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Synthesis and evaluation of 3-aminopropionyl substituted fentanyl analogues for opioid activity

Petrov, Ravil R.; Vardanyan, Ruben S.; Lee, Yeon S.; Ma, Shou-wu; Davis, Peg; Begay, Lucinda J.; Lai, Josephine Y.; Porreca, Frank; Hruby, Victor J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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An enkephalin analogue coupled to ‘aminofentanyl’ has been synthesized and tested for biological activities at the μ and δ opioid receptors. Aminofentanyl which represents a structural derivative of fentanyl has been synthesized by acylation of 1-(2-phenethyl)-4-(N-anilino)piperidine with phthaloyl protected β-alaninyl chloride in the presence of DIPEA, followed by deprotection with hydrazine hydrate. Aminofentanyl has also been successfully acylated with ethyl isocyanate, various acid anhydrides, to further investigate structure–activity relationships of these new fentanyl derivatives. Among the new derivatives compound 7 which carries a Tyr-D-Ala-Gly-Phe opioid message sequence showed good opioid affinity (1 nM at both δ and μ opioid receptors) and bioactivity (34.9 nM in MVD and 42 nM in GPI/LMMP bioassays).

N,N-diallyl-tyrosyl substitution confers antagonist properties on the kappa-selective opioid peptide [D-Pro10]dynorphin A(1-11).

Gairin, J. E.; Mazarguil, H.; Alvinerie, P.; Botanch, C.; Cros, J.; Meunier, J. C.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1988 EN
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1. In the search for kappa-opioid antagonists, we have designed two N,N-diallyl substituted analogues of the kappa-selective peptide [D-Pro10]dynorphin A (1-11)(DPDYN). In this study, we have examined (i) the binding properties of N,N-diallyl-DPDYN (analogue 1) and N,N-diallyl-[Aib2,3]DPDYN (analogue 2) at the three main types (mu, delta, kappa) of opioid binding sites, (ii) their binding sensitivity to Na+ ions (120 mM NaCl) and guanine nucleotide (50 microM Gpp(NH)p) at mu- and kappa-binding sites and (iii) their biological activity in two pharmacological bioassays specific for mu- and kappa-(guinea-pig ileum) and kappa-(rabbit vas deferens) opioid receptors. 2. Steric hindrance resulting from incorporation of two bulky allyl groups at the tyrosal nitrogen atom greatly altered the binding properties of DPDYN. A dramatic fall in apparent affinity for the three types (mu, delta, kappa) of site as well as selectivity for kappa-sites was observed for the two N,N-diallyl-substituted peptide analogues. 3. At kappa-sites of guinea-pig cerebellum and mu-sites of rabbit cerebellum, N,N-diallyl-substitution led to a complete loss of binding sensitivity to the inhibitory effect of 120 mM NaCl + 50 microM Gpp(NH)p compared to the high sensitivity of DPDYN. This may therefore suggest that the N...

2-Naphthalenesulphonyl L-aspartyl-(2-phenethyl)amide (2-NAP)--a selective cholecystokinin CCKA-receptor antagonist.

Hull, R. A.; Shankley, N. P.; Harper, E. A.; Gerkowitch, V. P.; Black, J. W.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1993 EN
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1. The in vitro pharmacological characterization of the sodium salt of 2-naphthalenesulphonyl 1-aspartyl-(2-phenethyl)amide [2-NAP], a hydrophilic compound derived from the C-terminal aspartate-phenylalanine dipeptide of cholecystokinin (CCK), is described. 2. 2-NAP behaved as a competitive antagonist of sulphated cholecystokinin octapeptide (CCK-8) at CCKA-receptors in both intact tissue bioassays (guinea-pig gall bladder, pancreas and ileum, human and rabbit gall bladder) and a radioligand displacement assay (guinea-pig pancreatic cells). The mean pKB, over assays, was 6.5. 3. Compared to the other assays, the rabbit gall bladder assay gave a significantly higher pKB estimate [7.0] for 2-NAP and a significantly lower estimate [8.9] for devazepide (formerly L-364,718 and MK-329), a well-characterized CCKA-receptor antagonist; these anomalous results suggest that a different class of CCKA-receptors may be involved. 4. 2-NAP, was found to be highly selective, having at least 300 fold greater affinity for CCKA-receptors than for 50 other pharmacological loci, including gastrin/CCKB, as estimated by bioassay or radioligand displacement.

