In social insects, recognition of nestmates from aliens is based on olfactory cues, and many studies have demonstrated that such cues are contained within the lipid layer covering the insect cuticle. These lipids are usually a complex mixture of tens of compounds in which aliphatic hydrocarbons are generally the major components. The experiments described here tested whether artificial changes in the cuticular profile through supplementation of naturally occurring alkanes and alkenes in honeybees affect the behaviour of nestmate guards. Compounds were applied to live foragers in microgram quantities and the bees returned to their hive entrance where the behaviour of the guard bees was observed. In this fashion we compared the effect of single alkenes with that of single alkanes; the effect of mixtures of alkenes versus that of mixtures of alkanes and the whole alkane fraction separated from the cuticular lipids versus the alkene fraction. With only one exception (the comparison between n-C(19) and (Z)9-C(19)), in all the experiments bees treated with alkenes were attacked more intensively than bees treated with alkanes. This leads us to conclude that modification of the natural chemical profile with the two different classes of compounds has a different effect on acceptance and suggests that this may correspond to a differential importance in the recognition signature.

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The novel synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphtalene carboxylic acid (AHPN/CD437) has been proven to be a potent inducer of apoptosis in a variety of tumor cell types. However, the mechanism of its action remains to be elucidated. Recent studies suggest that the lysosomal protease cathepsin D, when released from lysosomes to the cytosol, can initiate apoptosis. In this study, we examined whether cathepsin D and free radicals are involved in the CD437-induced apoptosis. Exposure of human leukemia HL-60 cells to CD437 resulted in rapid induction of apoptosis as indicated by caspase activation, phosphatidylserine exposure, mitochondrial alterations and morphological changes. Addition of the antioxidants atocopherol acetate effectively inhibited the CD437-induced apoptosis. Measurement of the intracellular free radicals indicated a rise in oxidative stress in CD437-treated cells, which could be attenuated by a-tocopherol acetate. Interestingly, pretreatment of cells with the cathepsin D inhibitor pepstatin A blocked the CD437-induced free radical formation and apoptotic effects, suggesting the involvement of cathepsin D. However, Western blotting revealed no difference in cellular quantity of any forms of cathepsin D between control cells and CD437-treated cells, whereas immunofluorescence analysis of the intracellular distribution of cathepsin D showed release of the enzyme from lysosomes to the cytosol. Labeling of lysosomes with lysosomotropic probes confirmed that CD437 could induce lysosomal leakage. The CD437-induced relocation of cathepsin D could not be prevented by a-tocopherol acetate, suggesting that the lysosomal leakage precedes free radical formation. Furthermore, a retinoic acid nuclear receptor (RAR) antagonist failed to block these effects of CD437, suggesting that the action of CD437 is RAR-independent. Taken together, these data suggest a novel lysosomal pathway for CD437-induced apoptosis, in which lysosomes are the primary target and cathepsin D and free radicals act as death mediators. Cell Death and Differentiation (2001) 8, 477 ± 485.

Previously, we showed that retinoic acid (RA) binds to the mannose-6-phosphate͞insulin-like growth factor II receptor (M6P͞IGF2R) with high affinity, suggesting that M6P͞IGF2R may be a receptor for RA. Here, we show that RA, after 2-3 h of incubation with cultured neonatal-rat cardiac fibroblasts, dramatically alters the intracellular distribution of M6P͞IGF2R as well as that of cathepsin B (a lysosomal protease bearing M6P). Immunof luorescence techniques indicate that this change in intracellular distribution is characterized by a shift of the proteins from the perinuclear area to cytoplasmic vesicles. The effect of RA was neither blocked by an RA nuclear receptor antagonist (AGN193109) nor mimicked by a selective RA nuclear-receptor agonist (TT-NPB). Furthermore, the RA-induced translocation of cathepsin B was not observed in M6P͞IGF2R-deficient P388D1 cells but occurred in stably transfected P388D1 cells expressing the receptor, suggesting that the effect of RA might be the result of direct interaction with M6P͞IGF2R, rather than the result of binding to the nuclear receptors. These observations not only support the idea that M6P͞IGF2R mediates an RAresponse pathway but also indicate a role for RA in control of intracellular trafficking of lysosomal enzymes. Therefore, our observations may have important implications for the understanding of the diverse biological effects of retinoids.The mannose-6-phosphate͞insulin-like growth factor II receptor (M6P͞IGF2R) is a 300-kDa multifunctional transmembrane glycoprotein. Recently, this receptor has been shown to play a fundamental role in the control of cell growth in fetal development (1-3) and carcinogenesis (4-10).M6P͞IGF2R is primarily (90-95%) localized in the intracellular compartments, particularly in the trans-Golgi network (TGN) and endosomes, with 5-10% of the receptor present on the cell surface (11). The primary function of M6P͞IGF2R is to sort and transport M6P-bearing glycoproteins from TGN to endosomes͞lysosomes (11). Normally, the newly synthesized soluble lysosomal enzymes are posttranslationally modified to contain M6P residues on their N-linked oligosaccharides. The acquired M6P residues enable the enzymes to bind to M6P receptors in the TGN. The receptors and their ligands cluster into clathrin-coated transport vesicles and travel to an acidic prelysosomal compartment where the low pH causes dissociation of the receptor-ligand complex. The free M6P receptors can travel to the plasma membrane or back to the TGN to reinitiate another cycle of biosynthetic enzyme transport. In