Cation-dependent mannose-6-phosphate receptor expression and distribution are influenced by estradiol in MCF-7 breast cancer cells.

Cancer cells secrete procathepsin D, and its secretion is enhanced by estradiol. Although alterations in the pro-enzyme intracellular transport have been reported, the mechanism by which it is secreted remains poorly understood. In this work, we have studied the influence of estradiol on the expression and distribution of the cation-dependent mannose-6-phosphate receptor (CD-MPR), which would be a key molecule to ensure the proper localization of the enzyme to lysosomes in breast cancer cells. Immunoblotting studies demonstrated that the expression of CD-MPR is higher in MCF-7 cells, as compared to other breast cancer and non-tumorigenic cells. This expression correlated with high levels of cathepsin D (CatD) in these cells. By immunofluorescence, this receptor mostly co-localized with a Golgi marker in all cell types, exhibiting an additional peripheral labelling in MCF-7 cells. In addition, CD-MPR showed great differences regarding to cation-independent mannose-6-phosphate receptor. On the other hand, the treatment with estradiol induced an increase in CD-MPR and CatD expression and a re-distribution of both proteins towards the cell periphery. These effects were blocked by the anti-estrogen tamoxifen. Moreover, a re-distribution of CD-MPR to plasma membrane-enriched fractions, analyzed by gradient centrifugation, was observed after estradiol treatment. We conclude that, in hormone-responsive breast cancer cells, CD-MPR and CatD are distributed together, and that their expression and distribution are influenced by estradiol. These findings strongly support the involvement of the CD-MPR in the pro-enzyme transport in MCF-7 cells, suggesting the participation of this receptor in the procathepsin D secretion previously reported in breast cancer cells.

Testosterone influences the expression and distribution of the cation-dependent mannose-6-phosphate receptor in rat epididymis. Implications in the distribution of enzymes.

The mammalian epididymis plays a role in sperm maturation through its secretory activity. Among the proteins secreted by the epithelium, there are significant amounts of acid hydrolases. In most cell types, the normal distribution of lysosomal enzymes is mediated by mannose-6-phosphate receptors (MPRs). In this study, we analysed the expression and distribution of the cation-dependent MPR (CD-MPR) in epididymis from control, castrated or castrated rats with testosterone replacement. It was observed that expression of CD-MPR increased due to castration in all regions of the epididymis, which was reversed by injection of testosterone. We also measured the activity of α-mannosidase and observed that the castration tends to increase the retention of this enzyme in the tissue, which is reversed by the hormone replacement. In corpus, this resulted in a reduced secretion of the enzyme. Immunohistochemistry showed that CD-MPR has a supranuclear location (different from the cation-independent MPR), most likely in principal cells, and low reactivity in other cell types. The signal in castrated animals was more intense and tended to redistribute towards the apical cytoplasm. Thus, we concluded that expression and distribution of CD-MPR is affected by decrease of testosterone in rat epididymis, and this could change the distribution of lysosomal enzymes.

Effect of dehydroleucodine on the reproductive tract of male mice.

The effects of a sesquiterpene lactone, dehydroleucodine, on the reproductive tract were investigated using adult male mice. Dehydroleucodine was dissolved in tap water and administered as drinking water for 30 days. All the parameters were compared with a control group that received only vehicle. Animals were killed by decapitation and the trunk blood, the testes and the epididymes were collected. Plasma concentrations of testosterone and oestradiol, and testicular weight and concentration of spermatids did not change by dehydroleucodine. Nevertheless, in epididymal cauda dehydroleucodine treatment caused a diminution in sperm number, a decrease in the amount of tubular fluid and a reduction in the activity of the hydrolytic enzyme N-acetyl-ß-d-glucosaminidase. However, the sperm motility was not altered by dehydroleucodine treatment, although sperm binding to zona-free oocytes increased significantly. These results suggest that dehydroleucodine, which has been implicated in the inhibition of aromatase P450, does not affect the plasma concentration of testosterone and oestradiol or testicular activity, whereas altering several epididymal parameters. The epididymis is thus a more sensitive target for dehydroleucodine action.

