zero-mortality value in a major ammonia release in 1973 in South Africa. Comparisons of secondary reports of ammonia lethality with original sources revealed discrepancies in contemporary sources, apparently resulting from failure to examine old documents or accurately translate foreign documents. The present investigation revealed that contemporary accident reconstructions yield ammonia lethality levels comparable to those in dozens of reports of animal bioassays, after adjustment of concentrations to human equivalent concentrations via U.S. Environmental Protection Agency (EPA) procedures. Ammonia levels potentially causing irreversible injury or impairing the ability of exposed people to escape from further exposure or from coincident perils similarly have been biased downwardly in contemporary sources. The EPA has identified ammonia as one of 366 extremely hazardous substances subject to community right-to-know provisions of the Superfund Act and emergency planning provisions of the Clean Air Act. The Clean Air Act defines emergency planning zones (EPZs) around industrial facilities exceeding a threshold quantity of ammonia on-site. This study suggests that EPZ areas around ammonia facilities can be reduced, thereby also reducing emergency planning costs, which will vary roughly with the EPZ radius squared.

  11. A statistical approach towards the derivation of predictive gene sets for potency ranking of chemicals in the mouse embryonic stem cell test.

    Science.gov (United States)

    Schulpen, Sjors H W; Pennings, Jeroen L A; Tonk, Elisa C M; Piersma, Aldert H

    2014-03-21

    The embryonic stem cell test (EST) is applied as a model system for detection of embryotoxicants. The application of transcriptomics allows a more detailed effect assessment compared to the morphological endpoint. Genes involved in cell differentiation, modulated by chemical exposures, may be useful as biomarkers of developmental toxicity. We describe a statistical approach to obtain a predictive gene set for toxicity potency ranking of compounds within one class. This resulted in a gene set based on differential gene expression across concentration-response series of phthalatic monoesters. We determined the concentration at which gene expression was changed at least 1.5-fold. Genes responding with the same potency ranking in vitro and in vivo embryotoxicity were selected. A leave-one-out cross-validation showed that the relative potency of each phthalate was always predicted correctly. The classical morphological 50% effect level (ID50) in EST was similar to the predicted concentration using gene set expression responses. A general down-regulation of development-related genes and up-regulation of cell-cycle related genes was observed, reminiscent of the differentiation inhibition in EST. This study illustrates the feasibility of applying dedicated gene set selections as biomarkers for developmental toxicity potency ranking on the basis of in vitro testing in the EST.

  12. Collaborative study for the validation of serological methods for potency testing of diphtheria toxoid vaccines - extended study: correlation of serology with in vivo toxin neutralisation.

    Science.gov (United States)

    Sesardic, D; Winsnes, R; Rigsby, P; Behr-Gross, M-E

    2004-01-01

    Phase I of BSP034 collaborative study was extended in two laboratories to include correlation of serology with in vivo toxin neutralisation test (TNT) using 2 separate sets of 20 serum pools, produced in-house. The study investigated the extent to which the in vitro methods for diphtheria antibodies, Vero cell assay and diphtheria enzyme-linked immunosorbent assay for diphtheria antitoxin (D-ELISA), can detect neutralising antibodies by comparison with TNT in guinea pigs. The study was also performed to compare the antibody neutralising potency obtained in relation to guinea pig (GP) or equine (DI) antitoxin standard. In addition, the study provided an opportunity to compare ELISA for tetanus antitoxin (T-ELISA) and TNT assay for detection of anti-tetanus antibodies, from the same set of serum pools. The data obtained show that antitoxin potency obtained by Vero cell assay, D-ELISA and T-ELISA using the same GP standard, highly correlated with neutralising potency as determined in respective TNT assays. Vero cell assay with DI provided estimates that also correlated with neutralising potency, but were of significantly lower titre. Since reference to DI standard is widely used in serodiagnosis, as well as in clinical studies where diphtheria antitoxin titres obtained in the Vero cell method are taken as surrogate markers for vaccine efficacy, it should be investigated if a similar difference is also observed for human serology.

  13. Development of a flow cytometric bead immunoassay and its assessment as a possible aid to potency evaluation of enterotoxaemia vaccines

    Directory of Open Access Journals (Sweden)

    Angela Buys

    2014-02-01

    Full Text Available Enterotoxaemia, an economically important disease of sheep, goats and calves, is caused by systemic effects of the epsilon toxin produced by the anaerobic bacterium Clostridium perfringens type D. The only practical means of controlling the occurrence of enterotoxaemia is to immunise animals by vaccination. The vaccine is prepared by deriving a toxoid from the bacterial culture filtrate and the potency of the vaccine is tested with the in vivo mouse neutralisation test (MNT. Due to ethical, economic and technical reasons, alternative in vitro assays are needed. In this study an indirect cytometric bead immunoassay (I-CBA was developed for use in vaccine potency testing and the results were compared with those obtained using an indirect enzyme-linked immunosorbent assay (I-ELISA and the MNT. Sera were collected from guinea pigs immunised with three different production batches of enterotoxaemia vaccine and the levels of anti-epsilon toxin antibodies were determined. Although the intra- and inter-assay variability was satisfactory, epsilon antitoxin levels determined by both the I-ELISA and indirect cytometric bead immunoassay (I-CBA tests were higher than those of the MNT assay. In contrast to the MNT, all of the serum samples were identified as having antitoxin levels above the required minimum (not less than 5 U/mL. These results indicate that the respective in vitro tests in their current formats are not yet suitable alternatives to the in vivo MNT. The growing demand for a more humane, cost-effective and efficient method for testing the potency of enterotoxaemia vaccines, however, provides a strong impetus for further optimisation and standardisation of the I-CBA assay but further analytical research is required.

  14. Ligand Exchange Kinetics of Environmentally Relevant Metals

    Energy Technology Data Exchange (ETDEWEB)

    Panasci, Adele Frances [Univ. of California, Davis, CA (United States)

    2014-07-15

    The interactions of ground water with minerals and contaminants are of broad interest for geochemists but are not well understood. Experiments on the molecular scale can determine reaction parameters (i.e. rates of ligand exchange, activation entropy, activation entropy, and activation volume) that can be used in computations to gain insight into reactions that occur in natural groundwaters. Experiments to determine the rate of isotopic ligand exchange for three environmentally relevant metals, rhodium (Rh), iron (Fe), and neptunium (Np), are described. Many environmental transformations of metals (e.g. reduction) in soil occur at trivalent centers, Fe(III) in particular. Contaminant ions absorb to mineral surfaces via ligand exchange, and the reversal of this reaction can be dangerous, releasing contaminants into the environment. Ferric iron is difficult to study spectroscopically because most of its complexes are paramagnetic and are generally reactive toward ligand exchange; therefore, Rh(III), which is diamagnetic and less reactive, was used to study substitution reactions that are analogous to those that occur on mineral oxide surfaces. Studies on both Np(V) and Np(VI) are important in their own right, as 237Np is a radioactive transuranic element with a half-life of 2 million years.

  15. Specific activity of radioiodine-labelled human chorionic gonadotropin ligand

    Energy Technology Data Exchange (ETDEWEB)

    Crespi, M. (South African Inst. for Medical Research, Sandringham. National Inst. for Virology); Kay, G.W.; Van der Walt, L.A. (South African Inst. for Medical Research, Johannesburg. Dept. of Pathology)

    1983-10-01

    The article deals with the determination of the specific activity of radioiodine-labelled human chorionic gonadotropin ligand. The iodiation of human chorionic gonadotropin and the counting efficiency of /sup 125/I are discussed.

  16. Bexarotene ligand pharmaceuticals.

    Science.gov (United States)

    Hurst, R E

    2000-12-01

    Bexarotene (LGD-1069), from Ligand, was the first retinoid X receptor (RXR)-selective, antitumor retinoid to enter clinical trials. The company launched the drug for the treatment of cutaneous T-cell lymphoma (CTCL), as Targretin capsules, in the US in January 2000 [359023]. The company filed an NDA for Targretin capsules in June 1999, and for topical gel in December 1999 [329011], [349982] specifically for once-daily oral administration for the treatment of patients with early-stage CTCL who have not tolerated other therapies, patients with refractory or persistent early stage CTCL and patients with refractory advanced stage CTCL. The FDA approved Targretin capsules at the end of December 1999 for once-daily oral treatment of all stages of CTCL in patients refractory to at least one prior systemic therapy, at an initial dose of 300 mg/m2/day. After an NDA was submitted in December 1999 for Targretin gel, the drug received Priority Review status for use as a treatment of cutaneous lesions in patients with stage IA, IB or IIA CTCL [354836]. The FDA issued an approvable letter in June 2000, and granted marketing clearance for CTCL in the same month [370687], [372768], [372769], [373279]. Ligand had received Orphan Drug designation for this indication [329011]. At the request of the FDA, Ligand agreed to carry out certain post-approval phase IV and pharmacokinetic studies [351604]. The company filed an MAA with the EMEA for Targretin Capsules to treat lymphoma in November 1999 [348944]. The NDA for Targretin gel is based on a multicenter phase III trial that was conducted in the US, Canada, Europe and Australia involving 50 patients and a multicenter phase I/II clinical program involving 67 patients. Targretin gel was evaluated for the treatment of patients with early stage CTCL (IA-IIA) who were refractory to, intolerant to, or reached a response plateau for at least 6 months on at least two prior therapies. Efficacy results exceeded the protocol-defined response

  17. The incidence of kidney injury for patients treated with a high-potency versus moderate-potency statin regimen after an acute coronary syndrome.

    Science.gov (United States)

    Sarma, Amy; Cannon, Christopher P; de Lemos, James; Rouleau, Jean L; Lewis, Eldrin F; Guo, Jianping; Mega, Jessica L; Sabatine, Marc S; O'Donoghue, Michelle L

    2014-05-01

    Observational studies have raised concerns that high-potency statins increase the risk of acute kidney injury. We therefore examined the incidence of kidney injury across 2 randomized trials of statin therapy. PROVE IT-TIMI 22 enrolled 4162 subjects after an acute coronary syndrome (ACS) and randomized them to atorvastatin 80 mg/day versus pravastatin 40 mg/day. A-to-Z enrolled 4497 subjects after ACS and randomized them to a high-potency (simvastatin 40 mg/day × 1 months, then simvastatin 80 mg/day) versus a delayed moderate-potency statin strategy (placebo × 4 months, then simvastatin 20 mg/day). Serum creatinine was assessed centrally at serial time points. Adverse events (AEs) relating to kidney injury were identified through database review. Across both trials, mean serum creatinine was similar between treatment arms at baseline and throughout follow-up. In A-to-Z, the incidence of a 1.5-fold or ≥ 0.3 mg/dL rise in serum creatinine was 11.4% for subjects randomized to a high-potency statin regimen versus 12.4% for those on a delayed moderate-potency regimen (odds ratio [OR], 0.91; 95% confidence interval [CI], 0.76 to 1.10; P=0.33). In PROVE IT-TIMI 22, the incidence was 9.4% for subjects randomized to atorvastatin 80 mg/day and 10.6% for subjects randomized to pravastatin 40 mg/day (OR, 0.88; 95% CI, 0.71 to 1.09; P=0.25). Consistent results were observed for different kidney injury thresholds and in individuals with diabetes mellitus or with moderate renal dysfunction. The incidence of kidney injury-related adverse events (AEs) was not statistically different for patients on a high-potency versus moderate-potency statin regimen (OR, 1.06; 95% CI, 0.68 to 1.67; P=0.78). For patients enrolled in 2 large randomized trials of statin therapy after ACS, the use of a high-potency statin regimen did not increase the risk of kidney injury.

  18. Parametric Effect of Sodium Hydroxide and Sodium Carbonate on the Potency of a Degreaser

    Directory of Open Access Journals (Sweden)

    Babatope Abimbola Olufemi

    2016-01-01

    Full Text Available Normal 0 false false false EN-US X-NONE X-NONE Experimental and statistical analysis was carried out on the comparative effect of sodium hydroxide and sodium carbonate on the potency of a laboratory produced degreaser in this work. The materials used include; octadecyl benzene sulphonic acid, sodium hydroxide, sodium carbonate, sodium metasilicate, carboxyl methyl cellulose (C.M.C, formadelhyde, perfume, colourant and distilled water. Different samples of degreaser were produced with varying composition of sodium hydroxide and sodium carbonate respectively. Statistical significance through methods like analysis of variance (ANOVA of some parameters on various concentrations of sodium hydroxide and sodium carbonate was investigated. The effect of the varying compositions of sodium hydroxide and sodium carbonate was also determined by using a gray scale (GS test, and also subjecting surfaces heavily stained with crude oil to determine and characterize the cleansing action of the degreaser. It was found that as the concentration of sodium hydroxide increases, the cleansing ability also increases, whereas the increase in concentration of sodium carbonate had no effect on the cleansing ability. The work would enable production of effective, useful and property controlled degreasers at moderate cost.

  19. Chemistry, Biology, and QSAR Studies of Substituted Biaryl Hydroxamates and Mercaptoacetamides as HDAC inhibitors - Nanomolar Potency Inhibitors of Pancreatic Cancer Cell Growth

    Science.gov (United States)

    Kozikowski, Alan P.; Chen, Yufeng; Gaysin, Arsen M.; Savoy, Doris N.; Billadeau, Daniel D.; Kim, Ki Hwan

    2009-01-01

    The histone deacetylases (HDACs) are able to regulate gene expression and inhibitors of the HDACs (HDACIs) hold promise in the treatment of cancer as well as a variety of neurodegenerative diseases. To investigate the possibility to achieve some measure of isoform selectivity in the inhibition of the HDACs, we prepared a small series of 2,4′-diaminobiphenyl ligands functionalized at the para-amino group with an appendage containing either a hydroxamate or a mercaptoacetamide group and coupled to an amino acid residue at the ortho-amino group. A smaller series of substituted phenylthiazoles was also explored. Some of these newly synthesized ligands show low nM potency in the HDAC inhibition assays and display micromolar to low nanomolar IC50 values when tested against five pancreatic cancer cell lines. The isoform selectivity of these ligands for the Class I HDACs (HDAC1-3 and 8) and Class IIb HDACs (HDAC6 and HDAC10) together with QSAR studies of their correlation with the lipophilicity are presented. Of particular interest is the HDAC6 selectivity of the mercaptoacetamides. PMID:18181121

  20. Chemistry, biology, and QSAR studies of substituted biaryl hydroxamates and mercaptoacetamides as HDAC inhibitors-nanomolar-potency inhibitors of pancreatic cancer cell growth.

    Science.gov (United States)

    Kozikowski, Alan P; Chen, Yufeng; Gaysin, Arsen M; Savoy, Doris N; Billadeau, Daniel D; Kim, Ki Hwan

    2008-03-01

    The histone deacetylases (HDACs) are able to regulate gene expression, and inhibitors of the HDACs (HDACIs) hold promise in the treatment of cancer as well as a variety of neurodegenerative diseases. To investigate the potential for isoform selectivity in the inhibition of HDACs, we prepared a small series of 2,4'-diaminobiphenyl ligands functionalized at the para-amino group with an appendage containing either a hydroxamate or a mercaptoacetamide group and coupled to an amino acid residue at the ortho-amino group. A smaller series of substituted phenylthiazoles was also explored. Some of these newly synthesized ligands show low-nanomolar potency in HDAC inhibition assays and display micromolar to low-nanomolar IC(50) values in tests against five pancreatic cancer cell lines. The isoform selectivity of these ligands for class I HDACs (HDAC1-3 and 8) and class IIb HDACs (HDAC6 and 10) together with QSAR studies of their correlation with lipophilicity are presented. Of particular interest is the selectivity of the mercaptoacetamides for HDAC6.

  1. Determining

    Directory of Open Access Journals (Sweden)

    Bahram Andarzian

    2015-06-01

    Full Text Available Wheat production in the south of Khuzestan, Iran is constrained by heat stress for late sowing dates. For optimization of yield, sowing at the appropriate time to fit the cultivar maturity length and growing season is critical. Crop models could be used to determine optimum sowing window for a locality. The objectives of this study were to evaluate the Cropping System Model (CSM-CERES-Wheat for its ability to simulate growth, development, grain yield of wheat in the tropical regions of Iran, and to study the impact of different sowing dates on wheat performance. The genetic coefficients of cultivar Chamran were calibrated for the CSM-CERES-Wheat model and crop model performance was evaluated with experimental data. Wheat cultivar Chamran was sown on different dates, ranging from 5 November to 9 January during 5 years of field experiments that were conducted in the Khuzestan province, Iran, under full and deficit irrigation conditions. The model was run for 8 sowing dates starting on 25 October and repeated every 10 days until 5 January using long-term historical weather data from the Ahvaz, Behbehan, Dezful and Izeh locations. The seasonal analysis program of DSSAT was used to determine the optimum sowing window for different locations as well. Evaluation with the experimental data showed that performance of the model was reasonable as indicated by fairly accurate simulation of crop phenology, biomass accumulation and grain yield against measured data. The normalized RMSE were 3%, 2%, 11.8%, and 3.4% for anthesis date, maturity date, grain yield and biomass, respectively. Optimum sowing window was different among locations. It was opened and closed on 5 November and 5 December for Ahvaz; 5 November and 15 December for Behbehan and Dezful;and 1 November and 15 December for Izeh, respectively. CERES-Wheat model could be used as a tool to evaluate the effect of sowing date on wheat performance in Khuzestan conditions. Further model evaluations

  2. Predicting protein-ligand affinity with a random matrix framework.

    Science.gov (United States)

    Lee, Alpha A; Brenner, Michael P; Colwell, Lucy J

    2016-11-29

    Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, we show that the similarity of an unknown ligand to the remaining, cleaned chemical features is a robust predictor of ligand-target affinity, performing as well or better than any algorithm in the published literature. We interpret our algorithm as deriving a model for the binding energy between a target receptor and the set of known ligands, where the underlying binding energy model is related to the classic Ising model in statistical physics.

  3. CS2164, a novel multi-target inhibitor against tumor angiogenesis, mitosis and chronic inflammation with anti-tumor potency.

    Science.gov (United States)

    Zhou, You; Shan, Song; Li, Zhi-Bin; Xin, Li-Jun; Pan, De-Si; Yang, Qian-Jiao; Liu, Ying-Ping; Yue, Xu-Peng; Liu, Xiao-Rong; Gao, Ji-Zhou; Zhang, Jin-Wen; Ning, Zhi-Qiang; Lu, Xian-Ping

    2017-03-01

    Although inhibitors targeting tumor angiogenic pathway have provided improvement for clinical treatment in patients with various solid tumors, the still very limited anti-cancer efficacy and acquired drug resistance demand new agents that may offer better clinical benefits. In the effort to find a small molecule potentially targeting several key pathways for tumor development, we designed, discovered and evaluated a novel multi-kinase inhibitor, CS2164. CS2164 inhibited the angiogenesis-related kinases (VEGFR2, VEGFR1, VEGFR3, PDGFRα and c-Kit), mitosis-related kinase Aurora B and chronic inflammation-related kinase CSF-1R in a high potency manner with the IC50 at a single-digit nanomolar range. Consequently, CS2164 displayed anti-angiogenic activities through suppression of VEGFR/PDGFR phosphorylation, inhibition of ligand-dependent cell proliferation and capillary tube formation, and prevention of vasculature formation in tumor tissues. CS2164 also showed induction of G2/M cell cycle arrest and suppression of cell proliferation in tumor tissues through the inhibition of Aurora B-mediated H3 phosphorylation. Furthermore, CS2164 demonstrated the inhibitory effect on CSF-1R phosphorylation that led to the suppression of ligand-stimulated monocyte-to-macrophage differentiation and reduced CSF-1R(+) cells in tumor tissues. The in vivo animal efficacy studies revealed that CS2164 induced remarkable regression or complete inhibition of tumor growth at well-tolerated oral doses in several human tumor xenograft models. Collectively, these results indicate that CS2164 is a highly selective multi-kinase inhibitor with potent anti-tumor activities against tumor angiogenesis, mitosis and chronic inflammation, which may provide the rationale for further clinical assessment of CS2164 as a therapeutic agent in the treatment of cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  4. Molecular modeling of sigma 1 receptor ligands: a model of binding conformational and electrostatic considerations.

    Science.gov (United States)

    Gund, Tamara M; Floyd, Jie; Jung, Dawoon

    2004-01-01

    We have performed molecular modeling studies on several sigma 1 specific ligands, including PD144418, spipethiane, haloperidol, pentazocine, and others to develop a pharmacophore for sigma 1 receptor-ligand binding, under the assumption that all the compounds interact at the same receptor binding site. The modeling studies have investigated the conformational and electrostatic properties of the ligands. Superposition of active molecules gave the coordinates of the hypothetical 5-point sigma 1 pharmacophore, as follows: R1 (0.85, 7.26, 0.30); R2 (5.47, 2.40, -1.51); R3 (-2.57, 4.82, -7.10); N (-0.71, 3.29, -6.40); carbon centroid (3.16, 4.83, -0.60), where R1, R2 were constructed onto the aromatic ring of each compound to represent hydrophobic interactions with the receptor; and R3 represents a hydrogen bond between the nitrogen atom and the receptor. Additional analyses were used to describe secondary binding sites to electronegative groups such as oxygen or sulfur atom. Those coordinates are (2.34, 5.08, -4.18). The model was verified by fitting other sigma 1 receptor ligands. This model may be used to search conformational databases for other possibly active ligands. In conjunction with rational drug design techniques the model may be useful in design and synthesis of novel sigma 1 ligands of high selectivity and potency. Calculations were performed using Sybyl 6.5.

  5. Species difference of CD137 ligand signaling in human and murine monocytes.

    Directory of Open Access Journals (Sweden)

    Qianqiao Tang

    Full Text Available BACKGROUND: Stimulation of CD137 ligand on human monocytes has been shown to induce DC differentiation, and these CD137L-DCs are more potent than classical DCs, in stimulating T cell responses in vitro. To allow an in vivo evaluation of the potency of CD137L-DCs in murine models we aimed at generating murine CD137L-DCs. METHODOLOGY/PRINCIPAL FINDINGS: When stimulated through CD137 ligand murine monocytes responded just as human monocytes with an increased adherence, morphological changes, proliferation and an increase in viable cell numbers. But CD137 ligand signaling did not induce expression of inflammatory cytokines and costimulatory molecules in murine monocytes and these cells had no T cell stimulatory activity. Murine monocytes did not differentiate to inflammatory DCs upon CD137 ligand signaling. Furthermore, while CD137 ligand signaling induces maturation of human immature classical DCs it failed to do so with murine immature classical DCs. CONCLUSIONS/SIGNIFICANCE: These data demonstrate that both human and murine monocytes become activated by CD137 ligand signaling but only human and not murine monocytes differentiate to inflammatory DCs.

  6. Early biochemical recurrence, urinary continence and potency outcomes following robot-assisted radical prostatectomy

    DEFF Research Database (Denmark)

    Berg, Kasper Drimer; Thomsen, Frederik Birkebæk; Hvarness, Helle

    2014-01-01

    OBJECTIVE: The aim of this study was to describe recovery of urinary continence and potency and report oncological and functional outcomes using the survival, continence and potency (SCP) system for patients undergoing robot-assisted radical prostatectomy (RARP). MATERIAL AND METHODS: From 2009...... with preoperative ESI, 77.6% (67.9-86.1) and 34.4% (24.1-47.5) maintained ESI 12 months postoperatively after bilateral and unilateral nerve-sparing surgery (NS), respectively. NS (p .... Using the SCP system and defining potency as ESI, functional and oncological success 12 months after surgery was achieved in 69 out of 135 (51.1%) preoperative continent and potent patients who underwent unilateral or bilateral NS, and did not require adjuvant treatment; when defining potency as IIEF...

  7. In vitro screening of the endocrine disrupting potency of brominated flame retardants and their metabolites

    NARCIS (Netherlands)

    Hamers, T.H.M.; Kamstra, J.H.; Sonneveld, E.; Murk, A.J.; Zegers, B.N.; Boon, J.P.; Brouwer, A.

    2004-01-01

    DEVELOPMENTAL AND REPRODUCTIVE TOXICITY vitro screening the endocrine disrupting potency brominated flame retardants and their metabolites Timo Hamers Jorke Kamstra Edwin Sonneveld Albertinka Murk Bart Zegers Jan Boon Abraham Brouwer Institute for Environmental Studies IVM Amsterdam BioDetection Sys

  8. The role of the 4''-hydroxyl on motilin agonist potency in the 9-dihydroerythromycin series.

    Science.gov (United States)

    Liu, Yaoquan; Carreras, Christopher W; Claypool, Mark; Myles, David C; Shaw, Simon J

    2011-06-15

    The role of the erythromycin 4''-hydroxyl group has been explored on the motilin agonist potential in the 9-dihydroerythromycin series of motilides. The compounds show potencies 2- to 4-fold superior to the corresponding hydroxylated compounds. The relationship is maintained when the 9-hydroxyl is alkylated to generate the corresponding 4''-deoxy-9-O-acetamido-9-dihydroerythromycins. However, concomitant with this increase in potency is an increase in hERG inhibition.

  9. Examining the profile of high-potency cannabis and its association with severity of cannabis dependence.

    Science.gov (United States)

    Freeman, T P; Winstock, A R

    2015-11-01

    Cannabis use is decreasing in England and Wales, while demand for cannabis treatment in addiction services continues to rise. This could be partly due to an increased availability of high-potency cannabis. Adults residing in the UK were questioned about their drug use, including three types of cannabis (high potency: skunk; low potency: other grass, resin). Cannabis types were profiled and examined for possible associations between frequency of use and (i) cannabis dependence, (ii) cannabis-related concerns. Frequent use of high-potency cannabis predicted a greater severity of dependence [days of skunk use per month: b = 0.254, 95% confidence interval (CI) 0.161-0.357, p cannabis was not associated with dependence (days of other grass use per month: b = 0.020, 95% CI -0.029 to 0.070, p = 0.436; days of resin use per month: b = 0.025, 95% CI -0.019 to 0.067, p = 0.245). Frequency of cannabis use (all types) did not predict severity of cannabis-related concerns. High-potency cannabis was clearly distinct from low-potency varieties by its marked effects on memory and paranoia. It also produced the best high, was preferred, and most available. High-potency cannabis use is associated with an increased severity of dependence, especially in young people. Its profile is strongly defined by negative effects (memory, paranoia), but also positive characteristics (best high, preferred type), which may be important when considering clinical or public health interventions focusing on cannabis potency.

  10. A fluorescence lifetime-based binding assay for acetylpolyamine amidohydrolases from Pseudomonas aeruginosa using a [1,3]dioxolo[4,5-f][1,3]benzodioxole (DBD) ligand probe.

    Science.gov (United States)

    Meyners, Christian; Wawrzinek, Robert; Krämer, Andreas; Hinz, Steffen; Wessig, Pablo; Meyer-Almes, Franz-Josef

    2014-08-01

    High-throughput assays for drug screening applications have to fulfill particular specifications. Besides the capability to identify even compounds with low potency, one of the major issues is to minimize the number of false-positive hits in a screening campaign in order to reduce the logistic effort for the subsequent cherry picking and confirmation procedure. In this respect, fluorescence lifetime (FLT) appears as an ideal readout parameter that is supposed to be robust against autofluorescent and light-absorbing compounds, the most common source of systematic false positives. The extraordinary fluorescence features of the recently discovered [1,3]dioxolo[4,5-f][1,3] benzodioxole dyes were exploited to develop an FLT-based binding assay with exceptionally robust readout. The assay setup was comprehensively validated and shown to comply not only with all requirements for a powerful high-throughput screening assay but also to be suitable to determine accurate binding constants for inhibitors against enzymes of the histone deacetylase family. Using the described binding assay, the first inhibitors against three members of this enzyme family from Pseudomonas aeruginosa were identified. The compounds were characterized in terms of potency and selectivity profile. The novel ligand probe should also be applicable to other homologues of the histone deacetylase family that are inhibited by N-hydroxy-N'-phenyloctandiamide.

  11. Probing the links between in vitro potency, ADMET and physicochemical parameters.

    Science.gov (United States)

    Gleeson, M Paul; Hersey, Anne; Montanari, Dino; Overington, John

    2011-03-01

    A common underlying assumption in current drug discovery strategies is that compounds with higher in vitro potency at their target(s) have greater potential to translate into successful, low-dose therapeutics. This has led to the development of screening cascades with in vitro potency embedded as an early filter. However, this approach is beginning to be questioned, given the bias in physicochemical properties that it can introduce early in lead generation and optimization, which is due to the often diametrically opposed relationship between physicochemical parameters associated with high in vitro potency and those associated with desirable absorption, distribution, metabolism, excretion and toxicity (ADMET) characteristics. Here, we describe analyses that probe these issues further using the ChEMBL database, which includes more than 500,000 drug discovery and marketed oral drug compounds. Key findings include: first, that oral drugs seldom possess nanomolar potency (50 nM on average); second, that many oral drugs have considerable off-target activity; and third, that in vitro potency does not correlate strongly with the therapeutic dose. These findings suggest that the perceived benefit of high in vitro potency may be negated by poorer ADMET properties.

  12. The octave potencies convention: a mathematical model of dilution and succussion.

    Science.gov (United States)

    Anick, David J

    2007-07-01

    Several hypothesized explanations for homeopathy posit that remedies contain a concentration of discrete information-carrying units, such as water clusters, nano-bubbles, or silicates. For any such explanation to be sustainable, dilution must reduce and succussion must restore the concentration of these units. Succussion can be modeled by a logistic equation, which leads to mathematical relationships involving the maximum concentration, the average growth of information-carrying units rate per succussion stroke, the number of succussion strokes, and the dilution factor (x, c, or LM). When multiple species of information-carrying units are present, the fastest-growing species will eventually come to dominate, as the potency is increased. An analogy is explored between iterated cycles dilution and succussion, in making homeopathic remedies, and iterated cycles of reseeding and growth, in bacterial cultures. Drawing on this analogy, the active ingredients in low and medium potency remedies may be present at early dilutions but only gradually come to 'dominate', while high potencies may develop from the occurrence of low-probability but faster-growing 'mutations.' Conclusions from this model include: 'x' and 'c' potencies are best compared by the amount of dilution, not the amount of succussion; the minimum number of succussion strokes needed per cycle is proportional to the logarithm of the dilution factor; and a plausible interpretation of why potencies at approximately regular ratios are traditionally used (the octave potencies convention).

