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We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.

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Polybutylcyanoacrylate (PBCA) nanoparticles were prepared and loaded with mitoxantrone, a highly effective anticancer drug. The proportion of mitoxantrone bound to the particles was analysed to be about 15 per cent of the initial drug concentration with the incorporation method and about 8 per cent with the adsorption method. Selected nanoparticle formulations were tested in leukaemia- or melanoma-bearing mice after intravenous injection. Efficacy and toxicity of mitoxantrone nanoparticles were compared with a drug solution and with a mitoxantrone-liposome formulation (small unilamellar vesicles with a negative surface charge). Furthermore, influence of an additional coating surfactant, poloxamine 1508, which has been shown to change body distribution of other polymeric nanoparticles, was investigated. It was shown that PBCA nanoparticles and liposomes influenced the efficacy of mitoxantrone in cancer therapy differently: liposomes prolonged survival time in P388 leukaemia, whereas nanoparticles led to a significant tumour volume reduction at the B16 melanoma. Neither nanoparticles nor liposomes were able to reduce the toxic side-effects caused by mitoxantrone, namely leucocytopenia. A slight additional influence of the coating surfactant was observed with only one preparation.

A suitable method for separating free drug and other residual compounds from drug-loaded, colloidal polymeric particles was developed. The objective of this separation procedure was to purify particle suspensions on a preparative scale by a rapid, simple, and exact method without changing the properties of the particles. The gel filtration method described in this paper using a middle-pressure chromatographic system is able to meet all these requirements. The separation process could be optimized by using only water as an eluent in combination with Sephadex G 50 medium. The purified particles appeared after 12-13 min in the eluate. Neither dispersity nor particle size or loading rate were influenced to a considerable extent.