Inhibition of interleukin 1 (IL-1) binding and bioactivity in vitro and modulation of acute inflammation in vivo by IL-1 receptor antagonist and anti-IL-1 receptor monoclonal antibody

Fonte: The Rockefeller University Press Publicador: The Rockefeller University Press
Tipo: Artigo de Revista Científica
Publicado em 01/04/1991 EN
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Recombinant human interleukin 1 receptor antagonist (IL-1ra) and 35F5, a neutralizing monoclonal antibody (mAb) to the type I mouse IL-1 receptor, were examined for their ability to bind to IL-1 receptors (IL- 1Rs) on various types of mouse cells and to block immune and inflammatory responses to IL-1 in vitro and in mice. IL-1ra competed for binding of 125I-IL-1 alpha to type I IL-1R present on EL-4 thymoma cells, 3T3 fibroblasts, hepatocytes, and Chinese hamster ovary cells expressing recombinant mouse type I IL-1R. The IC50 values for IL-1ra binding (ranging from 2 to 4 ng/ml) were similar to those of IL-1 alpha. In contrast, IL-1ra bound with very low affinity (IC50 values ranging from 10 to 200 micrograms/ml) to cells expressing type II IL- 1R, i.e., 70Z/3 pre-B cell line and polymorphonuclear leukocytes (PMN) derived from bone marrow and acute inflammatory exudates. The mAb 35F5 bound specifically to type I IL-1R; no inhibition of 125I-IL-1 alpha binding to cells having type II IL-1R was observed with very high concentrations of antibody. While neither IL-1ra nor 35F5 had intrinsic activity in bioassays using T helper D10.G4.1 cells and mouse thymocytes, both agents blocked the ability of IL-1 to stimulate proliferation of these cells. The effects of IL-1ra and 35F5 on acute inflammatory responses in mice were also evaluated. IL-1ra and 35F5 blocked the local accumulation of PMN after intraperitoneal injection of rIL-1 alpha. The response to IL-1 was inhibited when IL-1ra or 35F5 was administered simultaneously with or before administration of IL-1. IL-1ra and 35F5 also blocked PMN accumulation after intraperitoneal injection of lipopolysaccharide or proteose peptone...

Neutralizing antibodies to granulocyte–macrophage colony‐stimulating factor, interleukin‐1α and interferon‐α but not other cytokines in human immunoglobulin preparations

Wadhwa, M; Meager, A; Dilger, P; Bird, C; Dolman, C; Das, R G; Thorpe, R
Fonte: Blackwell Science Inc Publicador: Blackwell Science Inc
Tipo: Artigo de Revista Científica
Publicado em /01/2000 EN
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Human immunoglobulin preparations are used therapeutically for various disorders. Such therapy is generally safe but adverse effects occasionally occur in recipients. It has been suggested that antibodies to cytokines present in clinical immunoglobulin products may contribute to undesirable effects in recipients. Therefore, we investigated intravenous and intramuscular immunoglobulin products for the presence of cytokine‐specific neutralizing antibodies. Using validated bioassays, we detected neutralizing activity against human granulocyte–macrophage colony‐stimulating factor (GM‐CSF), interferon‐α2a (IFN‐α2a) and interleukin‐1α (IL‐1α) in immunoglobulin products. We found no neutralization of granulocyte colony‐stimulating factor, macrophage colony‐stimulating factor, stem cell factor, IL‐1β, IL‐2, IL‐3, IL‐4, IL‐6, IL‐9, IL‐10, IL‐12, tumour necrosis factor‐α, oncostatin M (OSM) and IFN‐γ. Most batches which neutralized IFN‐α2a activity also neutralized other IFN‐α subtypes, IFN‐ω and IFN‐β. Most products (94%) neutralized the biological activity of GM‐CSF. No correlation between batches and their ability to neutralize bioactivities of GM‐CSF, IFN‐α2a and IL‐1α was found. This neutralizing activity could be traced to plasma pools used for manufacture of immunoglobulins. The neutralization was mediated by specific cytokine antibodies contained within immunoglobulin products as it was present in specific immunoglobulin G (IgG) fractions eluted from cytokine affinity chromatography columns. Specific binding of such IgG fractions to cytokines in immunoblots and in enzyme‐linked immunosorbent assays (ELISAs) was observed. This contrasts with the broad non‐specific recognition of cytokine proteins observed using unfractionated immunoglobulins in ELISAs. This is the first comprehensive study showing the presence of neutralizing antibodies against GM‐CSF...