  13. The autoxidation activity of new mixed-ligand manganese and iron complexes with tripodal ligands

    NARCIS (Netherlands)

    van Gorkum, R.; Berding, J.; Tooke, D.M.; Spek, A.L.; Reedijk, J.; Bouwman, E.

    2007-01-01

    The activity of new manganese and iron complexes of dianionic tripodal ligands in the autoxidation of ethyl linoleate (EL) is reported. EL consumption rates were monitored using time-resolved FTIR and the degree of oligomerisation was determined by SEC. Almost all complexes showed the same trend in

  14. Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle

    NARCIS (Netherlands)

    Jamal, S.M.; Bouma, A.; Broek, van den J.; Stegeman, J.A.; Chenard, G.; Dekker, A.

    2008-01-01

    The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, typ

  15. High-throughput sequence analysis reveals structural diversity and improved potency among RNA inhibitors of HIV reverse transcriptase.

    Science.gov (United States)

    Ditzler, Mark A; Lange, Margaret J; Bose, Debojit; Bottoms, Christopher A; Virkler, Katherine F; Sawyer, Andrew W; Whatley, Angela S; Spollen, William; Givan, Scott A; Burke, Donald H

    2013-02-01

    Systematic evolution of ligands through exponential enrichment (SELEX) is a well-established method for generating nucleic acid populations that are enriched for specified functions. High-throughput sequencing (HTS) enhances the power of comparative sequence analysis to reveal details of how RNAs within these populations recognize their targets. We used HTS analysis to evaluate RNA populations selected to bind type I human immunodeficiency virus reverse transcriptase (RT). The populations are enriched in RNAs of independent lineages that converge on shared motifs and in clusters of RNAs with nearly identical sequences that share common ancestry. Both of these features informed inferences of the secondary structures of enriched RNAs, their minimal structural requirements and their stabilities in RT-aptamer complexes. Monitoring population dynamics in response to increasing selection pressure revealed RNA inhibitors of RT that are more potent than the previously identified pseudoknots. Improved potency was observed for inhibition of both purified RT in enzymatic assays and viral replication in cell-based assays. Structural and functional details of converged motifs that are obscured by simple consensus descriptions are also revealed by the HTS analysis. The approach presented here can readily be generalized for the efficient and systematic post-SELEX development of aptamers for down-stream applications.

  16. [Dmt(1)]DALDA analogues with enhanced μ opioid agonist potency and with a mixed μ/κ opioid activity profile.

    Science.gov (United States)

    Bai, Longxiang; Li, Ziyuan; Chen, Jiajia; Chung, Nga N; Wilkes, Brian C; Li, Tingyou; Schiller, Peter W

    2014-04-01

    Analogues of [Dmt(1)]DALDA (H-Dmt-d-Arg-Phe-Lys-NH2; Dmt=2',6'-dimethyltyrosine), a potent μ opioid agonist peptide with mitochondria-targeted antioxidant activity, were prepared by replacing Phe(3) with various 2',6'-dialkylated Phe analogues, including 2',6'-dimethylphenylalanine (Dmp), 2',4',6'-trimethylphenylalanine (Tmp), 2'-isopropyl-6'-methylphenylalanine (Imp) and 2'-ethyl-6'-methylphenylalanine (Emp), or with the bulky amino acids 3'-(1-naphthyl)alanine (1-Nal), 3'-(2-naphthyl)alanine (2-Nal) or Trp. Several compounds showed significantly increased μ agonist potency, retained μ receptor selectivity and are of interest as drug candidates for neuropathic pain treatment. Surprisingly, the Dmp(3)-, Imp(3)-, Emp(3)- and 1-Nal(3)-containing analogues showed much increased κ receptor binding affinity and had mixed μ/κ properties. In these cases, molecular dynamics studies indicated conformational preorganization of the unbound peptide ligands due to rotational restriction around the C(β)C(γ) bond of the Xxx(3) residue, in correlation with the observed κ receptor binding enhancement. Compounds with a mixed μ/κ opioid activity profile are known to have therapeutic potential for treatment of cocaine abuse.

  17. Methylphenidate and cocaine have a similar in vivo potency to block dopamine transporters in the human brain

    Energy Technology Data Exchange (ETDEWEB)

    Volkow, N.D. [Brookhaven National Lab., Upton, NY (United States)]|[State Univ. of New York, Stony Brook, NY (United States). Dept. of Psychiatry; Wang, G.J.; Fowler, J.S. [Brookhaven National Lab., Upton, NY (United States)] [and others

    1999-05-28

    The reinforcing effects of cocaine and methylphenidate have been linked to their ability to block dopamine transporters (DAT). Though cocaine and methylphenidate have similar in vitro affinities for DAT the abuse of methylphenidate in humans is substantially lower than of cocaine. To test if differences in in vivo potency at the DAT between these two drugs could account for the differences in their abuse liability the authors compared the levels of DAT occupancies that they had previously reported separately for intravenous methylphenidate in controls and for intravenous cocaine in cocaine abusers. DAT occupancies were measured with Positron Emission Tomography using [{sup 11}C]cocaine, as a DAT ligand, in 8 normal controls for the methylphenidate study and in 17 active cocaine abusers for the cocaine study. The ratio of the distribution volume of [{sup 11}C]cocaine in striatum to that in cerebellum, which corresponds to Bmax/Kd+1, was used as measure of DAT availability. Parallel measures were obtained to assess the cardiovascular effects of these two drugs. Methylphenidate and cocaine produced comparable dose-dependent blockage of DAT with an estimated ED{sub 50} for methylphenidate of 0.07 mg/kg and for cocaine of 0.13 mg/kg. Both drugs induced similar increases in heart rate and blood pressure but the duration of the effects were significantly longer for methylphenidate than for cocaine.

  18. 配位体交换-流动注射分析法测定水中有效氰化物%Determination of available cyanide in water by ligand exchange-flow injection analysis

    Institute of Scientific and Technical Information of China (English)

    乔小宇; 李华昌; 于力; 张萍

    2012-01-01

    A method for the determination of the available cyanide,namely, the total of cyanide including ions (CN-),HCN (aq),as well as zinc,copper,cadmium,mercury,nickel and silver cyanide complexes in water by ligand exchange reagents and CNSolution FS 3100 cyanide analyzer, has been studied. Gaseous HCN which was generated in the reaction module entered into alkaline absorption solution by gas diffusion diaphragm (GD) and was converted to cyanide ions (CN- ) ,and then was detected by ammeter in the flow testing pool. The interference of methanal, phenol, sodium hypochlorite, and sulfide to the determination of CN~ was inspected and removed. The experiment results indicated that, in the range of cyanide concentration between 2. 0-1 000 μg/L,r>0. 999 6;the detection limit of method was 0. 6 μg /L;the relative standard deviation (RSD) (n = 5) was smaller than 2. 5%,the recoveries of real samples were 92%-102%. It can be applied to detect the available cyanide in large a-mount of surface water,groundwater,drinking water and wastewater of petroleum chemical industry, metallurgy, electroplating, papermaking.%研究了采用配位体交换试剂和CNSolution FS 3100氰化物分析系统检测水中有效氰化物(即CN、HCN(aq)以及锌、铜、镉、汞、镍和银的氰络合物的总和)的分析方法.在反应模块中生成的气态HCN通过气体扩散膜片(GD)进入碱性吸收液中并转换为CN,然后进入流动检测池由安培计检测.考察了甲醛类化合物、苯酚化合物、次氯酸钠及硫化物等对CN测定的干扰及消除方法.实验结果表明,CN浓度在2.0~1 000 μg/L范围内,线性相关系数r>0.999 6;检出限为0.6 μg/L;RSD(n=5)<2.5%;实际样品加标回收率为92%~102%.该方法可应用于大批量地表水、地下水、饮用水及石油化工、冶金、电镀、造纸等行业废水中有效氰化物的测定.

  19. Kojic Acid Effect on the Inhibitory Potency of Tyrosinase

    Directory of Open Access Journals (Sweden)

    Forogh Azami

    2017-04-01

    Full Text Available In recent years, enzymatic activity of tyrosinase has been the focus of investigation due to its potential applications in medicine, agriculture and cosmetics. Tyrosinase, entitled polyphenol oxidase, is a key enzyme that catalyzes synthesis of melanin in plants, microorganisms and mammalian cells. Presence of some antioxidants can delay or inhibit the activity of this enzyme as well. In this survey, molecular docking calculation method using Autodock 4.0 software for prediction of binding energy of the protein with some antioxidant ligands was executed. The pose with the lowest energy of binding or inhibition constant was extracted at 298.15 K for kojic acid. Number of conformations in the cluster of rank was 13. The first and second boxes free energy and the inhibition constant were as follows: -5.60 kcalmol-1, 78.99 µM and -3.32 kcalmol-1, 3.66 µM, respectively. Since the first box presented a lower value of free energy, it was considered as the best mode of structure of kojic acid and the protein docking for further analysis. Thus, our present study could contribute to development and discernment of tyrosinase inhibitors in order to prevent hyper pigmentation.

  20. The potency of plant extracts as antimicrobials for the skin or hide preservation

    Science.gov (United States)

    Suparno, Ono; Afifah, Amalia; Panandita, Tania; Marimin, Purnawati, Rini

    2017-03-01

    Preservation of skin or hide uses antimicrobial that will be disposed in wastewater in the skin or hide processing resulting in the environmental pollution. Extracts of some types of plants contain some antimicrobial substances which are potential to be used as biocides for the preservation of skin or hide and are more environmentally friendly. The objectives of this study were to determine the phytochemical contents of moringa, cucumber tree or wuluh starfruit, cherry, and white leadtree or lamtoro leaves and to analyse the antibacterial activities of the plant extracts against microorganisms that cause spoilage of skin or hide. Phytochemical constituents of the dried plant leaves were extracted by 70% ethanol. The resulting extracts were analysed their phytochemical contents and antimicrobial activities against gram negative and gram positive bacteria (inhibition zone test) by well diffusion method, the minimum inhibitory concentration (MIC), and the minimum bactericidal concentration (MBC). Phytochemical test showed that the four leaf extracts contained alkaloids, saponins, tannins, flavonoids, steroids, and glycosides. The inhibition zones of the extracts against Escherichia coli were 5 mm for moringa leaf, 6 mm for cucumber tree leaf, 12 mm for cherry leaf, and 17 mm for white leadtree leaf. Inhibition zone of the extracts against Staphylococcus aureus were 2.5 mm for moringa leaf, 7 mm for cucumber tree leaf, 7.3 mm for cherry leaf, and 13 mm for white leadtree leaf. Inhibition zones of the extracts against Bacillus subtilis were 8 mm for moringa leaf, 9 mm for cucumber tree starfruit leaf, 14 mm for cherry leaf, and 15 mm for white leadtree leaf. The best MIC and MBC tests were demonstrated by white leadtree leaf extract against E. coli found at concentration of 1500 µg/ml, against S. aureus at concentration of 3000 µg/ml, and against B. subtilis at concentration of 3000 µg/ml. The ethanol extract of white leadtree leaf had the best antibacterial activity

  1. Melatonin: functions and ligands.

    Science.gov (United States)

    Singh, Mahaveer; Jadhav, Hemant R

    2014-09-01

    Melatonin is a chronobiotic substance that acts as synchronizer by stabilizing bodily rhythms. Its synthesis occurs in various locations throughout the body, including the pineal gland, skin, lymphocytes and gastrointestinal tract (GIT). Its synthesis and secretion is controlled by light and dark conditions, whereby light decreases and darkness increases its production. Thus, melatonin is also known as the 'hormone of darkness'. Melatonin and analogs that bind to the melatonin receptors are important because of their role in the management of depression, insomnia, epilepsy, Alzheimer's disease (AD), diabetes, obesity, alopecia, migraine, cancer, and immune and cardiac disorders. In this review, we discuss the mechanism of action of melatonin in these disorders, which could aid in the design of novel melatonin receptor ligands.

  2. P3 optimization of functional potency, in vivo efficacy and oral bioavailability in 3-aminopyrazinone thrombin inhibitors bearing non-charged groups at the P1 position.

    Science.gov (United States)

    Isaacs, Richard C A; Newton, Christina L; Cutrona, Kellie J; Mercer, Swati P; Dorsey, Bruce D; McDonough, Colleen M; Cook, Jacquelynn J; Krueger, Julie A; Lewis, S Dale; Lucas, Bobby J; Lyle, Elizabeth A; Lynch, Joseph J; Miller-Stein, Cynthia; Michener, Maria T; Wallace, Audrey A; White, Rebecca B; Wong, Bradley K

    2011-03-01

    Although the S3 pocket of the thrombin active site is lined with lipophilic amino acid residues, the accommodation of polarity within the lipophilic P3 moiety of small molecule inhibitors is possible provided that the polar functionality is capable of pointing away from the binding pocket outwards toward solvent while simultaneously allowing the lipophilic portion of the P3 ligand to interact with the S3 amino acid residues. Manipulation of this motif provided the means to effect optimization of functional potency, in vivo antithrombotic efficacy and oral bioavailability in a series of 3-aminopyrazinone thrombin inhibitors which contained non-charged groups at the P1 position. Copyright © 2010 Elsevier Ltd. All rights reserved.

  3. Dynamics of Antagonistic Potency of Rhodobacter capsulatus PG Lipopolysaccharide against Endotoxin-Induced Effects.

    Science.gov (United States)

    Kabanov, D S; Serov, D A; Zubova, S V; Grachev, S V; Prokhorenko, I R

    2016-03-01

    The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.

  4. The human skin/chick chorioallantoic membrane model accurately predicts the potency of cosmetic allergens.

    Science.gov (United States)

    Slodownik, Dan; Grinberg, Igor; Spira, Ram M; Skornik, Yehuda; Goldstein, Ronald S

    2009-04-01

    The current standard method for predicting contact allergenicity is the murine local lymph node assay (LLNA). Public objection to the use of animals in testing of cosmetics makes the development of a system that does not use sentient animals highly desirable. The chorioallantoic membrane (CAM) of the chick egg has been extensively used for the growth of normal and transformed mammalian tissues. The CAM is not innervated, and embryos are sacrificed before the development of pain perception. The aim of this study was to determine whether the sensitization phase of contact dermatitis to known cosmetic allergens can be quantified using CAM-engrafted human skin and how these results compare with published EC3 data obtained with the LLNA. We studied six common molecules used in allergen testing and quantified migration of epidermal Langerhans cells (LC) as a measure of their allergic potency. All agents with known allergic potential induced statistically significant migration of LC. The data obtained correlated well with published data for these allergens generated using the LLNA test. The human-skin CAM model therefore has great potential as an inexpensive, non-radioactive, in vivo alternative to the LLNA, which does not require the use of sentient animals. In addition, this system has the advantage of testing the allergic response of human, rather than animal skin.

  5. Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors.

    Science.gov (United States)

    Thorsell, Ann-Gerd; Ekblad, Torun; Karlberg, Tobias; Löw, Mirjam; Pinto, Ana Filipa; Trésaugues, Lionel; Moche, Martin; Cohen, Michael S; Schüler, Herwig

    2017-02-23

    Selective inhibitors could help unveil the mechanisms by which inhibition of poly(ADP-ribose) polymerases (PARPs) elicits clinical benefits in cancer therapy. We profiled 10 clinical PARP inhibitors and commonly used research tools for their inhibition of multiple PARP enzymes. We also determined crystal structures of these compounds bound to PARP1 or PARP2. Veliparib and niraparib are selective inhibitors of PARP1 and PARP2; olaparib, rucaparib, and talazoparib are more potent inhibitors of PARP1 but are less selective. PJ34 and UPF1069 are broad PARP inhibitors; PJ34 inserts a flexible moiety into hydrophobic subpockets in various ADP-ribosyltransferases. XAV939 is a promiscuous tankyrase inhibitor and a potent inhibitor of PARP1 in vitro and in cells, whereas IWR1 and AZ-6102 are tankyrase selective. Our biochemical and structural analysis of PARP inhibitor potencies establishes a molecular basis for either selectivity or promiscuity and provides a benchmark for experimental design in assessment of PARP inhibitor effects.

  6. Foot-and-mouth disease vaccine potency testing: the influence of serotype, type of adjuvant, valency, fractionation method, and virus culture on the dose-response curve in cattle.

    Science.gov (United States)

    Jamal, Syed M; Bouma, Annemarie; van den Broek, Jan; Stegeman, Arjan; Chénard, Gilles; Dekker, Aldo

    2008-11-25

    The aim of this study was to determine a relationship between vaccine potency (amount of PD50 per dose) and fraction of clinically protected cattle following homologous challenge with infectious foot-and-mouth disease (FMD) virus, and to determine the effect of method of fractionation, serotype, type of adjuvant, valency and type of virus culture on the dose-response curve. Data from 297 potency tests of FMD vaccines, comprising 4004 vaccinated cattle, performed at the FMD vaccine production facility in the Netherlands, were used for the present study. A generalised linear mixed effect model was used to analyse the results. Our study showed that the relation between FMD vaccine potency and fraction protected was also affected by the serotype and type of adjuvant. No common level of protection could be assigned to all FMD vaccines with the same amount of PD50 per dose, this information is essential when designing a new standard FMD vaccines control.

  7. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    G-quadruplex stabilizing compounds have recently received increased interest due to their potential application as anticancer therapeutics. A significant number of structurally diverse G-quadruplex ligands have been developed. Some of the most potent and selective ligands currently known are macr...

  8. Green synthesis of silver nanoparticles: characterization and determination of antibacterial potency

    Science.gov (United States)

    Annamalai, Jayshree; Nallamuthu, Thangaraju

    2016-02-01

    Silver ions (Ag+) and its compounds are highly toxic to microorganisms, exhibiting strong biocidal effects on many species of bacteria but have a low toxicity toward animal cells. In the present study, silver nanoparticles (SNPs) were biosynthesized using aqueous extract of Chlorella vulgaris as reducing agent and size of SNPs synthesized ranged between 15 and 47 nm. SNPs were characterized by UV-visible spectroscopy, scanning electron microscopy, transmission electron microscopy, X-ray diffraction and Fourier infrared spectroscopy, and analyzed for its antibacterial property against human pathogens. This approach of SNPs synthesis involving green chemistry process can be considered for the large-scale production of SNPs and in the development of biomedicines.

  9. Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.)

    NARCIS (Netherlands)

    Mateo Anson, N.; Berg, R. van den; Havenaar, R.; Bast, A.; Haenen, G.R.M.M.

    2008-01-01

    Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fracti

  10. Ferulic acid from aleurone determines the antioxidant potency of wheat grain (Triticum aestivum L.)

    NARCIS (Netherlands)

    Mateo Anson, N.; Berg, R. van den; Havenaar, R.; Bast, A.; Haenen, G.R.M.M.

    2008-01-01

    Grain is an important source of phytochemicals, which have potent antioxidant capacity. They have been implicated in the beneficial health effect of whole grains in reducing cardiovascular disease and type 2 diabetes. The aim of the present study was to identify the most important antioxidant fracti

  11. Rapid Turbidimetric Assay to Determine the Potency of Daptomycin in Lyophilized Powder

    Directory of Open Access Journals (Sweden)

    Eliane Gandolpho Tótoli

    2015-07-01

    Full Text Available Daptomycin is an important antimicrobial for clinical practice, mainly because it remains very active against Gram-positive resistant strains, such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci. Development of microbiological methods for the analysis of antimicrobials is highly recommended, since they can provide important information about their biological activities, which physicochemical methods are not able to provide. Considering that there are no studies in the literature describing microbiological methods for the analysis of daptomycin, the aim of this work was to validate a microbiological method for the quantitation of daptomycin by the turbidimetric assay. Staphylococcus aureus was used as the test microorganism, and the brain heart infusion broth was used as the culture medium. The validation of the method was performed according to the ICH guidelines, and it was shown to be linear, precise, robust, accurate and selective, over a concentration range of 8.0 to 18.0 µg mL−1. Student’s t-test showed the interchangeability of the proposed method with a previously-validated HPLC method. The developed turbidimetric method described in this paper is a convenient alternative for the routine quality control of daptomycin in its pharmaceutical dosage form.

  12. In silico modelling of permeation enhancement potency in Caco-2 monolayers based on molecular descriptors and random forest.

    Science.gov (United States)

    Welling, Søren H; Clemmensen, Line K H; Buckley, Stephen T; Hovgaard, Lars; Brockhoff, Per B; Refsgaard, Hanne H F

    2015-08-01

    Structural traits of permeation enhancers are important determinants of their capacity to promote enhanced drug absorption. Therefore, in order to obtain a better understanding of structure-activity relationships for permeation enhancers, a Quantitative Structural Activity Relationship (QSAR) model has been developed. The random forest-QSAR model was based upon Caco-2 data for 41 surfactant-like permeation enhancers from Whitehead et al. (2008) and molecular descriptors calculated from their structure. The QSAR model was validated by two test-sets: (i) an eleven compound experimental set with Caco-2 data and (ii) nine compounds with Caco-2 data from literature. Feature contributions, a recent developed diagnostic tool, was applied to elucidate the contribution of individual molecular descriptors to the predicted potency. Feature contributions provided easy interpretable suggestions of important structural properties for potent permeation enhancers such as segregation of hydrophilic and lipophilic domains. Focusing on surfactant-like properties, it is possible to model the potency of the complex pharmaceutical excipients, permeation enhancers. For the first time, a QSAR model has been developed for permeation enhancement. The model is a valuable in silico approach for both screening of new permeation enhancers and physicochemical optimisation of surfactant enhancer systems.

  13. The stress response and anesthetic potency of unilateral spinal anesthesia for total Hip Replacement in geriatric patients.

    Science.gov (United States)

    Zhu, Li; Tian, Chun; Li, Min; Peng, Ming-Qing; Ma, Kun-Long; Wang, Zhong-Lin; Ding, Jia-Hui; Cai, Yi

    2014-11-01

    Recently, some scholars suggested that it is important to keep a stablehemodynamic state and prevent the stress responses in geriatric patients undergoing total hip replacement (THR). We conducted this randomized prospective study to observe anesthetic potency of unilateral spinal anesthesia and stress response to it in geriatric patients during THR. We compared the effect of unilateral spinal and bilateral spinal on inhibition of stress response through measuring Norepinephrine (NE), epinephrine (E) and cortisol (CORT). Plasma concentrations of NE, E and CORT were determined in blood samples using ELISA (enzyme-linked immunosorbent assays) at three time points: To (prior to anesthesia) T1 (at the time point of skin closure), T2 (twenty-four hours after the operation). Sixty patients were randomly divided into two groups: group A (unilateral spinal anesthesia) and group B (conventional bilateral spinal anesthesia). 7.5tymg of hypobaric bupivacaine were injected into subarachnoid cavity at group A and 12mg hypobaric bupivacaine were given at group B. The onset time of sensory and motor block, loss of pinprick sensation, degree of motor block, regression of sensory and motor blocks and hemodynamic changes were also recorded. These data were used to evaluate anesthetic potency of spinal anesthesia. The results of this experiment show that unilateral spinal anesthesia can provide restriction of sensory and motor block, minimize the incidence of hypotension and prevent the stress responses undergoing THR. It is optimal anesthesia procedure for geriatric patients by rapid subarachnoid injection of small doses of bupivacaine.

  14. Similarity-potency trees: a method to search for SAR information in compound data sets and derive SAR rules.

    Science.gov (United States)

    Wawer, Mathias; Bajorath, Jürgen

    2010-08-23

    An intuitive and generally applicable analysis method, termed similarity-potency tree (SPT), is introduced to mine structure-activity relationship (SAR) information in compound data sets of any source. Only compound potency values and nearest-neighbor similarity relationships are considered. Rather than analyzing a data set as a whole, in part overlapping compound neighborhoods are systematically generated and represented as SPTs. This local analysis scheme simplifies the evaluation of SAR information and SPTs of high SAR information content are easily identified. By inspecting only a limited number of compound neighborhoods, it is also straightforward to determine whether data sets contain only little or no interpretable SAR information. Interactive analysis of SPTs is facilitated by reading the trees in two directions, which makes it possible to extract SAR rules, if available, in a consistent manner. The simplicity and interpretability of the data structure and the ease of calculation are characteristic features of this approach. We apply the methodology to high-throughput screening and lead optimization data sets, compare the approach to standard clustering techniques, illustrate how SAR rules are derived, and provide some practical guidance how to best utilize the methodology. The SPT program is made freely available to the scientific community.

  15. Implications of binding mode and active site flexibility for inhibitor potency against the salicylate synthase from Mycobacterium tuberculosis.

    Science.gov (United States)

    Chi, Gamma; Manos-Turvey, Alexandra; O'Connor, Patrick D; Johnston, Jodie M; Evans, Genevieve L; Baker, Edward N; Payne, Richard J; Lott, J Shaun; Bulloch, Esther M M

    2012-06-19

    MbtI is the salicylate synthase that catalyzes the first committed step in the synthesis of the iron chelating compound mycobactin in Mycobacterium tuberculosis. We previously developed a series of aromatic inhibitors against MbtI based on the reaction intermediate for this enzyme, isochorismate. The most potent of these inhibitors had hydrophobic substituents, ranging in size from a methyl to a phenyl group, appended to the terminal alkene of the enolpyruvyl group. These compounds exhibited low micromolar inhibition constants against MbtI and were at least an order of magnitude more potent than the parental compound for the series, which carries a native enolpyruvyl group. In this study, we sought to understand how the substituted enolpyruvyl group confers greater potency, by determining cocrystal structures of MbtI with six inhibitors from the series. A switch in binding mode at the MbtI active site is observed for inhibitors carrying a substituted enolpyruvyl group, relative to the parental compound. Computational studies suggest that the change in binding mode, and higher potency, is due to the effect of the substituents on the conformational landscape of the core inhibitor structure. The crystal structures and fluorescence-based thermal shift assays indicate that substituents larger than a methyl group are accommodated in the MbtI active site through significant but localized flexibility in the peptide backbone. These findings have implications for the design of improved inhibitors of MbtI, as well as other chorismate-utilizing enzymes from this family.

  16. PEGylated dendritic unimolecular micelles as versatile carriers for ligands of G protein-coupled receptors.

    Science.gov (United States)

    Kim, Yoonkyung; Hechler, Béatrice; Gao, Zhan-Guo; Gachet, Christian; Jacobson, Kenneth A

    2009-10-21

    Despite its widespread application in nanomedicine, poly(ethylene glycol) (PEG) is seldom used for covalent modification of ligands for G protein-coupled receptors (GPCRs) due to potential steric complications. In order to study the influence of PEG chains on the biological activity of GPCR ligands bound to a common macromolecular carrier, we prepared a series of G3 polyamidoamine (PAMAM) dendrimers derivatized with Alexa Fluor 488, varying numbers of PEG(550)/PEG(750)/PEG(2000), and nucleoside moieties derived from the A(2A) adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxylethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine). These dendrimer conjugates were purified by size exclusion chromatography and characterized by (1)H NMR and MALDI MS. In radioligand binding assays, some PAMAM-PEG conjugates showed enhanced subtype-selectivity at the human A(2A) AR compared to monomeric ligands of comparable affinity. The functional potency was measured in the A(2A) AR-mediated activation of adenylate cyclase and inhibition of ADP-induced platelet aggregation. Interestingly, the dendrimer conjugate 10c bearing 11 PEG(750) chains (out of theoretical 32 amino end groups) and 14 nucleoside moieties was 5-fold more potent in A(2A) AR-mediated stimulation of cyclic AMP formation than 10d with 4 PEG(2000) chains and 21 nucleosides, although the binding affinities of these 2 compounds were similar. Thus, a relatively small (≤10 nm) multivalent ligand 10c modified for water solubility maintained high potency and displayed increased A(2A) AR binding selectivity over the monomeric nucleosides. The current study demonstrates the feasibility of using short PEG chains in the design of carriers that target ligand-receptor interactions.

  17. Prescription of high-potency corticosteroid agents and clotrimazole-betamethasone dipropionate by pediatricians.

    Science.gov (United States)

    Fleischer, A B; Feldman, S R

    1999-10-01

    Family physicians, internists, and pediatricians are more likely to prescribe combination topical antifungal-topical corticosteroid preparations than are dermatologists. One such preparation, clotrimazole-betamethasone dipropionate, can cause atrophy because it has a high-potency corticosteroid component. We analyzed data from the National Ambulatory Medical Care Survey for visits to pediatricians from 1990 to 1994 and isolated visits at which a topical corticosteroid agent or clotrimazole-betamethasone dipropionate was prescribed. Pediatricians rarely prescribed single-agent, high-potency topical corticosteroid agents in managing patients with skin disorders. High-potency corticosteroid agents accounted for 5.0% of topical corticosteroid prescriptions, whereas 56.3% were low-potency and 38.7% were medium-potency agents. Of the 696,285 mentions of clotrimazole-betamethasone dipropionate, 56.4% (389,920) were for children aged newborn to 4 years; diagnoses were erythematodesquamatous dermatoses, diaper rash, tinea, well-baby visit, and candidiasis. In contrast, for dermatologists nationwide, no drug mention existed for this combination agent for children aged newborn to 4 years over the 5-year study period. Our results show that clotrimazole-betamethasone dipropionate is prescribed inappropriately by pediatricians, especially in the treatment of young children. Pediatricians rarely use high-potency topical corticosteroid agents, but most of their use of clotrimazole-betamethasone dipropionate is in the youngest children, in whom such corticosteroid use is least appropriate. This prescription pattern suggests that some pediatricians may be unaware that clotrimazole-betamethasone dipropionate has a high-potency cortico steroid component.