A novel icetexane diterpene, 5-epi-icetexone from Salvia gilliessi is active against Trypanosoma cruzi.

In this work the effect of a novel compound, 5-epi-icetexone (ICTX) obtained from Salvia gilliessi Benth. (Labiatae), is studied on cultured epimastigotes of Trypanosoma cruzi (Tulahuen). It was found that the compound exerts an antiproliferative effect on the parasites at concentrations between 2.8 and 4.2 microM, and similar sensitivity in other strains (Dm28c, CL-Brener and Y-strain). The compound was deleterious at concentrations higher than 4.2 microM, with an estimated IC50 of 6.5+/-0.75 microM, but with low cytotoxicity to mammalian cells. These effects were irreversible, even at short times of exposure to the drug. In solution, ICTX showed to be stable for at least 96 h at 29 degrees C. With cytostatic dose a little percentage of parasites was resistant to the action of ICTX, and they continued growing although with different kinetic. By electron transmission microscopy, at dose of 4.2 microM an external vesiculization was observed on the first day of exposure to the compound, but the parasite cytoplasm became plenty of vacuoles and exhibited nuclear disorganization from the second day of exposure. It was concluded that ICTX is active against T. cruzi and may act by multiple mechanisms. In future, this novel icetexane diterpene may be a good candidate for therapeutic use against Chagas' disease.

Developmental differences between cation-independent and cation-dependent mannose-6-phosphate receptors in rat brain at perinatal stages.

Mannose-6-phosphate receptors (MPRs) play a role in the selective transport of macromolecules bearing mannose-6-phosphate residue to lysosomes. To date, two types of MPRs have been described in most of cells and tissues: the cation-dependent (CD-MPR) and cation-independent mannose-6-phosphate receptor (CI-MPR). In order to elucidate their possible role in the central nervous system, the expression and binding properties of both MPRs were studied in rat brain along perinatal development. It was observed that the expression of CI-MPR decreases progressively from fetuses to adults, while the CD-MPR increases around the 10th day of birth, and maintains these values up to adulthood. Binding assays showed differences in the Bmax and KD values between the ages studied, and they did not correlate with the expression levels of both MPRs. Variations in lysosomal enzyme activities and expression of phosphomannosylated ligands during development correlated more with CD-MPR than with CI-MPR expression. These results suggest that both receptors play a different role in rat brain during perinatal development, being CD-MPR mostly involved in lysosome maturation.

Binding of AP-2 adaptor complex to brain membrane is regulated by phosphorylation of proteins.

Phosphorylation of proteins appears as a key process in early steps of clathrin coated vesicle formation. Here, we report that treatment of post-nuclear fraction with alkaline phosphatase induced redistribution of alpha subunits of AP-2 adaptor complex to cytosol and this effect was higher in the alpha2 subunit. A high serine phosphorylation status of alpha subunits correlated with the higher affinity of AP-2 to membranes. Using a simple binding assay, where membranes were incubated with either purified adaptors or cytosols, we observed an inhibitory effect of tyrphostin, a tyrosine kinase inhibitor, on the binding of AP-2 to membranes, but also an unexpected decrease induced by the phosphatase inhibitor cyclosporine. We also show an inhibitory effect of ATP mediated by cytosolic proteins, although it could not be related to the phosphorylation of AP-2, suggesting an action upstream a cascade of phosphorylations that participate in the regulation of the assembly of AP-2 to membranes.

Prolonged ethanol ingestion decreases alpha-mannosidase activity and induces its redistribution to the fluid phase in rat cauda epididymis.