Functional Characterization of HFR1, a High-Mannose N-Glycan-Specific Wheat Lectin Induced by Hessian Fly Larvae1[C][W]

Subramanyam, Subhashree; Smith, David F.; Clemens, James C.; Webb, Mary A.; Sardesai, Nagesh; Williams, Christie E.
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /07/2008 EN
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We previously cloned and characterized a novel jacalin-like lectin gene from wheat (Triticum aestivum) plants that responds to infestation by Hessian fly (Mayetiola destructor) larvae, a major dipteran pest of this crop. The infested resistant plants accumulated higher levels of Hfr-1 (for Hessian fly-responsive gene 1) transcripts compared with uninfested or susceptible plants. Here, we characterize the soluble and active recombinant His6-HFR1 protein isolated from Escherichia coli. Functional characterization of the protein using hemagglutination assays revealed lectin activity. Glycan microarray-binding assays indicated strong affinity of His6-HFR1 to Manα1-6(Manα1-3)Man trisaccharide structures. Resistant wheat plants accumulated high levels of HFR1 at the larval feeding sites, as revealed by immunodetection, but the avirulent larvae were deterred from feeding and consumed only small amounts of the lectin. Behavioral studies revealed that avirulent Hessian fly larvae on resistant plants exhibited prolonged searching and writhing behaviors as they unsuccessfully attempted to establish feeding sites. During His6-HFR1 feeding bioassays, Drosophila melanogaster larvae experienced significant delays in growth and pupation, while percentage mortality increased with progressively higher concentrations of His6-HFR1 in the diet. Thus...

Differential Antagonism of Activin, Myostatin and Growth and Differentiation Factor 11 by Wild-Type and Mutant Follistatin

Schneyer, Alan L.; Sidis, Yisrael; Gulati, Anisha; Sun, Jie L.; Keutmann, Henry; Krasney, Philip A.
Fonte: The Endocrine Society Publicador: The Endocrine Society
Tipo: Artigo de Revista Científica
EN
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Follistatin binds and neutralizes members of the TGFβ superfamily including activin, myostatin, and growth and differentiation factor 11 (GDF11). Crystal structure analysis of the follistatin-activin complex revealed extensive contacts between follistatin domain (FSD)-2 and activin that was critical for the high-affinity interaction. However, it remained unknown whether follistatin residues involved with myostatin and GDF11 binding were distinct from those involved with activin binding. If so, this would allow development of myostatin antagonists that would not inhibit activin actions, a desirable feature for development of myostatin antagonists for treatment of muscle-wasting disorders. We tested this hypothesis with our panel of point and domain swapping follistatin mutants using competitive binding analyses and in vitro bioassays. Our results demonstrate that activin binding and neutralization are mediated primarily by FSD2, whereas myostatin binding is more dependent on FSD1, such that deletion of FSD2 or adding an extra FSD1 in place of FSD2 creates myostatin antagonists with vastly reduced activin antagonism. However, these mutants also bind GDF11, indicating that further analysis is required for creation of myostatin antagonists that will not affect GDF11 activity that could potentially elicit GDF11-induced side effects in vivo.

Oxidation of Lanthionines Renders the Lantibiotic Nisin Inactive▿

Wilson-Stanford, Shawanda; Kalli, Anastasia; Håkansson, Kristina; Kastrantas, James; Orugunty, Ravi S.; Smith, Leif
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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The peptide antibiotic nisin A belongs to the group of antibiotics called lantibiotics. They are classified as lantibiotics because they contain the structural group lanthionine. Lanthionines are composed of a single sulfur atom that is linked to the β-carbons of two alanine moieties. These sulfur atoms are vulnerable to environmental oxidation. A mild oxidation reaction was performed on nisin A to determine the relative effects it would have on bioactivity. High-mass-accuracy Fourier transform ion cyclotron resonance mass spectrometry data revealed the addition of seven, eight, and nine oxygens. These additions correspond to the five lanthionines, two methionines, and two histidines that would be susceptible to oxidation. Subsequent bioassays revealed that the oxidized form of nisin A had a complete loss of bactericidal activity. In a competition study, the oxidized nisin did not appear to have an antagonistic affect on the bioactivity of nisin A, since the addition of an equal molar concentration of the oxidized variant did not have an influence on the bactericidal activity of the native antibiotic. Electron microscopy data revealed that the oxidized forms were still capable of assembling into large circular complexes, demonstrating that oxidation does not disrupt the lateral assembly mechanism of the antibiotic. Affinity thin-layer chromatography and fluorescence microscopy experiments suggested that the loss of activity is due to the inability of the oxidized form of nisin to bind to the cell wall precursor lipid II. Given the loss of bioactivity following oxidation...