  18. Predicting protein-ligand affinity with a random matrix framework

    OpenAIRE

    Lee, Alpha Albert; Brenner, MP; Colwell, Lucy Jane

    2016-01-01

    Rapid determination of whether a candidate compound will bind to a particular target receptor remains a stumbling block in drug discovery. We use an approach inspired by random matrix theory to decompose the known ligand set of a target in terms of orthogonal "signals" of salient chemical features, and distinguish these from the much larger set of ligand chemical features that are not relevant for binding to that particular target receptor. After removing the noise caused by finite sampling, ...

  19. Comparative study of estrogenic potencies of estradiol, tamoxifen, bisphenol-A and resveratrol with two in vitro bioassays.

    Science.gov (United States)

    Li, Wen; Seifert, Martin; Xu, Ying; Hock, Bertold

    2004-05-01

    This study was undertaken to compare the sensitivity of two in vitro screening test methods and to determine the accuracy of predicted response to spiked laboratory water samples. A newly developed enzyme-linked receptor assay (ELRA) and a widely used yeast estrogen screen (YES) assay were selected to evaluate the estrogenic responses. Four natural, pharmaceutical, xenobiotic or phytobiotic chemicals: 17beta-estradiol (E2), tamoxifen, bisphenol-A and resveratrol were examined, and 17beta-E2 was used as a positive control. 17beta-E2 can strongly induce estrogenic response in both test systems, however, ELRA was found to be more sensitive to 17beta-E2 with a detection limit of 0.07 microg/l compared to 0.88 microg/l in YES assay. Similar results were obtained for bisphenol-A and resveratrol, and their estrogen potencies relative to E2 (100%) determined by ELRA were at least 5.6 times greater than produced by YES assay. ELRA was unable to distinguish the anti-estrogen tamoxifen and YES assay is also poor at distinguishing. Comparison of response to spiked laboratory water samples show that ELRA can give accurate determination to all four chemicals with recoveries among 70-120%, while YES can only give accurate determination to 17beta-E2 and bisphenol-A with recoveries among 69-112%. The comparative results provide evidence that ELRA is more suitable for rapid screening estrogenic potency of the environmental samples. Combination of ELRA and mammalian cellular assay will constitute an advantageous test to specify agonistic or antagonistic effects.

  20. Detection of iron(III)-binding ligands originating from marine phytoplankton using cathodic stripping voltammetry.

    Science.gov (United States)

    Hasegawa, Hiroshi; Maki, Teruya; Asano, Kohnosuke; Ueda, Kentaro; Ueda, Kazumasa

    2004-01-01

    The sample preparation and analytical methodology are described for detecting biologically produced iron(III)-binding ligands in laboratory cultures of coastal marine phytoplankton. The iron(III)-binding ligands from the culture media were purified by passage through a column packing with a hydrophobic absorbent. The concentrations and stability constants of the ligands were determined by adsorptive cathodic stripping voltammetry with competitive ligand equilibration. The analytical results of the cultivated cultures suggest that eukaryotic phytoplankton would produce iron(III)-binding ligands in analogy with other microorganisms.

  1. Experimental evidence in support of the biological effects and physical basis of homeopathic potencies

    Directory of Open Access Journals (Sweden)

    Nirmal Sukul

    2012-09-01

    Full Text Available Background: Homeopathic potencies 12 cH and above cross the Avogadro number and, for this, do not contain any original drug molecules. Two major problems involved in the scientific study of potencies are (1 understanding the physical basis of potencies and (2 demonstrating the biological effects of potencies. The present study aims to address these questions. Methods and Results: In course of our experimental studies spanned over more than 30 years we have demonstrated significant effects of homeopathic potencies on man, animals and plants. We have also showed that potencies could be differentiated through their electronic spectra, and this difference in spectra can be attributed to the electron transfer interaction. In a molecular complex, electron of one molecule absorbs a quantum of visible radiation and is excited, not to a higher energy level of this molecule, but to one of the vacant high energy levels of the neighboring molecules. This process is known as electron or charge transfer interaction. This has been demonstrated in Iodine Ó© in two different solvents of CCl4 and aqueous ethanol (Sukul N C, Environ Ecol 17,866-872, 1999. We have further demonstrated that the effect of a homeopathic potency can be transmitted from one part of a plant to another, and also from one plant to another through water. I am presenting here a few selected cases of our experimental studies. Potentized Nux vomica significantly reduced ethanol consumption in rats by 73.7%and ethanol-induced sleep time in albino mice by 44.4%. Causticum 30 C and Rhus tox 30 C produced anti-inflamatory and anti-nocicptive effect on adjuvant arthritis in albino rats. Potentized homeopathic drugs reduced microfilaraemia by 28 to 100% and filariasis in two villages of West Bengal endemic for Bancroftian filaiasis. Potentized Cina and Thuja ameliorated trichinellosis in mice reducing larval population in muscles by 84% and 68%, respectively. Potencies of Agaricus and Nux

  2. Rational design and asymmetric synthesis of potent and neurotrophic ligands for FK506-binding proteins (FKBPs).

    Science.gov (United States)

    Pomplun, Sebastian; Wang, Yansong; Kirschner, Alexander; Kozany, Christian; Bracher, Andreas; Hausch, Felix

    2015-01-01

    To create highly efficient inhibitors for FK506-binding proteins, a new asymmetric synthesis for pro-(S)-C(5) -branched [4.3.1] aza-amide bicycles was developed. The key step of the synthesis is an HF-driven N-acyliminium cyclization. Functionalization of the C(5)  moiety resulted in novel protein contacts with the psychiatric risk factor FKBP51, which led to a more than 280-fold enhancement in affinity. The most potent ligands facilitated the differentiation of N2a neuroblastoma cells with low nanomolar potency.

  3. Proteochemometric modelling coupled to in silico target prediction: an integrated approach for the simultaneous prediction of polypharmacology and binding affinity/potency of small molecules.

    Science.gov (United States)

    Paricharak, Shardul; Cortés-Ciriano, Isidro; IJzerman, Adriaan P; Malliavin, Thérèse E; Bender, Andreas

    2015-01-01

    The rampant increase of public bioactivity databases has fostered the development of computational chemogenomics methodologies to evaluate potential ligand-target interactions (polypharmacology) both in a qualitative and quantitative way. Bayesian target prediction algorithms predict the probability of an interaction between a compound and a panel of targets, thus assessing compound polypharmacology qualitatively, whereas structure-activity relationship techniques are able to provide quantitative bioactivity predictions. We propose an integrated drug discovery pipeline combining in silico target prediction and proteochemometric modelling (PCM) for the respective prediction of compound polypharmacology and potency/affinity. The proposed pipeline was evaluated on the retrospective discovery of Plasmodium falciparum DHFR inhibitors. The qualitative in silico target prediction model comprised 553,084 ligand-target associations (a total of 262,174 compounds), covering 3,481 protein targets and used protein domain annotations to extrapolate predictions across species. The prediction of bioactivities for plasmodial DHFR led to a recall value of 79% and a precision of 100%, where the latter high value arises from the structural similarity of plasmodial DHFR inhibitors and T. gondii DHFR inhibitors in the training set. Quantitative PCM models were then trained on a dataset comprising 20 eukaryotic, protozoan and bacterial DHFR sequences, and 1,505 distinct compounds (in total 3,099 data points). The most predictive PCM model exhibited R (2) 0 test and RMSEtest values of 0.79 and 0.59 pIC50 units respectively, which was shown to outperform models based exclusively on compound (R (2) 0 test/RMSEtest = 0.63/0.78) and target information (R (2) 0 test/RMSEtest = 0.09/1.22), as well as inductive transfer knowledge between targets, with respective R (2) 0 test and RMSEtest values of 0.76 and 0.63 pIC50 units. Finally, both methods were integrated to predict the protein

  4. Assessment of toxic potency of complex mixtures of PAHs from Lincoln Creek, Milwaukee, WI

    Energy Technology Data Exchange (ETDEWEB)

    Villeneuve, D.; Crunkilton, R.; DeVita, W. [Univ. of Wisconsin, Stevens Point, WI (United States)

    1995-12-31

    An assay of cytochrome P4501A catalytic activity in PLHC-1 fish hepatoma cells was used to evaluate the toxic potency of dialysates from triolein filled semipermeable polymeric membrane devices (SPMDS) exposed for variable durations and under various flow regimes to water from Lincoln Creek. Toxic potency was expressed as 2,3,7,8 tetrachlorodibenzo-p-dioxin equivalents (TCDD-EQ) calculated from bioassay results. Dose-dependent responses in measured ethoxyresorufin-o-deethylase (EROD) activity of PLHC-1 cells exposed to SPMD dialysates were shown. Toxic potency of dialysates, expressed as bioassay derived TCDD equivalents, increased with duration of SPMD exposure in Lincoln Creek from 2.0 pg/uL for a 2 day exposure to 19.5 pg/uL for a 30 day exposure. This corresponded to an increase in dialysate polycyclic aromatic hydrocarbon (PAH) concentration from 8.82 ug/g after a 2 day exposure to 24.14 ug/g after 30 days. Dialysates from SPMDs exposed to Lincoln Creek stormflow had higher toxic potencies and total PAH concentrations than those exposed to baseflow only, These results suggest that levels of PAH contamination, particularly those associated with stormflow, in Lincoln Creek have potential to accumulate in fish to levels significant enough to elicit a measurable biological response (cytochrome P4501 A induction) at a potency level approaching 0.08% that of TCDD.

  5. Team Potency and Its Impact on Performance via Self-efficacy and Adaptability

    Directory of Open Access Journals (Sweden)

    Rodrigo Bastos Monteiro

    2016-01-01

    Full Text Available In sales, working together as a team for achieving individual performance is a relevant element. In this paper, we suggest a theoretical framework that analyzes the impact of team potency on subjective performance, according to two mechanisms: self-efficacy and adaptability. The hypotheses suggest that (a team potency has a positive relationship with self-efficacy and adaptability; self-efficacy and adaptability impact performance (b directly and (c indirectly, through a mediating role; and (d interpersonal climate quality moderates these associations. We did a national survey with 290 salespeople organized in 101 teams from a water purification company. We used multilevel analyses and results suggested that sales team potency has a main effect on self-efficacy and adaptability. Second, results showed that self-efficacy and adaptability explain subjective performance. Third, self-efficacy and adaptability mediate the association between team potency and individual performance. Fourth, we did not find support for a moderating role of interpersonal climate quality on team potency. Final remarks and future research are discussed in the paper.

  6. Fate and antibacterial potency of anticoccidial drugs and their main abiotic degradation products

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, Martin [Section of Toxicology and Environmental Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)], E-mail: mah@farma.ku.dk; Krogh, Kristine A. [Section of Toxicology and Environmental Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark); Brandt, Asbjorn [Section of Veterinary Medicines, Danish Medicines Agency, Axel Heides Gade 1, DK-2300 Copenhagen (Denmark); Christensen, Jan H. [Section of Soil and Environmental Chemistry, Department of Basic Sciences and Environment, Faculty of Life Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg (Denmark); Halling-Sorensen, Bent [Section of Toxicology and Environmental Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen (Denmark)

    2009-02-15

    The antibacterial potency of eight anticoccidial drugs was tested in a soil bacteria bioassay (pour plate method), EC{sub 50}-values between 2.4 and 19.6 {mu}M were obtained; however, one compound, nicarbazin exhibited an EC{sub 50}-value above the maximum tested concentration (21 {mu}M, 9.1 mg L{sup -1}). The potency of mixtures of two of the compounds, narasin and nicarbazin, was synergistic (more than additive) with 10-fold greater antibacterial potency of the mixture than can be explained by their individual EC{sub 50}-values. The influence of pH, temperature, oxygen concentration and light on the transformation of robenidine and salinomycin was investigated. Robenidine was transformed by photolysis (DT{sub 50} of 4.1 days) and was unstable at low pH (DT{sub 50} of approximately 4 days); salinomycin was merely transformed at low pH, the latter into an unknown number of products. The antibacterial potency of the mixtures of transformation products of robenidine after photolysis and at low pH was comparable with that of the parent compound. Finally five photo-transformation products of robenidine were structural elucidated by accurate mass measurements, i-FIT values (isotopic pattern fit) and MS/MS fragmentation patterns. - Five photo-transformation products of robenidine were structural elucidated. This mixture was found to have similar antibacterial potency as the parent compound.

  7. Glutamate receptor ligands

    DEFF Research Database (Denmark)

    Guldbrandt, Mette; Johansen, Tommy N; Frydenvang, Karla Andrea;

    2002-01-01

    Homologation and substitution on the carbon backbone of (S)-glutamic acid [(S)-Glu, 1], as well as absolute stereochemistry, are structural parameters of key importance for the pharmacological profile of (S)-Glu receptor ligands. We describe a series of methyl-substituted 2-aminoadipic acid (AA......-ray crystallographic analyses, chemical correlation, and CD spectral analyses. The effects of the individual stereoisomers at ionotropic and metabotropic (S)-Glu receptors (iGluRs and mGluRs) were characterized. Compounds with S-configuration at the alpha-carbon generally showed mGluR2 agonist activity of similar...... limited effect on pharmacology. Structure-activity relationships at iGluRs in the rat cortical wedge preparation showed a complex pattern, some compounds being NMDA receptor agonists [e.g., EC(50) =110 microM for (2S,5RS)-5-methyl-AA (6a,b)] and some compounds showing NMDA receptor antagonist effects [e...

  8. Bioactive potency of epidermal mucus extracts from greasy grouper, Epinephelus tauvina (Forsskal, 1775

    Directory of Open Access Journals (Sweden)

    Ganesh Manikantan

    2016-07-01

    Full Text Available Objective: To study the bio-potency of epidermal mucus from Epinephelus tauvina. Methods: Mucus was extracted with acidic, organic and aqueous solvents. Protein, carbohydrate, lipid, amino acid and fatty acid content of mucus extracts were quantified by UV-spectrophotometer, high performance liquid chromatography and gas chromatographymass spectrometer, respectively. Antimicrobial activity was tested against five human and fish pathogens by using agar well diffusion method. The molecular weight of peptides was determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The haemolytic activity of extracts was tested against chick, goat, cow and human red blood cell. Results: Protein contributed with maximum of 26.25% in crude mucus. Arginine was recorded maximum of (133.9 nmol/mL in crude mucus. 2,4,6-Decatrienoic acid and bis (a-chloroethyl sulfone were confirmed in organic extract. The antimicrobial activity of acidic extract was significant. Among the human pathogens, maximum zone of inhibition [(26.0 ± 0.3 mm] was observed against Proteus mirabilis. Whereas, among fish pathogens maximum zone of inhibition [(25.0 ± 0.1 mm] was observed against Vibrio parahemolyticus. The activity of other two extracts was not remarkable. The molecular weight of peptides ranged from 115.5– 37.1 kDa in acidic extract was determined. Chicken and goat blood were found to be highly vulnerable to the lysis. Conclusions: The whole mucus could be a promising source with numerous bioactivepotency. Consequently, this preliminary information suggested that mucus is a source of novel antimicrobial agents for fish and human health related applications.

  9. Human 15-LOX-1 active site mutations alter inhibitor binding and decrease potency.

    Science.gov (United States)

    Armstrong, Michelle; van Hoorebeke, Christopher; Horn, Thomas; Deschamps, Joshua; Freedman, J Cody; Kalyanaraman, Chakrapani; Jacobson, Matthew P; Holman, Theodore

    2016-11-01

    Human 15-lipoxygenase-1 (h15-LOX-1 or h12/15-LOX) reacts with polyunsaturated fatty acids and produces bioactive lipid derivatives that are implicated in many important human diseases. One such disease is stroke, which is the fifth leading cause of death and the first leading cause of disability in America. The discovery of h15-LOX-1 inhibitors could potentially lead to novel therapeutics in the treatment of stroke, however, little is known about the inhibitor/active site interaction. This study utilizes site-directed mutagenesis, guided in part by molecular modeling, to gain a better structural understanding of inhibitor interactions within the active site. We have generated eight mutants (R402L, R404L, F414I, F414W, E356Q, Q547L, L407A, I417A) of h15-LOX-1 to determine whether these active site residues interact with two h15-LOX-1 inhibitors, ML351 and an ML094 derivative, compound 18. IC50 values and steady-state inhibition kinetics were determined for the eight mutants, with four of the mutants affecting inhibitor potency relative to wild type h15-LOX-1 (F414I, F414W, E356Q and L407A). The data indicate that ML351 and compound 18, bind in a similar manner in the active site to an aromatic pocket close to F414 but have subtle differences in their specific binding modes. This information establishes the binding mode for ML094 and ML351 and will be leveraged to develop next-generation inhibitors.

  10. Luteinizing hormone reduction by the male potency herb, Butea superba Roxb.

    Directory of Open Access Journals (Sweden)

    S. Malaivijitnond

    2010-09-01

    Full Text Available To determine if Butea superba Roxb., a traditional Thai male potency herb, has androgenic activity in 60-day-old male Wistar rats, we measured its effects on the pituitary-testicular axis and sex organs. Intact and orchidectomized adult male rats were subdivided into five groups (10 rats/group: distilled water, Butea superba (BS-10, BS-50, BS-250, and testosterone propionate (TP. They received 0, 10, 50, and 250 mg·kg body weight-1·day-1 BS in distilled water by gavage and 6 mg·kg body weight-1·day-1 TP sc, respectively, during the 30-day treatment period. Blood was collected every 15 days and luteinizing hormone (LH, follicle-stimulating hormone (FSH and testosterone were measured. Changes of weight and histological appearance of sex organs were determined at the end of the 30-day treatment and 15-day post-treatment periods. TP treatment reduced serum FSH and LH levels and significantly increased the weight of the seminal vesicles and epididymis, in accordance with histopathological changes, in both intact and orchidectomized rats. No changes in serum testosterone, LH, and FSH levels were observed in any of the intact rats treated with BS, but a significant increase in seminal vesicle weight was observed only in the BS-250 group. Although a significant reduction in serum LH was detected in the BS-50 and BS-250 groups of orchidectomized rats, no significant change in weight or histology of sex organs was observed. Thus, we conclude that B. superba needs endogenous testosterone to work synergistically to stimulate the accessory sex organ of intact animals and can potentially exhibit an LH reduction effect in orchidectomized animals.

  11. Competition for ligands between FGFR1 and FGFR4 regulates Xenopus neural development.

    Science.gov (United States)

    Yamagishi, Masahiro; Okamoto, Harumasa

    2010-01-01

    Cell-surface-localized receptors and their extracellular ligands usually comprise distinct families and promote diversity of signal transduction regulation. The number of available ligand molecules is often the limiting factor for receptor activation during interpretation of the signal by the responding cell. Limited ligand availability in a particular area of tissue should lead to local competition between different members of a receptor family for binding and subsequent activation. Fibroblast growth factor receptor (FGFR) 4 (FGFR4) is a less potent activator of downstream pathways than FGFR1, the major subtype of FGFR. Regional expression of Xenopus FGFR1 and FGFR4 (XFGFR1 and XFGFR4, respectively) overlap in the anterior part of prospective and developing neural tissue. In this paper we show that XFGFR1 and XFGFR4 have opposing effects on the positioning of expression domains of mid- and hindbrain markers when the expression levels of the receptors are altered. We present a line of evidence to support our hypothesis that competition between XFGFR1 and XFGFR4 for ligands is required for correct positioning of marker expression. Local competition between receptors with different potencies should provide an efficient means for a cell to interpret the ligand signal correctly, and may constitute a more general mechanism for regulating signal transduction.

  12. Identification of Soft Matter Binding Peptide Ligands Using Phage Display.

    Science.gov (United States)

    Günay, Kemal Arda; Klok, Harm-Anton

    2015-10-21

    Phage display is a powerful tool for the selection of highly affine, short peptide ligands. While originally primarily used for the identification of ligands to proteins, the scope of this technique has significantly expanded over the past two decades. Phage display nowadays is also increasingly applied to identify ligands that selectively bind with high affinity to a broad range of other substrates including natural and biological polymers as well as a variety of low-molecular-weight organic molecules. Such peptides are of interest for various reasons. The ability to selectively and with high affinity bind to the substrate of interest allows the conjugation or immobilization of, e.g., nanoparticles or biomolecules, or generally, facilitates interactions at materials interfaces. On the other hand, presentation of peptide ligands that selectively bind to low-molecular-weight organic materials is of interest for the development of sensor surfaces. The aim of this article is to highlight the opportunities provided by phage display for the identification of peptide ligands that bind to synthetic or natural polymer substrates or to small organic molecules. The article will first provide an overview of the different peptide ligands that have been identified by phage display that bind to these "soft matter" targets. The second part of the article will discuss the different characterization techniques that allow the determination of the affinity of the identified ligands to the respective substrates.

  13. The effect of electrical lighting power and irradiance on indoor-grown cannabis potency and yield.

    Science.gov (United States)

    Potter, David J; Duncombe, Paul

    2012-05-01

    The floral development and potencies [Δ(9) -tetrahydrocannabinol (THC) contents] of cannabis plants were compared when grown indoors under high-pressure sodium lamps consuming electrical power at three densities (270, 400, and 600 W/m(2)). After a 3-week vegetative phase, plants were grown for 8 weeks, with lamps maintaining an artificial day length of 12 h. Foliar and floral yields were measured. Gas chromatography was used to measure the content of the psychoactive cannabinoid THC. Mean yields per unit of electrical power in each lighting regime ranged from 0.9 to 1.6 g/W, the highest being achieved in the lowest irradiance regime. The individual potencies of the separated leaf and flower materials were not affected by increasing irradiance. However, there was a corresponding increase in the overall potency of the aerial plant tissue. This was because of the plants in brighter conditions producing a higher proportion of floral material.

  14. The potency of cannabis in New Zealand from 1976 to 1996.

    Science.gov (United States)

    Poulsen, H A; Sutherland, G J

    2000-01-01

    The potency of cannabis plant and cannabis products seized in New Zealand over the period of 20 years is studied. The earlier part of the study includes mainly imported cannabis oil and cannabis resin, and both imported and locally grown cannabis plant, that was seized by the police. The later part of the study includes little imported material. Cannabis plant is now locally grown, cannabis oil is locally manufactured and imported cannabis resin is rarely seized. The average potency of the cannabis plant available to the user has not increased over the 20 years period. Cannabis leaf contains on average 1% THC and the female flowering heads on average 3.5% THC. The average potency of cannabis oil has dropped from its peak at 34% THC in 1985 to 13% THC in 1995.

  15. In vitro potency and combination testing of antimicrobial agents against Neisseria gonorrhoeae.

    Science.gov (United States)

    Bharat, Amrita; Martin, Irene; Zhanel, George G; Mulvey, Michael R

    2016-03-01

    Antimicrobial resistant Neisseria gonorrhoeae is a major concern to public health due to decreased susceptibility to frontline antimicrobials. To find agents that are active against N. gonorrhoeae, we tested antimicrobials alone or in combination by Etest gradient strips. The potencies (as assessed by minimum inhibitory concentrations) of twenty-five antimicrobials were evaluated against nine reference strains of N. gonorrhoeae (WHO F, G, K, L, M, N, O, P and ATCC 49226). Potency was greatest for netilmicin, quinupristin-dalfopristin, ceftriaxone, ertapenem and piperacillin-tazobactam. Combinations of azithromycin, moxifloxacin, or gentamicin with ceftriaxone, doripenem, or aztreonam were tested against reference isolates and the fractional inhibitory concentration index (FICI) was calculated. All nine combinations resulted in indifference (>0.5 FICI ≤ 4). Combinations with FICI gonorrhoeae. These data on antimicrobials with higher potency and combinations that did not show antagonism can help to guide larger scale susceptibility studies for antimicrobial resistant N. gonorrhoeae.

  16. Ligand efficiency-based support vector regression models for predicting bioactivities of ligands to drug target proteins.

    Science.gov (United States)

    Sugaya, Nobuyoshi

    2014-10-27

    The concept of ligand efficiency (LE) indices is widely accepted throughout the drug design community and is frequently used in a retrospective manner in the process of drug development. For example, LE indices are used to investigate LE optimization processes of already-approved drugs and to re-evaluate hit compounds obtained from structure-based virtual screening methods and/or high-throughput experimental assays. However, LE indices could also be applied in a prospective manner to explore drug candidates. Here, we describe the construction of machine learning-based regression models in which LE indices are adopted as an end point and show that LE-based regression models can outperform regression models based on pIC50 values. In addition to pIC50 values traditionally used in machine learning studies based on chemogenomics data, three representative LE indices (ligand lipophilicity efficiency (LLE), binding efficiency index (BEI), and surface efficiency index (SEI)) were adopted, then used to create four types of training data. We constructed regression models by applying a support vector regression (SVR) method to the training data. In cross-validation tests of the SVR models, the LE-based SVR models showed higher correlations between the observed and predicted values than the pIC50-based models. Application tests to new data displayed that, generally, the predictive performance of SVR models follows the order SEI > BEI > LLE > pIC50. Close examination of the distributions of the activity values (pIC50, LLE, BEI, and SEI) in the training and validation data implied that the performance order of the SVR models may be ascribed to the much higher diversity of the LE-based training and validation data. In the application tests, the LE-based SVR models can offer better predictive performance of compound-protein pairs with a wider range of ligand potencies than the pIC50-based models. This finding strongly suggests that LE-based SVR models are better than pIC50-based

  17. Observations of the effect of atmospheric processes on the genotoxic potency of airborne particulate matter

    DEFF Research Database (Denmark)

    Feilberg, Anders; Nielsen, Torben; Binderup, Mona-Lise

    2002-01-01

    In this study, the relationship between genotoxic potency and the occurrence of polycyclic aromatic hydrocarbons (PAH), including benzo(a)pyrene (BaP), and nitro-PAH in urban and semi-rural air masses has been investigated. The Salmonella/microsome assay has been used as a measure of genotoxic...... potency. We find that the ratios of BaP/ mutagenicity and PAH/mutagenicity are highly variable. The processes responsible for the variation are formation and degradation of mutagens and transport of polluted air masses from heavily industrialized regions, Air masses from Central Europe are shown...

  18. The spermicidal potency of Coca-Cola and Pepsi-Cola.

    Science.gov (United States)

    Hong, C Y; Shieh, C C; Wu, P; Chiang, B N

    1987-09-01

    The inhibitory effect of Old Coke, caffeine-free New Coke, New Coke, Diet Coke and Pepsi-Cola on human sperm motility was studied with a trans-membrane migration method. None of them could decrease sperm motility to less than 70% of control within one hour. A previous study which claimed a marked variation of spermicidal potencies among different formulations of Coca-Cola could not be confirmed. Even if cola has a spermicidal effect, its potency is relatively weak as compared with other well-known spermicidal agents.

  19. Evaluation of the potency activity of aphrodisiac in Eurycoma longifolia Jack.

    Science.gov (United States)

    Ang, H H; Ikeda, S; Gan, E K

    2001-08-01

    The butanol, methanol, water and chloroform extracts of the roots of Eurycoma longifolia Jack were studied using various tests of potency of treated male rats. The results showed that E. longifolia produced a dose-dependent, recurrent and significant increase in the episodes of penile reflexes as evidenced by increases in quick flips, long flips and erections of the treated male rats during the 30 min observation period. These results provide further evidence that E. longifolia increases the aphrodisiac potency activity in treated animals.

  20. Rationale for further medical and health research on high-potency sweeteners.

    Science.gov (United States)

    Schiffman, Susan S

    2012-10-01

    High-potency or artificial sweeteners have historically been considered inert compounds without physiological consequences other than taste sensations. However, recent data suggest that some of these sweeteners have biological effects that may impact human health. Furthermore, there are significant gaps in our current knowledge of the pharmacokinetics of these sweeteners, their potential for "sweetener-drug interactions" and their impact on appetite and body weight regulation. Nine research needs are described that address some of the major unknown issues associated with ingestion of high-potency sweeteners.

  1. Estrogenic potencies of resorcylic acid lactones and 17 beta-estradiol in female rats.

    Science.gov (United States)

    Everett, D J; Perry, C J; Scott, K A; Martin, B W; Terry, M K

    1987-01-01

    Uterotrophic response in sexually immature female rats has been used to rank the relative estrogenic potencies of six resorcylic acid lactones (RALs) and to compare their activities with that of 17 beta-estradiol. On oral administration, the estrogenic potency relative to 17 beta-estradiol is as follows: 7 alpha-zearalenol, 10 times less; zeranol, 150 times less; taleranol, 350 times less; zearalanone, 400 times less; zearalenone, 650 times less; 7 beta-zearalenol, 3500 times less. On subcutaneous administration, zeranol is 500 times less estrogenic than 17 beta-estradiol.

  2. Desvenlafaxine succinate identifies novel antagonist binding determinants in the human norepinephrine transporter.

    Science.gov (United States)

    Mason, John N; Deecher, Darlene C; Richmond, Rhonda L; Stack, Gary; Mahaney, Paige E; Trybulski, Eugene; Winneker, Richard C; Blakely, Randy D

    2007-11-01

    Desvenlafaxine succinate (DVS) is a recently introduced antagonist of the human norepinephrine and serotonin transporters (hNET and hSERT, respectively), currently in clinical development for use in the treatment of major depressive disorder and vasomotor symptoms associated with menopause. Initial evaluation of the pharmacological properties of DVS (J Pharmacol Exp Ther 318:657-665, 2006) revealed significantly reduced potency for the hNET expressed in membranes compared with whole cells when competing for [(3)H]nisoxetine (NIS) binding. Using hNET in transfected human embryonic kidney-293 cells, this difference in potency for DVS at sites labeled by [(3)H]NIS was found to distinguish DVS, the DVS analog rac-(1-[1-(3-chloro-phenyl)-2-(4-methylpiperazin-1-yl)-ethyl]cyclohexanol (WY-46824), methylphenidate, and the cocaine analog 3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester (RTI-55) from other hNET antagonists, such as NIS, mazindol, tricyclic antidepressants, and cocaine. These differences seem not to arise from preparation-specific perturbations of ligand intrinsic affinity or antagonist-specific surface trafficking but rather from protein conformational alterations that perturb the relationships between distinct hNET binding sites. In an initial search for molecular features that differentially define antagonist binding determinants, we document that Val148 in hNET transmembrane domain 3 selectively disrupts NIS binding but not that of DVS.

