Molecular modeling of A1 and A2A adenosine receptors: comparison of rhodopsin- and beta2-adrenergic-based homology models through the docking studies

Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. The homology models of adenosine A1 and A2A receptors were constructed. The high-resolution X-ray structure of bovine rhodopsin and crystal structure of beta2-adrenergic receptor were used as templates. The binding sites of the A1 and A2A ARs were constructed by using data obtained from mutagenesis experiments as well as docking simulations of the respective AR antagonsists DPCPX and XAC. To compare rhodopsin- and beta2-adrenergic-based models, the binding mode of A1 (KW-3902, LUF-5437) and A2A (KW-6002, ZM-241385) ARs antagonists were also examined. The differences in the binding ability of both models were noted during the study. The beta2-adrenergic-based A2A AR model was much more capable to stabilize the ligand in the binding site cavity than the corresponding rhodopsin-based A2A AR model, however, such differences were not so clear in case of A1 AR models. It was suggested that for the A1 AR it is possible to use the crystal structure of rhodopsin as a template as well as beta2-adrenergic receptor, but for A2A AR, with the now available beta2-adrenergic receptor X-ray structure, docking studies should be avoided on the rhodopsin-based model. However, taking into account that the beta2AR shares about 31% of the residues with the AR in comparison to 21% in case of bRho, we suggest using beta2-adrenergic-based models for the A1 and A2A ARs for further in silico ligand screening also because of their generally better ability to stabilize ligands inside the binding pocket.     

A walk around the A3-coupling

In recent years, the transition-metal catalyzed three-component coupling of an aldehyde, an alkyne and an amine, commonly called A(3)-coupling, has been established as a convenient and general approach towards propargylamines. Furthermore, the A(3)-coupling has found a broad application as a key step in the construction of various nitrogen-containing heterocycles, biologically active compounds and natural products. Several interesting modifications of the A(3)-coupling as well as different tandem reactions involving A(3)-coupling have been developed. This tutorial review aims to highlight the current achievements in the field of A(3)-couplings and related transformations.     

Identification and biotyping of clinical isolates of Acinetobacter

A total of 343 Acinetobacter strains, most isolated from hospital patients, were identified using a 16-test system (acid production from glucose, gelatin hydrolysis and utilization of 14 carbon sources) associated with tests for growth at 37, 41 and 44°C. Of 299 nosocomial isolates, 253 were identified as A. baumannii, 20 as Acinetobacter genospcies 3, 8 as A. haemolyticus, 8 as A. lwoffii, 4 as A. johnsonii and 6 as other (presently) unnamed species. A biotyping system based on the utilization of levulinate, citraconate, L-phenylanine, phenylacetate, 4-hydroxybenzoate and L-tartrate allowed recognition of 17 biotypes among 247 A. baumannii isolates. This biotyping system should be useful in epidemiological studies of Acinetobacter strains.     

Antibody purification by affinity chromatography based on small molecule affinity ligands identified by SPR-based screening of chemical microarrays

Libraries of small molecules were searched for Fc-fragment selective binders to a recombinant human antibody ("MDJ8″, IgG(1)-subtype, κ-light chain) via SPR-based screening of chemical microarrays. Identified hit structures were immobilised on NHS-activated Sepharose for the determination of MDJ8 binding and selectivity versus typical proteineous impurities represented by the spend cell culture supernatant. Columns were packed and the most promising ligands further characterized in terms of binding constants, binding kinetics, as well as dynamic and equilibrium binding capacities. The performance of the best ligand, 2A10, was compared to standard Protein A chromatography. Using ligand 2A10 antibody capture from unprocessed cell culture supernatants was possible at similar recovery yield (>90%), purity (>80%), and eluting concentration (approximately 1 g/L) as with Protein A. Affinity constants (K(d)) of 2A10 were an order of magnitude higher than for the Protein A material, but still in the nM-range, while maximum binding capacities and binding kinetics were in the same order of magnitude. Ligand 2A10 was also able to capture a murine monoclonal antibody, again with similar efficiency as Protein A, as well as a number of humanised therapeutic antibodies. Antibody elution from the 2A10 column was possible using the Protein A standard protocol, i.e. 100mM glycine HCl pH 3.0, but also at near physiological pH, when some organic solvent or modifiers were present. Ligand 2A10 thus constitutes a cheaper, more robust alternative to Protein A as possible generic antibody binder. Moreover, the outlined approach to ligand selection could in principle by used to create suitable affinity ligands for other high value biotech products.     

