trum of possible biological actions of the bioactive compound quercetin is made using multiple gene expression analysis. Quercetin is a flavonoid that can inhibit proliferation of tumor cells and reduce the number of aberrant crypt foci, although increase of number of colon tumors was also reported. Aim of the study In order to elucidate possible mechanisms involved in its mode of action the effect of quercetin on expression of 4000 human genes in Caco-2 cells was studied and related to functional effects. Methods Caco-2 cells were exposed to 5 or 50 µM quercetin for 48 hours, differential expression of 4000 human genes was studied using microarrays and related to functional effects. Differentially expressed genes were categorized in seven functional groups: cell cycle and differentiation, apoptosis, tumor suppressor genes and oncogenes, cell adhesion and cell-cell interaction, transcription, signal transduction and energy metabolism. Also, cell proliferation and cell cycle distribution were measured. Results Quercetin (5 µM) downregulated expression of cell cycle genes (for example CDC6, CDK4 and cyclin D1), downregulated cell proliferation and induced cell cycle arrest in Caco-2 cells. After exposure to 50 µM quercetin cell proliferation decreased to 51.3 % of control, and further decrease of the percentage of cells in the G1 phase coincided with an increase of the percentage of cells in the sub-G1 phase. Quercetin upregulated expression of several tumor suppressor genes. In addition, genes involved in signal transduction pathways like beta catenin/ TCF signalling and MAPK signal transduction were influenced by quercetin. Conclusions This study shows that large-scale gene expression analysis in combination with functional assays yields a considerable amount of information on (anti-)carcinogenic potential of food components like quercetin.

Penicillin biosynthesis by Penicillium chrysogenum is a compartmentalized process. The first catalytic step is mediated by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase), a high molecular mass enzyme that condenses the amino acids L-alpha-aminoadipate, L-cysteine, and L-valine into the tripeptide ACV. ACV synthetase has previously been localized to the vacuole where it is thought to utilize amino acids from the vacuolar pools. We localized ACV synthetase by subcellular fractionation and immuno-electron microscopy under conditions that prevented proteolysis and found it to co-localize with isopenicillin N synthetase in the cytosol, while acyltransferase localizes in microbodies. These data imply that the key enzymatic steps in penicillin biosynthesis are confined to only two compartments, i.e., the cytosol and microbody.

The enzymes of the penicillin biosynthetic pathway in Penicillium chrysogenum are located in different subcellular compartments. Consequently, penicillin pathway precursors and the biologically active penicillins have to cross one or more membranes. The final enzymatic step that is mediated by acyltransferase takes place in a microbody. The pH of the microbody lumen in penicillin producing cells has been determined with fluorescent probes and mutants of the green fluorescent protein and found to be slightly alkaline.

cDNA clones of dsRNAs associated with La France disease of Agaricus bisporus were isolated. Clones corresponding to L1 and L5 dsRNAs were sequenced. The deduced amino acid sequence of L1 dsRNA (1078 amino acids, Mr 121K) showed significant homology with RNA-dependent RNA polymerases of other dsRNA viruses. The deduced amino acid sequence of L5 dsRNA (724 amino acids, Mr 82K) showed no homology with known proteins. Amino acid sequences of tryptic digests of three virion-associated proteins were determined. The 34-nm virion-associated protein of Mr 115K was encoded by the L1 dsRNA, thus identifying this protein as the RNA-dependent RNA polymerase. The virion-associated protein of Mr 90K was encoded by the previously sequenced L3 dsRNA. A cDNA clone of the previously sequenced M2 dsRNA was expressed in Escherichia coli and antibodies raised against this protein reacted only with a protein present in the cytoplasm of diseased A. bisporus fruit bodies but not in the 34-nm virions.

In industrial fermentations, Penicillium chrysogenumuses sulfate as the source of sulfur for the biosynthesis of penicillin. By a PCR-based approach, two genes, sutA andsutB, whose encoded products belong to the SulP superfamily of sulfate permeases were isolated. Transformation of a sulfate uptake-negative sB3 mutant of Aspergillus nidulans with the sutB gene completely restored sulfate uptake activity. The sutA gene did not complement the A. nidulans sB3 mutation, even when expressed under control of the sutB promoter. Expression of both sutA and sutB in P. chrysogenum is induced by growth under sulfur starvation conditions. However, sutA is expressed to a much lower level than is sutB. Disruption of sutB resulted in a loss of sulfate uptake ability. Overall, the results show that SutB is the major sulfate permease involved in sulfate uptake byP. chrysogenum.