  3. In vitro stability, potency, and dissolution of duloxetine enteric-coated pellets after exposure to applesauce, apple juice, and chocolate pudding.

    Science.gov (United States)

    Wells, Kevin A; Losin, William G

    2008-07-01

    Difficulty swallowing is a common problem in the clinical setting, particularly in elderly patients, and can significantly affect an individual's ability to maintain a proper level of nutrition. The purpose of this in vitro study was to determine if mixing duloxetine enteric-coated pellets in food substances is an acceptable alternative method for administering this oral formulation to patients with swallowing difficulties. To determine whether administration in food substances with varying pH values (applesauce and apple juice, pH = approximately 3.5; chocolate pudding, pH = approximately 5.5-6.0) affects the enteric coating of the formulation, duloxetine pellets (ie, the contents of a 20-mg duloxetine capsule) were exposed to applesauce, apple juice, and chocolate pudding at room temperature and tested in triplicate for potency and impurities; for dissolution, 6 replicates were tested. To assess product stability and integrity of the enteric coating, potency, impurities, and dissolution tests of the pellets were conducted and compared with pellets not exposed to food. The duloxetine pellets were extracted from the food material using a solution of 0.1 normal (N) hydrochloric acid (HCl) prepared from concentrated HCl (commercially available) and deionized water. For the potency and impurities tests, a 40:60 solution of acetonitrile and pH 8.0 phosphate buffer was used as the sample solvent to extract the active pharmaceutical ingredient from the formulation to prepare the samples for testing. The amount of active pharmaceutical ingredient released (in vitro dissolution) from the pellets after exposure to the food substances was determined using 2 media solutions, 0.1 N HCl followed by pH 6.8 phosphate buffer. Applesauce and chocolate pudding were selected as vehicles for oral administration, while apple juice was intended to be used as a wash for a nasogastric tube. Mean (SD) potency results for the 20-mg capsule strength were 20.256 (0.066), 20.222 (0.163), and

  4. Effects of Formulation on Microbicide Potency and Mitigation of the Development of Bacterial Insusceptibility.

    Science.gov (United States)

    Cowley, Nicola L; Forbes, Sarah; Amézquita, Alejandro; McClure, Peter; Humphreys, Gavin J; McBain, Andrew J

    2015-10-01

    Risk assessments of the potential for microbicides to select for reduced bacterial susceptibility have been based largely on data generated through the exposure of bacteria to microbicides in aqueous solution. Since microbicides are normally formulated with multiple excipients, we have investigated the effect of formulation on antimicrobial activity and the induction of bacterial insusceptibility. We tested 8 species of bacteria (7 genera) before and after repeated exposure (14 passages), using a previously validated gradient plating system, for their susceptibilities to the microbicides benzalkonium chloride, benzisothiozolinone, chlorhexidine, didecyldimethyl ammonium chloride, DMDM-hydantoin, polyhexamethylene biguanide, thymol, and triclosan in aqueous solution (nonformulated) and in formulation with excipients often deployed in consumer products. Susceptibilities were also assessed following an additional 14 passages without microbicide to determine the stability of any susceptibility changes. MICs and minimum bactericidal concentrations (MBC) were on average 11-fold lower for formulated microbicides than for nonformulated microbicides. After exposure to the antimicrobial compounds, of 72 combinations of microbicide and bacterium there were 19≥4-fold (mean, 8-fold) increases in MIC for nonformulated and 8≥4-fold (mean, 2-fold) increases in MIC for formulated microbicides. Furthermore, there were 20≥4-fold increases in MBC (mean, 8-fold) for nonformulated and 10≥4-fold (mean, 2-fold) increases in MBC for formulated microbicides. Susceptibility decreases fully or partially reverted back to preexposure values for 49% of MICs and 72% of MBCs after further passage. In summary, formulated microbicides exhibited greater antibacterial potency than unformulated actives and susceptibility decreases after repeated exposure were lower in frequency and extent.

  5. Analysis of Cannabis Seizures in NSW, Australia: Cannabis Potency and Cannabinoid Profile

    Science.gov (United States)

    Li, Kong M.; Arnold, Jonathon C.; McGregor, Iain S.

    2013-01-01

    Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ9-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous systematic analysis of the cannabis being used. In the present study we examined the cannabinoid content of 206 cannabis samples that had been confiscated by police from recreational users holding 15 g of cannabis or less, under the New South Wales “Cannabis Cautioning” scheme. A further 26 “Known Provenance” samples were analysed that had been seized by police from larger indoor or outdoor cultivation sites rather than from street level users. An HPLC method was used to determine the content of 9 cannabinoids: THC, CBD, cannabigerol (CBG), and their plant-based carboxylic acid precursors THC-A, CBD-A and CBG-A, as well as cannabichromene (CBC), cannabinol (CBN) and tetrahydrocannabivarin (THC-V). The “Cannabis Cautioning” samples showed high mean THC content (THC+THC-A = 14.88%) and low mean CBD content (CBD+CBD-A = 0.14%). A modest level of CBG was detected (CBG+CBG-A = 1.18%) and very low levels of CBC, CBN and THC-V (cannabis with very low CBD content. The implications for public health outcomes and harm reduction strategies are discussed. PMID:23894589

  6. Potency of a human monoclonal antibody to diphtheria toxin relative to equine diphtheria anti-toxin in a guinea pig intoxication model.

    Science.gov (United States)

    Smith, Heidi L; Cheslock, Peter; Leney, Mark; Barton, Bruce; Molrine, Deborah C

    2016-08-17

    Prompt administration of anti-toxin reduces mortality following Corynebacterium diphtheriae infection. Current treatment relies upon equine diphtheria anti-toxin (DAT), with a 10% risk of serum sickness and rarely anaphylaxis. The global DAT supply is extremely limited; most manufacturers have ceased production. S315 is a neutralizing human IgG1 monoclonal antibody to diphtheria toxin that may provide a safe and effective alternative to equine DAT and address critical supply issues. To guide dose selection for IND-enabling pharmacology and toxicology studies, we dose-ranged S315 and DAT in a guinea pig model of diphtheria intoxication based on the NIH Minimum Requirements potency assay. Animals received a single injection of antibody premixed with toxin, were monitored for 30 days, and assigned a numeric score for clinical signs of disease. Animals receiving ≥ 27.5 µg of S315 or ≥ 1.75 IU of DAT survived whereas animals receiving ≤ 22.5 µg of S315 or ≤ 1.25 IU of DAT died, yielding a potency estimate of 17 µg S315/IU DAT (95% CI 16-21) for an endpoint of survival. Because some surviving animals exhibited transient limb weakness, likely a systemic sign of toxicity, DAT and S315 doses required to prevent hind limb paralysis were also determined, yielding a relative potency of 48 µg/IU (95% CI 38-59) for this alternate endpoint. To support advancement of S315 into clinical trials, potency estimates will be used to evaluate the efficacy of S315 versus DAT in an animal model with antibody administration after toxin exposure, more closely modeling anti-toxin therapy in humans.

  7. Recombinant HA1 produced in E. coli forms functional oligomers and generates strain-specific SRID potency antibodies for pandemic influenza vaccines

    Science.gov (United States)

    Khurana, Surender; Larkin, Christopher; Verma, Swati; Joshi, Manju B.; Fontana, Juan; Steven, Alasdair C.; King, Lisa R.; Manischewitz, Jody; McCormick, William; Gupta, Rajesh K.; Golding, Hana

    2011-01-01

    Vaccine production and initiation of mass vaccination is a key factor in rapid response to new influenza pandemic. During the 2009–2010 H1N1 pandemic, several bottlenecks were identified, including the delayed availability of vaccine potency reagents. Currently, antisera for the single-radial immunodiffusion (SRID) potency assay are generated in sheep immunized repeatedly with HA released and purified after bromelain-treatment of influenza virus grown in eggs. This approach was a major bottleneck for pandemic H1N1 (H1N1pdm09) potency reagent development in 2009. Alternative approaches are needed to make HA immunogens for generation of SRID reagents in the shortest possible time. In this study, we found that properly folded recombinant HA1 globular domain (rHA1) from several type A viruses including H1N1pdm09 and two H5N1 viruses could be produced efficiently by using a bacterial expression system and subsequent purification. The rHA1 proteins were shown to form functional oligomers of trimers, similar to virus derived HA, and elicited high titer of neutralizing antibodies in rabbits and sheep. Importantly, the immune sera formed precipitation rings with reference antigens in the SRID assay in a dose-dependent manner. The HA contents in multiple H1N1 vaccine products from different manufacturers (and in several lots) as determined with the rHA1-generated sheep sera were similar to the values obtained with a traditionally generated sheep serum from NIBSC. We conclude that bacterially-expressed recombinant HA1 proteins can be produced rapidly and used to generate SRID potency reagents shortly after new influenza strains with pandemic potential are identified. PMID:21704111

  8. SuperLigands – a database of ligand structures derived from the Protein Data Bank

    Directory of Open Access Journals (Sweden)

    Preissner Robert

    2005-05-01

    Full Text Available Abstract Background Currently, the PDB contains approximately 29,000 protein structures comprising over 70,000 experimentally determined three-dimensional structures of over 5,000 different low molecular weight compounds. Information about these PDB ligands can be very helpful in the field of molecular modelling and prediction, particularly for the prediction of protein binding sites and function. Description Here we present an Internet accessible database delivering PDB ligands in the MDL Mol file format which, in contrast to the PDB format, includes information about bond types. Structural similarity of the compounds can be detected by calculation of Tanimoto coefficients and by three-dimensional superposition. Topological similarity of PDB ligands to known drugs can be assessed via Tanimoto coefficients. Conclusion SuperLigands supplements the set of existing resources of information about small molecules bound to PDB structures. Allowing for three-dimensional comparison of the compounds as a novel feature, this database represents a valuable means of analysis and prediction in the field of biological and medical research.

  9. SuperLigands – a database of ligand structures derived from the Protein Data Bank

    Science.gov (United States)

    Michalsky, Elke; Dunkel, Mathias; Goede, Andrean; Preissner, Robert

    2005-01-01

    Background Currently, the PDB contains approximately 29,000 protein structures comprising over 70,000 experimentally determined three-dimensional structures of over 5,000 different low molecular weight compounds. Information about these PDB ligands can be very helpful in the field of molecular modelling and prediction, particularly for the prediction of protein binding sites and function. Description Here we present an Internet accessible database delivering PDB ligands in the MDL Mol file format which, in contrast to the PDB format, includes information about bond types. Structural similarity of the compounds can be detected by calculation of Tanimoto coefficients and by three-dimensional superposition. Topological similarity of PDB ligands to known drugs can be assessed via Tanimoto coefficients. Conclusion SuperLigands supplements the set of existing resources of information about small molecules bound to PDB structures. Allowing for three-dimensional comparison of the compounds as a novel feature, this database represents a valuable means of analysis and prediction in the field of biological and medical research. PMID:15943884

  10. Structure-Based Design of Potent and Ligand-Efficient Inhibitors of CTX-M Class A [beta]-Lactamase

    Energy Technology Data Exchange (ETDEWEB)

    Nichols, Derek A.; Jaishankar, Priyadarshini; Larson, Wayne; Smith, Emmanuel; Liu, Guoqing; Beyrouthy, Racha; Bonnet, Richard; Renslo, Adam R.; Chen, Yu (USF); (UCSF); (Clermont)

    2012-07-11

    The emergence of CTX-M class A extended-spectrum {beta}-lactamases poses a serious health threat to the public. We have applied structure-based design to improve the potency of a novel noncovalent tetrazole-containing CTX-M inhibitor (K{sub i} = 21 {mu}M) more than 200-fold via structural modifications targeting two binding hot spots, a hydrophobic shelf formed by Pro167 and a polar site anchored by Asp240. Functional groups contacting each binding hot spot independently in initial designs were later combined to produce analogues with submicromolar potencies, including 6-trifluoromethyl-3H-benzoimidazole-4-carboxylic acid [3-(1H-tetrazol-5-yl)-phenyl]-amide, which had a K{sub i} value of 89 nM and reduced the MIC of cefotaxime by 64-fold in CTX-M-9 expressing Escherichia coli. The in vitro potency gains were accompanied by improvements in ligand efficiency (from 0.30 to 0.39) and LipE (from 1.37 to 3.86). These new analogues represent the first nM-affinity noncovalent inhibitors of a class A {beta}-lactamase. Their complex crystal structures provide valuable information about ligand binding for future inhibitor design.

  11. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Computer program for analysis of the electrical power consumption billing through the A-load and potency hyperboloid in a cold-storage slaughter house; Programa computacional para analise do faturamento do consumo de energia eletrica atraves do A-hiperboloide de carga e potencia em uma empresa frigorifica abatedora de bovinos

    Energy Technology Data Exchange (ETDEWEB)

    Cardim, Delcio [UNESP, Botucatu, SP (Brazil). Faculdade de Ciencias Agronomicas. Programa de Pos-Graduacao em Agronomia]. E-mail: delciocardim@uol.com.br; Gabriel, Luiz Roberto Almeida [UNESP, Presidente Prudente, SP (Brazil). Dept. de Matematica]. E-mail: gabriel@prudente.unesp.br

    2001-12-01

    The purpose of this work was to project and develop a computer program to analyze the billing of the electrical power consumption and the demand of active and reactive potency of a cold-storage slaughter house, by verifying their efficient and rational use of the electrical power. From the electrical standards of a enterprise, it is obtained,during peak and non-peak times, the potency factor (FP), the load factor (FC) and the average active consumption C{sub m}. Basing on the load factor (FC) and potency factor (FP) dta of slaughter house in a specific period, and by means of mathematical models, which govern the electrical consumption billing and demand of both the active and the reactive potency, it was possible to generate the surface denominated A-Load and Potency Hyperboloid. Then, the areas and volumes that were determined by this Hyperboloid were calculated. The results that were obtained through the volumes of the solids, determined by the hyperboloid and the areas of the regions determined by this surface, were used to analyze the situation of the considered slaughter house, regarding the electrical power consumption billing and demand of both the active and the reactive potency. Borland Delphi 5.0 Microsoft Windows environment was used of elaborating the compute program. (author)

  13. Estrogenic potency of benzophenone UV filters in breast cancer cells: proliferative and transcriptional activity substantiated by docking analysis.

    Directory of Open Access Journals (Sweden)

    Gwenneg Kerdivel

    Full Text Available The results from recent studies show that some benzophenones (BPs and their hydroxylated metabolites can function as weak estrogens (E2 in the environment. However, little is known about the structure-activity relationship of these molecules. We have examined the effects of exposure to ten different BPs on the proliferation of estrogen receptor (ER-positive breast cancer cells and on the transcriptional activity of E2-target genes. We analyzed two genes that are tightly linked with estrogen-mediated proliferation, the CXCL12 and amphiregulin genes and two classical estrogen-responsive genes, the pS2 and progesterone receptor. Significant differences in the BPs efficiency to induce cell proliferation and endogenous E2-target gene expressions were observed. Using ERE-, Sp1-, AP1- and C3-reporter genes that contain different ER-binding sites in their promoter, we also showed significant differences in the BPs efficiency in activation of the ER transactivation. Together, our analyzes showed that the most active molecule is 4-hydroxy-BP. Docking analysis of the interaction of BPs in the ligand-binding pocket of ERα suggests that the minimum structural requirement for the estrogenic activity of BPs is a hydroxyl (OH group in the phenyl A-ring that allows interaction with Glu-353, Arg-394 or Phe-404, which enhances the stability between BPs and ERα. Our modeling also indicates a loss of interaction between the OH groups of the phenyl B-ring and His-524. In addition, the presence of some OH groups in the phenyl B-ring can create repulsion forces, which may constrain helix 12 in an unfavorable position, explaining the differential estrogenic effects of BPs. These results, together with our analysis of BPs for their potency in activation of cell proliferation and ER-mediated transcription, report an improved understanding of the mechanism and structure-activity relationship of BPs.

  14. Improving therapeutic HPV peptide-based vaccine potency by enhancing CD4+ T help and dendritic cell activation

    Directory of Open Access Journals (Sweden)

    Hung Chien-Fu

    2010-11-01

    Full Text Available Abstract Background Effective vaccination against human papillomavirus (HPV represents an opportunity to control cervical cancer. Peptide-based vaccines targeting HPV E6 and/or E7 antigens while safe, will most likely require additional strategies to enhance the vaccine potency. Methods We tested the HPV-16 E7 peptide-based vaccine in combination with a strategy to enhance CD4+ T help using a Pan HLA-DR epitope (PADRE peptide and a strategy to enhance dendritic cell activation using the toll-like receptor 3 ligand, poly(I:C. Results We observed that mice vaccinated with E7 peptide-based vaccine in combination with PADRE peptide and poly(I:C generated better E7-specific CD8+ T cell immune responses as well as significantly improved therapeutic anti-tumor effects against TC-1 tumors compared to E7 peptide-based vaccine with either PADRE peptide or poly(I:C alone. Furthermore, we found that intratumoral vaccination with the E7 peptide in conjunction with PADRE peptide and poly(I:C generates a significantly higher frequency of E7-specific CD8+ T cells as well as better survival compared to subcutaneous vaccination with the same regimen in treated mice. Conclusions The combination of PADRE peptide and poly(I:C with antigenic peptide is capable of generating potent antigen-specific CD8+ T cell immune responses and antitumor effects in vaccinated mice. Our study has significant clinical implications for peptide-based vaccination.

  15. A Structural Switch between Agonist and Antagonist Bound Conformations for a Ligand-Optimized Model of the Human Aryl Hydrocarbon Receptor Ligand Binding Domain

    Directory of Open Access Journals (Sweden)

    Arden Perkins

    2014-10-01

    Full Text Available The aryl hydrocarbon receptor (AHR is a ligand-activated transcription factor that regulates the expression of a diverse group of genes. Exogenous AHR ligands include the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, which is a potent agonist, and the synthetic AHR antagonist N-2-(1H-indol-3ylethyl-9-isopropyl-2- (5-methylpyridin-3-yl-9H-purin-6-amine (GNF351. As no experimentally determined structure of the ligand binding domain exists, homology models have been utilized for virtual ligand screening (VLS to search for novel ligands. Here, we have developed an “agonist-optimized” homology model of the human AHR ligand binding domain, and this model aided in the discovery of two human AHR agonists by VLS. In addition, we performed molecular dynamics simulations of an agonist TCDD-bound and antagonist GNF351-bound version of this model in order to gain insights into the mechanics of the AHR ligand-binding pocket. These simulations identified residues 307–329 as a flexible segment of the AHR ligand pocket that adopts discrete conformations upon agonist or antagonist binding. This flexible segment of the AHR may act as a structural switch that determines the agonist or antagonist activity of a given AHR ligand.

  16. Design of HIV-1 protease inhibitors with pyrrolidinones and oxazolidinones as novel P1'-ligands to enhance backbone-binding interactions with protease: synthesis, biological evaluation, and protein-ligand X-ray studies

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Leshchenko-Yashchuk, Sofiya; Anderson, David D.; Baldridge, Abigail; Noetzel, Marcus; Miller, Heather B.; Tie, Yunfeng; Wang, Yuan-Fang; Koh, Yasuhiro; Weber, Irene T.; Mitsuya, Hiroaki; (GSU); (Purdue); (NCI); (Kumamoto University School of Medicine)

    2009-09-02

    Structure-based design, synthesis, and biological evaluation of a series of novel HIV-1 protease inhibitors are described. In an effort to enhance interactions with protease backbone atoms, we have incorporated stereochemically defined methyl-2-pyrrolidinone and methyl oxazolidinone as the P1{prime}-ligands. These ligands are designed to interact with Gly-27{prime} carbonyl and Arg-8 side chain in the S1{prime}-subsite of the HIV protease. We have investigated the potential of these ligands in combination with our previously developed bis-tetrahydrofuran (bis-THF) and cyclopentanyltetrahydrofuran (Cp-THF) as the P2-ligands. Inhibitor 19b with a (R)-aminomethyl-2-pyrrolidinone and a Cp-THF was shown to be the most potent compound. This inhibitor maintained near full potency against multi-PI-resistant clinical HIV-1 variants. A high resolution protein-ligand X-ray crystal structure of 19b-bound HIV-1 protease revealed that the P1{prime}-pyrrolidinone heterocycle and the P2-Cp-ligand are involved in several critical interactions with the backbone atoms in the S1{prime} and S2 subsites of HIV-1 protease.

  17. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    NARCIS (Netherlands)

    Gibbs, S.; Corsini, E.; Spiekstra, S.W.; Galbiati, V.; Fuchs, H.W.; Degeorge, G.; Troese, M.; Hayden, P.; Deng, W.; Roggen, E.

    2013-01-01

    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low

  18. Opportunities and strategies to further reduce animal use for Leptospira vaccine potency testing.

    Science.gov (United States)

    Walker, A; Srinivas, G B

    2013-09-01

    Hamsters are routinely infected with virulent Leptospira for two purposes in the regulation of biologics: the performance of Codified potency tests and maintenance of challenge culture for the Codified potency tests. Options for reducing animal use in these processes were explored in a plenary lecture at the "International Workshop on Alternative Methods for Leptospira Vaccine Potency Testing: State of the Science and the Way Forward" held at the Center for Veterinary Biologics in September 2012. The use of validated in vitro potency assays such as those developed by the U.S. Department of Agriculture for Leptospira (L.) canicola, Leptospira grippotyphosa, Leptospira pomona, and Leptospira icterohaemorrhagiae rather than the Codified hamster vaccination-challenge assay was encouraged. Alternatives such as reduced animal numbers in the hamster vaccination-challenge testing were considered for problematic situations. Specifically, the merits of sharing challenge controls, reducing group sizes, and eliminating animals for concurrent challenge dose titration were assessed. Options for maintaining virulent, stable cultures without serial passage through hamsters or with decreased hamster use were also discussed. The maintenance of virulent Leptospira without the use of live animals is especially difficult since a reliable means to maintain virulence after multiple in vitro passages has not yet been identified. Published by Elsevier Ltd.

  19. Discovery of CC chemokine receptor-3 (CCR3) antagonists with picomolar potency.

    Science.gov (United States)

    De Lucca, George V; Kim, Ui Tae; Vargo, Brian J; Duncia, John V; Santella, Joseph B; Gardner, Daniel S; Zheng, Changsheng; Liauw, Ann; Wang, Zhang; Emmett, George; Wacker, Dean A; Welch, Patricia K; Covington, Maryanne; Stowell, Nicole C; Wadman, Eric A; Das, Anuk M; Davies, Paul; Yeleswaram, Swamy; Graden, Danielle M; Solomon, Kimberly A; Newton, Robert C; Trainor, George L; Decicco, Carl P; Ko, Soo S

    2005-03-24

    Starting with our previously described(20) class of CC chemokine receptor-3 (CCR3) antagonist, we improved the potency by replacing the phenyl linker of 1 with a cyclohexyl linker and by replacing the 4-benzylpiperidine with a 3-benzylpiperidine. The resulting compound, 32, is a potent and selective antagonist of CCR3. SAR studies showed that the 3-acetylphenyl urea of 32 could be replaced with heterocyclic ureas or heterocyclic-substituted phenyl ureas and still maintain the potency (inhibition of eotaxin-induced chemotaxis) of this class of compounds in the low-picomolar range (IC(50) = 10-60 pM), representing some of the most potent CCR3 antagonists reported to date. The potency of 32 for mouse CCR3 (chemotaxis IC(50) = 41 nM) and its oral bioavailability in mice (20% F ) were adequate to assess the efficacy in animal models of allergic airway inflammation. Oral administration of 32 reduced eosinophil recruitment into the lungs in a dose-dependent manner in these animal models. On the basis of its overall potency, selectivity, efficacy, and safety profile, the benzenesulfonate salt of 32, designated DPC168, entered phase I clinical trials.

  20. 75 FR 8937 - Development of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH...

    Science.gov (United States)

    2010-02-26

    ... AGENCY Development of a Relative Potency Factor (RPF) Approach for Polycyclic Aromatic Hydrocarbon (PAH...) Approach for Polycyclic Aromatic Hydrocarbon (PAH) Mixtures'' (EPA/635/R-08/012A). The draft document was... 27, 2010. The listening session on the draft document for PAH mixtures will be held on April 7,...

  1. Validation of self-reported cannabis dose and potency: an ecological study

    NARCIS (Netherlands)

    van der Pol, P.; Liebregts, N.; de Graaf, R.; Korf, D.J.; van den Brink, W.; van Laar, M.

    2013-01-01

    Aims To assess the reliability and validity of self-reported cannabis dose and potency measures. Design Cross-sectional study comparing self-reports with objective measures of amount of cannabis and delta-9-tetrahydrocannabinol (THC) concentration. Setting Ecological study with assessments at

  2. Replacing the NIH test for rabies vaccine potency testing: a synopsis of drivers and barriers

    NARCIS (Netherlands)

    Schiffelers, M.J.; Blaauboer, B.J.; Bakker, W.; Hendriksen, C.F.M.

    2014-01-01

    Approximately 70% of animal use is utilized to demonstrate quality control of vaccines. Especially rabies vaccine potency testing, using the NIH challenge test, involves objections in terms of scientific relevance, animal welfare concern and costs. Several 3R models have been proposed to refine, red

  3. Benzothiophene inhibitors of MK2. Part 2: improvements in kinase selectivity and cell potency.

    Science.gov (United States)

    Anderson, David R; Meyers, Marvin J; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Long, Scott A; Pierce, Betsy S; Mahoney, Matthew W; Mourey, Robert J; Parikh, Mihir D

    2009-08-15

    Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

  4. Benzothiophene inhibitors of MK2. Part 2: Improvements in kinase selectivity and cell potency

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, David R.; Meyers, Marvin J.; Kurumbail, Ravi G.; Caspers, Nicole; Poda, Gennadiy I.; Long, Scott A.; Pierce, Betsy S.; Mahoney, Matthew W.; Mourey, Robert J.; Parikh, Mihir D.; Pfizer

    2010-10-01

    Optimization of kinase selectivity for a set of benzothiophene MK2 inhibitors provided analogs with potencies of less than 500 nM in a cell based assay. The selectivity of the inhibitors can be rationalized by examination of X-ray crystal structures of inhibitors bound to MK2.

  5. Social Capital, Team Efficacy and Team Potency: The Mediating Role of Team Learning Behaviors

    Science.gov (United States)

    van Emmerik, Hetty; Jawahar, I. M.; Schreurs, Bert; de Cuyper, Nele

    2011-01-01

    Purpose: Drawing on social capital theory and self-identification theory, this study aims to examine the associations of two indicators of social capital, personal networks and deep-level similarity, with team capability measures of team efficacy and team potency. The central focus of the study is to be the hypothesized mediating role of team…

  6. Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Webb, Nicholas E; Montefiori, David C; Lee, Benhur

    2015-09-29

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose-response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120-gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

  7. Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay.

    Science.gov (United States)

    Fernández-Salas, Ester; Wang, Joanne; Molina, Yanira; Nelson, Jeremy B; Jacky, Birgitte P S; Aoki, K Roger

    2012-01-01

    Botulinum neurotoxin serotype A (BoNT/A), a potent therapeutic used to treat various disorders, inhibits vesicular neurotransmitter exocytosis by cleaving SNAP25. Development of cell-based potency assays (CBPAs) to assess the biological function of BoNT/A have been challenging because of its potency. CBPAs can evaluate the key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity; and therefore could replace the current mouse bioassay. Primary neurons possess appropriate sensitivity to develop potential replacement assays but those potency assays are difficult to perform and validate. This report describes a CBPA utilizing differentiated human neuroblastoma SiMa cells and a sandwich ELISA that measures BoNT/A-dependent intracellular increase of cleaved SNAP25. Assay sensitivity is similar to the mouse bioassay and measures neurotoxin biological activity in bulk drug substance and BOTOX® product (onabotulinumtoxinA). Validation of a version of this CBPA in a Quality Control laboratory has led to FDA, Health Canada, and European Union approval for potency testing of BOTOX®, BOTOX® Cosmetic, and Vistabel®. Moreover, we also developed and optimized a BoNT/A CBPA screening assay that can be used for the discovery of novel BoNT/A inhibitors to treat human disease.

  8. Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays

    NARCIS (Netherlands)

    Murk, A.J.; Legler, J.; Lipzig, van M.M.H.; Meerman, J.H.N.; Belfroid, A.C.; Spenkelink, A.; Burg, van der B.; Rijs, G.B.J.; Vethaak, D.

    2002-01-01

    A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the E

  9. Antipsychotic-induced extrapyramidal syndromes - Risperidone compared with low- and high-potency conventional antipsychotic drugs

    NARCIS (Netherlands)

    Schillevoort, [No Value; de Boer, A; Herings, RMC; Roos, RAC; Jansen, PAF; Leufkens, HGM

    2001-01-01

    Aim: To compare the risk of extrapyramidal syndromes (EPS) between patients using risperidone and those using low-potency conventional antipsychotic drugs (APDs) in outpatient clinical practice, as measured by the use of anticholinergic medication. We tried to replicate results from previous clinica

  10. Synthesis and pharmacological evaluation of dual acting ligands targeting the adenosine A2A and dopamine D2 receptors for the potential treatment of Parkinson's disease.