Exploring organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton channel

We describe the use of organosilanes as inhibitors and structural probes of a membrane protein, the M2 proton channel from influenza A virus. Organosilane amine inhibitors were found to be generally as potent as their carbon analogues in targeting WT A/M2 and more potent against the drug-resistant A/M2-V27A mutant. In addition, intermolecular NOESY spectra with dimethyl-substituted organosilane amine inhibitors clearly located the drug binding site at the N-terminal lumen of the A/M2 channel close to V27.     

Organic–Organic Heterostructures: Concepts and Applications

We review basic concepts as well as recent examples and applications of organic–organic heterostructures. We organize the different types of heterostructures according to material A deposited on material B (A/B), A co‐deposited with B (A:B), heterostructures in the monolayer regime including nanostructuring concepts and systems involving self‐assembled monolayers, as well as various other architectures, including superlattices. While most examples are related to small‐molecule organic semiconductors, many of the ideas can be applied to other systems. The central theme is growth and structure as well as optical and electronic properties. Finally, we comment on implications for device applications.     

Il17A Autoantibody Induced by Recombinant Protein Ag85A-Il17A

IL17A is a widely studied cytokine which plays an important role in autoimmune diseases as well as asthma. In this study, murine IL17a gene fragment was inserted into pET28a-Ag85A and it was expressed as a fusion protein named Ag85A-IL17A. Recombinant protein Ag85A-IL17A was expressed as an open reading frame of 1,287 bp, encoding a polypeptide of 429 amino acid residues with a predicted molecular mass of 50 kDa. It was expressed in Escherichia coli. BL21 and purified by metal affinity chromatography using a nickel–nitrilotriacetic acid column. Bioinformatics revealed that Ag85A-IL17A contained B cell epitopes of IL17A. Purified proteins Ag85A-IL17A and Ag85A were injected with adjuvants into mice then antibody responses in sera were measured by enzyme-linked immunosorbent assay and western blot assay. Experiments showed that purified protein Ag85-IL17A could bind to a standard IL17A antibody and could induce IL17A autoantibody in mice.     

氟烷基取代的双酚A类衍生物的合成

利用相转移催化剂十六烷基三甲基溴化铵(CTMAB)催化的连二亚硫酸钠引发的苯酚衍生物与含氟碘代烷的氟烷基化反应, 合成了含氟烷基取代的酚类化合物. 研究了双酚A与含氟碘代烷的氟烷基化反应, 得到了一类重要的含氟单体, 即含氟烷基取代的双酚A衍生物. 通过改变含氟碘代烷与双酚A的摩尔比, 可以得到单取代、二取代和四取代的含氟烷基双酚A衍生物.     

The effects of anion variation and ligand derivatization on silver coordination networks based upon weaker interactions

