Beads labelled using surface enhanced resonance Raman scattering (SERRS) are highly sensitive and specific tags, with potential applications in biological assays, including molecular diagnostics. The beads consist of a nucleus containing dye labelled silver-nanoparticle aggregates surrounded by a polymer core. The nuclei generate strong SERRS signals. To illustrate the coding advantage created by the sharp, molecularly specific SERRS signals, four specially designed SERRS dyes have been used as labels and three of these have been combined in a multiplex analysis. These dyes use specific groups such as benzotriazole and 8-hydroxyquinoline to improve binding to the surface of the silver particles. The aggregation state of the particles is held constant by the polymer core, this nucleus also contains many dye labels, yielding a very high Raman scattering intensity for each bead. To functionalise these beads for use in biological assays an outer polymer shell can be added, which allows the attachment of oligonucleotide probes. Oligonucleotide modified beads can then be used for detection of specific oligonucleotide targets. The specificity of SERRS will allow for the detection of multiple targets within a single assay.
Inorganic UV absorbers such as titanium oxide and zinc oxide are in common use in formulations of sunscreens and cosmetics. These materials strongly absorb UV, and show no UV induced degradation over time. However, the dissipation mechanism of the UV energy is not often considered. It is demonstrated that the primary de-excitation mechanism of inorganic sunscreen components is via the surface of the particle and leads to the creation of free radicals. Substantial free radical generation is observed under simulated solar energy with all sunscreen grades of titanium oxide and zinc oxide. Free radicals are implicated in a number of potential health issues such as skin aging. Doping trap centres into titanium oxide and zinc oxide results in the total elimination of free radical generation under simulated solar energy. The trap centres provide sites within the oxide via which it is energetically favourable for photogenerated charge carriers to de-excite. Thus the charge is confined on sites within the host lattice and cannot migrate to the particle surface and create free radicals. The absorbed UV energy is converted to long wavelength light and heat. This effect is demonstrated by electron spin resonance, a primary measure of free radical generation, and by a plasmid nicking assay. The plasmid nicking assay demonstrates the effect of free radicals on supercoiled DNA.MST/6109
The effect of manganese doping on the free radical generation rate, free radical scavenging and UVA absorption properties of micronised sunscreen grade titania has been studied with respect to enhancement of the UVA photostability of test sunscreen formulations containing the organic UVA absorber Parsol 1789. Manganese doping has been shown to increase the UVA:UVB absorption ratio of titania, reduce free radical generation rates by over 90%, and provide free radical scavenging behaviour. Adding manganese-doped titania to a test formulation incorporating Parsol 1789 shows that manganese doping increases UVA attenuation stability by up to 3 times the amount achieved by comparable commercial undoped titania materials. HPLC data shows this to be related to an improved stabilisation of the organic sunscreen components. Manganese doped titania shows improved efficacy over undoped titania in sunscreen formulations containing organic UV absorbers.
Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutaratedependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with sitedirected mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (⌬310 mutant) enhanced ring-expansion of penicillin G by ϳ2-fold. A double mutant, ⌬310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The ⌬310/N305L/ M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K m ؍ 2.00 ؎ 0.47 compared with 6.02 ؎ 0.97 mM for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.
Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the a-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.
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