    Science.gov (United States)

    Jörg, Manuela; May, Lauren T; Mak, Frankie S; Lee, Kiew Ching K; Miller, Neil D; Scammells, Peter J; Capuano, Ben

    2015-01-22

    A relatively new strategy in drug discovery is the development of dual acting ligands. These molecules are potentially able to interact at two orthosteric binding sites of a heterodimer simultaneously, possibly resulting in enhanced subtype selectivity, higher affinity, enhanced or modified physiological response, and reduced reliance on multiple drug administration regimens. In this study, we have successfully synthesized a series of classical heterobivalent ligands as well as a series of more integrated and "drug-like" dual acting molecules, incorporating ropinirole as a dopamine D2 receptor agonist and ZM 241385 as an adenosine A2A receptor antagonist. The best compounds of our series maintained the potency of the original pharmacophores at both receptors (adenosine A2A and dopamine D2). In addition, the integrated dual acting ligands also showed promising results in preliminary blood-brain barrier permeability tests, whereas the classical heterobivalent ligands are potentially more suited as pharmacological tools.

  11. Functional potencies of dopamine agonists and antagonists at human dopamine D₂ and D₃ receptors.

    Science.gov (United States)

    Tadori, Yoshihiro; Forbes, Robert A; McQuade, Robert D; Kikuchi, Tetsuro

    2011-09-01

    We measured the functional agonist potencies of dopamine agonists including antiparkinson drugs, and functional antagonist potencies of antipsychotics at human dopamine D(2) and D(3) receptors. In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cells expressing low and high densities of human dopamine D(2L) and D(2S) receptors (hD(2L)-Low, hD(2L)-High, hD(2S)-Low and hD(2S)-High, respectively) and human dopamine D(3) Ser-9 and D(3) Gly-9 receptors (hD(3)-Ser-9 and hD(3)-Gly-9, respectively). Cabergoline, bromocriptine, pergolide, (±)-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT), talipexole, pramipexole, R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-olhydrochloride (PD128907) and ropinirole behaved as dopamine D(2) and D(3) receptor full agonists and showed higher potencies in hD(2L)-High and hD(2S)-High compared to hD(2L)-Low and hD(2S)-Low. In hD(3)-Ser-9 and hD(3)-Gly-9 compared to hD(2L)-Low and hD(2S)-Low, dopamine, ropinirole, PD128907, and pramipexole potencies were clearly higher; talipexole and 7-OH-DPAT showed slightly higher potencies; pergolide showed slightly lower potency; and, cabergoline and bromocriptine potencies were lower. Aripiprazole acted as an antagonist in hD(2L)-Low; a low intrinsic activity partial agonist in hD(2S)-Low; a moderate partial agonist in hD(3)-Ser-9 and hD(3)-Gly-9; a robust partial agonist in hD(2L)-High; and a full agonist in hD(2S)-High. Amisulpride, sulpiride and perphenazine behaved as preferential antagonists; and chlorpromazine and asenapine behaved as modest preferential antagonists; whereas fluphenazine, haloperidol, and blonanserin behaved as non-preferential antagonists in hD(2S)-Low and hD(2S)-High compared to hD(3)-Ser-9 and hD(3)-Gly-9. These findings may help to elucidate the basis of therapeutic benefit observed with these drugs, with

  12. Cationic Gold Clusters Ligated with Differently Substituted Phosphines: Effect of Substitution on Ligand Reactivity and Binding

    Energy Technology Data Exchange (ETDEWEB)

    Johnson, Grant E.; Olivares, Astrid M.; Hill, David E.; Laskin, Julia

    2015-01-01

    We present a systematic study of the effect of the number of methyl (Me) and cyclohexyl (Cy) functional groups in monodentate phosphine ligands on the solution-phase synthesis of ligated sub-nanometer gold clusters and their gas-phase fragmentation pathways. Small mixed ligand cationic gold clusters were synthesized using ligand exchange reactions between pre-formed triphenylphosphine ligated (PPh3) gold clusters and monodentate Me- and Cy-substituted ligands in solution and characterized using electrospray ionization mass spectrometry (ESI-MS) and collision-induced dissociation (CID) experiments. Under the same experimental conditions, larger gold-PPh3 clusters undergo efficient exchange of unsubstituted PPh3 ligands for singly Me- and Cy-substituted PPh2Me and PPh2Cy ligands. The efficiency of ligand exchange decreases with an increasing number of Me or Cy groups in the substituted phosphine ligands. CID experiments performed for a series of ligand-exchanged gold clusters indicate that loss of a neutral Me-substituted ligand is preferred over loss of a neutral PPh¬3 ligand while the opposite trend is observed for Cy-substituted ligands. The branching ratio of the competing ligand loss channels is strongly correlated with the electron donating ability of the phosphorous lone pair as determined by the relative proton affinity of the ligand. The results indicate that the relative ligand binding energies increase in the order PMe3 < PPhMe2 < PPh2Me < PPh3< PPh2Cy < PPhCy2< PCy3. Furthermore, the difference in relative ligand binding energies increases with the number of substituted PPh3-mMem or PPh3-mCym ligands (L) exchanged onto each cluster. This study provides the first experimental determination of the relative binding energies of ligated gold clusters containing differently substituted monophosphine ligands, which are important to controlling their synthesis and reactivity in solution. The results also indicate that ligand substitution is an important

  13. Visualization of Metal-to-Ligand and Ligand-to-Ligand Charge Transfer in Metal-Ligand Complexes

    Institute of Scientific and Technical Information of China (English)

    Yong Ding; Jian-xiu Guo; Xiang-si Wang; Sha-sha Liu; Feng-cai Ma

    2009-01-01

    Three methods including the atomic resolved density of state, charge difference density, and the transition density matrix are used to visualize metal to ligand charge transfer (MLCT) in ruthenium(Ⅱ) ammine complex. The atomic resolved density of state shows that there is density of Ru on the HOMOs. All the density is localized on the ammine, which reveals that the excited electrons in the Ru complex are delocalized over the ammine ligand. The charge difference density shows that all the holes are localized on the Ru and the electrons on the ammine. The localization explains the MLCT on excitation. The transition density matrix shows that there is electron-hole coherence between Ru and ammine. These methods are also used to examine the MLCT in Os(bpy)(p0p)Cl ("Osp0p"; bpy=2,2'-bipyridyl; p0p=4,4'-bipyridyl) and the ligand-to-ligand charge transfer (LLCT) in Alq3. The calculated results show that these methods are powerful to examine MLCT and LLCT in the metal-ligand system.

  14. Validation of self-reported cannabis dose and potency: an ecological study.

    Science.gov (United States)

    van der Pol, Peggy; Liebregts, Nienke; de Graaf, Ron; Korf, Dirk J; van den Brink, Wim; van Laar, Margriet

    2013-10-01

    To assess the reliability and validity of self-reported cannabis dose and potency measures. Cross-sectional study comparing self-reports with objective measures of amount of cannabis and delta-9-tetrahydrocannabinol (THC) concentration. Ecological study with assessments at participants' homes or in a coffee shop. Young adult frequent cannabis users (n = 106) from the Dutch Cannabis Dependence (CanDep) study. The objectively measured amount of cannabis per joint (dose in grams) was compared with self-reported estimates using a prompt card and average number of joints made from 1 g of cannabis. In addition, objectively assessed THC concentration in the participant's cannabis was compared with self-reported level of intoxication, subjective estimate of cannabis potency and price per gram of cannabis. Objective estimates of doses per joint (0.07-0.88 g/joint) and cannabis potency (1.1-24.7%) varied widely. Self-reported measures of dose were imprecise, but at group level, average dose per joint was estimated accurately with the number of joints made from 1 g [limit of agreement (LOA) = -0.02 g, 95% confidence interval (CI) = -0.29; 0.26], whereas the prompt card resulted in serious underestimation (LOA = 0.14 g, 95% CI = -0.10; 0.37). THC concentration in cannabis was associated with subjective potency ['average' 3.77% (P = 0.002) and '(very) strong' 5.13% more THC (P < 0.001) than '(very) mild' cannabis] and with cannabis price (about 1% increase in THC concentration per euro spent on 1 g of cannabis, P < 0.001), but not with level of intoxication. Self-report measures relating to cannabis use appear at best to be associated weakly with objective measures. Of the self-report measures, number of joints per gram, cannabis price and subjective potency have at least some validity. © 2013 Society for the Study of Addiction.

  15. Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays.

    Science.gov (United States)

    Murk, Albertinka J; Legler, Juliette; van Lipzig, Marola M H; Meerman, John H N; Belfroid, Angelique C; Spenkelink, Albertus; van der Burg, Bart; Rijs, Gerard B J; Vethaak, Dick

    2002-01-01

    A study was performed to optimize sample preparation and application of three in vitro assays for measuring estrogenic potency in environmental extracts. The three assays applied were an estrogen receptor (ER)-binding assay and two reporter gene effect assays: a yeast estrogen screen (YES) and the ER-mediated chemically activated luciferase gene expression (ER-CALUX) assay. All assays were able to detect estrogenicity, but the amounts of material needed for the assays differed greatly between the three assays (ER-binding assay > YES > ER-CALUX). In addition, in the ER-binding assay, both agonists and antagonists give an estrogenic response, resulting in higher estradiol equivalency (EEQ) levels than both the ER-CALUX and the YES assay for the same samples. The EEQs found in wastewater treatment plants (WTPs) with the ER-CALUX assay were in the range of 4 to 440 and 0.11 to 59 pmol/L for influent and effluent, respectively. Water extracts from four large rivers had levels ranging from 0.25 to 1.72 pmol/L. Extracts from suspended matter and sludge contained estrogenic potency of 0.26 to 2.49 and 1.6 to 41 pmol EEQ/g dry weight, respectively. In WTPs, the average reduction of estrogenic potency in effluent compared to influent was 90 to 95% in municipal WTPs and about 50% in industrial WTPs. In influent, 30% of the ER-CALUX activity could not be explained by the calculated potencies based on chemical analysis of a number of known (xeno)estrogens; in effluent the unexplained fraction was 80%. These first results of analyzing estrogenic potency in WTP water and surface water in The Netherlands indicate that further studies are warranted to investigate the actual risks for aquatic systems.

  16. Riboswitch structure in the ligand-free state.

    Science.gov (United States)

    Liberman, Joseph A; Wedekind, Joseph E

    2012-01-01

    Molecular investigations of riboswitches bound to small-molecule effectors have produced a wealth of information on how these molecules achieve high affinity and specificity for a target ligand. X-ray crystal structures have been determined for the ligand-free state for representatives of the preQ₁-I, S-adenosylmethionine I, lysine, and glycine aptamer classes. These structures in conjunction with complimentary techniques, such as in-line probing, NMR spectroscopy, Förster resonance energy transfer, small-angle scattering, and computational simulations, have demonstrated that riboswitches adopt multiple conformations in the absence of ligand. Despite a number of investigations that support ligand-dependent folding, mounting evidence suggests that free-state riboswitches interact with their effectors in the sub-populations of largely prefolded states as embodied by the principle of conformational selection, which has been documented extensively for protein-mediated ligand interactions. Fundamental riboswitch investigations of the bound and free states have advanced our understanding of RNA folding, ligand recognition, and how these factors culminate in communication between an aptamer and its expression platform. An understanding of these topics is essential to comprehend riboswitch gene regulation at the molecular level, which has already provided a basis to understand the mechanism of action of natural antimicrobials.

  17. Benzothiophene inhibitors of MK2. Part 1: structure-activity relationships, assessments of selectivity and cellular potency.

    Science.gov (United States)

    Anderson, David R; Meyers, Marvin J; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Long, Scott A; Pierce, Betsy S; Mahoney, Matthew W; Mourey, Robert J

    2009-08-15

    Identification of potent benzothiophene inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2), structure-activity relationship (SAR) studies, selectivity assessments against CDK2, cellular potency and mechanism of action are presented. Crystallographic data provide a rationale for the observed MK2 potency as well as selectivity over CDK2 for this class of inhibitors.

  18. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

    NARCIS (Netherlands)

    Hamers, T.H.M.; Brink, van den P.J.; Mos, L.; Linden, van der S.C.; Legler, J.; Koeman, J.H.; Murk, A.J.

    2003-01-01

    In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local

  19. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

    NARCIS (Netherlands)

    Hamers, T.H.M.; Brink, van den P.J.; Mos, L.; Linden, van der S.C.; Legler, J.; Koeman, J.H.; Murk, A.J.

    2003-01-01

    In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricul

  20. Singular Value Decomposition and Ligand Binding Analysis

    Directory of Open Access Journals (Sweden)

    André Luiz Galo

    2013-01-01

    Full Text Available Singular values decomposition (SVD is one of the most important computations in linear algebra because of its vast application for data analysis. It is particularly useful for resolving problems involving least-squares minimization, the determination of matrix rank, and the solution of certain problems involving Euclidean norms. Such problems arise in the spectral analysis of ligand binding to macromolecule. Here, we present a spectral data analysis method using SVD (SVD analysis and nonlinear fitting to determine the binding characteristics of intercalating drugs to DNA. This methodology reduces noise and identifies distinct spectral species similar to traditional principal component analysis as well as fitting nonlinear binding parameters. We applied SVD analysis to investigate the interaction of actinomycin D and daunomycin with native DNA. This methodology does not require prior knowledge of ligand molar extinction coefficients (free and bound, which potentially limits binding analysis. Data are acquired simply by reconstructing the experimental data and by adjusting the product of deconvoluted matrices and the matrix of model coefficients determined by the Scatchard and McGee and von Hippel equation.

  1. Reversible Size Control of Silver Nanoclusters via Ligand-exchange

    KAUST Repository

    Bootharaju, Megalamane Siddaramappa

    2015-05-21

    The properties of atomically monodisperse noble metal nanoclusters (NCs) are intricately intertwined with their precise molecular formula. The vast majority of size-specific NC syntheses start from the reduction of the metal salt and thiol ligand mixture. Only in gold was it recently shown that ligand-exchange could induce the growth of NCs from one atomically precise species to another; a process of yet unknown reversibility. Here, we present a process for the ligand-exchange-induced growth of atomically precise silver NCs, in a biphasic liquid-liquid system, which is particularly of interest because of its complete reversibility and ability to occur at room temperature. We explore this phenomenon in-depth using Ag35(SG)18 [SG= glutathionate] and Ag44(4-FTP)30 [4-FTP= 4-fluorothiophenol] as model systems. We show that the ligand-exchange conversion of Ag35(SG)18 into Ag44(4-FTP)30 is rapid (< 5 min) and direct, while the reverse process proceeds slowly through intermediate cluster sizes. We adapt a recently developed theory of reverse Ostwald ripening to model the NCs’ interconvertibility. The model’s predictions are in good agreement with the experimental observations, and they highlight the importance of small changes in the ligand-metal binding energy in determining the final equilibrium NC size. Based on the insight provided by this model, we demonstrated experimentally that by varying the choice of ligands, ligand-exchange can be used to obtain different sized NCs. The findings in this work establish ligand-exchange as a versatile tool for tuning cluster sizes.

  2. Assessment of chemical skin-sensitizing potency by an in vitro assay based on human dendritic cells.

    Science.gov (United States)

    Lambrechts, Nathalie; Vanheel, Hanne; Nelissen, Inge; Witters, Hilda; Van Den Heuvel, Rosette; Van Tendeloo, Viggo; Schoeters, Greet; Hooyberghs, Jef

    2010-07-01

    The skin-sensitizing potential of chemicals is an important concern for public health and thus a significant end point in the hazard identification process. To determine skin-sensitizing capacity, large research efforts focus on the development of assays, which do not require animals. As such, an in vitro test has previously been developed based on the differential expression of CREM and CCR2 transcripts in CD34(+) progenitor-derived dendritic cells (CD34-DC), which allows to classify chemicals as skin (non-)sensitizing. However, skin sensitization is not an all-or-none phenomenon, and up to now, the assessment of relative potency can only be derived using the in vivo local lymph node assay (LLNA). In our study, we analyzed the feasibility to predict the sensitizing potency, i.e., the LLNA EC3 values, of 15 skin sensitizers using in vitro data from the CD34-DC-based assay. Hereto, we extended the in vitro-generated gene expression data set by an additional source of information, the concentration of the compound that causes 20% cell damage (IC20) in CD34-DC. We statistically confirmed that this IC20 is linearly independent from the gene expression changes but that it does correlate with LLNA EC3 values. In a further analysis, we applied a robust linear regression with both IC20 and expression changes of CREM and CCR2 as explanatory variables. For 13 out of 15 compounds, a high linear correlation was established between the in vitro model and the LLNA EC3 values over a range of four orders of magnitude, i.e., from weak to extreme sensitizers.

  3. Discovery of novel ligands for mouse olfactory receptor MOR42-3 using an in silico screening approach and in vitro validation.

    Directory of Open Access Journals (Sweden)

    Selvan Bavan

    Full Text Available The ligands for many olfactory receptors remain largely unknown despite successful heterologous expression of these receptors. Understanding the molecular receptive range of olfactory receptors and deciphering the olfactory recognition code are hampered by the huge number of odorants and large number of olfactory receptors, as well as the complexity of their combinatorial coding. Here, we present an in silico screening approach to find additional ligands for a mouse olfactory receptor that allows improved definition of its molecular receptive range. A virtual library of 574 odorants was screened against a mouse olfactory receptor MOR42-3. We selected the top 20 candidate ligands using two different scoring functions. These 40 odorant candidate ligands were then tested in vitro using the Xenopus oocyte heterologous expression system and two-electrode voltage clamp electrophysiology. We experimentally confirmed 22 of these ligands. The candidate ligands were screened for both agonist and antagonist activity. In summary, we validated 19 agonists and 3 antagonists. Two of the newly identified antagonists were of low potency. Several previously known ligands (mono- and dicarboxylic acids are also confirmed in this study. However, some of the newly identified ligands were structurally dissimilar compounds with various functional groups belonging to aldehydes, phenyls, alkenes, esters and ethers. The high positive predictive value of our in silico approach is promising. We believe that this approach can be used for initial deorphanization of olfactory receptors as well as for future comprehensive studies of molecular receptive range of olfactory receptors.

  4. The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)*

    Science.gov (United States)

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases. PMID:24860101

  5. Teratogenic potency of valproate analogues evaluated by quantitative estimation of cellular morphology in vitro.

    Science.gov (United States)

    Berezin, V; Kawa, A; Bojic, U; Foley, A; Nau, H; Regan, C; Edvardsen, K; Bock, E

    1996-10-01

    To develop a simple prescreening system for teratogenicity testing, a novel in vitro assay was established using computer assisted microscopy allowing automatic delineation of contours of stained cells and thereby quantitative determination of cellular morphology. The effects of valproic acid (VPA) and analogues with high as well as low teratogenic activities-(as previously determined in vivo)-were used as probes for study of the discrimination power of the in vitro model. VPA, a teratogenic analogue (+/-)-4-en-VPA, and a non-teratogenic analogue (E)-2-en-VPA, as well as the purified (S)- and (R)-enantiomers of 4-yn-VPA (teratogenic and non-teratogenic, respectively), were tested for their effects on cellular morphology of cloned mouse fibroblastoid L-cell lines, neuroblastoma N2a cells, and rat glioma BT4Cn cells, and were found to induce varying increases in cellular area: Furthermore, it was demonstrated that under the chosen conditions the increase in area correlated statistically significantly with the teratogenic potency of the employed compounds. Setting the cellular area of mouse L-cells to 100% under control conditions, the most pronounced effect was observed for (S)-4-yn-VPA (211%, P = < 0.001) followed by VPA (186%, P < 0.001), 4-en-VPA (169%, P < 0.001) and non-teratogenic 2-en-VPA (137%, P < 0.005) and (R)-4-yn-VPA (105%). This effect was independent of the choice of substrata, since it was observed on L-cells grown on plastic, fibronectin, laminin and Matrigel. However, when VPA-treated cells were exposed to an arginyl-glycyl-aspartate (RGD)-containing peptide to test whether VPA treatment was able to modulate RGD-dependent integrin interactions with components of the extracellular matrix, hardly any effect could be observed, whereas control cells readily detached from the substratum, indicating a changed substrate adhesion of the VPA-treated cells. The data thus indicate that measurement of cellular area may serve as a simple in vitro test in the

  6. Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol.

    Science.gov (United States)

    Van den Belt, K; Berckmans, P; Vangenechten, C; Verheyen, R; Witters, H

    2004-02-10

    The estrogenic activity of compounds was evaluated in a comparative approach both with in vitro and in vivo assays. By comparing simultaneously obtained experimental data, we evaluated the differences in response sensitivity (by EC10) and concentration-response relationships (including EC50) in order to get an idea about the predictive value of in vitro assays for in vivo estrogenic potencies or effects in fish. Two human estrogen receptor-based assays, the MVLN-assay (transformed MCF-7 human breast cancer cell line) and the yeast estrogen screen (YES-screen) were used for the in vitro evaluation of the estrogenic potencies. An in vivo model with the female zebrafish (Danio rerio) with plasma vitellogenin (VTG) as a biomarker for exposure and the ovarian somatic index (OSI) as an effect endpoint was used for the in vivo work. Compounds tested were 17beta-estradiol (E2), estrone (E1), 17alpha-ethynylestradiol (EE2) and the alkylphenolic compound nonylphenol (NP). All compounds were found to be estrogenic in both in vitro assays and were able to induce VTG and to reduce the ovarian somatic index in female zebrafish. The MVLN-assay appeared up to 15 times more sensitive than the YES-screen. Concentration-response relationships, determined by EC10 and EC50 (concentration of test compound causing 10% or 50% effect compared to control) for VTG and OSI were of the same order of magnitude, indicating that VTG induction as an exposure biomarker can be predictive for effects on ovaries in females. We further demonstrated that for E1 and NP, the in vitro observed estrogenic potencies, based on EC50 values, were of the same order of magnitude as the in vivo estrogenic potencies. For EE2, a difference between in vitro and in vivo relative estrogenic potency was observed, being about 25 times more potent in vivo than could be expected based on the in vitro results. These experimental results showed the suitability of in vitro assays for screening purposes with qualitative

  7. Vasopeptidase-activated latent ligands of the histamine receptor-1.

    Science.gov (United States)

    Gera, Lajos; Roy, Caroline; Charest-Morin, Xavier; Marceau, François

    2013-11-01

    Whether peptidases present in vascular cells can activate prodrugs active on vascular cells has been tested with 2 potential latent ligands of the histamine H1 receptor (H1R). First, a peptide consisting of the antihistamine cetirizine (CTZ) condensed at the N-terminus of ε-aminocaproyl-bradykinin (εACA-BK) was evaluated for an antihistamine activity that could be revealed by degradation of the peptide part of the molecule. CTZ-εACA-BK had a submicromolar affinity for the BK B2 receptor (B2R; IC50 of 590 nM, [(3)H]BK binding competition), but a non-negligible affinity for the human H1 receptor (H1R; IC50 of 11 μM for [(3)H]pyrilamine binding). In the human isolated umbilical vein, a system where both endogenous B2R and H1R mediate strong contractions, CTZ-εACA-BK exerted mild antagonist effects on histamine-induced contraction that were not modified by omapatrilat or by a B2R antagonist that prevents endocytosis of the BK conjugate. Cells expressing recombinant ACE or B2R incubated with CTZ-εACA-BK did not release a competitor of [(3)H]pyrilamine binding to H1Rs. Thus, there is no evidence that CTZ-εACA-BK can release free cetirizine in biological environments. The second prodrug was a blocked agonist, L-alanyl-histamine, potentially activated by aminopeptidase N (APN). This compound did not compete for [(3)H]pyrilamine binding to H1Rs. The human umbilical vein contractility assay responded to L-alanyl-histamine (EC50 54.7 μM), but the APN inhibitor amastatin massively (17-fold) reduced its apparent potency. Amastatin did not influence the potency of histamine as a contractile agent. One of the 2 tested latent H1R ligands, L-alanyl-histamine, supported the feasibility of pro-drug activation by vascular ectopeptidases.

  8. Synthesis and structural determination of zinc complexes based on an anilido-aldimine ligand containing an O-donor pendant arm: zinc alkoxide derivative as an efficient initiator for ring-opening polymerization of cyclic esters.

    Science.gov (United States)

    Wang, Chao-Hsiang; Li, Chen-Yu; Huang, Bor-Hunn; Lin, Chu-Chieh; Ko, Bao-Tsan

    2013-08-14

    Zinc complexes bearing the anilido-aldiminate AA(OMe) ligand (AA(OMe)-H = (E)-2,6-diisopropyl-N-(2-(((2-methoxyethyl)imino)methyl)phenyl)aniline) were synthesized in a stepwise method and were structurally characterized. The reaction of AA(OMe)-H (1) with one equivalent of diethyl zinc (ZnEt2) furnishes a three-coordinated and mononuclear zinc complex [(AA(OMe))ZnEt] (2). Further reaction of 2 with a stoichiometric amount of benzyl alcohol (BnOH) affords a four-coordinated and dinuclear zinc benzylalkoxide complex [(AA(OMe))Zn(μ-OBn)]2 (3). In the presence of two equivalents of AA(OMe)-H with ZnEt2, a homoleptic and four-coordinated zinc complex [(AA(OMe))2Zn] (4) is formed. The geometry around the zinc centres of 3 and 4 are both distorted tetrahedrals, while 2 adopts a different coordination mode with a slightly distorted trigonal planar geometry. The variable-temperature (1)H NMR studies of 3 illustrate that 3 exhibits a dinuclear structure in solution at low temperature as well as in the solid state. While raising the temperature, it drifts towards dissociation to form a mononuclear zinc benzylalkoxide species, which coexists in solution. The ring-opening polymerizations of ε-caprolactone (ε-CL) and β-butyrolactone (β-BL) catalyzed by complexes 3 and 4 are investigated. The ε-CL and β-BL polymerizations initiated by zinc alkoxide 3 were demonstrated to have living characteristics and to proceed in a controlled manner with narrow polydispersity indices (PDIs high monomer-to-initiator ratio (1200/1) initiated by 3 has also been reported.

  9. A race for RAGE ligands.

    Science.gov (United States)

    Schleicher, Erwin D

    2010-08-01

    In experimental animals a causal involvement of the multiligand receptor for advanced glycation end products (RAGE) in the development of diabetic vascular complications has been demonstrated. However, the nature of RAGE ligands present in patients with diabetic nephropathy has not yet been defined; this leaves open the relevance of the RAGE system to the human disease.

  10. Novel histamine H3 receptor antagonists: affinities in an H3 receptor binding assay and potencies in two functional H3 receptor models.

    OpenAIRE

    Schlicker, E.; Kathmann, M; Reidemeister, S.; Stark, H.; Schunack, W

    1994-01-01

    1. We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring-containing alkyl residue [corrected]. 2. The specific binding o...

  11. Nutrient Potency of Rice Straw Processed with Amofer as Cattle Feed Stuff in East Kalimantan

    Directory of Open Access Journals (Sweden)

    Hamdi Mayulu

    2015-12-01

    Full Text Available Forage demand can be supplied from rice straw which processed with certain feed technology innovation. Some advantages can be derived under this technology i.e. increasing nutrient content, optimizing the utilization, improving the efficiency and reducing production cost by minimizing feed cost. Ammoniation fermentation (amofer as an applied technology could give solution over the limitation of rice straw availability. The objective of this research was to determine the potency of rice straw processed with amofer (amofer-rice straw as raw material to formulate complete feed. The experiment was carried out with randomized block design with three treatments and six replications. Amofer treatment was carried out by adding urea 3% from the total material and then placed into plastic jar +12 liter and then incubated under an-aerobic process for 18 days. Fermentation material i.e. biology starter by 1% of total material was added at the ninth day. Dry Matter (DM content resulted from each treatments was 87,28%, 85,96% and 84,61% for T1, T2 and T3, respectively. Crude Protein (CP content was T1=24,48%, T2=21,04%; and T3=24,46%, Crude Fiber (CF content was T1=31,30%; T2=31,30%; and T3=31,39%. Total Digestible Nutrient (TDN for version 1 was  T1=57,29; T2=56,19; and T3=56,89 and version 2 was T1=53,11%; T2=52,28%; and T3=51,10%. The average value of Non Nitrogen Free Extract (NNFE was T1=23,49%; T2=28,08%; and T3=26,57%. The utilization of amofer as applicable technology is considered as the most appropriate method to increase the quality of rice straw by significantly increase nutrient content in term of crude protein (CP and reduce crude fiber (CF content. This increasing quality can be seen from the result of proximate analysis, NNFE and TDN content

  12. Charge-Transfer Effects in Ligand Exchange Reactions of Au25 Monolayer-Protected Clusters.

    Science.gov (United States)

    Carducci, Tessa M; Blackwell, Raymond E; Murray, Royce W

    2015-04-16

    Reported here are second-order rate constants of associative ligand exchanges of Au25L18 nanoparticles (L = phenylethanethiolate) of various charge states, measured by proton nuclear magnetic resonance at room temperature and below. Differences in second-order rate constants (M(-1) s(-1)) of ligand exchange (positive clusters ∼1.9 × 10(-5) versus negative ones ∼1.2 × 10(-4)) show that electron depletion retards ligand exchange. The ordering of rate constants between the ligands benzeneselenol > 4-bromobenzene thiol > benzenethiol reveals that exchange is accelerated by higher acidity and/or electron donation capability of the incoming ligand. Together, these observations indicate that partial charge transfer occurs between the nanoparticle and ligand during the exchange and that this is a rate-determining effect in the process.