This article presents a series of silver(I) coordination networks based upon nonchelating bidentate thioether ligands. Frameworks using AgOTs as the silver(I) starting material form two-dimensional frameworks and are quite stable as shown by differential scanning calorimetry/thermogravimetric analysis (DSC/TGA) data. The networks are sufficiently robust as to maintain the same layered motif when the basic skeleton of the ligand is sequentially derivatized with -OEt, OBu, and OHex groups. Crystal structures of the AgOTs complexes of the underivatized and bis(hexoxy) derivatives, compounds 5 and 8, respectively, are presented as well as powder X-ray diffraction (PXRD) data of the other complexes. For 5, C20H20S3O3Ag, crystal data are as follows: monoclinic, space group P2(1)/n, a = 11.8117(5) A, b = 7.8813(5) A, c = 22.3316(10) A, beta = 102.245(5) degrees, V = 2031.6(2) A(3), Z = 4. For 8, C30H44S3O6Ag, crystal data are as follows: triclinic, space group Ponebar a = 8.445(4) A, b = 10.855(5) A, c = 19.308(9) A, alpha = 84.53(1) degrees, beta = 78.76(1) degrees, gamma = 68.43(1) degrees V = 1613.9(13) A(3), Z = 2. Changing the silver(I) starting material to AgPF6 results in a shift to a one-dimensional structure, 9, as shown by X-ray crystallography and in highly compromised stability. For 9, C14H16S2N2PF6Ag, crystal data are as follows: monoclinic, space group P2/n, a = 11.9658(11) A, b = 3.9056(4) A, c = 19.6400(18) A, beta = 92.87(1) degrees, V = 916.70(15) A(3), Z = 4.     

Investigation of high current density on zinc electrodeposition and anodic corrosion in zinc electrowinning

Journal of Solid State Electrochemistry - In this work, the effects of high current density (500 A/m2, 600 A/m2, 700 A/m2, 800 A/m2) on zinc electrodeposition as well as the anodic corrosion...     

Synthesis and biological activities of 4-phenyl-5-pyridyl-1,3-thiazole derivatives as selective adenosine A3 antagonists

To investigate the potency of an adenosine A3 receptor (A3AR) antagonist as an anti-asthmatic drug, a novel series of 4-phenyl-5-pyridyl-1,3-thiazole derivatives was synthesized and evaluated in human adenosine A1, A2A and A3 receptor and rat adenosine A3 receptor binding assays. From investigation of the SAR study, compound 7af was identified as a highly potent human and rat A3AR antagonist. This compound inhibited IB-MECA-induced plasma protein extravasation in the skin of rats and showed good oral absorption. Also, compound 7af significantly inhibited antigen-induced hyper-responsiveness to acetylcholine in actively sensitized Brown Norway rats. These results show that 4-phenyl-5-pyridyl-1,3-thiazole derivatives are potential candidates to enable the evaluation of A3AR antagonists. Further evaluation of this class of compounds may afford a novel anti-inflammatory agent such as an anti-asthmatic drug.     

Synthesis and anti-tumor activity of new steroidal nuclear analogues of aragusterol A

3alpha,7alpha-Dihydroxy-5-epiaragusterol A (3) was synthesized from bile acid (cholic acid) as a new steroidal nuclear analogue of antitumor marine steroid aragusterol A. 7alpha-Hydroxyaragusterol A (4) was also derived from xestokerol B. The in vitro anti-proliferative activity of each of these analogues toward KB cells as well as in vivo anti-tumor activity of 5-epiaragusterol A (2) previously synthesized by the authors and 3 were assessed.     

Phellifuropyranone A: a new furopyranone compound isolated from fruit bodies of wild Phellinus linteus

A new furopyranone, phellifuropyranone A, was isolated from fruit bodies of wild Phellinus linteus as well as phelligridin G, and their chemical structures were determined by various spectroscopic methods including measurement of NMR spectra. Phellifuropyranone A together with meshimakobnol A and meshimakobnol B showed antiproliferative activity against mouse melanoma cells and human lung cancer cells in vitro.     