Identification of the Potent Phytoestrogen Glycinol in Elicited Soybean (Glycine max)

Boué, Stephen M.; Tilghman, Syreeta L.; Elliott, Steven; Zimmerman, M. Carla; Williams, K. Y.; Payton-Stewart, Florastina; Miraflor, Allen P.; Howell, Melanie H.; Shih, Betty Y.; Carter-Wientjes, Carol H.; Segar, Chris; Beckman, Barbara S.; Wiese, Thomas
Fonte: The Endocrine Society Publicador: The Endocrine Society
Tipo: Artigo de Revista Científica
EN
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The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent estrogen antagonists in vitro and in vivo. The discovery of the glyceollins’ ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 μm, which correlated with comparable colony formation of MCF-7 cells at 10 μm. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor-1). Competitive binding assays revealed a high affinity of glycinol for both ERα (ΙC50 = 13.8 nm) and ERβ (ΙC50 = 9.1 nm). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ERα and ERβ. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods.

Downregulation of a Chitin Deacetylase-Like Protein in Response to Baculovirus Infection and Its Application for Improving Baculovirus Infectivity ▿

Jakubowska, Agata K.; Caccia, Silvia; Gordon, Karl H.; Ferré, Juan; Herrero, Salvador
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
EN
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Several expressed sequence tags (ESTs) with homology to chitin deacetylase-like protein (CDA) were selected from a group of Helicoverpa armigera genes whose expression changed after infection with H. armigera single nucleopolyhedrovirus (HearNPV). Some of these ESTs coded for a midgut protein containing a chitin deacetylase domain (CDAD). The expressed protein, HaCDA5a, did not show chitin deacetylase activity, but it showed a strong affinity for binding to chitin. Sequence analysis showed the lack of any chitin binding domain, described for all currently known peritrophic membrane (PM) proteins. HaCDA5a has previously been detected in the H. armigera PM. Such localization, together with its downregulation after pathogen infection, led us to hypothesize that this protein might be responsible for the homeostasis of the PM structure and that, by reduction of its expression, the insect may reduce PM permeability, decreasing the entrance of baculovirus. To test this hypothesis, we constructed a recombinant nucleopolyhedrovirus to express HaCDA5a in insect cells and tested its influence on PM permeability as well as the influence of HaCDA5a expression on the performance of the baculovirus. The experiments showed that HaCDA5a increased PM permeability...

Nucleic acid-based fluorescent probes and their analytical potential

Juskowiak, Bernard
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
EN
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It is well known that nucleic acids play an essential role in living organisms because they store and transmit genetic information and use that information to direct the synthesis of proteins. However, less is known about the ability of nucleic acids to bind specific ligands and the application of oligonucleotides as molecular probes or biosensors. Oligonucleotide probes are single-stranded nucleic acid fragments that can be tailored to have high specificity and affinity for different targets including nucleic acids, proteins, small molecules, and ions. One can divide oligonucleotide-based probes into two main categories: hybridization probes that are based on the formation of complementary base-pairs, and aptamer probes that exploit selective recognition of nonnucleic acid analytes and may be compared with immunosensors. Design and construction of hybridization and aptamer probes are similar. Typically, oligonucleotide (DNA, RNA) with predefined base sequence and length is modified by covalent attachment of reporter groups (one or more fluorophores in fluorescence-based probes). The fluorescent labels act as transducers that transform biorecognition (hybridization, ligand binding) into a fluorescence signal. Fluorescent labels have several advantages...