  13. Quantitative Analysis of Multivalent Ligand Presentation on Gold Glyconanoparticles and Their Effects on Protein Binding

    Science.gov (United States)

    Wang, Xin; Ramström, Olof; Yan, Mingdi

    2010-03-01

    Bio-functionalized nanomaterials, which combine functions of biological ligands and unique properties of nano-sized building blocks, have exhibited increased potential applications in biosensing, therapeutics, and diagnostics. Glyconanoparitcles carrying a monolayer of carbohydrate ligands on nanoparticles provide an excellent platform for sensitive protein recognitions. Using Au nanoparticles as the scaffold, multivalent interactions between glycan ligands and proteins have been demonstrated. However, quantitative analysis especially the binding affinity of the resulting glyconanoparticles is challenging to determine. Here we present a new characterization technique, based on fluorescent competition binding assays, for measuring dissociation constants for glyconanoparticles-protein interactions. Au nanoparticles coupled with a series of un-derivatized carbohydrates were prepared by a photocoupling chemistry. Dramatic binding affinity enhancement was observed due to the high ligand density on nanoparticles, which was highly relevant to ligand display, controlled by the linker type, chain length, ligand size and density.

  14. Potency trends of Δ9-THC and other cannabinoids in confiscated cannabis preparations from 1993 to 2008.

    Science.gov (United States)

    Mehmedic, Zlatko; Chandra, Suman; Slade, Desmond; Denham, Heather; Foster, Susan; Patel, Amit S; Ross, Samir A; Khan, Ikhlas A; ElSohly, Mahmoud A

    2010-09-01

    The University of Mississippi has a contract with the National Institute on Drug Abuse (NIDA) to carry out a variety of research activities dealing with cannabis, including the Potency Monitoring (PM) program, which provides analytical potency data on cannabis preparations confiscated in the United States. This report provides data on 46,211 samples seized and analyzed by gas chromatography-flame ionization detection (GC-FID) during 1993-2008. The data showed an upward trend in the mean Δ(9)-tetrahydrocannabinol (Δ(9)-THC) content of all confiscated cannabis preparations, which increased from 3.4% in 1993 to 8.8% in 2008. Hashish potencies did not increase consistently during this period; however, the mean yearly potency varied from 2.5-9.2% (1993-2003) to 12.0-29.3% (2004-2008). Hash oil potencies also varied considerably during this period (16.8 ± 16.3%). The increase in cannabis preparation potency is mainly due to the increase in the potency of nondomestic versus domestic samples.

  15. A Semi-quantitative Serological Method to Assess the Potency of Inactivated Rabies Vaccine for Veterinary Use

    Institute of Scientific and Technical Information of China (English)

    Ye Liu; Shoufeng Zhang; Fei Zhang; Rongliang Hu

    2012-01-01

    Potency is one of the most important indexes of inactivated vaccines.A number of methods have been established to assay the potency,of which the NIH test and single-dose mouse protection test are the "prescribed methods".Here,we report a method to semi-quantitatively assay the potency of an inactivated rabies vaccine,which uses fewer animals and takes less time to complete.Depending on the quality requirements of a vaccine(e.g.minimum potency),a rabies reference vaccine is,for example,diluted to the minimum potency,and 50 μL of the dilution is taken to inoculate 10 mice.The same amount of the test rabies vaccine is inoculated into another 10 mice.After two weeks,all mice are bled and serum samples are assayed for viral neutralizing antibody by the fluorescent antibody virus neutralization(FAVN) test.By comparing the median and interquartile range of antibody titers of the reference vaccine with those of the test vaccine,the test vaccine potency can be semi-quantitatively judged as to whether it is in accord with the required quality.The reliability of this method was also confirmed in dogs.The procedure can be recommended for batch potency testing during inactivated rabies vaccine production.

  16. Estimating Potency in High-Throughput Screening Experiments by Maximizing the Rate of Change in Weighted Shannon Entropy.

    Science.gov (United States)

    Shockley, Keith R

    2016-06-15

    High-throughput in vitro screening experiments can be used to generate concentration-response data for large chemical libraries. It is often desirable to estimate the concentration needed to achieve a particular effect, or potency, for each chemical tested in an assay. Potency estimates can be used to directly compare chemical profiles and prioritize compounds for confirmation studies, or employed as input data for prediction modeling and association mapping. The concentration for half-maximal activity derived from the Hill equation model (i.e., AC50) is the most common potency measure applied in pharmacological research and toxicity testing. However, the AC50 parameter is subject to large uncertainty for many concentration-response relationships. In this study we introduce a new measure of potency based on a weighted Shannon entropy measure termed the weighted entropy score (WES). Our potency estimator (Point of Departure, PODWES) is defined as the concentration producing the maximum rate of change in weighted entropy along a concentration-response profile. This approach provides a new tool for potency estimation that does not depend on the assumption of monotonicity or any other pre-specified concentration-response relationship. PODWES estimates potency with greater precision and less bias compared to the conventional AC50 assessed across a range of simulated conditions.

  17. Sanctioning Large-Scale Domestic Cannabis Production - Potency, Yield and Professionalism

    DEFF Research Database (Denmark)

    Møller, Kim; Lindholst, Christian

    2014-01-01

    Domestically cultivated cannabis, referred to as sinsemilla, constitutes a growing share of the illicit drug markets in the Scandinavian countries. In this study we present forensic evidence of THC content in sinsemilla and resin confiscated by the Danish police from 2008 to 2012. The purpose...... is to establish a mean potency, a yield estimate for professionally grown plants, and an assessment of how „professionalism” applies to domestic cultivation. We find a mean THC content in our sample normally distributed around 12% for sinsemilla (n=35) and 11.7% for resin (n=99). This is markedly higher than...... the assumption of THC content in sinsemilla between 6-8% as is currently applied in Norway and Sweden. It is also higher than the previous estimate of resin potency. The yield in sinsemilla plants is found to be 46 grams per plant. This is much lower than the estimate applied in criminal cases, which indicates...

  18. Evaluation of monoquaternary pyridinium oximes potency to reactivate tabun-inhibited human acetylcholinesterase.

    Science.gov (United States)

    Odzak, Renata; Calić, Maja; Hrenar, Tomica; Primozic, Ines; Kovarik, Zrinka

    2007-04-20

    Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH(3), Py-4-Br, Py-4-Cl and Py-4-NO(2)) were synthesized in order to examine their potency as reactivators of tabun-inhibited human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Within 24h, the reactivation of tabun-inhibited AChE reached 80% with Py-4-CH(3), Py-4-Br and Py-4-Cl, 40% with Py-4-NO(2), and 30% with Py-4-H. The overall reactivation rate constants were up to 5.0min(-1)M(-1). All oximes inhibited human AChE reversibly, and the inhibition potency increased in the following order Py-4-Brtabun-phosphorylated human AChE.

  19. Combination of isothermal titration calorimetry and time-resolved luminescence for high affinity antibody-ligand interaction thermodynamics and kinetics

    Science.gov (United States)

    Aweda, Tolulope A.; Meares, Claude F.

    2011-01-01

    For experiments using synthetic ligands as probes for biological experiments, it is useful to determine the specificity and affinity of the ligands for their receptors. As ligands with higher affinities are developed (KA >108 M−1; KD calorimetry measures heat produced or consumed during ligand binding, and also provides the equilibrium binding constant. However, as normally practiced, its range is limited. Displacement titration, where a competing weaker ligand is used to lower the apparent affinity of the stronger ligand, can be used to determine the binding affinity as well as the complete thermodynamic data for ligand-antibody complexes with very high affinity. These equilibrium data have been combined with kinetic measurements to yield the rate constants as well. We describe this methodology, using as an example antibody 2D12.5, which captures yttrium S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate. PMID:21964396

  20. Conformational energy range of ligands in protein crystal structures: The difficult quest for accurate understanding.

    Science.gov (United States)

    Peach, Megan L; Cachau, Raul E; Nicklaus, Marc C

    2017-02-24

    In this review, we address a fundamental question: What is the range of conformational energies seen in ligands in protein-ligand crystal structures? This value is important biophysically, for better understanding the protein-ligand binding process; and practically, for providing a parameter to be used in many computational drug design methods such as docking and pharmacophore searches. We synthesize a selection of previously reported conflicting results from computational studies of this issue and conclude that high ligand conformational energies really are present in some crystal structures. The main source of disagreement between different analyses appears to be due to divergent treatments of electrostatics and solvation. At the same time, however, for many ligands, a high conformational energy is in error, due to either crystal structure inaccuracies or incorrect determination of the reference state. Aside from simple chemistry mistakes, we argue that crystal structure error may mainly be because of the heuristic weighting of ligand stereochemical restraints relative to the fit of the structure to the electron density. This problem cannot be fixed with improvements to electron density fitting or with simple ligand geometry checks, though better metrics are needed for evaluating ligand and binding site chemistry in addition to geometry during structure refinement. The ultimate solution for accurately determining ligand conformational energies lies in ultrahigh-resolution crystal structures that can be refined without restraints.

  1. Copper binding ligands: production by marine plankton and characterization by ESI-MS

    Science.gov (United States)

    Orians, K.; Ross, A.; Lawrence, M.; Ikonomou, M.

    2003-04-01

    Organic complexation affects the bioavailability and distribution of copper in the surface ocean. The cyanobacterium Synechococcus sp. PCC 7002 was cultured in the lab and subjected to near-toxic Cu concentrations. Strong Cu-binding ligands were produced under these conditions, as found for other species of Synechococcus. The copper-binding ligand produced had a log K'cond. (log conditional stability constant) of 12.2, similar to the natural ligands found in the surface ocean. The amount of ligand produced was proportional to the amount of copper present. Isolation and concentration of these compounds for characterization by electrospray mass spectrometry (ESI-MS) provides information about the structure of the organic ligands and their metal-ion complexes. Using model ligands, we'll show that ligands can be characterized by ESI-MS and that the location of the copper binding site can be determined in complex molecules. We'll also present results of copper-complexing ligands extracted from the coastal waters of British Columbia. Ligand concentrations are higher at low salinity and in surface waters, indicating that these ligands are produced in surface waters and/or delivered to the region via the Fraser River. Analysis of the extracts with highest UV absorbance identified two Cu2+ ligands of molecular weight 259 and 264. The mass and isotopic distributions are consistent with dipeptides and tripeptides containing two metal-binding amino groups. This result is consistent with the findings of other studies attempting to characterize Cu2+ ligands in seawater. The structure of the identified ligand is similar to that of rhodotorulic acid (a microbial siderophore), glutathione, and phytochelatins, indicating that small peptides and related compounds can act as strong, specific metal chelators in natural waters

  2. Understanding the Molecular Determinant of Reversible Human Monoamine Oxidase B Inhibitors Containing 2H-chromen-2-One Core: Structure-Based and Ligand-Based Derived 3-D QSAR Predictive Models.

    Science.gov (United States)

    Mladenovic, Milan; Patsilinakos, Alexandros; Pirolli, Adele; Sabatino, Manuela; Ragno, Rino

    2017-03-14

    Monoamine oxidase B (MAO B) catalyzes the oxidative deamination of aryalkylamines neurotransmitters with concomitant reduction of oxygen to hydrogen peroxide. Consequently, the enzyme's malfunction can induce oxidative damage to mitochondrial DNA and mediates development of Parkinson's disease. Thus, MAO B emerges as a promising target for developing pharmaceuticals potentially useful to treat this vicious neurodegenerative condition. Aiming to contribute to the development of drugs with the reversible mechanism of MAO B inhibition only, herein, an extended in silico-in vitro procedure for the selection of novel MAO B inhibitors is demonstrated, including: (1) definition of optimized and validated structure-based (SB) 3-D QSAR models derived from available co-crystallized inhibitor-MAO B complexes; (2) elaboration of structure-activity relationships (SAR) features for either irreversible or reversible MAO B inhibitors to characterize and improve coumarin-based inhibitor activity (Protein Data Bank ID: 2V61) as the most potent reversible lead compound; (3) definition of structure-based (SB) and ligand-based (LB) alignment rules assessments by which virtually any untested potential MAO B inhibitor might be evaluated; (4) predictive ability validation of the best 3-D QSAR model through SB/LB modeling of four coumarin-based external test sets (267 compounds); (5) design and SB/LB alignment of novel coumarin-based scaffolds experimentally validated through synthesis and biological evaluation in vitro. Due to the wide range of molecular diversity within the 3-D QSARs training set and derived features, the selected N probe-derived 3-D QSAR model proves to be a valuable tool for virtual screening (VS) of novel MAO B inhibitors and a platform for design, synthesis and evaluation of novel active structures. Accordingly, six highly active and selective MAO B inhibitors (picomolar to low nanomolar range of activity) were disclosed as a result of rational SB/LB 3-D QSAR design

  3. Formazanate Ligands as Structurally Versatile, Redox-Active Analogues of beta-Diketiminates in Zinc Chemistry

    NARCIS (Netherlands)

    Chang, Mu-Chieh; Roewen, Peter; Travieso-Puente, Raquel; Lutz, Martin; Otten, Edwin

    2015-01-01

    A range of tetrahedral bis(formazanate)zinc complexes with different steric and electronic properties of the formazanate ligands were synthesized. The solid-state structures for several of these were determined by X-ray crystallography, which showed that complexes with symmetrical, unhindered ligand

  4. Integrin receptors and ligand-gated channels.

    Science.gov (United States)

    Morini, Raffaella; Becchetti, Andrea

    2010-01-01

    Plastic expression of different integrin subunits controls the different stages of neural development, whereas in the adult integrins regulate synaptic stability. Evidence of integrin-channel crosstalk exists for ionotropic glutamate receptors. As is often the case in other tissues, integrin engagement regulates channel activity through complex signaling pathways that often include tyrosine phosphorylation cascades. The specific pathways recruited by integrin activation depend on cerebral region and cell type. In turn, ion channels control integrin expression onto the plasma membrane and their ligand binding affinity. The most extensive studies concern the hippocampus and suggest implications for neuronal circuit plasticity. The physiological relevance of these findings depends on whether adhesion molecules, aside from determining tissue stability, contribute to synaptogenesis and the responsiveness of mature synapses, thus contributing to long-term circuit consolidation. Little evidence is available for other ligand-gated channels, with the exception of nicotinic receptors. These exert a variety of functions in neurons and non neural tissue, both in development and in the adult, by regulating cell cycle, synaptogenesis and synaptic circuit refinement. Detailed studies in epidermal keratinocytes have shed some light on the possible mechanisms through which ACh can regulate cell motility, which may be of general relevance for morphogenetic processes. As to the control of mature synapses, most results concern the integrinic control of nicotinic receptors in the neuromuscular junction. Following this lead, a few studies have addressed similar topics in adult cerebral synapses. However, pursuing and interpreting these results in the brain is especially difficult because of the complexity of the nicotinic roles and the widespread contribution of nonsynaptic, paracrine transmission. From a pathological point of view, considering the well-known contribution of both

  5. Ghana/JICA/WHO training course in polio diagnostic procedures and vaccine potency testing.

    Science.gov (United States)

    Kobayakawa, T

    1993-12-01

    The Ghana/Japan International Cooperation Agency/WHO training course in polio diagnostic procedures and vaccine potency testing was initiated at the Noguchi Memorial Institute for Medical Research in Ghana for 5 consecutive years from 1992. The purpose of the course is to create virologists and technicians competent in the laboratory methods to support the polio eradication initiative in Africa. The participants are invited mainly from anglophone countries in the region.

  6. Monitoring blend potency in a tablet press feed frame using near infrared spectroscopy.

    Science.gov (United States)

    Ward, Howard W; Blackwood, Daniel O; Polizzi, Mark; Clarke, Hugh

    2013-06-01

    A near-infrared (NIR) probe was installed into the feed frame of a rotary tablet press to monitor API concentration as a function of time. A series of step change experimental trials were completed, where a placebo blend was initially charged into the feed frame, and an active blend was layered above. The compression process was initiated, and process parameters, such as mass throughput rate, and feed frame paddle wheel speed were systematically varied. For the range of mass throughput rates studied, excellent correlations were shown between the NIR signal and weight corrected tablet potency from stratified tablet samples. A similar correlation was demonstrated for higher feed frame paddle wheel speeds. However, for lower feed frame paddle wheel speeds, a bias was observed between weight corrected tablet potency and the NIR signal. This finding suggests that compression process parameters, such as paddle wheel rotational speed and NIR probe location, must be optimized for different tablet press geometries to ensure that the NIR process signal can be related to tablet potency. This emerging application of Process Analytical Technology (PAT) may be used to identify powder segregation events during discharge of powder from an intermediate bulk container (IBC) or as a development tool to further understand powder mixing dynamics occurring within the feed frame. This may also be used as a diagnostic tool for fault detection during tablet compression. Finally, this PAT application may also be integrated with the tablet press control system as a gating or reject device for sub or super-potent tablets or enable Real-Time-Release testing (RTRt) through the continuous monitoring of the potency and homogeneity of powder circulating within the feed frame.

  7. Analysis of Cannabis Seizures in NSW, Australia: Cannabis Potency and Cannabinoid Profile

    OpenAIRE

    Wendy Swift; Alex Wong; Li, Kong M.; Arnold, Jonathon C.; McGregor, Iain S.

    2013-01-01

    Recent analysis of the cannabinoid content of cannabis plants suggests a shift towards use of high potency plant material with high levels of Δ(9)-tetrahydrocannabinol (THC) and low levels of other phytocannabinoids, particularly cannabidiol (CBD). Use of this type of cannabis is thought by some to predispose to greater adverse outcomes on mental health and fewer therapeutic benefits. Australia has one of the highest per capita rates of cannabis use in the world yet there has been no previous...

  8. Learned flavor preferences: the variable potency of post-oral nutrient reinforcers

    OpenAIRE

    Ackroff, Karen

    2008-01-01

    The notion that preferences for flavors paired with various nutrients can be attributed simply to their energy content (“flavor-calorie learning”) is belied by variation in nutrient reinforcing potency. Fructose, fat and ethanol, all regarded as powerful contributors to food and fluid preferences, are less potent than glucose when their orosensory effects are bypassed. Conditioning studies in animals infused with nutrients as they consume target flavor solutions have shown that the weaker rei...

  9. Rationale for Further Medical and Health Research on High-Potency Sweeteners

    OpenAIRE

    Schiffman, Susan S.

    2012-01-01

    High-potency or artificial sweeteners have historically been considered inert compounds without physiological consequences other than taste sensations. However, recent data suggest that some of these sweeteners have biological effects that may impact human health. Furthermore, there are significant gaps in our current knowledge of the pharmacokinetics of these sweeteners, their potential for “sweetener–drug interactions” and their impact on appetite and body weight regulation. Nine research n...

  10. N-Aryl-oxazolidin-2-imine Muscle Selective Androgen Receptor Modulators Enhance Potency through Pharmacophore Reorientation

    Energy Technology Data Exchange (ETDEWEB)

    Nirschl, Alexandra A.; Zou, Yan; Krystek, Jr., Stanley R.; Sutton, James C.; Simpkins, Ligaya M.; Lupisella, John A.; Kuhns, Joyce E.; Seethala, Ramakrishna; Golla, Rajasree; Sleph, Paul G.; Beehler, Blake C.; Grover, Gary J.; Egan, Donald; Fura, Aberra; Vyas, Viral P.; Li, Yi-Xin; Sack, John S.; Kish, Kevin F.; An, Yongmi; Bryson, James A.; Gougoutas, Jack Z.; DiMarco, John; Zahler, Robert; Ostrowski, Jacek; Hamann, Lawrence G.; (BMS)

    2010-11-09

    A novel selective androgen receptor modulator (SARM) scaffold was discovered as a byproduct obtained during synthesis of our earlier series of imidazolidin-2-ones. The resulting oxazolidin-2-imines are among the most potent SARMs known, with many analogues exhibiting sub-nM in vitro potency in binding and functional assays. Despite the potential for hydrolytic instability at gut pH, compounds of the present class showed good oral bioavailability and were highly active in a standard rodent pharmacological model.

  11. GluR2 ligand-binding core complexes

    DEFF Research Database (Denmark)

    Kasper, C; Lunn, M-L; Liljefors, T

    2002-01-01

    X-ray structures of the GluR2 ligand-binding core in complex with (S)-Des-Me-AMPA and in the presence and absence of zinc ions have been determined. (S)-Des-Me-AMPA, which is devoid of a substituent in the 5-position of the isoxazolol ring, only has limited interactions with the partly hydrophobic...

  12. Traditional marijuana, high-potency cannabis and synthetic cannabinoids: increasing risk for psychosis.

    Science.gov (United States)

    Murray, Robin M; Quigley, Harriet; Quattrone, Diego; Englund, Amir; Di Forti, Marta

    2016-10-01

    Epidemiological evidence demonstrates that cannabis use is associated with an increased risk of psychotic outcomes, and confirms a dose-response relationship between the level of use and the risk of later psychosis. High-potency cannabis and synthetic cannabinoids carry the greatest risk. Experimental administration of tetrahydrocannabinol, the active ingredient of cannabis, induces transient psychosis in normal subjects, but this effect can be ameliorated by co-administration of cannabidiol. This latter is a constituent of traditional hashish, but is largely absent from modern high-potency forms of cannabis. Argument continues over the extent to which genetic predisposition is correlated to, or interacts with, cannabis use, and what proportion of psychosis could be prevented by minimizing heavy use. As yet, there is not convincing evidence that cannabis use increases risk of other psychiatric disorders, but there are no such doubts concerning its detrimental effect on cognitive function. All of the negative aspects are magnified if use starts in early adolescence. Irrespective of whether use of cannabis is decriminalized or legalized, the evidence that it is a component cause of psychosis is now sufficient for public health messages outlining the risk, especially of regular use of high-potency cannabis and synthetic cannabinoids. © 2016 World Psychiatric Association.

  13. Introduction of mismatches in a random shRNA-encoding library improves potency for phenotypic selection.

    Directory of Open Access Journals (Sweden)

    Yongping Wang

    Full Text Available RNA interference (RNAi is a mechanism for interfering with gene expression through the action of small, non-coding RNAs. We previously constructed a short-hairpin-loop RNA (shRNA encoding library that is random at the nucleotide level [1]. In this library, the stems of the hairpin are completely complementary. To improve the potency of initial hits, and therefore signal-to-noise ratios in library screening, as well as to simplify hit-sequence retrieval by PCR, we constructed a second-generation library in which we introduced random mismatches between the two halves of the stem of each hairpin, on a random template background. In a screen for shRNAs that protect an interleukin-3 (IL3 dependent cell line from IL3 withdrawal, our second-generation library yielded hit sequences with significantly higher potencies than those from the first-generation library in the same screen. Our method of random mutagenesis was effective for a random template and is likely suitable, therefore, for any DNA template of interest. The improved potency of our second-generation library expands the range of possible unbiased screens for small-RNA therapeutics and biologic tools.

  14. Potency trends of delta9-THC and other cannabinoids in confiscated marijuana from 1980-1997.

    Science.gov (United States)

    ElSohly, M A; Ross, S A; Mehmedic, Z; Arafat, R; Yi, B; Banahan, B F

    2000-01-01

    The analysis of 35,312 cannabis preparations confiscated in the USA over a period of 18 years for delta-9-tetrahydrocannabinol (delta9-THC) and other major cannabinoids is reported. Samples were identified as cannabis, hashish, or hash oil. Cannabis samples were further subdivided into marijuana (loose material, kilobricks and buds), sinsemilla, Thai sticks and ditchweed. The data showed that more than 82% of all confiscated samples were in the marijuana category for every year except 1980 (61%) and 1981 (75%). The potency (concentration of delta9-THC) of marijuana samples rose from less than 1.5% in 1980 to approximately 3.3% in 1983 and 1984, then fluctuated around 3% till 1992. Since 1992, the potency of confiscated marijuana samples has continuously risen, going from 3.1% in 1992 to 4.2% in 1997. The average concentration of delta9-THC in all cannabis samples showed a gradual rise from 3% in 1991 to 4.47% in 1997. Hashish and hash oil, on the other hand, showed no specific potency trends. Other major cannabinoids [cannabidiol (CBD), cannabinol (CBN), and cannabichromene (CBC)] showed no significant change in their concentration over the years.

  15. Reduction of animal suffering in rabies vaccine potency testing by introduction of humane endpoints.

    Science.gov (United States)

    Takayama-Ito, Mutsuyo; Lim, Chang-Kweng; Nakamichi, Kazuo; Kakiuchi, Satsuki; Horiya, Madoka; Posadas-Herrera, Guillermo; Kurane, Ichiro; Saijo, Masayuki

    2017-03-01

    Potency controls of inactivated rabies vaccines for human use are confirmed by the National Institutes of Health challenge test in which lethal infection with severe neurological symptoms should be observed in approximately half of the mice inoculated with the rabies virus. Weight loss, decreased body temperature, and the presence of rabies-associated neurological signs have been proposed as humane endpoints. The potential for reduction of animal suffering by introducing humane endpoints in the potency test for inactivated rabies vaccine for human use was investigated. The clinical signs were scored and body weight was monitored. The average times to death following inoculation were 10.49 and 10.99 days post-inoculation (dpi) by the potency and challenge control tests, respectively, whereas the average times to showing Score-2 signs (paralysis, trembling, and coma) were 6.26 and 6.55 dpi, respectively. Body weight loss of more than 15% appeared at 5.82 and 6.42 dpi. The data provided here support the introduction of obvious neuronal signs combined with a body weight loss of ≥15% as a humane endpoint to reduce the time of animal suffering by approximately 4 days. Copyright © 2017 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  16. Sterol superlattice affects antioxidant potency and can be used to assess adverse effects of antioxidants.

    Science.gov (United States)

    Olsher, Michelle; Chong, Parkson Lee-Gau

    2008-11-01

    We have developed a fluorescence method to examine how membrane sterol lateral organization affects the potency of antioxidants, and used this information to evaluate possible adverse effects of lipid-soluble antioxidants seen in recent clinical studies. In the presence of an antioxidant, the lag time (tau) produced during free radical-induced sterol oxidation in lipid vesicles reflects the potency of the antioxidant. The ascorbic acid-induced tau value varies with sterol mol% in a biphasic manner, showing a minimum at the critical sterol mole fraction for maximal superlattice formation (C r), in ascorbic acid concentrations antioxidant potency. In contrast, the biphasic change in tau at C r was observed only at doses of ascorbyl palmitate antioxidant than its water-soluble counterpart as judged by the tau value, it can easily perturb sterol lateral organization by insertion into membrane bilayers, which could impose detrimental effects on cells. The threshold antioxidant concentration (C th) to abolish biphasic change in tau at C r may vary with antioxidant and could be used to assess potential adverse effects of other lipid-soluble antioxidants.

  17. A common genetic influence on human intensity ratings of sugars and high-potency sweeteners.

    Science.gov (United States)

    Hwang, Liang-Dar; Zhu, Gu; Breslin, Paul A S; Reed, Danielle R; Martin, Nicholas G; Wright, Margaret J

    2015-08-01

    The perception of sweetness varies among individuals but the sources of this variation are not fully understood. Here, in a sample of 1,901 adolescent and young adults (53.8% female; 243 MZ and 452 DZ twin pairs, 511 unpaired individuals; mean age 16.2±2.8, range 12–26 years), we studied the variation in the perception of sweetness intensity of two monosaccharides and two high-potency sweeteners: glucose, fructose, neohesperidine dihydrochalcone (NHDC), and aspartame. Perceived intensity for all sweeteners decreased with age (2–5% per year) and increased with the history of otitis media (6–9%). Males rated aspartame slightly stronger than females (7%). We found similar heritabilities for sugars (glucose: h2=0.31, fructose: h2=0.34) and high-potency sweeteners (NHDC: h2=0.31, aspartame: h2=0.30); all were in the modest range. Multivariate modeling showed that a common genetic factor accounted for >75% of the genetic variance in the four sweeteners, suggesting that individual differences in perceived sweet intensity, which are partly due to genetic factors, may be attributed to a single set of genes. This study provided evidence of the shared genetic pathways between the perception of sugars and high-potency sweeteners.

  18. Traditional marijuana, high‐potency cannabis and synthetic cannabinoids: increasing risk for psychosis

    Science.gov (United States)

    Murray, Robin M.; Quigley, Harriet; Quattrone, Diego; Englund, Amir; Di Forti, Marta

    2016-01-01

    Epidemiological evidence demonstrates that cannabis use is associated with an increased risk of psychotic outcomes, and confirms a dose‐response relationship between the level of use and the risk of later psychosis. High‐potency cannabis and synthetic cannabinoids carry the greatest risk. Experimental administration of tetrahydrocannabinol, the active ingredient of cannabis, induces transient psychosis in normal subjects, but this effect can be ameliorated by co‐administration of cannabidiol. This latter is a constituent of traditional hashish, but is largely absent from modern high‐potency forms of cannabis. Argument continues over the extent to which genetic predisposition is correlated to, or interacts with, cannabis use, and what proportion of psychosis could be prevented by minimizing heavy use. As yet, there is not convincing evidence that cannabis use increases risk of other psychiatric disorders, but there are no such doubts concerning its detrimental effect on cognitive function. All of the negative aspects are magnified if use starts in early adolescence. Irrespective of whether use of cannabis is decriminalized or legalized, the evidence that it is a component cause of psychosis is now sufficient for public health messages outlining the risk, especially of regular use of high‐potency cannabis and synthetic cannabinoids. PMID:27717258

  19. Enthalpy of ligand substitution in cis organopalladium complexes with monodentate ligands.

    Science.gov (United States)

    Salas, Gorka; Casares, Juan A; Espinet, Pablo

    2009-10-21

    The enthalpy for the substitution reaction cis-[PdRf(2)(THF)(2)] + 2 L -->cis-[PdRf(2)L(2)] + 2THF (THF = tetrahydrofuran) has been measured in THF by calorimetric methods for Rf = 3,5-dichloro-2,4,6-trifluorophenyl, L = PPh(3), AsPh(3), SbPh(3), PMePh(2), PCyPh(2), PMe(3), AsMePh(2), or L(2) = dppe (1,2-bis(diphenylphosphino)ethane), dppf (1,1'-bis(diphenylphosphino)ferrocene). The values determined show that the substitution enthalpy has a strong dependence on the electronic and steric properties of the ligand. The study of the consecutive substitution reactions cis-[PdRf(2)(THF)(2)] + L -->cis-[PdRf(2)L(THF)] + THF, and cis-[PdRf(2)L(THF)] + L -->cis-[PdRf(2)L(2)] + THF has been carried our for L = PPh(3) and L = PCyPh(2). The first substitution is clearly more favorable for the bulkier leaving ligand, but the second gives practically the same DeltaH value for both cases, indicating that the differences in steric hindrance happen to compensate the electronic differences for both ligands. The X-ray structures of cis-[PdRf(2)(PMePh(2))(2)], cis-[PdRf(2)(dppe)] and cis-[PdRf(2)(dppf)] are reported.

