Molecular photoprotection of human keratinocytes in vitro by the naturally occurring mycosporine-like amino acid palythine.

BACKGROUND: Solar ultraviolet radiation (UVR) induces molecular and genetic changes in the skin, which result in skin cancer, photoageing and photosensitivity disorders. The use of sunscreens is advocated to prevent such photodamage; however, most formulations contain organic and inorganic UVR filters that are nonbiodegradable and can damage fragile marine ecosystems. Mycosporine-like amino acids (MAAs) are natural UVR-absorbing compounds that have evolved in marine species for protection against chronic UVR exposure in shallow-water habitats. OBJECTIVES: To determine if palythine, a photostable model MAA, could offer protection against a range of UVR-induced damage biomarkers that are important in skin cancer and photoageing. METHODS: HaCaT human keratinocytes were used to assess the photoprotective potential of palythine using a number of end points including cell viability, DNA damage (nonspecific, cyclobutane pyrimidine dimers and oxidatively generated damage), gene expression changes (linked to inflammation, photoageing and oxidative stress) and oxidative stress. The antioxidant mechanism was investigated using chemical quenching and Nrf2 pathway activation assays. RESULTS: Palythine offered statistically significant protection (P < 0·005) against all end points tested even at extremely low concentrations (0·3% w/v). Additionally, palythine was found to be a potent antioxidant, reducing oxidatively generated stress, even when added after exposure. CONCLUSIONS: Palythine is an extremely effective multifunctional photoprotective molecule in vitro that has potential to be developed as a natural and biocompatible alternative to currently approved UVR filters.

Changes in clinical indications for community antibiotic prescribing for children in the UK from 1996 to 2006: will the new NICE prescribing guidance on upper respiratory tract infections just be ignored?

OBJECTIVE: To analyse changes in clinical indications for community antibiotic prescribing for children in the UK between 1996 and 2006 and relate these findings to the new NICE guidelines for the treatment of upper respiratory tract infections in children. STUDY DESIGN: Retrospective cohort study. METHOD: The IMS Health Mediplus database was used to obtain annual antibiotic prescribing rates and associated clinical indications in 0-18-year-old patients between 1 January 1996 and 31 December 2006 in the UK. RESULTS: Antibiotic prescribing declined by 24% between 1996 and 2000 but increased again by 10% during 2003-2006. Respiratory tract infection was the most common indication for which an antibiotic was prescribed, followed by "abnormal signs and symptoms", ear and skin infections. Antibiotic prescriptions for respiratory tract infections have decreased by 31% (p<0.01) mainly because of reduced prescribing for lower respiratory tract infections (56% decline, p<0.001) and specific upper respiratory tract infections including tonsillitis/pharyngitis (48% decline, p<0.001) and otitis (46% decline, p<0.001). Prescribing for non-specific upper respiratory tract infection increased fourfold (p<0.001). Prescribing for "abnormal signs and symptoms" increased significantly since 2001 (40% increase, p<0.001). CONCLUSION: There has been a marked decrease in community antibiotic prescribing linked to lower respiratory tract infection, tonsillitis, pharyngitis and otitis. Overall prescribing is now increasing again but is associated with non-specific upper respiratory tract infection diagnoses. General practitioners may be avoiding using diagnoses where formal guidance suggests antibiotic prescribing is not indicated. The new NICE guidance on upper respiratory tract infections is at risk of being ignored.

Has UK guidance affected general practitioner antibiotic prescribing for otitis media in children?

BACKGROUND: Since 1997, UK guidance has advocated limiting antibiotic prescribing for otitis media. It is not known whether this has influenced general practitioner prescribing practice. Aims and objectives To investigate the trends in diagnoses and antibiotic prescribing for otitis media in children in relation to guidance. METHODS: We used the General Practice Research Database to conduct time-trend analyses of diagnoses and antibiotic prescribing for otitis media in 3 months to 15 years old, between 1990 and 2006. RESULTS: A total of 1 210 237 otitis media episodes were identified in 464 845 children; two-thirds (68%; 818 006) received antibiotics. Twenty-two percent (267 335) were classified as acute, 85% (227 335) of which received antibiotics. Overall, antibiotic prescribing for otitis media declined by 51% between 1995 and 2000. Much of this reduction predated guidance. During this period, prescribing for otitis media coded as acute increased by 22%. Children diagnosed with acute otitis media were more likely to receive antibiotics than otitis media not coded as acute (P < 0.05). From 2000 prescribing plateaued, despite publication of further guidance. Otitis media diagnoses consistently paralleled prescribing. CONCLUSIONS: The reduction in antibiotic prescribing for otitis media predated guidance. The simultaneous decrease in prescribing for non-acute otitis media and increase for acute otitis media suggest diagnostic transfer, possibly to justify the decision to treat.

