Synthesis of coumarin-polyamine-based molecular probe for the detection of hydroxyl radicals generated by gamma radiation

To develop a molecular probe for detection of hydroxyl radicals in the vicinity of DNA, the coumarin-polyamine complexes, N(1),N(12)-bis[2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (5) and tris[2-(2-oxo-2H-chromene-3-carboxamido)ethyl]amine (7), and their hydroxylated derivatives, N(1),N(12)-bis[7-hydroxy-2-oxo-2H-chromene-3-carbonyl]-1,12-diamine-4,9-diazadodecane (6) and tris[2-(7-hydroxy-2-oxo-2H-chromene-3-carboxamido)ethyl]amine (8), have been synthesized. Using computer-generated molecular modeling, the derivatives have been docked onto DNA dodecamer d(CGCGAATTCGCG)(2), the ligand-DNA complexes have been minimized, and the free binding energies (DeltaG(binding)) and inhibition constants (K(i)) have been calculated. Compound 7 is not water-soluble at the concentrations required for the project. When aqueous solutions of 5 are irradiated with gamma rays, the relationship between induced fluorescence and dose is linear in the range of 0 to 10 Gy. The fluorescence emission spectrum of irradiated 5 is similar to that of its dihydroxy derivative 6, indicating conversion of 5 to 6, and induction of fluorescence records formation of hydroxyl radicals in aqueous solution. The dicoumarin-polyamine 5, a novel compound for the detection of hydroxyl radicals close to DNA, is a sensitive and quantitative probe with potential for applications in biological systems.     

Exocyclic amino groups of flanking guanines govern sequence-dependent adduct conformations and local structural distortions for minor groove-aligned benzo[a]pyrenyl-guanine lesions in a GG mutation hotspot context

The environmental carcinogen benzo[a]pyrene (BP) is metabolized to reactive diol epoxides that bind to cellular DNA by predominantly forming N²-guanine adducts (G*). Mutation hotspots for these adducts are frequently found in 5′-···GG··· dinucleotide sequences, but their origins are poorly understood. Here we used high resolution NMR and molecular dynamics simulations to investigate differences in G* adduct conformations in 5′-···CG*GC··· and 5′-···CGG*C··· sequence contexts in otherwise identical 12-mer duplexes. The BP rings are positioned 5′ along the modified strand in the minor groove in both cases. However, subtle orientational differences cause strong distinctions in structural distortions of the DNA duplexes, because the exocyclic amino groups of flanking guanines on both strands compete for space with the BP rings in the minor groove, acting as guideposts for placement of the BP. In the 5′-···CGG*C··· case, the 5′-flanking G · C base pair is severely untwisted, concomitant with a bend deduced from electrophoretic mobility. In the 5′-···CG*GC··· context, there is no untwisting, but there is significant destabilization of the 5′-flanking Watson–Crick base pair. The minor groove width opens near the lesion in both cases, but more for 5′-···CGG*C···. Differential sequence-dependent removal rates of this lesion result and may contribute to the mutation hotspot phenomenon.     

Organization of the aerotaxis receptor aer in the membrane of Escherichia coli

The Aer receptor guides Escherichia coli to specific oxygen and energy-generating niches. The input sensor in Aer is a flavin adenine dinucleotide-binding PAS domain, which is separated from a HAMP/signaling output domain by two membrane-spanning segments that flank a short (four-amino-acid) periplasmic loop. In this study, we determined the overall membrane organization of Aer by introducing combinations of residues that allowed us to differentiate intradimeric collisions from interdimeric collisions. Collisions between proximal residues in the membrane anchor were exclusively intra- or interdimeric but, with one exception, not both. Cross-linking profiles were consistent, with a rigid rather than flexible periplasmic loop and a tilted TM2 helix that crossed TM2' at residue V197C, near the center of the lipid bilayer. The periplasmic loop formed a stable neighborhood that (i) included a maximum of three Aer dimers, (ii) did not swap neighbors over time, and (iii) appeared to be constrained by interactions in the cytosolic signaling domain.     

Small conformationally restricted piperidine N-arylsulfonamides as orally active gamma-secretase inhibitors

The design and development of a new class of small 2,6-disubstituted piperidine N-arylsulfonamide gamma-secretase inhibitors is reported. Lowering molecular weight including the use of conformational constraint led to compounds with less CYP 3A4 liability compared to early leads. Compounds active orally in lowering Abeta levels in Tg CRND8 mice were identified as potential treatments for Alzheimer's disease.     

Smear cytology in the intra-operative assessment of periodontoid pseudotumour of the craniocervical junction

OBJECTIVE: Periodontoid pseudotumours are lesions of presumed degenerative aetiology which typically occur in elderly patients with cervical myelopathy. The clinical and radiological differential diagnosis includes neoplastic and inflammatory lesions, and the exclusion of such lesions may be of value in the intra-operative management of the patient. This audit aimed to examine the value of intra-operative smear cytology in the management of this condition. METHODS: The intra-operative smear cytological features of four cases of periodontoid pseudotumour were reviewed. RESULTS: In each case the intra-operative cytological interpretation was in keeping with a non-neoplastic and non-inflammatory process. CONCLUSION: These results suggest that intra-operative smear cytology can provide information on the value in the intra-operative management of patients with this lesion.     