Expression and characterization of antimicrobial peptides Retrocyclin-101 and Protegrin-1 in chloroplasts to control viral and bacterial infections

Lee, Seung-Bum; Li, Baichuan; Jin, Shuangxia; Daniell, Henry
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/2011 EN
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Retrocyclin-101 (RC101) and Protegrin-1 (PG1) are two important antimicrobial peptides that can be used as therapeutic agents against bacterial and/or viral infections, especially those caused by the HIV-1 or sexually transmitted bacteria. Because of their antimicrobial activity and complex secondary structures, they have not yet been produced in microbial systems and their chemical synthesis is prohibitively expensive. Therefore, we created chloroplast transformation vectors with the RC101 or PG1 coding sequence, fused with GFP to confer stability, furin or Factor Xa cleavage site to liberate the mature peptide from their fusion proteins and a His-tag to aid in their purification. Stable integration of RC101 into the tobacco chloroplast genome and homoplasmy were confirmed by Southern blots. RC101 and PG1 accumulated up to 32%–38% and 17%~26% of the total soluble protein. Both RC101 and PG1 were cleaved from GFP by corresponding proteases in vitro, and Factor Xa–like protease activity was observed within chloroplasts. Confocal microscopy studies showed location of GFP fluorescence within chloroplasts. Organic extraction resulted in 10.6-fold higher yield of RC101 than purification by affinity chromatography using His-tag. In planta bioassays with Erwinia carotovora confirmed the antibacterial activity of RC101 and PG1 expressed in chloroplasts. RC101 transplastomic plants were resistant to tobacco mosaic virus infections...

Synthesis of Novel Estrogen Receptor Antagonists Using Metal-Catalyzed Coupling Reactions and Characterization of Their Biological Activity

Jiang, Xiang-Rong; Wang, Pan; Smith, Carolyn L.; Zhu, Bao Ting
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Estrogen receptor (ER) antagonists are valuable in the treatment of ER-positive human breast cancer. In this study, we designed and synthesized nine new derivatives of 17β-estradiol (E2) with a bulky side chain attached to its C-7α position, and determined their ER antagonistic activity using in vitro bioassays. Four of the derivatives showed a strong inhibition of ERα transactivation activity in a luciferase reporter assay and blocked ERα interactions with coactivators. Similarly, these derivatives also strongly inhibited the growth of the ERα-positive human breast cancer cells. Computational docking analysis was conducted to model the interaction of these antagonists with the human ERα, and showed that they could tightly bind to the ERα in a similar manner as ICI-182,780, a pure ER antagonist. These results provide an example that attachment of a bulky side chain to the C-7α position of E2 can produce ER antagonists with comparable ER affinity as ICI-182,780.

Comparative Studies on Acetylcholinesterase Characteristics between the Aphids, Sitobion avenae and Rhopalosiphum padi

Lu, Y. H.; He, Y. P.; Gao, X. W.
Fonte: University of Wisconsin Library Publicador: University of Wisconsin Library
Tipo: Artigo de Revista Científica
Publicado em 31/01/2013 EN
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The aphids Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) (Hemiptera: Aphidiae) are serious pests on grain crops and usually coexist on late period of wheat growth in China. Bioassays showed that R. padi was more susceptible than S. avenae to pirimicarb that is used for wheat aphid control, and the determination of acetylcholinesterase (AChE, EC 3.1.1.7) sensitivity showed that the sensitivity of AChE to pirimicarb was significantly higher in R. padi than in S. avenae (Lu and Gao 2009). AChE is the target enzyme of the carbamates, including pirimicarb, hence, to understand the mechanism responsible for the tolerance difference to carbamate insecticides of S. avenae and R. padi, we purified AChE from both aphid species using procainamide affinity column and characterized the AChE. The purification factor and yield from S. avenae (234.7-fold and 92.9%) were far higher than that from R. padi 17.3-fold and 13.9%. The results of substrate and inhibitor specificities of purified enzyme from both S. avenae and R. padi indicated that the purified enzyme was a typical AChE. The crude AChE extract from S. avenae was 5.4-, 4.3- and 8.1-fold less sensitive to inhibition by pirimicarb, methomyl and thiodicarb, respectively, than that from R. padi...