Polyketide synthase genes and the natural products potential of Dictyostelium discoideum.

MOTIVATION: The genome of the social amoeba Dictyostelium discoideum contains an unusually large number of polyketide synthase (PKS) genes. An analysis of the genes is a first step towards understanding the biological roles of their products and exploiting novel products. RESULTS: A total of 45 Type I iterative PKS genes were found, 5 of which are probably pseudogenes. Catalytic domains that are homologous with known PKS sequences as well as possible novel domains were identified. The genes often occurred in clusters of 2-5 genes, where members of the cluster had very similar sequences. The D.discoideum PKS genes formed a clade distinct from fungal and bacterial genes. All nine genes examined by RT-PCR were expressed, although at different developmental stages. The promoters of PKS genes were much more divergent than the structural genes, although we have identified motifs that are unique to some PKS gene promoters.

Physical and microbiological stability of an extemporaneous tacrolimus suspension for paediatric use.

OBJECTIVE: An extemporaneous suspension of tacrolimus for paediatric use has recently been developed but poor bioavailability and erratic plasma concentrations were observed during clinical use. It was not clear whether this was due to changes in the physical properties of the suspension during storage. The aim of this work was to investigate the physical and microbiological stability over the recommended 8-week shelf-life of this extemporaneous tacrolimus suspension. METHODS: Suspensions (0.5 mg/mL) were custom made by a special manufacturer under Good Manufacturing Practice conditions. The procedure involved mixing tacrolimus capsule contents into Ora Plus and Simple Syrup (1 : 1) using a mortar and pestle followed by an homogenization step. The particle sizes of the suspensions were measured using a MasterSizer. A light microscope equipped with polarizers was used to visualize any particle size changes or crystal growth. Viable bacterial and fungal contamination was assessed using standard colony count techniques on solid media. The suspensions were kept at 22-26 degrees C and evaluated weekly. RESULTS: The volume mean diameter d((4,3)) from laser diffraction did not change significantly. Light microscopy did not reveal any significant change in particle size or crystal growth. Contamination by viable and culturable micro-organisms could not be detected. CONCLUSION: The suspension was physically (particle size) and microbiologically stable during the 8-week study period suggesting other factors including poor dosing could be responsible for the pharmacokinetic variation observed during clinical use which warrants further investigation.

Evidence for apparent gene instability in the rifamycin-producing oligoketide synthase. Implications for combinatorial biosynthesis and heterologous gene expression.

The oligoketide ('polyketide') synthase leading to the formation of proansamycin X in Amycolatopsis mediterranei also prematurely releases a range of acyclic intermediates from the enzyme complex. We intended to study the chemical biology of this ectopic chain release using RifA as a model protein system; however, we were unable to clone the rifA gene in its entirety. Restriction analysis of cosmid clones revealed that rifA is subject to random deletions at high frequency, especially in central regions of the locus. Examination of the gene sequence in this region reveals a high concentration of inverted repeats; we suggest that these sequences are subject to alteration in secondary structure when cloned outside the environment of the A. mediterranei genome, leading to recombination and deletion.

Engineering of complex polyketide biosynthesis--insights from sequencing of the monensin biosynthetic gene cluster.

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation.

A chain initiation factor common to both modular and aromatic polyketide synthases.

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.

Detection and identification of novel actinomycetes.

The actinomycetes are well known as a group of filamentous, Gram-positive bacteria that produce many useful secondary metabolites, including antibiotics and enzymes. Although they have been intensively studied for both theoretical and practical objectives, there is much scope for developing our basic knowledge of the means of detection and isolation of these microbes. This session concentrated on new methods for the detection and identification of novel actinomycetes from a range of environments. Approaches to the detection of actinomycetes ranged from investigations of neglected habitats and extreme environments (e.g. alkaline soils and oil drills) to the analysis of DNA extracted from the environment and use of specific phages. The continuing problems of the identification of actinomycete isolates were also considered. Topics discussed included use of phage typing, DNA probes, and correlation between phenetic and genotypic species of Streptomyces.