  20. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    Science.gov (United States)

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

  1. Alkali metal salts of formazanate ligands : diverse coordination modes as a result of the nitrogen-rich [NNCNN] ligand backbone

    NARCIS (Netherlands)

    Travieso-Puente, Raquel; Chang, Mu-Chieh; Otten, Edwin

    2014-01-01

    Alkali metal salts of redox-active formazanate ligands were prepared, and their structures in the solid-state and in solution are determined. The nitrogen-rich [NNCNN] backbone of formazanates results in a varied coordination chemistry, with both the internal and terminal nitrogen atoms available fo

  2. Surface reactivity, cytotoxicity, and transforming potency of iron-covered compared to untreated refractory ceramic fibers.

    Science.gov (United States)

    Elias, Zoé; Poirot, Odile; Danière, Marie-Céleste; Terzetti, Francine; Binet, Stéphane; Tomatis, Maura; Fubini, Bice

    2002-12-13

    Untreated and iron-coated refractory ceramic fibers (RCFs) 1, 3, and 4 were examined for their potential to generate free radicals and to catalyze hydrogen peroxide decomposition in cell-free assays and were compared for cytotoxic and transforming potencies in Syrian hamster embryo (SHE) cell system. Coating with a high quantity of iron increased the capability of RCFs to generate hydroxyl radicals and to catalyze the decomposition of hydrogen peroxide. In the SHE cells, the untreated RCFs had varying ability to induce inhibition of cell proliferation, cytotoxicity (as measured by the colony-forming efficiency, CE) and morphological transformation, in a concentration-dependent manner. According to cytotoxic and transforming potencies, they ranged as follows: RCF3 > RCF1 > RCF4. The lethal concentration 50 (LC50; decrease of CE to 50% of controls after 7 d of treatment) expressed per number of RCF3 and RCF1/cm(2) of culture dish was 2.5 x 10(4) and 3.7 x 10(4), respectively, whereas RCF4 was not cytotoxic up to the highest concentration tested (23.7 x 10(4) fibers/cm(2)). At LC50, RCF3 was 1.4-fold more transforming than RCF1, and the weakest, RCF4, induced less than 1% transformation. Iron coating of RCF1 and RCF3 markedly attenuated their cytostatic, cytotoxic, and transforming potencies without a linear concentration-transformation relationship. In contrast, iron coating of RCF4 affected slightly its low transforming potency, although the growth inhibitory effect was reduced. The observed decrease rather than increase in the cytotoxic and transforming potencies of the active samples RCF1 and RCF3 by their coating with large amounts of ferric iron suggests that it is not the quantity or any form of iron on the surface of fibers but the iron, even in trace, in a particular redox and coordinate state that might play a role in the fiber's surface reactivity with regard to the biological material. Surface chemical functions involved in the interaction with the cell

  3. The relative teratogenic index and teratogenic potency: proposed components of developmental toxicity risk assessment.

    Science.gov (United States)

    Fabro, S; Shull, G; Brown, N A

    1982-01-01

    Teratogenicity tests should provide answers to three questions: (1) Can the agent induce developmental defects? ("teratogenic potential"); (2) What are the effective doses? ("teratogenic potency"); and (3) Are effective doses below adult toxic doses? ("teratogenic hazard"). The answers to (2) and (3) should be quantitative in nature, but there are no accepted parameters to express these properties. In this paper we propose parameters for the description of teratogenic potency and hazard in quantitative terms. Derivation and calculation of the parameters are illustrated by the analysis of adult lethality and teratogenicity data of eight structurally related anhydrides and imides, following testing in the CD-1 mouse. Teratogenicity was evaluated following treatment on Days 8-10 of gestation, using an average of four dose groups per compound and at least 10 dams per group. Adult lethality was estimated following a similar 3-day dosage schedule with an average of 6 dose groups per compound and at least 8 animals per group. Dose-response relationships of teratogenicity were fitted to a probit model from which tD50 (median effective dose), and other effective doses were computed. It is proposed that tD05, as a minimum teratogenic dose, best represents teratogenic potency. In this study, potency ranged from 0.17 mmol/kg/day for phenytoin to 5.2 mmol/kg/day for ethosuximide. In order to measure teratogenic hazard a ratio between adult toxic (lethality was chosen as the most appropriate measure) and teratogenic responses was made. Since the dose-response slopes of lethality and teratogenicity were different, a simple ratio between median effective doses could not be used. It is shown that a ratio of LD01 to tD05 provides a "Relative Teratogenic Index" (RTI) which reflects the teratogenic hazard of a test agent. The following RTI values (LD01/tD05) were computed in this study: phthalic anhydride, 0.9; phensuximide, 1.0; succinic anhydride, 1.0; ethosuximide, 1.2; phenytoin

  4. Effect of ligands on thermal dissipation from gold nanorods.

    Science.gov (United States)

    Alper, Joshua; Hamad-Schifferli, Kimberly

    2010-03-16

    Thermal interface conductance was measured for soluble gold nanorods (NRs) coated with mercaptocarboxylic acids (HS-(CH(2))(n)COOH, n = 5, 10, 15), thiolated polyethylene glycols (MW = 356, 1000, 5000), and HS-(CH(2))(15)-COOH-coated NRs further coated with alternating layers of poly(diallyldimethylammonium chloride) and poly(sodium styrenesulfonate). Ferguson analysis determined ligand thickness. The thermal-diffusion-dominated regime of transient absorption spectra was fit to a continuum heat diffusion finite element model to obtain the thermal interface conductance, G, which varied with ligand chemistry but not molecule length. The results suggest that the ability to exclude water from the NR surface governs ligand G values.

  5. The chemistry of separations ligand degradation by organic radical cations

    Energy Technology Data Exchange (ETDEWEB)

    Mezyk, S.P.; Horne, G.P. [California State University at Long Beach, Long Beach, CA 90840 (United States); Mincher, B.J.; Zalupski, P.R. [Idaho National Laboratory, Idaho Falls, ID 83415 (United States); Cook, A.R.; Wishart, J.F. [Chemistry Department, Brookhaven National Laboratory, New York, 11973 (United States)

    2016-07-01

    Solvent based extractions of used nuclear fuel use designer ligands in an organic phase extracting ligand complexed metal ions from an acidic aqueous phase. These extractions will be performed in highly radioactive environments, and the radiation chemistry of all these complexing agents and their diluents will play a major role in determining extraction efficiency, separation factors, and solvent-recycle longevity. Although there has been considerable effort in investigating ligand damage occurring in acidic water radiolysis conditions, only minimal fundamental kinetic and mechanistic data has been reported for the degradation of extraction ligands in the organic phase. Extraction solvent phases typically use normal alkanes such as dodecane, TPH, and kerosene as diluents. The radiolysis of such diluents produce a mixture of radical cations (R{sup .+}), carbon-centered radicals (R{sup .}), solvated electrons, and molecular products such as hydrogen. Typically, the radical species will preferentially react with the dissolved oxygen present to produce relatively inert peroxyl radicals. This isolates the alkane radical cation species, R{sup .+} as the major radiolytically-induced organic species that can react with, and degrade, extraction agents in this phase. Here we report on our recent studies of organic radical cation reactions with 2 ligands: CMPO and TODGA. Elucidating these parameters, and combining them with the known acidic aqueous phase chemistry, will allow a full, fundamental, understanding of the impact of radiation on solvent extraction based separation processes to be achieved. (authors)

  6. Novel ligands that target the mitochondrial membrane protein mitoNEET

    Science.gov (United States)

    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  7. How reliable are ligand-centric methods for Target Fishing?

    Directory of Open Access Journals (Sweden)

    Antonio ePeon

    2016-04-01

    Full Text Available Computational methods for Target Fishing (TF, also known as Target Prediction or Polypharmacology Prediction, can be used to discover new targets for small-molecule drugs. This may result in repositioning the drug in a new indication or improving our current understanding of its efficacy and side effects. While there is a substantial body of research on TF methods, there is still a need to improve their validation, which is often limited to a small part of the available targets and not easily interpretable by the user. Here we discuss how target-centric TF methods are inherently limited by the number of targets that can possibly predict (this number is by construction much larger in ligand-centric techniques. We also propose a new benchmark to validate TF methods, which is particularly suited to analyse how predictive performance varies with the query molecule. On average over approved drugs, we estimate that only five predicted targets will have to be tested to find two true targets with submicromolar potency (a strong variability in performance is however observed. In addition, we find that an approved drug has currently an average of eight known targets, which reinforces the notion that polypharmacology is a common and strong event. Furthermore, with the assistance of a control group of randomly-selected molecules, we show that the targets of approved drugs are generally harder to predict.

  8. Controlled-deactivation cannabinergic ligands.

    Science.gov (United States)

    Sharma, Rishi; Nikas, Spyros P; Paronis, Carol A; Wood, Jodianne T; Halikhedkar, Aneetha; Guo, Jason Jianxin; Thakur, Ganesh A; Kulkarni, Shashank; Benchama, Othman; Raghav, Jimit Girish; Gifford, Roger S; Järbe, Torbjörn U C; Bergman, Jack; Makriyannis, Alexandros

    2013-12-27

    We report an approach for obtaining novel cannabinoid analogues with controllable deactivation and improved druggability. Our design involves the incorporation of a metabolically labile ester group at the 2'-position on a series of (-)-Δ(8)-THC analogues. We have sought to introduce benzylic substituents α to the ester group which affect the half-lives of deactivation through enzymatic activity while enhancing the affinities and efficacies of individual ligands for the CB1 and CB2 receptors. The 1'-(S)-methyl, 1'-gem-dimethyl, and 1'-cyclobutyl analogues exhibit remarkably high affinities for both CB receptors. The novel ligands are susceptible to enzymatic hydrolysis by plasma esterases in a controllable manner, while their metabolites are inactive at the CB receptors. In further in vitro and in vivo experiments key analogues were shown to be potent CB1 receptor agonists and to exhibit CB1-mediated hypothermic and analgesic effects.

  9. Privileged chiral ligands and catalysts

    CERN Document Server

    Zhou, Qi-Lin

    2011-01-01

    This ultimate ""must have"" and long awaited reference for every chemist working in the field of asymmetric catalysis starts with the core structure of the catalysts, explaining why a certain ligand or catalyst is so successful. It describes in detail the history, the basic structural characteristics, and the applications of these ""privileged catalysts"". A novel concept that gives readers a much deeper insight into the topic.

  10. Tumor targeting via integrin ligands

    Directory of Open Access Journals (Sweden)

    Udaya Kiran eMarelli

    2013-08-01

    Full Text Available Selective and targeted delivery of drugs to tumors is a major challenge for an effective cancer therapy and also to overcome the side effects associated with current treatments. Overexpression of various receptors on tumor cells is a characteristic structural and biochemical aspect of tumors and distinguishes them from physiologically normal cells. This abnormal feature is therefore suitable for selectively directing anticancer molecules to tumors by using ligands that can preferentially recognize such receptors. Several subtypes of integrin receptors that are crucial for cell adhesion, cell signaling, cell viability and motility have been shown to have an upregulated expression on cancer cells. Thus, ligands that recognize specific integrin subtypes represent excellent candidates to be conjugated to drugs or drug carrier systems and be targeted to tumors. In this regard, integrins recognizing the RGD cell adhesive sequence have been extensively targeted for tumor specific drug delivery. Here we review key recent examples on the presentation of RGD-based integrin ligands by means of distinct drug delivery systems, and discuss the prospects of such therapies to specifically target tumor cells.

  11. A proline-based aminophenol ligand: synthesis, iron complexation, magnetic, electronic and redox investigation.

    Science.gov (United States)

    Sheykhi, Hamid; Safaei, Elham

    2014-01-24

    A new proline-based aminophenol ligand was synthesized by a convenient procedure. The ligand was characterized by (1)H NMR, (13)C NMR and IR spectroscopies, elemental analysis and optical activity measurements. Mononuclear iron(III) complex (FeL(Pro)) of this ligand was synthesized and characterized by IR, UV-vis, ESI-MS, magnetic susceptibility studies and cyclic voltammetry techniques. The equilibrium formation constant of FeL(Pro) and the pure UV-vis spectral profile of the complex was determined by multivariate hard modeling method. The molecular structure of FeL(Pro) determined by ESI-MS consist of two aminophenolate ligands. The variation of magnetic susceptibility with temperature indicates paramagnetic iron(III) in the monomeric complex. FeL(Pro) complex undergo metal-centered reduction, and ligand-centered oxidation. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. A model for aryl hydrocarbon receptor-activated gene expression shows potency and efficacy changes and predicts squelching due to competition for transcription co-activators.

    Directory of Open Access Journals (Sweden)

    Ted W Simon

    Full Text Available A stochastic model of nuclear receptor-mediated transcription was developed based on activation of the aryl hydrocarbon receptor (AHR by 2,3,7,8-tetrachlorodibenzodioxin (TCDD and subsequent binding the activated AHR to xenobiotic response elements (XREs on DNA. The model was based on effects observed in cells lines commonly used as in vitro experimental systems. Following ligand binding, the AHR moves into the cell nucleus and forms a heterodimer with the aryl hydrocarbon nuclear translocator (ARNT. In the model, a requirement for binding to DNA is that a generic coregulatory protein is subsequently bound to the AHR-ARNT dimer. Varying the amount of coregulator available within the nucleus altered both the potency and efficacy of TCDD for inducing for transcription of CYP1A1 mRNA, a commonly used marker for activation of the AHR. Lowering the amount of available cofactor slightly increased the EC50 for the transcriptional response without changing the efficacy or maximal response. Further reduction in the amount of cofactor reduced the efficacy and produced non-monotonic dose-response curves (NMDRCs at higher ligand concentrations. The shapes of these NMDRCs were reminiscent of the phenomenon of squelching. Resource limitations for transcriptional machinery are becoming apparent in eukaryotic cells. Within single cells, nuclear receptor-mediated gene expression appears to be a stochastic process; however, intercellular communication and other aspects of tissue coordination may represent a compensatory process to maintain an organism's ability to respond on a phenotypic level to various stimuli within an inconstant environment.

  13. Discovery of high potency, single-chain insulin analogs with a shortened B-chain and nonpeptide linker.

    Science.gov (United States)

    Kaur, Zachary P; Ochman, Alexander R; Mayer, John P; Gelfanov, Vasily M; DiMarchi, Richard D

    2013-08-16

    A series of novel, single chain insulin analogs containing polyethylene glycol based connecting segments were synthesized by native chemical ligation and tested for biological activity. While the full length single chain insulin analogs exhibited low potency, deletion of amino acids B26-B30 unexpectedly generated markedly higher activity. This observation is unprecedented in all previous studies of single chain insulin analogs and is consistent with the presumption that in the native hormone this sequence must translocate to achieve high potency insulin receptor interaction. Optimization of the sequence yielded an insulin analog with potency and selectivity comparable to that of native insulin. These results establish a basis for discovery of novel higher potency, single chain insulin analogs of shortened length.

  14. Antecedents of team potency and team effectiveness: an examination of goal and process clarity and servant leadership.

    Science.gov (United States)

    Hu, Jia; Liden, Robert C

    2011-07-01

    Integrating theories of self-regulation with team and leadership literatures, this study investigated goal and process clarity and servant leadership as 3 antecedents of team potency and subsequent team effectiveness, operationalized as team performance and organizational citizenship behavior. Our sample of 304 employees represented 71 teams in 5 banks. Results showed that team-level goal and process clarity as well as team servant leadership served as 3 antecedents of team potency and subsequent team performance and team organizational citizenship behavior. Furthermore, we found that servant leadership moderated the relationships between both goal and process clarity and team potency, such that the positive relationships between both goal and process clarity and team potency were stronger in the presence of servant leadership.

  15. Polymer-pendant ligand chemistry. 1. Reactions of organoarsonic acids and arsenic acid with catechol ligands bonded to polystryene-divinylbenzene and regeneration of the ligand site by a simple hydrolysis procedure

    Energy Technology Data Exchange (ETDEWEB)

    Fish, R.H.; Tannous, R.S.

    1985-12-18

    A novel method is reported for reactions of organoarsonic acids and arsenic acid, known to be present in oil shale and its pyrolysis products, with catechol ligands bonded to either 2% or 20% cross-linked methylated polystyrene-divinylbenzene (PS-DVB) resins. A previous study with catechol-bonded ligands on PS-DVB resins dealt with their reactions with metal ions in aqueous solution and showed a selectivity toward Hg/sup 2 +/ ions. As far as we have been able to determine, reactions of this polymer-supported ligand with organometallic compounds or inorganic anions have not been reported. 9 references, 2 figures, 1 table.

  16. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location

    Energy Technology Data Exchange (ETDEWEB)

    Hamers, T.; Brink, P.J. van den; Mos, L.; Linden, S.C. van der; Legler, J.; Koeman, J.H.; Murk, A.J

    2003-05-01

    Estrogenic potency of rainwater correlated well with organochlorine concentrations, but could not be attributed to specific pesticides. - In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between SPRING and SUMMER were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in HORT than in BACK and BULB. Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for

  17. A solvated ligand rotamer approach and its application in computational protein design.

    Science.gov (United States)

    Huang, Xiaoqiang; Yang, Ji; Zhu, Yushan

    2013-03-01

    The structure-based design of protein-ligand interfaces with respect to different small molecules is of great significance in the discovery of functional proteins. By statistical analysis of a set of protein-ligand complex structures, it was determined that water-mediated hydrogen bonding at the protein-ligand interface plays a crucial role in governing the binding between the protein and the ligand. Based on the novel statistic results, a solvated ligand rotamer approach was developed to explicitly describe the key water molecules at the protein-ligand interface and a water-mediated hydrogen bonding model was applied in the computational protein design context to complement the continuum solvent model. The solvated ligand rotamer approach produces only one additional solvated rotamer for each rotamer in the ligand rotamer library and does not change the number of side-chain rotamers at each protein design site. This has greatly reduced the total combinatorial number in sequence selection for protein design, and the accuracy of the model was confirmed by two tests. For the water placement test, 61% of the crystal water molecules were predicted correctly in five protein-ligand complex structures. For the sequence recapitulation test, 44.7% of the amino acid identities were recovered using the solvated ligand rotamer approach and the water-mediated hydrogen bonding model, while only 30.4% were recovered when the explicitly bound waters were removed. These results indicated that the developed solvated ligand rotamer approach is promising for functional protein design targeting novel protein-ligand interactions.

  18. The Characteristics and the Modern Research of Potency Cold%药性寒的特点及现代研究

    Institute of Scientific and Technical Information of China (English)

    肖开; 苗明三

    2013-01-01

    Objective:To investigate the relationship between the potency cold and functionality,the pharmacological effects,Chinese medicine attending,modern medicine attending,Smell,Meridian,chemical composition.Methods:The application of "traditional Chinese medicine functions-pharmacology-clinical database system",the query" cold "with the frequency of association between the pharmacological effects,Smell,Meridian,the function,Chinese medicine attending,modern medicine attending,chemical composition,based on the number of the frequency,take the highest frequency covers 2/3 as the basic category,the highest frequency covers the remaining 2/ 3 as joint areas,remaining as expansion areas.Results:the basic functions of the potency cold are heat,detoxification,cooling blood,swelling;basic pharmacological effects of potency cold are anti-bacterial,anti-inflammatory,anti-tumor,antipyretic,diuretic,affect the cardiovascular system,lowering blood pressure,sedation,analgesic,anti-cancer; the TCM Indications of potency cold are carbuncles,sore throat,jaundice,erysipelas,external treatment of eczema,boils,eczema,Wind hot cold,the hot leaching,the hot leaching astringent pain,pharyngitis,nose bleeding,red eyes,sore,vomiting blood,Mumps,blood in the stool,edema fullness; the modern medicine attending of potency cold are burns,eczema,psoriasis,dysentery,hepatitis,coronary heart disease,hypertension,hyperlipidemia,constipation gallbladder inflammation,herpes zoster,cervical erosion,flat warts,gastric ulcer,chronic bronchitis,chronic bronchitis,urticaria,mumps,whooping cough;the basic Smell often combined with potency cold is sweet and bitter; the basic Meridian of potency cold are liver,lung,stomach,heart,kidney; the basic chemical composition of potency cold are organic acids,alkaloids,carbohydrates,inorganic substances,amino acids,glycosides,llavonoids,esters,tannins,volatile oils.Conclusion:To determine the relationship between the potency cold and functionality,the pharmacological

  19. Multistep continuous-flow synthesis in medicinal chemistry: discovery and preliminary structure-activity relationships of CCR8 ligands.

    Science.gov (United States)

    Petersen, Trine P; Mirsharghi, Sahar; Rummel, Pia C; Thiele, Stefanie; Rosenkilde, Mette M; Ritzén, Andreas; Ulven, Trond

    2013-07-08

    A three-step continuous-flow synthesis system and its application to the assembly of a new series of chemokine receptor ligands directly from commercial building blocks is reported. No scavenger columns or solvent switches are necessary to recover the desired test compounds, which were obtained in overall yields of 49-94%. The system is modular and flexible, and the individual steps of the sequence can be interchanged with similar outcome, extending the scope of the chemistry. Biological evaluation confirmed activity on the chemokine CCR8 receptor and provided initial structure-activity-relationship (SAR) information for this new ligand series, with the most potent member displaying full agonist activity with single-digit nanomolar potency. To the best of our knowledge, this represents the first published example of efficient use of multistep flow synthesis combined with biological testing and SAR studies in medicinal chemistry.

  20. FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.

    Science.gov (United States)

    Kreiter, Sebastian; Diken, Mustafa; Selmi, Abderraouf; Petschenka, Jutta; Türeci, Özlem; Sahin, Ugur

    2016-01-01

    Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

  1. Biophysical and physicochemical methods differentiate highly ligand-efficient human D-amino acid oxidase inhibitors.

    Science.gov (United States)

    Lange, Jos H M; Venhorst, Jennifer; van Dongen, Maria J P; Frankena, Jurjen; Bassissi, Firas; de Bruin, Natasja M W J; den Besten, Cathaline; de Beer, Stephanie B A; Oostenbrink, Chris; Markova, Natalia; Kruse, Chris G

    2011-10-01

    Many early drug research efforts are too reductionist thereby not delivering key parameters such as kinetics and thermodynamics of target-ligand binding. A set of human D-Amino Acid Oxidase (DAAO) inhibitors 1-6 was applied to demonstrate the impact of key biophysical techniques and physicochemical methods in the differentiation of chemical entities that cannot be adequately distinguished on the basis of their normalized potency (ligand efficiency) values. The resulting biophysical and physicochemical data were related to relevant pharmacodynamic and pharmacokinetic properties. Surface Plasmon Resonance data indicated prolonged target-ligand residence times for 5 and 6 as compared to 1-4, based on the observed k(off) values. The Isothermal Titration Calorimetry-derived thermodynamic binding profiles of 1-6 to the DAAO enzyme revealed favorable contributions of both ΔH and ΔS to their ΔG values. Surprisingly, the thermodynamic binding profile of 3 elicited a substantially higher favorable contribution of ΔH to ΔG in comparison with the structurally closely related fused bicyclic acid 4. Molecular dynamics simulations and free energy calculations of 1, 3, and 4 led to novel insights into the thermodynamic properties of the binding process at an atomic level and in the different thermodynamic signatures of 3 and 4. The presented holistic approach is anticipated to facilitate the identification of compounds with best-in-class properties at an early research stage.

  2. Multifunctional ligands in medicinal inorganic chemistry--current trends and future directions.

    Science.gov (United States)

    Chiang, Linus; Jones, Michael R; Ferreira, Cara L; Storr, Tim

    2012-01-01

    This review will highlight recent advances in ligand design for innovative applications in medicinal inorganic chemistry. Ligands that effectively bind metal ions and also include specific features to enhance targeting, reporting, and overall efficacy are driving innovation in areas of disease diagnosis and therapy. Increasing the potency of therapeutic compounds, while limiting side-effects, is a common goal in medicinal chemistry. In an effort to achieve this goal, compounds are being developed that either target a disease site, or are activated by a disease specific biological process. Metal complexes containing targeting functions and/or bioactive ligands, as well as agents that are activated by specific enzymes, or changes in pH and pO2, provide new avenues for drug development. Radiodiagnostic compounds, magnetic resonance imaging agents, and optical probes containing transition metals offer versatility unavailable to organic imaging agents. In certain cases, dual modality agents have been developed, and will be highlighted. Finally, we will discuss targeted metal binding compounds for treatment of metal overload disorders, and the recent application to neurodegenerative disease.

  3. Potencies of antagonists chemically related to iodoproxyfan at histamine H3 receptors in mouse brain cortex and guinea-pig ileum: evidence for H3 receptor heterogeneity?

    Science.gov (United States)

    Schlicker, E; Kathmann, M; Bitschnau, H; Marr, I; Reidemeister, S; Stark, H; Schunack, W

    1996-04-01

    We determined the affinities of 16 newly synthesized H3 receptor antagonists in an H3 receptor binding assay and the potencies of 12 of these compounds at functional H3 receptors in the mouse brain cortex and guinea-pig ileum. The compounds differ from histamine in that the C-C-N side chain is replaced by a chain of the structure C-C-C-O. The two major aims of the study were (1) to investigate whether the two functional H3 receptors are pharmacologically different and (2) to derive structure-activity relationships. The specific binding of 3H-Na-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the 16 compounds at pKi values ranging from 7.30 to 9.48. In superfused mouse brain cortex slices preincubated with 3H-noradrenaline, the electrically evoked tritium overflow was slightly decreased by iodoproxyfan and its deiodo analogue; this effect was counteracted by the H3 receptor antagonist clobenpropit. The other compounds did not affect the evoked tritium overflow by themselves. The concentration-response curve of histamine for its inhibitory effect on the electrically evoked tritium overflow was shifted to the right by the 12 compounds with apparent pA2 values ranging from 7.02 to 9.00. The 12 compounds also shifted to the right the concentration-response curve of R-a-methylhistamine for its inhibitory effect on the electrically induced contraction in guinea-pig ileum strips; the apparent pA2 values ranged from 5.97 to 9.00. Iodoproxyfan decreased the electrically induced contraction by itself and this effect was counteracted by the H3 receptor antagonist thioperamide. The apparent pA2 values in the two functional H3 receptor models showed a highly significant correlation (r = 0.882; P H3 receptor antagonist potency. The two functional H3 receptors in the mouse brain cortex and the guinea-pig ileum may be slightly different; further studies are necessary to clarify whether this difference is due to H3 receptor heterogeneity

  4. Adaptive immune response to whole cell pertussis vaccine reflects vaccine quality: A possible complementation to the Pertussis Serological Potency test.

    Science.gov (United States)

    Hoonakker, M E; Verhagen, L M; van der Maas, L; Metz, B; Uittenbogaard, J P; van de Waterbeemd, B; van Els, C A C M; van Eden, W; Hendriksen, C F M; Sloots, A; Han, W G H

    2016-08-17

    Whole cell Bordetella pertussis (wP) vaccines are still used in many countries to protect against the respiratory disease pertussis. The potency of whole-cell pertussis vaccine lots is determined by an intracerebral challenge test (the Kendrick test). This test is criticized due to lack of immunological relevance of the read-out after an intracerebral challenge with B. pertussis. The alternative in vivo test, which assesses specific antibody levels in serum after wP vaccination, is the Pertussis Serological Potency test (PSPT). Although the PSPT focuses on a parameter that contributes to protection, the protective immune mechanisms after wP vaccination includes more elements than specific antibody responses only. In this study, additional parameters were investigated, i.e. circulating pro-inflammatory cytokines, antibody specificity and T helper cell responses and it was evaluated whether they can be used as complementary readout parameters in the PSPT to assess wP lot quality. By deliberate manipulation of the vaccine preparation procedure, a panel of high, intermediate and low quality wP vaccines were made. The results revealed that these vaccines induced similar IL-6 and IP10 levels in serum 4h after vaccination (innate responses) and similar antibody levels directed against the entire bacterium. In contrast, the induced antibody specificity to distinct wP antigens differed after vaccination with high, intermediate and low quality wP vaccines. In addition, the magnitude of wP-induced Th cell responses (Th17, Th1 and Th2) was reduced after vaccination with a wP vaccine of low quality. T cell responses and antibody specificity are therefore correlates of qualitative differences in the investigated vaccines, while the current parameter of the PSPT alone was not sensitive enough to distinguish between vaccines of different qualities. This study demonstrates that assessment of the magnitude of Th cell responses and the antigen specificity of antibodies induced by w

  5. Phenol-pyrazole ligands in the design of manganese(III) compounds : synthesis, structural characterization and study of the magnetic properties

    NARCIS (Netherlands)

    Viciano Chumillas, Marta

    2009-01-01

    In this thesis project, new manganese(III) compounds containing phenol-pyrazole ligands are presented. Small variations on the phenol-pyrazole ligand have been performed to investigate the role of the ligand in the formation of new complexes. The reaction conditions are also crucial to determine the

  6. Evolutionarily conserved paired immunoglobulin-like receptor α (PILRα) domain mediates its interaction with diverse sialylated ligands.

