A quantitative real-time RT-PCR system was established to identify which secreted aspartyl proteinase (SAP) genes are most highly expressed and potentially contribute to Candida albicans infection of human epithelium in vitro and in vivo. C. albicans SC5314 SAP1-10 gene expression was monitored in organotypic reconstituted human epithelium (RHE) models, monolayers of oral epithelial cells, and patients with oral (n517) or vaginal (n517) candidiasis. SAP gene expression was also analysed in Dsap1-3, Dsap4-6, Defg1 and Defg1/cph1 mutants to determine whether compensatory SAP gene regulation occurs in the absence of distinct proteinase gene subfamilies. In monolayers, RHE models and patient samples SAP9 was consistently the most highly expressed gene in wild-type cells. SAP5 was the only gene significantly upregulated as infection progressed in both RHE models and was also highly expressed in patient samples. Interestingly, the SAP4-6 subfamily was generally more highly expressed in oral monolayers than in RHE models. SAP1 and SAP2 expression was largely unchanged in all model systems, and SAP3, SAP7 and SAP8 were expressed at low levels throughout. In Dsap1-3, expression was compensated for by increased expression of SAP5, and in Dsap4-6, expression was compensated for by SAP2: both were observed only in the oral RHE. Both Dsap1-3 and Dsap4-6 mutants caused RHE tissue damage comparable to the wild-type. However, addition of pepstatin A reduced tissue damage, indicating a role for the Sap family as a whole in inducing epithelial damage. With the hypha-deficient mutants, RHE tissue damage was significantly reduced in both Defg1/cph1 and Defg1, but SAP5 expression was only dramatically reduced in Defg1/cph1 despite the absence of hyphal growth in both mutants. This indicates that hypha formation is the predominant cause of tissue damage, and that SAP5 expression can be hyphaindependent and is not solely controlled by the Efg1 pathway but also by the Cph1 pathway. This is believed to be the first study to fully quantify SAP gene expression levels during human mucosal infections; the results suggest that SAP5 and SAP9 are the most highly expressed proteinase genes in vivo. However, the overall contribution of the Sap1-3 and Sap4-6 subfamilies individually in inducing epithelial damage in the RHE models appears to be low.Abbreviations: IVET, in vitro expression technology; LDH, lactate dehydrogenase; RHE, reconstituted human epithelium; Sap, secreted aspartyl protease.

The in vivo expression of Candida albicans secreted aspartyl proteinase (SAP1-SAP8) and phospholipase B (PLB1 and PLB2) genes was analyzed in 137 human subjects with oral and vaginal candidiasis or carriage. Total RNA was isolated from whole unstimulated saliva or vaginal swabs, and the expression of SAP1-8 and PLB1-2 was evaluated by reverse-transcriptase polymerase chain reaction using specific primer sets. A spectrum of SAP gene expression profiles was obtained from different C. albicans strains during symptomatic disease and asymptomatic carriage. SAP2 and SAP5 were the most common genes expressed during both infection and carriage. SAP1, SAP3, SAP4, SAP7, SAP8, and PLB1 expression was correlated with oral disease, whereas SAP1, SAP3, and SAP6-SAP8 expression was correlated with vaginal disease. Furthermore, SAP1, SAP3, and SAP8 were preferentially expressed in vaginal, rather than oral, infections. This study demonstrates the differential expression of the hydrolytic enzyme genes in humans and correlates the expression of specific Candida species virulence genes with active disease and anatomical location.

It is widelv accepted that primary school children will approach sciencc with strong 'alternative conccptions' about the variables at play which, unlcss directly challenged, will circumscribe learning. Iixtcnsivc discussion concerning thc form the challenges should take has led t o the conclusion that learning will be maximized if children cxplore their conceptions while working with peers whosc alternative conceptions arc different. At present, however, there is little research t o support this, and the small amount that does exist says littlc about thc process by which learning is cffected. The current study attempted to redress this in the context of motion down an incline. Individual pre-tests were administered t o 113 A-to 12-yearold children t o establish their alternative conccptions. O n the bisis of thcir pre-test responses, and in order to establish adequate controls. the children wcre put into groups of four according t o whcther their conceptions wcre different o r similar. Thc children worked in their groups on tasks designed t o elicit the cxploration of alternative conceptions, and werc subsequently post-tcsted. Thc pattcrn of pre-to post-test change gave some support t o the notion that learning is maximized when alternativc conceptions differ. I Iowevcr, it gavc few grounds for thinking that lcarning involvcs the internalization of conceptions that the grnups jointly construct. Rather, it suggested a proccss of private conflict resolution, for which thc catalyst was discussion held during the groups but continuing long after thcir coinplction.In the past, research into the acquisition of conceptual knowledge in science hy primary school children was seldom contemplated. Educationalists were mainly concerned with the secondary age range, and when conceptual knowledge was studied by psychologists, the logical and social domains were the central focus. Recently, however, there has been a change, and it is not hard to see why. After a decade of debate, the National Curriculum (Department of Eiducation & Science, 1989) has stipulated that 'the knowledge and understanding of science' be taught from the first years of schooling. Thus, primary teachers have been charged with tinding appropriate methods, and there was a widely held impression that research Kcqucsts for reprints.

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