Mannose 6-phosphate receptor targeting and its applications in human diseases.

The cation-independent mannose 6-phosphate receptor is a multifunctional protein which binds at the cell surface to two distinct classes of ligands, the mannose 6-phosphate (M6P) bearing proteins and IGF-II. Its major function is to bind and transport M6P-enzymes to lysosomes, but it can also modulate the activity of a variety of extracellular M6P-glycoproteins (i.e., latent TGFbeta precursor, urokinase-type plasminogen activator receptor, Granzyme B, growth factors, Herpes virus). The purpose of this review is to highlight the synthesis and potential use of high affinity M6P analogues able to target this receptor. Several M6P analogues with phosphonate, carboxylate or malonate groups display a higher affinity and a stronger stability in human serum than M6P itself. These derivatives could be used to favour the delivery of specific therapeutic compounds to lysosomes, notably in enzyme replacement therapies of lysosomal diseases or in neoplastic drug targeting. In addition, their potential applications in preventing clinical disorders, which are associated with the activities of other M6P-proteins involved in wound healing, cell growth or viral infection, will be discussed.

Differential expression of estrogen receptor-alpha and -beta messenger RNAs as a potential marker of ovarian carcinogenesis.

Although estrogen receptor (ER)-alpha is expressed in both benign and malignant ovarian tumors, the role of ER in ovarian carcinogenesis of epithelial tumors is still unknown. In view of the recent characterization of ER-beta, a second form of ER that seems to be highly expressed in ovaries, we reexamined this issue by studying the relative expression of ER-alpha and -beta in human ovarian tumor progression. We developed a competitive PCR assay based on coamplification of the two ERs in target nucleotide sequences displaying a high homology (exons 3 and 4). Coamplification experiments with varying amounts of plasmids containing ER-alpha and -beta cDNAs showed that this assay was reliable for discriminating as little as a 2-fold difference in the initial ER-alpha:ER-beta cDNA ratio. The relative expression of ER-alpha compared with ER-beta mRNAs was studied in human ovarian cancer cell lines (n = 5) and in normal ovaries (n = 6), then in human benign and malignant tumor samples including ovarian cysts (n = 24), borderline tumors (n = 3), and cancers (n = 10). In normal ovaries, ER-beta mRNA was the predominant ER form, whereas in ovarian cancer cell lines ER-alpha mRNA was markedly increased as compared with ER-beta. In benign and borderline tumors, ER-beta mRNA was detected in 78% of tumors, whereas ER-alpha mRNA was detected in 29%. In ovarian carcinomas, both ER-alpha and -beta mRNAs were expressed in 80% of tumors. The ER-alpha:ER-beta mRNA ratio was >1 in only one cyst sample (4%). In contrast, the ER-alpha:ER-beta mRNA ratio was markedly increased in ovarian cancers because 60% showed an ER-alpha:ER-beta mRNA >1. In situ hybridization experiments showed overlapping tissular distribution of ER-beta and -alpha expression in cancers and cysts, with a main localization in the epithelium and only a low level of expression in stromal cells. In summary, we found an increase in the ER-alpha:ER-beta mRNA ratio in ovarian carcinomas as compared with normal ovaries and cysts. These data suggest that overexpression of ER-alpha relative to ER-beta mRNA may be a marker of ovarian carcinogenesis.

Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite schistosoma mansoni.

The insect moulting hormones, ecdysone and 20 hydroxyecdysone, were detected by the combined use of radioimmunoassay and high performance liquid chromatography in the human parasite Schistosoma mansoni. On day 11 after infection only the ecdysone form is present, but, on day 40 after infection the ratio between ecdysone and 20 hydroxyecdysone changes with anatomic localization of the adult worms in mammalian host. In the eggs, the ratio of these two hormones is identical to the ratio found in sexually mature worms located in mesenteric veins. These data demonstrate for the first time that S. mansoni synthesizes the steroid hormones ecdysone and 20 OH ecdysone which are potent molecules in stimulating growth and vitello-genesis of this gonochoric trematode.

Excretion of ecdysteroids by schistosomes as a marker of parasite infection.

Ecdysteroids produced by schistosomes are released in biological fluids of infected hosts. In the sera, the concentration of ecdysteroids correlates with the permissiveness of the host to schistosome infection and its detection is available in the absence of positive parasitological tests. In the urine, ecdysteroid concentration decreases markedly after chemotherapy. 20-Hydroxyecdysone and its epimer were identified in the urine of infected patients using mass spectrometry. These data demonstrate for the first time that ecdysteroids are released by organisms. Moreover, they are potent molecules of parasite infection and can be used for parasite diagnosis.

Quantitation of androgen receptor messenger RNA from genital skin fibroblasts by reverse transcription--competitive polymerase chain reaction.