A 104kDa Aedes aegypti aminopeptidase N is a putative receptor for the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis

Chen, Jianwu; Likitvivatanavong, Supaporn; Aimanova, Karlygash G.; Gill, Sarjeet S.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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The Cry11Aa protein produced in Bacillus thuringiensis subsp. israelensis, a bacterial strain used worldwide for the control of Aedes aegypti larvae, binds midgut brush border membrane vesicles (BBMV) with an apparent Kd of 29.8 nM. Previously an aminopeptidase N (APN), named AaeAPN2, was identified as a putative Cry11Aa toxin binding protein by pull-down assays using biotinylated Cry11Aa toxin (Chen et al., (2009) Insect Biochem Mol Biol., 39: 688–696). Here we show this protein localizes to the apical membrane of epithelial cells in proximal and distal regions of larval caeca. The AaeAPN2 protein binds Cry11Aa with high affinity, 8.6 nM. The full-length and fragments of AaeAPN2 were cloned and expressed in Escherichia coli. The toxin-binding region was identified and further competitive assays demonstrated that Cry11Aa binding to BBMV was efficiently competed by the full-length AaeAPN2 and the fragments of AaeAPN2b and AaeAPN2e. In bioassays against Ae. aegypti larvae, the presence of full-length and a partial fragment (AaeAPN2b) of AaeAPN2 enhanced Cry11Aa larval mortality. Taken together, we conclude that AaeAPN2 is a binding protein and plays a role in Cry11Aa toxicity.

Integrated microfluidic and imaging platform for a kinase activity radioassay to analyze minute patient cancer samples

Fang, Cong; Wang, Yanju; Vu, Nam T.; Lin, Wei-Yu; Hsieh, Yao-Te; Rubbi, Liudmilla; Phelps, Michael E.; Muschen, Markus; Kim, Yong-Mi; Chatziioannou, Arion F.; Tseng, Hsian-Rong; Graeber, Thomas G.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Oncogenic kinase activity and the resulting aberrant growth and survival signaling is a common driving force of cancer. Accordingly, many successful molecularly targeted anti-cancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane (PDMS) microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric 32P-ATP labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity micro beads. We also developed a solid state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radio bioassays. We demonstrate that the resulting integrated device can measure ABL kinase activity from BCR-ABL positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples.

Mass spectrometric studies on effects of counter ions of TMPyP4 on binding to human telomeric DNA and RNA G-quadruplexes

Bai, Li-Ping; Liu, Jie; Han, Li; Ho, Hing-Man; Wang, Renxiao; Jiang, Zhi-Hong
Fonte: Springer Berlin Heidelberg Publicador: Springer Berlin Heidelberg
Tipo: Artigo de Revista Científica
EN
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A comparative study on human telomeric DNA G-quadruplex binding of meso-5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) between its two salt forms, i.e., tetratosylate and tetrachloride, was conducted by using ESI-TOF-MS, UV-melting measurement, and molecular modeling methods. Besides cation TMPyP4, the tosyl anion was found to bind to human telomeric DNA G-quadruplex with multiple binding stoichiometries from 1:1 to 3:1 observed in ESI-TOF-MS spectra, indicating that the stabilization activity of TMPyP4 tetratosylate on G-quadruplex is derived from a synergetic effect of both TMPyP4 cation and tosyl anion. A molecular modeling study suggests that a tosyl anion fills up the vacant space between TMPyP4 cation and DNA G-quadruplex and thus stabilizes the complex by 3.8 kcal/mol. Therefore, it is estimated that TMPyP4 tetratosylate’s activity might not reflect the real effect of TMPyP4 cation in some bioassays related to G-quadruplex stabilization. This was verified by the results of less binding affinity of TMPyP4 tetrachloride with DNA G-quadruplex obtained from ESI-TOF-MS measurement, and of 2.27 °C less thermal stabilization of TMPyP4 tetrachloride for DNA G-quadruplex, compared to its tetratosylate under the same conditions. Our study demonstrated the influence of counter ions of TMPyP4 on G-quadruplex binding...