The role of glycosidases in mammalian epididymal fluid is still a controvertial subject. There exists a body of evidence in favour of a function in remodeling the sperm surface as one step in gamete maturation, whilst others argue in favor of an extraepididymal role for these enzymes. In this study we measured the activity and distribution of four glycosidases in rat cauda epididymis after prolonged ethanol ingestion, a condition associated with fertility disturbances. We found that alpha-mannosidase is the most sensitive enzyme to the stress caused by alcohol, since its activity in epididymis significantly decreased and partly redistributed from the spermatozoa to the fluid phase. From these results we suggested that alcohol treatment affects the expression of the enzyme and possibly induces a loss of interaction with the affinity sites on the sperm surface. Although other enzymes also underwent changes due to the alcohol treatment, we focussed on the importance of alpha-mannosidase in the fertilizing capability of spermatozoa.

Compartmentalization of lysosomal enzymes in cauda epididymis of normal and castrated rats.

The mammalian epididymis is an organ particularly rich in acid hydrolases, consistent with a developed lysosomal apparatus. However, some of these enzymes could also play a role in an extracellular environment, since they are actively secreted by the epithelium. In this study the authors measured the activity of five acid hydrolases distributed between the epithelium, fluid, small vesicles, and spermatozoa of the rat cauda epididymis in adult rats, and compared with that distribution under conditions of deprivation of luminal testosterone and testicular compounds (hemicastration). Lysosomal enzymes are differently compartmentalized in rat cauda epididymis. Most of beta-galactosidase (beta-GAL) and aryl sulfatase (approximately 70%) were found in soluble form within the fluid. Some 60% of N-acetyl-beta-D-glucosaminidase (beta-NAG) and alpha-mannosidase (alpha-MAN) become transiently bound to sperm, and beta-glucuronidase (beta-GLU) was mostly concentrated in the epithelium. After remotion of testis this distribution changed, as the retention of alpha-MAN, beta-GAL, beta-GLU, and beta-NAG by the epididiymal tissue increased. The increase of beta-GLU followed an increase of synthesis of the enzyme. The distribution of enzymes in the epididymis from the contralateral side was similar to that in normal rats. The different roles for each enzyme in the epididymis are discussed.

The sesquiterpene lactone dehydroleucodine reversibly inhibits Allium cepa L. root growth.

Here, we prove that dehydroleucodine, a sesquiterpene lactone, at low concentrations (25-100 microM) slowed down the Allium cepa L root growth by 22-70% respectively neither affecting cell viability nor cell size. Removal of the drug after 24 h incubation restored the normal growth rate of the roots. Higher concentrations (200 microM) of dehydroleucodine were deleterious for the roots. As cell size did not change, it is most likely that dehydroleucodine affected some event of cell division cycle making it longer. Thus, dehydroleucodine could be a useful tool to slow down cell proliferation.

Purification of proteins from rat sperm membranes that interact with ligands other than phosphomannosyl residues.

In this study proteins were purified from rat sperm membranes which might be the high affinity sites for ligands of epididymal fluid other than the mannose-6-phosphate receptors. The sperm membrane proteins were solubilized and passed over an affinity column containing epididymal fluid proteins coupled to a matrix. Two bands in the range of 45-55 kDa were eluted from the column with fructose-6-phosphate but not with mannose-6-phosphate. Although the molecular weight of these proteins are similar to those of the cation-dependent phosphomannosyl receptors they are not related. These two proteins may correspond either to two different receptors or to forms of the same receptor that recognize ligands from rat epididymal fluid. Sequencing and identification of these proteins will be the aim of future studies.

Changes in the content of rat epididymal fluid induced by prolonged treatment with tamoxifen.

Prolonged treatment with tamoxifen induces changes in the male reproductive tract in rats. In this study changes in the protein content of the rat epididymal fluid as a consequence of prolonged treatment with tamoxifen are reported. Among five lysosomal enzymes measured in the epididymal fluid, alpha-mannosidase (alpha-MAN) significantly diminished, but other enzymes did not. Electrophoretic analysis of fluids showed that proteins of estimated molecular weight 25, 60, 80-85 and 180 kDa decreased in the treated rats. We also detected an increase in the binding of beta-galactosidase (beta-GAL) to caudal spermatozoa in treated rats. These changes may be related in part to the loss of fertilizing capacity of spermatozoa after tamoxifen treatment.