To gain further information concerning the regulation by androgen of AR mRNA expression in cultured genital skin fibroblasts (GSF), we first developed a quantitative reverse transcription-competitive polymerase chain reaction (RT-PCR). This method used an ethidium bromide stain analysis of the PCR products for the accurate quantitation of low levels of human androgen receptor (hAR) mRNA in GSF. To control for variations due to sample preparation, and to minimize the disparity of the reverse transcriptase efficiency between samples after the RT procedure, we produced an initial PCR for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, and then adjusted the amount of cDNA to that of this housekeeping gene. Competitive PCR for hAR was then immediately performed on normalized cDNA with a competitor DNA that exhibited a 13 bp deletion as compared to the 163 bp for the target fragment, and the PCR products were easily separated by 3.5% agarose gel electrophoresis. This quantitation procedure involved no additional steps, such as enzymatic cleavage of the PCR products, nor the use of radioactivity. In GSF from individuals, we found that the normal amount of AR mRNA was 5.6 attomoles/microg RNA, (+/-1.0, s.e.m.) with an intra- and an inter-assay of 8.4 and 14.7%, respectively. We observed a biphasic pattern of AR mRNA expression in normal human GSF in the presence of physiological concentration of androgen. Quantitative RT-PCR of AR mRNA may be useful for studying AR mRNA expression in experimental or clinical conditions.

[Probable genetic independence of four hereditary characteristics peculiar to Mongoloid populations]. / Indépendance génétique probable de quatre particularité héréditaires caractéristiques des populations mongoles.

Several hereditary characteristics are found in Mongoloid populations (Asian and Amerindian) with a much higher frequency than in other populations. These are the mongoloid facial features, the presence of alcohol dehydrogenase beta 2, the presence of inactive mitochondrial aldehyde dehydrogenase 2, and the high rate of urinary excretion of beta aminoisobutyric acid. Analyses carried out in France and Switzerland have been made to research a possible correlation between these diverse characteristics. This correlation does not seem to exist. With regard to the excretion of beta aminoisobutyric acid, the hypothetical role of mercury in dental fillings has been checked in Caucasoid subjects. If this role is not completely excluded, it is far from being statistically significant.

Targeting multiplicity: the key factor for anti-cancer nanoparticles.

In this mini-review, we focus on different strategies to bring nanotools specifically to cancer cells. We discuss about a better targeting of tumor, combining the characteristics of tumor environment, the increase in nanoparticles life time, the biomarkers overexpressed on cancer cells and different physical methods for non invasive therapies. Here we detail the necessity of a synergy between passive and active targeting for an actual specificity of cancer cells.

Heat shock cognate 70 protein secretion as a new growth arrest signal for cancer cells.

Earlier studies indicated that density-arrested cancer cells released an unidentified growth inhibitor whose secretion was prevented by overexpression of the lysosomal protease cathepsin D (cath D). In this study, this growth inhibitor was purified by affinity chromatography and identified as the heat shock cognate 70 protein (hsc70) based on its peptide microsequencing and specific antibody recognition. Among intracellular proteins, including other heat shock proteins, only constitutive hsc70 was secreted in response to the high-cell density. Moreover, hsc70 secretion from cancer cells was generated by serum deprivation, whereas its cellular concentration did not change. Prevention of Hsc70 secretion by cath D overexpression was associated with the formation of multilayer cell cultures, thus indicating a loss of contact inhibition. In addition, we showed that supplementing the culture medium with purified hsc70 inhibited cell proliferation in the nanomolar range. Conversely, removal of this extracellular hsc70 from the medium by either retention on ADP-agarose or competition at the Hsc70 binding site restored cell proliferation. Hsc70 appears active in human breast cancer cells and hypersecreted by direct cath D inhibition. These results suggest a new role of this secreted hsc70 chaperone in cell proliferation that might account for the higher tumor growth of cancer cells overexpressing cath D.

Microanalysis of serotonin by high pressure liquid chromatography with electrochemical detection.

Serotonin (5-HT) analysis by high pressure liquid chromatography with electrochemical detection (HPLC-ED) has been designed to perform micro assays below the picogram range. We demonstrated that the oxidation potential of 5-HT was tightly linked to the pH of the buffer used as eluent phase. The electrochemistry of 5-HT in pH 4.00 acetate buffer and in pH 6.80 phosphate buffer allowed quantification down to 2 x 10(-18) g. The oxidization current was linked to the 5-HT amount added. The electrochemical response of 5-HT was different for low amounts in the 10(-18) g range and for amounts above the picogram range. Two curves were obtained according to the electrochemical response of 5-HT: the first one was for the 10(-18) g range and the second one was for the picogram range and above. It thus appeared between concentration ranges that assays are not be feasible. This method has been used to analyse 20 microns frozen slices of rat spinal cord. These data open new insights into scarce quantification of 5-HT without the use of radio-elements.