Specific GABA(A) agonists and partial agonists

The GABA(A) receptor system is implicated in a number of neurological and psychiatric diseases, making GABA(A) receptor ligands interesting as potential therapeutic agents. Only a few different classes of structures are currently known as ligands for the GABA recognition site on the hetero-pentameric GABA(A) receptor complex, reflecting the very strict structural requirements for GABA(A) receptor recognition and activation. Within the series of compounds showing agonist activity at the GABA(A) receptor site that have been developed, most of the ligands are structurally derived from the GABA(A) agonists muscimol, THIP, or isoguvacine, which we developed in the initial stages of the project. Using recombinant GABA(A) receptors, functional selectivity was demonstrated for a number of compounds, including THIP, showing highly subunit-dependent potency and maximal response. In light of the interest in partial GABA(A) receptor agonists as potential therapeutics, structure-activity studies of a number of analogs of 4-PIOL, a low-efficacy partial GABA(A) agonist derived from THIP, have been performed. In this connection, a series of GABA(A) ligands has been developed that exhibit pharmacological profiles from moderately potent low-efficacy partial GABA(A) agonist activity to potent and selective antagonist effects. Very little information is available on direct-acting GABA(A) receptor agonists in clinical studies. However, the results of clinical studies on the effect of the partial GABA(A) agonist THIP on human sleep patterns show that the functional consequences of a direct-acting agonist are different from those seen after the administration of GABA(A) receptor modulators, such as benzodiazepines and barbiturates.     

Evaluation of the Expression of Amyloid Precursor Protein and the Ratio of Secreted Amyloid Beta 42 to Amyloid Beta 40 in SH-SY5Y Cells Stably Transfected with Wild-Type,Single-Mutant and Double-Mutant Forms of the APP Gene for the Study of Alzheimer’s Disease Pathology

Neuroblastoma cell lines such as SH-SY5Y are the most frequently utilized models in neurodegenerative research, and their use has advanced the understanding of the pathology of neurodegeneration over the past few decades. In Alzheimer’s disease (AD), several pathogenic mutations have been described, all of which cause elevated levels of pathological hallmarks such as amyloid-beta (Aβ). Although the genetics of Alzheimer’s disease is well known, familial AD only accounts for a small number of cases in the population, with the rest being sporadic AD, which contains no known mutations. Currently, most of the in vitro models used to study AD pathogenesis only examine the level of Aβ42 as a confirmation of successful model generation and only perform comparisons between wild-type APP and single mutants of the APP gene. Recent findings have shown that the Aβ42/40 ratio in cerebrospinal fluid (CSF) is a better diagnostic indicator for AD patients than is Aβ42 alone and that more extensive Aβ formation, such as accumulation of intraneuronal Aβ, Aβ plaques, soluble oligomeric Aβ (oAβ), and insoluble fibrillar Aβ (fAβ) occurs in TgCRND8 mice expressing a double-mutant form (Swedish and Indiana) of APP, later leading to greater progressive impairment of the brain. In this study, we generated SH-SY5Y cells stably transfected separately with wild-type APP, the Swedish mutation of APP, and the Swedish and Indiana mutations of APP and evaluated the APP expression as well as the Aβ42/40 ratio in those cells. The double-mutant form of APP (Swedish/Indiana) expressed markedly high levels of APP protein and showed a high Aβ2/40 ratio compared to wild-type and single-mutant cells.     

Molecularly asymmetric triblock copolymers as a single-molecule route to ordered bidisperse polymer brushes

The conditions signaling the formation of bidisperse brushes in ordered block copolymers are investigated as an A(2) block is progressively grown onto an A(1)B diblock copolymer to form a series of molecularly asymmetric, isomorphic A(1)BA(2) triblock copolymers. Small-angle scattering and self-consistent field theory confirm that the microphase-ordered period decreases when the A(2) block is short relative to the A(1) block, but then increases as A(1)+A(2) bidisperse brushes develop. The mechanical properties systematically follow the spatial distribution of the A(2) block.     