Novel symmetric amphiphilic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) with high plasmid DNA binding affinity as a biodegradable gene carrier

This study communicates the molecular design, preparation, and biological application of novel symmetric amphiphilic polycationic dendritic poly(L-lysine)-b-poly(L-lactide)-b-dendritic poly(L-lysine) D2-LLA15-D2 bearing two two-generation poly(L-lysine) PLL dendrons D2 and a central hydrophobic biodegradable poly(L-lactide) block LLA15. First, an amino-protected precursor of L1-OH was designed and synthesized and was further employed to prepare L1-LLA15 with an organic 4-(dimethylamino)-pyridine-mediated living-ring-opening polymerization of l-lactide. Subsequently, the hydroxy end-capped L1-LLA15 was coupled to synthesize a new triblock L1-LLA15-L1 with two one-generation amino-protected PLL dendrons L1. Furthermore, with a repeated trifluoroacetic-acid-mediated amino deprotection-protection cycle, new amphiphilic triblock D2-LLA15-D2 was successfully prepared. By means of NMR, mass spectrometry, and gel permeation chromatography, these synthetic precursors and final amphiphilic product were characterized to bear well-defined triblock structures. In addition, this synthesized amphiphilic triblock polycationic macromolecule was applied as a new polycationic plasmid DNA carrier, and its DNA binding affinity was examined via an agarose electrophoresis and a fluorescence titration assay along with two important references of hydrophilic dendritic D2-HEX-D2 and double-hydrophilic D2-PEG-4K-D2 bearing the same two D2 dendrons; much enhanced DNA binding affinity was interestingly revealed for the new amphiphilic structural D2-LLA15-D2. Moreover, the assembled polyplex microparticles of plasmid DNA/polycationic carrier were further analyzed by dynamic light scattering and transmission electron microscopy, indicating their averaged nanoparticle size around 150-200 nm. As for the cytotoxicity of the new D2-LLA15-D2, MTT assays were conducted with a human hepatocellular carcinoma cell line (SMMC-7721), indicating a very low cytotoxicity as compared with commercial linear PLL-23K and PEI-2K, and a DNase I degradation of the assembled polyplex particles was also done in the HBS buffer solution to evaluate their stabilities. Finally, employing the new amphiphilic D2-LLA15-D2 as gene carrier, in vitro gene transfection experiments were conducted with the SMMC-7721 cell line, indicating a transfection efficiency increase of at least 10 times higher than that of the naked plasmid DNA under a N/P charge ratio of 10. Therefore, these interesting results may provide a new possible way to construct efficient polycationic macromolecular gene carriers with low toxicity and less expensive low-generation PLL dendrons.     

Length‐weight and length‐length relationships of two tropical fish Secutor megalolepis (Mochizuki & Hayashi, 1989) and Rhabdamia gracilis (Bleeker, 1856) from Sabah,Malaysia

Length‐weight relationships (LWRs) and length‐length relationships (LLRs) for two tropical fish species from Marudu Bay, Sabah were studied. Specimens were caught with the traditional ‘Bagan net’ fishing gear from October 2012 to September 2013, using the light trap method. The maximum total length for Rhabdamia gracilis (7.4 cm) reported in this study is a new record for this species. No information regarding length‐weight relationships was reported previously in FishBase for either Secutor megalolepis or Rhabdamia gracilis.     

Multiplexed Integrating Plasmids for Engineering of the Erythromycin Gene Cluster for Expression in Streptomyces spp. and Combinatorial Biosynthesis

Bacteria in the genus Streptomyces and its close relatives are prolific producers of secondary metabolites with antibiotic activity. Genome sequencing of these bacteria has revealed a rich source of potentially new antibiotic pathways, whose products have never been observed. Moreover, these new pathways can provide novel genes that could be used in combinatorial biosynthesis approaches to generate unnatural analogues of existing antibiotics. We explore here the use of multiple orthologous integrating plasmid systems, based on the int/attP loci from phages TG1, SV1, and ϕBT1, to express the polyketide synthase (PKS) for erythromycin in a heterologous Streptomyces host. Streptomyces strains containing the three polyketide synthase genes eryAI, eryAII, and eryAIII expressed from three different integrated plasmids produced the aglycone intermediate, 6-deoxyerythronolide B (6-dEB). A further pair of integrating plasmids, both derived from the ϕC31 int/attP locus, were constructed carrying a gene cassette for glycosylation of the aglycone intermediates, with or without the tailoring gene, eryF, required for the synthesis of erythronolide B (EB). Liquid chromatography-mass spectrometry of the metabolites indicated the production of angolosaminyl-6-dEB and angolosaminyl-EB. The advantages of using multiplexed integrating plasmids for engineering expression and for combinatorial biosynthesis were demonstrated.