Drug-resistant epimastigotes of Trypanosoma cruzi and persistence of this phenotype after differentiation into amastigotes.

In vitro benznidazole resistance was induced in cloned T. cruzi epimastigotes using a continuous drug pressure protocol. Stocks were selected according to their previous genetic characterization by multilocus enzyme electrophoresis. All the resistant clones were able to grow in long term cultivation in the presence of at least 50 microM benznidazole, which is the drug plasma level during chemotherapy in man. The highest level of resistance achieved was 220 microM. After differentiation of the epimastigote into the amastigote forms, the drug-resistance level was not affected. In both, the resistant epimastigotes and the resistant amastigotes, growth curves exhibited a lower growth rate than the sensitive counterparts without affecting the viability of the parasites. These data could be significant in basic research, to study the drug-resistance phenotype on relevant chemoresistant clones of T. cruzi, and to follow this phenotype after in vivo cycles.

Fundamental aspects and potential roles of ecdysteroids in schistosomes an update overview.

The human digenic trematodeSchistosoma mansoni produces ecdysteroid hormones. Both ecdysone (22R)-2ß,3ß,14,22,25-pentahydroxy-cholest-7-en-6-one and 20-hydroxyecdysone (22R)-2ß,3ß, 14,20,22,25-hexahydroxycholest-7-en-6-one were detected during the critical development stages of this bisexual parasite: during the exponential growth phase to adult stage (day 11 postinfection) and during the sexual maturation of adult and egg laying (day 40 postinfection). The parasites released their ecdysteroid hormones in the biological fluids of the infected vertebrates as soon as six days postinfection. In addition, the time course of the ecdysteroid titer was correlated with the susceptibility or the innate resistance of the host to schistosome infection. Moreover, we demonstrated that after a schistomicide therapy, ecdysteroids from urine of infected children decreased markedly four days after drug administration. We also have demonstrated that immunization of rodents with an ecdysone-BSA complex led to the reduction of the worm burden. By in vitro studies, we have shown that the ecdysterone antibodies were able to kill the juvenile worms within 24 hr. In addition, we have demonstrated that the enzymes Superoxide dismutase (SOD, EC 1.15.1.1) were present in schistosomes and that the total Superoxide dismutase activities in both males and females could be correlated with the 20-hydroxyecdysone within parasites.

Antimineralocorticoid 11beta-substituted spirolactones exhibit androgen receptor agonistic activity: a structure function study.

In humans, spironolactone and mespirenone are well known antimineralocorticoids without C-11beta substituents. These compounds display antagonist properties by acting through the human androgen receptor (hAR). In contrast, we demonstrate here that synthetic mineralocorticoid antagonists bearing hydrophobic C-11beta substituents and C-17gamma-lactone are potent hAR agonists in vitro. The three-dimensional construction of both the ligand binding domain (LBD) of the hAR and the human mineralocorticoid receptor (hMR), based on the crystal structure of the LBD of the human progesterone receptor, revealed not only that the interactions with the steroidal A- and D-rings seemed to be crucial for stabilization of active hMR or hAR conformation, but that other steroidal substitutions could influence the agonist versus antagonist activity of ligands. The docking of synthetic compounds bearing C-11beta hydrophobic substituents within the ligand binding pocket of hAR demonstrated that precise positions of the steroid, such as C-11 and C-17, are in close contact with some residues on the receptor, C-11 with Gly 708 and C-17 with Asn705 and Thr877. These contacts are crucial for the stabilization of the active receptor conformation. Mutation of Asn705 by alanine altered the 11beta-substituted spirolactone-mediated trans-activation function of hAR, suggesting an anchoring of the C-17-lactone carbonyl group (C-22) with this residue. The stabilizing effect of the H12 helix in its active conformation is also induced by hydrophobic contacts between the Gly708 and C-11beta substituents, as recently observed with the A773G-hMR mutant in the presence of similar drugs. The study of the role of these substituents suggests efficient new directions for the drug design of selective androgen agonists.

Detection of ecdysteroids in the human trematode, Schistosoma mansoni.

Freeze fracture study of Schistosoma mansoni membrane differentiation enabled us to describe the characteristic features of the tegumental membrane complex at various stages of development of this parasite. The observation of a membrane exuviation during the 10-20 day period of infection of the definitive host led us to search for possible hormonal control. The development of an appropriate radioimmunoassay has allowed the detection of significant production of ecdysteroids in a human platyhelminth. This hormonal material undergoes level variation during the development of post-cercarial stages of S. mansoni and its precise chemical characterization is presently being investigated.