Modification of a Small β‐Barrel Protein,To Give Pseudo‐Amyloid Structures,Inhibits Amyloid β‐Peptide Aggregation

Aggregation of amyloid β‐peptide (Aβ) is closely related to the pathogenesis of Alzheimer’s disease (AD). Although much effort has been devoted to the construction of molecules that inhibit the aggregation of Aβ1‐42, high doses are needed for the inhibition of Aβ aggregation in many cases. Previously, we reported that designed green fluorescent protein (GFP) analogues that gives pseudo‐Aβ β‐sheet structures can work as an aggregation inhibitor against Aβ. To further test this design strategy, we constructed protein analogues that mimic Aβ β‐sheet structures of amyloids by using insulin‐like growth factor 2 receptor domain 11 (IGF2R‐d11) as a scaffold. A designed protein, named IG11KK, which has a parallel configuration of Aβ‐like β sheets, can bind more preferentially to oligomeric Aβ1‐42 than the monomer. Moreover, IG11KK suppressed the aggregation of Aβ1‐42 efficiently, even though lower concentrations of IG11KK than Aβ were used. The aggregation kinetics of Aβ in the presence of the designed proteins revealed that IG11KK can work as an inhibitor not only for the early to middle stages, but also in the latter stage of Aβ aggregation owing to its favorable binding to oligomeric structures of Aβ. The design strategy using β‐barrel proteins such as IGF2R‐d11 and GFP is useful in generating excellent inhibitors of protein misfolding and amyloid formation.     

Development of a Microsphere-Based Immunoassay Authenticating A2 Milk and Species Purity in the Milk Production Chain

Processed milk and milk products produced from bovine milk, commonly contain β-casein A1 (βCA1) and β-casein A2 (βCA2). Since the presence of βCA1 is linked to milk intolerance and digestion problems, A2A2 milk, which only contains βCA2, is proposed as a healthier alternative. To support this health claim, the purity of A2A2-milk has to be guaranteed. In the presented study, a multiplex immunoassay, able to distinguish between βCA2 and βCA1, was developed and real-life applicability was shown on raw milk samples from genotyped A1A1, A1A2 and A2A2 cows. Because of its ability to discriminate between βCA2 and βCA1, this newly developed method was able to detect the addition of common bovine A1A2 milk to A2A2 milk, as low as 1%. Besides the detection of A2A2 milk purity, the developed assay can also be implemented as a rapid phenotyping method at dairy farms to replace the more invasive DNA-based screening. Additionally, the developed method was capable of detecting the addition of common bovine milk up to 1% in sheep, goat, buffalo, horse and donkey milk, which conforms to EU recommendations. In conclusion, a newly developed multiplex method capable of reliably detecting the dilution of A2A2 milk of multiple species, with common bovine milk up to 1%, is presented.     

Chemical constituents of roots of Epimedium wushanense and evaluation of their biological activities

Seven flavonoids named diphylloside A, epimedoside A, epimedin C, icariin, epimedoside C, icarisoside A, desmethylanhydroicaritin, as well as the oleanolic acid, were isolated from the roots of Epimedium wushanense for the first time. These flavonoids manifested significant antioxidant activity in vitro. Scavenging effects of two flavonoids were comparable to that of Vitamin C. Antibacterial experiment has shown that the diphylloside A, icarisoside A and desmethylanhydroicaritin have significant activity towards Pseudomonas aeruginosa.     

Differentiation between Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus based on carbohydrates in lipopolysaccharide

In the present study, the closely related facultative, Gram-negative rods, Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, were distinguished taxonomically by means of their carbohydrate composition in phenol-extracted lipopolysaccharide. Both A. actinomycetemcomitans and H. aphrophilus lipopolysaccharide contained rhamnose, fucose, galactose, glucose, L-glycero-D-mannoheptose, galactosamine, and glucosamine. The content of galactose was approximately twice as high in lipopolysaccharide from H. aphrophilus as in lipopolysaccharide from A. actinomycetemcomitans. D-Glycero-D-mannoheptose was detected exclusively in lipopolysaccharide from A. actinomycetemcomitans where it constituted 11.8-16.7% of the sugar content. This aldoheptose may therefore serve as a marker for chemotaxonomic differentiation between A. actinomycetemcomitans and H. aphrophilus. The present study also describes fragmentation of methylheptoside derivatives of trifluoroacetic acid (D-glycero- and L-glycero-D-mannoheptose) from A. actinomycetemcomitans as suggested by mass spectrometry.