Antiandrogen oligonucleotides: active principles in hair- and skin-derived culture cells.

Most drugs used for treatment of androgen-related dermatological disorders are not completely satisfactory in terms of clinical efficacy and potential secondary effects. There is, therefore, a need for a new generation of specific antiandrogens. This paper focuses on an oligonucleotide antisense pharmacological strategy. Acceptor sites were first disclosed by mapping the human Androgen Receptor (AR) mRNA conformation using an mRNA walking approach, oligonucleotide binding, and S1 protection assays. Antisense-sensitive regions were localized by RNAse H degradation and AR in vitro translation inhibition. Oligonucleotides were then designed and assessed, in primary cultures of human hair dermal papillae and skin derived fibroblasts, for their capability to down-regulate AR expression. Some of them were able to inhibit more than 60 to 80% of the AR expression. These could be a new class of antiandrogen oligonucleotides pharmacologically active in hair and skin derived cells, suitable for the treatment of dermatological disorders.

The cloned C-terminal region of a Trypanosoma cruzi P ribosomal protein harbors two antigenic determinants.

A DNA strategy was designed to characterize the antigenic site(s) within a lambda gt11 cloned 35-amino-acid antigenic peptide, identified with antibodies from patients with chronic Chagas' heart disease (cChHD) and systemic lupus erythematosus (SLE) as the C-terminal portion of a Trypanosoma cruzi P ribosomal protein. The 198-bp cDNA insert was digested with AluI, resulting in two DNA segments that were recloned in lambda gt11. To identify specific antigenic determinants, the recombinant phage and the purified recombinant antigens were probed with sera from clinically characterized subjects. Chronic ChHD and SLE sera defined a linear epitope common to both diseases within the 15 C-terminal residues of the parasite P ribosomal protein. It is also shown that the cloned 35-amino-acid peptide contained an antigenic site unique to cChHD.

Further studies on the phosphorylation-regulated cAMP-phosphodiesterase from the dimorphic fungus Mucor rouxii.

A partially purified preparation (200-fold) of cAMP phosphodiesterase (PDE) was obtained from Mucor rouxii grown and extracted under conditions minimizing endogenous proteolysis. Four purification steps were applied: batch DEAE-Sepharose, DEAE-Sepharose chromatography, Sephadex G-150 super-fine gel filtration and sucrose gradient centrifugation. The final PDE preparation was activatable by cAMP-dependent phosphorylation and controlled trypsin treatment. A careful correlation of protein patterns with PDE activity was done throughout the whole procedure by analyzing the active fractions of each step by mini-polyacrylamide non-denaturing gel electrophoresis. The final preparation displayed four major protein bands, none of which corresponded to PDE, although PDE activity comigrated with two of them. Some properties of this preparation were studied. Vmax increased around 10-15 fold by activation of PDE by phosphorylation or proteolysis; Km values were unaffected. PDE had Stokes radius of 3.5 nm, sedimentation coefficient of 4.3 S and molecular weight of 70,000 daltons. The treatment of sucrose gradient fractions with [gamma-32P] ATP and cAMP-dependent protein kinase catalytic subunit and further analysis through minigels showed that none of the visible bands was phosphorylated, and that among the four phosphorylated bands there was one that cosedimented and comigrated with PDE activity. Trypsin treatment of the phosphorylated samples removed the label but did not modify the staining pattern.