Comparative studies on acetylcholinesterase characteristics between the aphids, sitobion avenae and Rhopalosiphum padi

Lu, Y. H.; He, Y. P.; Gao, X. W.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica Formato: text/html
EN
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The aphids Sitobion avenae (Fabricius) and Rhopalosiphum padi (Linnaeus) (Hemiptera: Aphidiae) are serious pests on grain crops and usually coexist on late period of wheat growth in China. Bioassays showed that R. padi was more susceptible than S. avenae to pirimicarb that is used for wheat aphid control, and the determination of acetylcholinesterase (AChE, EC 3.1.1.7) sensitivity showed that the sensitivity of AChE to pirimicarb was significantly higher in R. padi than in S. avenae (Lu and Gao 2009). AChE is the target enzyme of the carbamates, including pirimicarb, hence, to understand the mechanism responsible for the tolerance difference to carbamate insecticides of S. avenae and R. padi, we purified AChE from both aphid species using procainamide affinity column and characterized the AChE. The purification factor and yield from S. avenae (234.7-fold and 92.9%) were far higher than that from R. padi 17.3-fold and 13.9%. The results of substrate and inhibitor specificities of purified enzyme from both S. avenae and R. padi indicated that the purified enzyme was a typical AChE. The crude AChE extract from S. avenae was 5.4-...

Preparation of derivatives of steviol and isosteviol and evaluation of some biological activities

Ullah, Asad
Fonte: Universidade Federal do Paraná Publicador: Universidade Federal do Paraná
Tipo: Tese de Doutorado Formato: 339f. : il. algumas color., tabs., grafs.; application/pdf
INGLêS
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Orientador : Prof. Brás Heleno de Oliveira; Tese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Exatas, Programa de Pós-Graduação em Química. Defesa: Curitiba, 17/04/2015; Inclui referências; Área: Orgnic Chemistry; Resumo: Em relação à contínua necessidade de procurar novas moléculas bioativas e do crescente interesse pelas atividades biológicas de diterpenos tetracíclicos, o principal objetivo deste trabalho foi sintetizar novos derivados de esteviol, isoesteviol e avaliar suas in vitro bioatividades contra alvos biológicos selecionados. Os novos derivados foram 2, 4-dinitrofenil-hidrazina (2, 4-DNPH), 4- nitro-fenil-hidrazona (4-NPH), e também mais simples hidrazona, hidrazona isopropílico. Uma série de éster de benzilo e éster de fenacilo p-metoxi também foram preparados. Estes derivados foram avaliados quanto à sua in vitro antitumoral, anti-maláricos, anti-Trypanosoma cruzi, anti-Corynebacterium diphtheriae e anti-leishmaniose atividades. Os antitumorais bioensaios foram avaliados contra três células tumorais: Carcinoma do pulmão (A549), glioma do cérebro humano (T98MG), glioblastoma humano-astrocitoma (U8MG). Os resultados mostraram que os análogos de isoesteviol possuindo fragmentos de hidrazona e oxima em C16 são citotóxicos. Os ensaios anti-maláricos mostraram que os derivado isoesteviol com 2...

Atividade bioinseticida e mecanismo de ação de vicilinas de sementes Erythrina velutina sobre moscas-das frutas Ceratitis capitata

Macedo, Leonardo Lima Pepino de
Fonte: Universidade Federal do Rio Grande do Norte; BR; UFRN; Programa de Pós-Graduação em Bioquímica; Bioquímica; Biologia Molecular Publicador: Universidade Federal do Rio Grande do Norte; BR; UFRN; Programa de Pós-Graduação em Bioquímica; Bioquímica; Biologia Molecular
Tipo: Dissertação Formato: application/pdf
POR
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The fruit fly Ceratitis capitata is considered the most destructive pest of the world fruitculture. Many pest management practices, mainly based on agrochemicals, have been developed to allow the world-wide commerce of fruit. Solutions to decrease the use of synthetic insecticides in agriculture are based on the development of new target-specific compounds which cause less damage to the environment, especially vegetal proteins with insecticidal effects. The aim of this work was to evaluate the deleterious effect of a purified vicilin of E. velutina (EvV) seeds to C. capitata larvae and adult insects and to investigate the mechanisms involved in these effects. EvV was purified, characterized and its deleterious effect was tested in bioassay systems. EvV mechanism of action was determined by immunodetection techniques and fluorescence localization in chitin structures that are present in C. capitata digestory system. EvV is a glycoprotein with affinity to chitin. Its molecular weight, of 216,57 kDa, was determined by gel filtration chromatography in FPLC system. Using SDS-PAGE, it was possible to observe EvV dissociation in two main subunits of 54,8 and 50,8 kDa. When it was submitted to eletrophoresis in native conditions, EvV presented only one band of acid characteristic. The WD50 and LD50 values found in the bioassays were 0...