    Science.gov (United States)

    Sun, Yonglian; Senger, Kate; Baginski, Tomasz K; Mazloom, Anita; Chinn, Yvonne; Pantua, Homer; Hamidzadeh, Kajal; Ramani, Sree Ranjani; Luis, Elizabeth; Tom, Irene; Sebrell, Andrew; Quinones, Gabriel; Ma, Yan; Mukhyala, Kiran; Sai, Tao; Ding, Jiabing; Haley, Benjamin; Shadnia, Hooman; Kapadia, Sharookh B; Gonzalez, Lino C; Hass, Philip E; Zarrin, Ali A

    2012-05-04

    Paired immunoglobulin-like receptor (PILR) α is an inhibitory receptor that recognizes several ligands, including mouse CD99, PILR-associating neural protein, and Herpes simplex virus-1 glycoprotein B. The physiological function(s) of interactions between PILRα and its cellular ligands are not well understood, as are the molecular determinants of PILRα/ligand interactions. To address these uncertainties, we sought to identify additional PILRα ligands and further define the molecular basis for PILRα/ligand interactions. Here, we identify two novel PILRα binding partners, neuronal differentiation and proliferation factor-1 (NPDC1), and collectin-12 (COLEC12). We find that sialylated O-glycans on these novel PILRα ligands, and on known PILRα ligands, are compulsory for PILRα binding. Sialylation-dependent ligand recognition is also a property of SIGLEC1, a member of the sialic acid-binding Ig-like lectins. SIGLEC1 Ig domain shares ∼22% sequence identity with PILRα, an identity that includes a conserved arginine localized to position 97 in mouse and human SIGLEC1, position 133 in mouse PILRα and position 126 in human PILRα. We observe that PILRα/ligand interactions require conserved PILRα Arg-133 (mouse) and Arg-126 (human), in correspondence with a previously reported requirement for SIGLEC1 Arg-197 in SIGLEC1/ligand interactions. Homology modeling identifies striking similarities between PILRα and SIGLEC1 ligand binding pockets as well as at least one set of distinctive interactions in the galactoxyl-binding site. Binding studies suggest that PILRα recognizes a complex ligand domain involving both sialic acid and protein motif(s). Thus, PILRα is evolved to engage multiple ligands with common molecular determinants to modulate myeloid cell functions in anatomical settings where PILRα ligands are expressed.

  7. Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant.

    Science.gov (United States)

    Zeitlin, Larry; Pettitt, James; Scully, Corinne; Bohorova, Natasha; Kim, Do; Pauly, Michael; Hiatt, Andrew; Ngo, Long; Steinkellner, Herta; Whaley, Kevin J; Olinger, Gene G

    2011-12-20

    No countermeasures currently exist for the prevention or treatment of the severe sequelae of Filovirus (such as Ebola virus; EBOV) infection. To overcome this limitation in our biodefense preparedness, we have designed monoclonal antibodies (mAbs) which could be used in humans as immunoprotectants for EBOV, starting with a murine mAb (13F6) that recognizes the heavily glycosylated mucin-like domain of the virion-attached glycoprotein (GP). Point mutations were introduced into the variable region of the murine mAb to remove predicted human T-cell epitopes, and the variable regions joined to human constant regions to generate a mAb (h-13F6) appropriate for development for human use. We have evaluated the efficacy of three variants of h-13F6 carrying different glycosylation patterns in a lethal mouse EBOV challenge model. The pattern of glycosylation of the various mAbs was found to correlate to level of protection, with aglycosylated h-13F6 providing the least potent efficacy (ED(50) = 33 μg). A version with typical heterogenous mammalian glycoforms (ED(50) = 11 μg) had similar potency to the original murine mAb. However, h-13F6 carrying complex N-glycosylation lacking core fucose exhibited superior potency (ED(50) = 3 μg). Binding studies using Fcγ receptors revealed enhanced binding of nonfucosylated h-13F6 to mouse and human FcγRIII. Together the results indicate the presence of Fc N-glycans enhances the protective efficacy of h-13F6, and that mAbs manufactured with uniform glycosylation and a higher potency glycoform offer promise as biodefense therapeutics.

  8. The intravenous to oral relative milligram potency ratio of morphine during chronic dosing in cancer pain.

    Science.gov (United States)

    Lasheen, Wael; Walsh, Declan; Mahmoud, Fade; Sarhill, Nabeel; Rivera, Nilo; Davis, Mellar; Lagman, Ruth; Legrand, Susan

    2010-01-01

    Morphine (M) is the opioid analgesic of choice for severe cancer pain. The IV to PO M equipotent switch ratio (CR) is controversial. We designed this prospective observational cohort to confirm the efficacy and safety of M IV to PO CR of 1:3. Consecutive cancer patients admitted to an inpatient palliative medicine unit were screened for inclusion. Pain was managed by palliative medicine specialists. They were blinded to the patient data collected, and the calculated CR. The switch was considered successful if the following criteria were met: (1) Pain adequately controlled: pain rated as none or mild (2) Number of RD less than 4 (for non incident pain) per 24 hours (3) No limiting side effects. We used Day 3 ATC M dose for CR calculations. The major outcome measures were the IV : PO CR ratio, morphine doses (mg/day), pain severity, number of PRN doses, and day 1 and day 3side effects. Descriptive statistics were used to report mean, median, standard deviation and range of different variables. Two hundred and fifty six consecutive admissions were screened, and 106 were eligible for the study. Sixty two underwent a successful M route switch and were included in this analysis. A ratio of 1:3 was safely implemented over a wide M dose range. About 80% were successfully switched with a calculated CR of 1:3. 20% required an oral M dose adjustment after route switch either to better pain control or reduce side effects with a resultant higher (e.g. 1:4) or lower (e.g. 1:2) calculated potency ratios respectively. A potency ratio of 1:3 was safe as evaluated by common M side-effects, the dose also easy to calculate. The 1: 3 M IV to PO relative milligram potency ratio appears correct and practical for most patients over a wide M dose range.

  9. Acidosis induced by carbon dioxide insufflation decreases heparin potency: a risk factor for thrombus formation.

    Science.gov (United States)

    Gorter, Karin A M; Stehouwer, Marco C; Van Putte, Bart P; Vlot, Eline A; Urbanus, Rolf T

    2017-04-01

    Since the introduction of CO2 insufflation during open heart surgery in our hospital, we incidentally observed thrombus formation in the dissected heart, in the pericardium and in the cardiotomy reservoir of the cardiopulmonary bypass system. Furthermore, we measured very high levels of pCO2, causing severe acidosis, in stagnant blood in the pericardium and cardiotomy reservoir. In this in vitro study, we assessed the influence of acidosis and hypothermia on heparin potency and thrombin formation. We assessed heparin potency in function of pH (pH 5.0-7.4) and temperature (24-37°C) by comparing the activated partial thromboplastin time in platelet-poor plasma between samples with and without unfractionated heparin. We measured thrombin formation in platelet-poor plasma by means of fluorescent, calibrated, automated thrombography in function of pH (pH 5.0-7.4) and temperature (24-37°C). The parameters of interest were the endogenous thrombin potential and the peak amount of thrombin generation. The major finding of this study is the significant decrease in the efficiency of unfractionated heparin in delaying thrombus formation at acidotic (pH 5.0-7.0) conditions (p=0.034-0.05). Furthermore, we found that thrombin formation is significantly increased at hypothermic (24-34°C) conditions (p=acidosis may lead to a decreased heparin potency. Acidosis, as induced by CO2 insufflation, may predispose patients to incidental thrombus formation in stagnant blood in the open thorax and in the cardiotomy reservoir. Hypothermia might further increase this risk. Therefore, we recommend reconsidering the potential advantages and disadvantages of using CO2 insufflation during cardiopulmonary bypass.

  10. Structure-Activity Relationship Studies on a Macrocyclic Agouti-Related Protein (AGRP) Scaffold Reveal Agouti Signaling Protein (ASP) Residue Substitutions Maintain Melanocortin-4 Receptor Antagonist Potency and Result in Inverse Agonist Pharmacology at the Melanocortin-5 Receptor.

    Science.gov (United States)

    Ericson, Mark D; Freeman, Katie T; Schnell, Sathya M; Fleming, Katlyn A; Haskell-Luevano, Carrie

    2017-10-04

    The melanocortin system consists of five reported receptors, agonists from the proopiomelanocortin gene transcript, and two antagonists, agouti-signaling protein (ASP) and agouti-related protein (AGRP). For both ASP and AGRP, the hypothesized Arg-Phe-Phe pharmacophores are on exposed β-hairpin loops. In this study, the Asn and Ala positions of a reported AGRP macrocyclic scaffold (c[Pro-Arg-Phe-Phe-Asn-Ala-Phe-DPro]) were explored with 14-compound and 8-compound libraries, respectively, to generate more potent, selective melanocortin receptor antagonists. Substituting diaminopropionic acid (Dap), DDap, and His at the Asn position yielded potent MC4R ligands, while replacing Ala with Ser maintained MC4R potency. Since these substitutions correlate to ASP loop residues, an additional Phe to Ala substitution was synthesized and observed to maintain MC4R potency. Seventeen compounds also possessed inverse agonist activity at the MC5R, the first report of this pharmacology. These findings are useful in developing molecular probes to study negative energy balance conditions and unidentified functions of the MC5R.

  11. Intermediate affinity and potency of clozapine and low affinity of other neuroleptics and of antidepressants at H3 receptors.

    Science.gov (United States)

    Kathmann, M; Schlicker, E; Göthert, M

    1994-12-01

    It was the aim of the present study to determine the affinities of four neuroleptics and five antidepressants for histamine H3 receptors. In rat brain cortex membranes, the specifically bound [3H]-N alpha-methylhistamine was monophasically displaced by clozapine (pKi 6.15). The other drugs did not completely displace the radioligand even at 100 microM; the pKi values were: haloperidol (4.91); sulpiride (4.73); amitriptyline (4.56); desipramine (4.15); levomepromazine (4.14); fluovoxamine (4.13); maprotiline (4.09); moclobemide (H3 receptor model, i.e., in superfused mouse brain cortex slices preincubated with [3H]-noradrenaline. The electrically evoked tritium overflow was not affected by clozapine 0.5-32 microM. However, clozapine shifted the concentration-response curve of histamine for its inhibitory effect on the evoked overflow to the right, but did not affect the maximum effect of histamine. The Schild plot yielded a pA2 value of 6.33. In conclusion, clozapine shows an intermediate affinity and potency (as a competitive antagonist) at H3 receptors. The Ki value of clozapine at H3 receptors resembles its Ki value at D2 receptors (the target of the classical neuroleptics), but is higher than its Ki values at D4, 5-HT2 or muscarinic acetylcholine receptors, which according to current hypotheses, might be involved in the atypical profile of clozapine.

  12. In ovo injection of prebiotics and synbiotics affects the digestive potency of the pancreas in growing chickens.

    Science.gov (United States)

    Pruszynska-Oszmalek, E; Kolodziejski, P A; Stadnicka, K; Sassek, M; Chalupka, D; Kuston, B; Nogowski, L; Mackowiak, P; Maiorano, G; Jankowski, J; Bednarczyk, M

    2015-08-01

    The purpose of the study was to examine the effect of 2 prebiotics and 2 synbiotics on the digestive potency of pancreas in 1-, 3-, 7-, 14-, 21-, and 34-day-old cockerels. Prebiotics (inulin and Bi²tos) and synbiotics (inulin + Lactococcus lactis subsp. lactis and Bi²tos + Lactococcus lactis subsp. cremoris) were injected in ovo into the air cell on the 12th d embryonic development. Their application increased the activity of amylase, lipase, and trypsin in the pancreas. The most pronounced changes were observed at the end of the investigated rearing period (d 34). The strongest stimulative effects on amylase were shown by both synbiotics, on lipase synbiotic Bi²tos + Lactococcus lactis subsp. cremoris, and on trypsin all the used prebiotics and synbiotics. Simultaneously, neither the absolute nor the relative mass of the pancreas in comparison to control group were changed. Also, the injected in ovo compounds did not cause a deterioration in the posthatching condition of the chicken liver, as determined by measurement of the activity of marker enzymes in the blood (alanine aminotransferase and aspartate aminotransferase). Treatment with the prebiotics and synbiotics did not change the feed conversion ratio but Bi²tos (galacto-oligosaccharide) and inulin (fructan) + Lactococcus lactis subsp. lactis significantly increased final BW.

  13. A newly described scorpion species, Leiurus abdullahbayrami (Scorpion: Buthidae, and the lethal potency and in vivo effects of its venom

    Directory of Open Access Journals (Sweden)

    O Ozkan

    2011-01-01

    Full Text Available Currently, medically significant scorpion species belong to the Buthidae family and are represented by the genera Androctonus, Buthus, Mesobuthus, Hottentotta, Parabuthus, Tityus, Centruroides, Leiurus. Although Leiurus was originally considered a monotypic genus, four additional species have since been described. Leiurus abdullahbayrami (previously identified as L. quinquestriatus in Turkey was classified as a new Leiurus species. This is the first report conducted on the lethality and biologic effects of L. abdullahbayrami scorpion venom in mice. In this study, the electrophoretic protein pattern of its venom was also determined. Two protein bands with molecular masses of 4 and 6 kDa were more strongly detected than other protein bands in the venom sample. Electrophoresis showed that L. abdullahbayrami scorpion venom possesses both short- and long-chain neurotoxins. The median lethal dose of this venom was found to be 0.19 mg/kg by subcutaneous (SC injection in mice. Animals experimentally envenomed with L. abdullahbayrami venom exhibited hyperexcitability, agitation, aggressive behavior, squeaking and fighting, tachypnea, weakness, convulsions, and death due to cardiac and respiratory failure. In further studies, the potency of antivenom should be investigated in relation to the scorpion venom. Molecular and pharmacological studies are also required to identify and characterize L. abdullahbayrami scorpion venom.

  14. Establishment of alternative potency test for botulinum toxin type A using compound muscle action potential (CMAP) in rats.

    Science.gov (United States)

    Torii, Yasushi; Goto, Yoshitaka; Nakahira, Shinji; Ginnaga, Akihiro

    2014-11-01

    The biological activity of botulinum toxin type A has been evaluated using the mouse intraperitoneal (ip) LD50 test. This method requires a large number of mice to precisely determine toxin activity, and, as such, poses problems with regard to animal welfare. We previously developed a compound muscle action potential (CMAP) assay using rats as an alternative method to the mouse ip LD50 test. In this study, to evaluate this quantitative method of measuring toxin activity using CMAP, we assessed the parameters necessary for quantitative tests according to ICH Q2 (R1). This assay could be used to evaluate the activity of the toxin, even when inactive toxin was mixed with the sample. To reduce the number of animals needed, this assay was set to measure two samples per animal. Linearity was detected over a range of 0.1-12.8 U/mL, and the measurement range was set at 0.4-6.4 U/mL. The results for accuracy and precision showed low variability. The body weight was selected as a variable factor, but it showed no effect on the CMAP amplitude. In this study, potency tests using the rat CMAP assay of botulinum toxin type A demonstrated that it met the criteria for a quantitative analysis method.

  15. An epidermal equivalent assay for identification and ranking potency of contact sensitizers

    Energy Technology Data Exchange (ETDEWEB)

    Gibbs, Susan, E-mail: S.Gibbs@VUMC.nl [Department of Dermatology, VU University Medical Centre, Dept of Oral Cell Biology, ACTA, Amsterdam (Netherlands); Corsini, Emanuela [Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano (Italy); Spiekstra, Sander W. [Department of Dermatology, VU University Medical Centre, Dept of Oral Cell Biology, ACTA, Amsterdam (Netherlands); Galbiati, Valentina [Laboratory of Toxicology, DiSFeB, Università degli Studi di Milano (Italy); Fuchs, Horst W. [CellSystems GmbH, Troisdorf (Germany); DeGeorge, George; Troese, Matthew [MB Research Labs, Spinnerstown, PA (United States); Hayden, Patrick; Deng, Wei [MatTek Corporation, Ashland, MA (United States); Roggen, Erwin [3Rs Management and Consultancy (Denmark)

    2013-10-15

    The purpose of this study was to explore the possibility of combining the epidermal equivalent (EE) potency assay with the assay which assesses release of interleukin-18 (IL-18) to provide a single test for identification and classification of skin sensitizing chemicals, including chemicals of low water solubility or stability. A protocol was developed using different 3D-epidermal models including in house VUMC model, epiCS® (previously EST1000™), MatTek EpiDerm™ and SkinEthic™ RHE and also the impact of different vehicles (acetone:olive oil 4:1, 1% DMSO, ethanol, water) was investigated. Following topical exposure for 24 h to 17 contact allergens and 13 non-sensitizers a robust increase in IL-18 release was observed only after exposure to contact allergens. A putative prediction model is proposed from data obtained from two laboratories yielding 95% accuracy. Correlating the in vitro EE sensitizer potency data, which assesses the chemical concentration which results in 50% cytotoxicity (EE-EC{sub 50}) with human and animal data showed a superior correlation with human DSA{sub 05} (μg/cm{sup 2}) data (Spearman r = 0.8500; P value (two-tailed) = 0.0061) compared to LLNA data (Spearman r = 0.5968; P value (two-tailed) = 0.0542). DSA{sub 05} = induction dose per skin area that produces a positive response in 5% of the tested population Also a good correlation was observed for release of IL-18 (SI-2) into culture supernatants with human DSA{sub 05} data (Spearman r = 0.8333; P value (two-tailed) = 0.0154). This easily transferable human in vitro assay appears to be very promising, but additional testing of a larger chemical set with the different EE models is required to fully evaluate the utility of this assay and to establish a definitive prediction model. - Highlights: • A potential epidermal equivalent assay to label and classify sensitizers • Il-18 release distinguishes sensitizers from non sensitizers • IL-18 release can rank sensitizer potency

  16. Sanctioning Large-Scale Domestic Cannabis Production - Potency, Yield and Professionalism

    DEFF Research Database (Denmark)

    Møller, Kim; Lindholst, Christian

    2014-01-01

    Domestically cultivated cannabis, referred to as sinsemilla, constitutes a growing share of the illicit drug markets in the Scandinavian countries. In this study we present forensic evidence of THC content in sinsemilla and resin confiscated by the Danish police from 2008 to 2012. The purpose...... that courts do not apply a yield-percentage estimate. The specificities of domestic cannabis cultivation also relate to the sanction criteria „professionalism”. Firstly, the number of plants found can provide for calculation of an aggregate quantum. Secondly, this can be related to the formal quantum......-scale cannabis cases would improve by applying a 1:1 potency level between sinsemilla and resin....

  17. Increased potency of an inactivated trivalent polio vaccine with oil-in-water emulsions.

    Science.gov (United States)

    Baldwin, Susan L; Fox, Christopher B; Pallansch, Mark A; Coler, Rhea N; Reed, Steven G; Friede, Martin

    2011-01-17

    The use of inactivated poliovirus vaccines (IPV) will be required to achieve, world-wide eradication of polio. The current expense of IPV is however prohibitive for, some countries, and therefore efforts to decrease the costs of the vaccine are a high, priority. Our results show that the addition of oil-in-water emulsion adjuvants to an, inactivated trivalent poliovirus vaccine are dose-sparing and are capable of enhancing, neutralizing antibody titers in the rat potency model. Copyright © 2010 Elsevier Ltd. All rights reserved.

  18. An Extensive Survey of the Phytochemistry and Therapeutic Potency of Ocimum sanctum (Queen of Herbs

    Directory of Open Access Journals (Sweden)

    *S. K. Bariyah

    2013-03-01

    Full Text Available Ocimum sanctum, known as Queen of Herbs, is an important member of the family Lamiaceae due to its use in herbal medication centuries back, especially, in India and other parts of the sub-continent. It is still a subject of immense importance in modern medical research and it is due to the chemical constituents present in it like flavonoids, terpenoids, alkaloids, saponins, vitamins, minerals, proteins, carbohydrates and many others. It has shown a wide range of therapeutic potencies like antimicrobial, anticataleptic, antitoxic, immunomodulatory, analgesic, antidiabetic and cardioprotective activities. The aim of the present review is to present an extensive survey on the phytochemistry and pharmacological applications of the herb.

  19. The solidarization potencies of Buddhist axiological system and humanistic grounding of its priorities

    Directory of Open Access Journals (Sweden)

    Stefaniv Maryana Ivanivna

    2016-10-01

    Full Text Available The article is devoted to the analysis of the value system of the Buddhist religion in the context of current globalization trends. The author considers the stereotypes about the confrontation in the parameters “West – East” and offers the alternative view on the model of Buddhist values and its solidarization potencies. The author extrapolates her ideas on the intellectual and practical sense of a famous Buddhist philosopher Daisaku Ikeda. The author emphasizes the importance of a practical way of aesthetic and ethical projects initiated by Ikeda and justifies their humanistic social significance.

  20. Potency of Intraguild Predation to Disrupt the Optimum Functions of Predatory Arthropods: An Ecological Perspective

    Directory of Open Access Journals (Sweden)

    Nugroho Susetya Putra

    2006-12-01

    Full Text Available Some empirical studies have revealed the ecological consequence of interspecific relationships among predatory arthropods that is the disruption of natural functions of indigenous species of predators on their preys, i.e. phytophagous arthropods. In relation to many efforts on the utilization of introduced species of natural enemies, particularly predatory arthropods, the effects oflntraguild Predation (IGP on key predators should be considered carefully to optimize their potency. In addition, understanding the impact of biological traits of each species of predatory arthropods on their ability to adapt from being predated by other species might be important to predict their composition and possibilities for being exist in ecosystem.

  1. Acceleromyography and mechanomyography for establishing potency of neuromuscular blocking agents: a randomized-controlled trial

    DEFF Research Database (Denmark)

    Claudius, C; Viby-Mogensen, J; Skovgaard, Lene Theil

    2009-01-01

    and opioid. Neuromuscular blockade was induced with rocuronium 100, 150, 200 or 250 microg/kg. Neuromuscular monitoring was performed with AMG (TOF-Watch SX) with pre-load (Hand Adapter) at one arm and MMG (modified TOF-Watch SX) on the other, using 0.1 Hz single twitch stimulation. Dose......) for this purpose. The aim of this study was to compare AMG and MMG for establishing dose-response relationship and potency, using rocuronium as an example. METHODS: We included 40 adult patients in this randomized-controlled single-dose response study. Anaesthesia was induced and maintained with propofol...

  2. Gestodene: a review of its pharmacology, potency and tolerability in combined contraceptive preparations.

    Science.gov (United States)

    Stanczyk, Frank Z; Archer, David F

    2014-04-01

    Combined progestin-estrogen pills are an established and reliable contraceptive option used by women worldwide. Combined oral contraceptives (COCs) containing the progestins--gestodene, desogestrel or norgestimate--were developed to minimize androgenic side effects and are considered an effective, well-tolerated contraceptive option. Gestodene achieves contraceptive efficacy with the lowest dose of any progestin in a COC, and has an established and favorable short- and long-term tolerability profile. In this review we present an overview of the pharmacology, potency and tolerability of gestodene.

  3. A retrospective evaluation of the efficacy of intravenous bumetanide and comparison of potency with furosemide

    Directory of Open Access Journals (Sweden)

    Nappi JM

    2013-03-01

    Full Text Available Background: The potency of intravenous bumetanide to furosemide using a ratio of 1:40 has been suggested; however, there are little data supporting this ratio. Recent drug shortages required the use of bumetanide in a large patient population, enabling further characterization of the efficacy of IV bumetanide.Objective: The primary objective of this study was to estimate a dose-response effect of IV bumetanide on urine output (UOP in all patients that received 48 hours of therapy as well as in a subgroup of patients with heart failure (HF. This subgroup was used to compare the potency of bumetanide with furosemide. A secondary safety objective described electrolyte replacement required during therapy. Methods: This was a single-center retrospective study examining the dose-response effect of IV bumetanide in patients receiving at least 48 hours of intermittent (iIV or continuous (cIV dosing, measured by UOP per mg of drug received (mL/mg. The potency of IV bumetanide was compared with furosemide in a subset of patients with HF using pre-existing data. The safety of IV bumetanide was analyzed by quantifying electrolyte replacement received during the study period.Results: The primary outcome was higher in the iIV group (n=93 at 1273 ± 844 mL/mg compared with the cIV group (n=16 at 749 ± 370 mL/mg (P=0.002. Among patients with HF who received furosemide (iIV n=30, cIV n=26 or bumetanide (iIV n=30, cIV n=3, a potency ratio of 41:1 was found for the iIV group and 34:1 for all patients with HF. There was no significant difference in electrolyte replacement between groups.Conclusion: A greater response was seen with intermittent bumetanide compared with continuous infusion bumetanide. This study supports the 40:1 dose equivalence ratio (furosemide:bumetanide in patients with HF receiving at least 48 hours of intravenous intermittent bumetanide.

  4. Degradable terpolymers with alkyl side chains demonstrate enhanced gene delivery potency and nanoparticle stability.

    Science.gov (United States)

    Eltoukhy, Ahmed A; Chen, Delai; Alabi, Christopher A; Langer, Robert; Anderson, Daniel G

    2013-03-13

    Degradable, cationic poly(β-amino ester)s (PBAEs) with alkyl side chains are developed for non-viral gene delivery. Nanoparticles formed from these PBAE terpolymers exhibit significantly enhanced DNA transfection potency and resistance to aggregation. These hydrophobic PBAE terpolymers, but not PBAEs lacking alkyl side chains, support interaction with PEG-lipid conjugates, facilitating their functionalization with shielding and targeting moieties and accelerating the in vivo translation of these materials. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. THE EFFECT OF DIGESTIVE JUICES ON THE POTENCY OF BOTULINUS TOXIN.

    Science.gov (United States)

    Bronfenbrenner, J J; Schlesinger, M J

    1924-03-31

    Botulinus toxin resists a degree of acidity equivalent to that of the stomach even when exposed thereto for 24 hours at 37 degrees C. It is less resistant to alkali, however, as shown by the fact that in a medium of weakly alkaline reaction its potency is reduced to less than one-tenth in 24 hours. It is unaffected by peptic and tryptic digestion. Because of its instability in mildly alkaline solutions and resistance to acid, there is reason to believe that the toxin is in the main absorbed from the upper portion of the digestive tract; that is to say, from the stomach and upper duodenum.

  6. Estrogenic and esterase-inhibiting potency in rainwater in relation to pesticide concentrations, sampling season and location.

    Science.gov (United States)

    Hamers, Timo; van den Brink, Paul J; Mos, Lizzy; van der Linden, Sander C; Legler, Juliette; Koeman, Jan H; Murk, Albertinka J

    2003-01-01

    In a year-round monitoring program (1998), pesticide composition and toxic potency of the mix of pollutants present in rainwater were measured. The goal of the study was to relate atmospheric deposition of toxic potency and pesticide composition to each other and to sampling period and local agricultural activity. Rainwater was collected in 26 consecutive periods of 14 days in a background location (BACK) and in two locations representative for different agricultural practices, i.e. intensive greenhouse horticulture (HORT) and flower bulb culture (BULB). Samples were chemically analyzed for carbamate (CARB), organophosphate (OP) and organochlorine (OC) pesticides and metabolites. Esterase inhibiting potency of rainwater extracts was measured in a specially developed bio-assay with honeybee esterases and was expressed as an equivalent concentration of the model inhibitor dichlorvos. Estrogenic potency of the extracts was measured in the ER-CALUX reporter gene assay and was expressed as an equivalent concentration of estradiol. Multivariate principal component analysis (PCA) techniques proved to be valuable tools to analyze the numerous pesticide concentrations in relation to toxic potency, sampling location, and sampling season. Pesticide composition in rainwater depended much more on sampling season than on sampling location, but differences between and were mainly attributed to local differences in agricultural practice. On average, the esterase inhibiting potency exceeded the maximum permissible concentration set for dichlorvos in The Netherlands, and was significantly higher in than in and . Esterase inhibition correlated significantly with OP and CARB concentrations, as expected given the working mechanism of these insecticides. The estrogenic potency incidentally exceeded NOEC levels reported for aquatic organisms and was highest in . Although estrogenic potency of rainwater correlated with OC concentrations, the ER-CALUX responses could not be attributed to

  7. Ligand-dependent exciton dynamics and photovoltaic properties of PbS quantum dot heterojunction solar cells.

    Science.gov (United States)

    Chang, Jin; Ogomi, Yuhei; Ding, Chao; Zhang, Yao Hong; Toyoda, Taro; Hayase, Shuzi; Katayama, Kenji; Shen, Qing

    2017-03-01

    The surface chemistry of colloidal quantum dots (QDs) plays an important role in determining the photoelectric properties of QD films and the corresponding quantum dot heterojunction solar cells (QDHSCs). To investigate the effects of the ligand structure on the photovoltaic performance and exciton dynamics of QDHSCs, PbS QDHSCs were fabricated by the solid state ligand exchange method with mercaptoalkanoic acid as the cross-linking ligand. Temperature-dependent photoluminescence and ultrafast transient absorption spectra show that the electronic coupling and charge transfer rate within QD ensembles were monotonically enhanced as the ligand length decreased. However, in practical QDHSCs, the second shortest ligand 3-mercaptopropionic acid (MPA) showed higher power conversion efficiency than the shortest ligand thioglycolic acid (TGA). This could be attributed to the difference in their surface trap states, supported by thermally stimulated current measurements. Moreover, compared with the non-conjugated ligand MPA, the conjugated ligand 4-mercaptobenzoic acid (MBA) introduces less trap states and has a similar charge transfer rate in QD ensembles, but has poor photovoltaic properties. This unexpected result could be contributed by the QD-ligand orbital mixing, leading to the charge transfer from QDs to ligands instead of charge transfer between adjacent QDs. This work highlights the significant effects of ligand structures on the photovoltaic properties and exciton dynamics of QDHSCs, which would shed light on the further development of QD-based photoelectric devices.