Phenytoin metabolism by human cytochrome P450 : involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Conifer defence against insects: microarray gene expression profiling of Sitka spruce (Picea sitchensis) induced by mechanical wounding or feeding by spruce budworms (Choristoneura occidentalis) or white pine weevils (Pissodes strobi) reveals large

Conifers are resistant to attack from a large number of potential herbivores or pathogens. Previous molecular and biochemical characterization of selected conifer defence systems support a model of multigenic, constitutive and induced defences that act on invading insects via physical, chemical, biochemical or ecological (multitrophic) mechanisms. However, the genomic foundation of the complex defence and resistance mechanisms of conifers is largely unknown. As part of a genomics strategy to characterize inducible defences and possible resistance mechanisms of conifers against insect herbivory, we developed a cDNA microarray building upon a new spruce (Picea spp.) expressed sequence tag resource. This first-generation spruce cDNA microarray contains 9720 cDNA elements representing c. 5500 unique genes. We used this array to monitor gene expression in Sitka spruce (Picea sitchensis) bark in response to herbivory by white pine weevils (Pissodes strobi, Curculionidae) or wounding, and in young shoot tips in response to western spruce budworm (Choristoneura occidentalis, Lepidopterae) feeding. Weevils are stem-boring insects that feed on phloem, while budworms are foliage feeding larvae that consume needles and young shoot tips. Both insect species and wounding treatment caused substantial changes of the host plant transcriptome detected in each case by differential gene expression of several thousand array elements at 1 or 2 d after the onset of treatment. Overall, there was considerable overlap among differentially expressed gene sets from these three stress treatments. Functional classification of the induced transcripts revealed genes with roles in general plant defence, octadecanoid and ethylene signalling, transport, secondary metabolism, and transcriptional regulation. Several genes involved in primary metabolic processes such as photosynthesis were down-regulated upon insect feeding or wounding, fitting with the concept of dynamic resource allocation in plant defence. Refined expression analysis using gene-specific primers and real-time PCR for selected transcripts was in agreement with microarray results for most genes tested. This study provides the first large-scale survey of insect-induced defence transcripts in a gymnosperm and provides a platform for functional investigation of plant-insect interactions in spruce. Induction of spruce genes of octadecanoid and ethylene signalling, terpenoid biosynthesis, and phenolic secondary metabolism are discussed in more detail.

The quest for golden bananas: Investigating carotenoid regulation in a Fe’i group Musa cultivar

The regulation of carotenoid biosynthesis in a high-carotenoid-accumulating Fe’i group Musa cultivar, “Asupina”, has been examined and compared to that of a low-carotenoid-accumulating cultivar, “Cavendish”, to understand the molecular basis underlying carotenogenesis during banana fruit development. Comparisons in the accumulation of carotenoid species, expression of isoprenoid genes, and product sequestration are reported. Key differences between the cultivars include greater carotenoid cleavage dioxygenase 4 (CCD4) expression in “Cavendish” and the conversion of amyloplasts to chromoplasts during fruit ripening in “Asupina”. Chromoplast development coincided with a reduction in dry matter content and fruit firmness. Chromoplasts were not observed in “Cavendish” fruits. Such information should provide important insights for future developments in the biofortification and breeding of banana.

The use of online counselling within an Australian secondary school setting: a practitioner’s viewpoint

This paper proposes that the provision of online counselling services for young people accessed through their local school website has the potential to assist students with mental health issues as well as increasing their help seeking behaviours. It stems from the work of the authors who trialled an online counselling service within one Australian secondary school. In Australia, online counselling with the adult population is now an accepted part of the provision of mental health services. Online provision of mental health information for young people is also well accepted. However, online counselling for young people is provided by only a few community organisations such as Kids Help Line within Australia. School based counselling services which are integral to most secondary schools in Australia, seem slow to provide this service in spite of initial interest and enthusiasm by individual school counsellors. This discussion is the product of reflection on the potential benefits of this trial with a consideration of relevant research of the issues raised. It highlights the need for further research into the use of computer mediated communication in the provision of counselling within a school setting.

Secondary teachers’ conceptions of teaching and learning

This paper presents a phenomenographic analysis of the conceptions of teaching and learning held by a sample of 16 secondary school teachers in two Australian schools. It provides descriptions of four categories, derived from pooled data, of the ways in which these teachers thought about teaching and about learning, their teaching strategies, and their focus on student or content. The categories for teaching and learning are described with each teacher allocated to the category most typical of their conceptions of teaching and of learning. The lack of congruence, in some cases, between the conceptions of teaching and of learning held by these teachers is discussed.