Androgen 5-alpha-reductase type 2 is highly expressed and active in rat spinal cord motor neurones.

Spinal cord motoneurones express high levels of androgen receptor. However, in responsive tissue, the effects of testosterone is often mediated by the more potent androgenic derivative 5-alpha-dihydrotestosterone (DHT). This compound is formed in androgen target cells by the enzyme 5-alpha-reductase. Two isoforms of the 5-alpha-reductase, with limited degree of homology, have been cloned, type 1 and type 2. The low affinity-constitutive type 1 isoenzyme is widely distributed in the body; the high affinity-androgen regulated 5-alpha-reductase type 2 is confined to androgen-dependent structures and shows a peculiar pH optimum at acidic values. We have previously shown that high levels of 5-alpha-reductase activity are detectable in rat spinal cord. Here, using reverse transcriptase-polymerase chain reaction, we show that both isoforms are expressed in the whole spinal cord of the rat. The enzymatic pH optimum measured in immortalized spinal cord motoneurones (NSC34) is typical of the type 2 isoenzyme. Using in situ hybridization technique, we found that 5-alpha-reductase type 2 is confined to the motoneuronal cells of the anterior horns of the rat spinal cord, the cells that also are known to express high levels of androgen receptor. Because of the close association of androgen receptor and 5-alpha alpha-reductase type 2, motoneuronal cells should be considered as target cells for androgens.

Polyglutamine tract expansion of the androgen receptor in a motoneuronal model of spinal and bulbar muscular atrophy.

Spinobulbar muscular atrophy (SBMA) is a late-onset disorder characterized by progressive muscle loss, degeneration of motoneurons in the spinal cord and brainstem, and partial androgen insensitivity. SBMA is directly correlated with the expansion of CAG repeats encoding a polyglutamine tract (polyQ) of extended length. The identification of polyQ expansion in SBMA led to the discovery of an entire class of neurodegenerative disorders. In fact, at least eight different diseases, including Huntington's disease, share a common molecular mechanism involving an expansion of a polyQ tract within different proteins. The elongated polyQ tract causes a toxic gain of function in the mutant protein and is associated with the formation of intracellular aggregates, whose pathogenetic role has not been fully established yet. Our observations in a motoneuron cell line (NSC34), indicate that the expression of the androgen receptor (AR) carrying the elongated polyQ tract (AR-Q48) has a toxic effect in aggregate-independent manner. In fact, in basal condition, AR-Q48 shows a cytoplasmic diffuse distribution, yet it reduces the viability of transfected NSC34. In contrast, testosterone treatment, while inducing aggregation of the mutant AR, also increases cell viability. Aggregates in NSC34 are localized mainly in the perinuclear region and occasionally in the neuropil, whereas no nuclear aggregate has ever been found. Further observations of the minor subset of cells showing neuropil aggregates, reveal an alteration of the neurite morphology, suggesting a different role of the two types of cytoplasmic aggregates.

Determination of the adenosine A(1) agonist N(6)-cyclopentyladenosine in rat blood by solid-phase extraction and HPLC.

A solid-phase extraction procedure has been developed for the isolation of the adenosine A1 receptor agonist N(6)-cyclopentyladenosine from rat blood. The biological samples were spiked with N(6)-cyclopentyladenosine and the analogue N(6)-cyclohexladenosine (internal standard), diluted with sodium hydroxide, loaded onto disposable cartridges with subsequent desorption with methanol and analysis by HPLC. The performance of columns pre-packed with different C18-bonded silica phases or with a polymeric reversed-phase sorbent (Oasis HLB) was assessed. The highest extraction efficiencies (recovery rates>83.3%) for the two N6-alkyl substituted adenosines were achieved by the Oasis HLB cartridges. In addition, the polymeric sorbent provided reproducible recoveries (relative standard deviation<4.8%), whereas large variations (relative standard deviation values, 9--16.3%) in the extraction yields were observed using the conventional silica-based C18 cartridges. The described sample preparation method is rapid, simple, selective and it is suitable for pharmacokinetic studies.

5Alpha-reductase type 2 and androgen receptor expression in gonadotropin releasing hormone GT1-1 cells.

Gonadal steroids are potent modulators of gonadotropin releasing hormone (GnRH) secretion, and androgen binding sites and 5alpha-reductase activity have been found in the immortalized GnRH secreting cell line GT1-1, suggesting the existence of a direct androgenic control of GnRH dynamics. Two isoforms of the 5alpha-reductase have been cloned with very different biochemical/functional properties: 5alpha-reductase type 1 (widely distributed in the body) and 5alpha-reductase type 2 (confined in androgen target structures). We have analysed whether, in GT1-1, androgen binding sites are linked to "classical" androgen receptor, and which 5alpha-reductase isoform is active. Reverse transcriptase-polymerase chain reaction analysis showed that the mRNAs coding for androgen receptor and for the two 5alpha-reductase isoforms are all expressed in GT1-1 cells. However, the 5alpha-reductase enzymatic reaction showed a peak of activity at a narrow pH around 5.5, the optimum for the 5alpha-reductase type 2. The affinity for testosterone, of the enzyme present in GT1-1 cells, was very similar to that observed for the recombinant type 2 isozyme expressed in yeasts. The data indicate that GT1-1 cells (i) express a "classical" androgen receptor and (ii) contain the 5alpha-reductase type 2 isoform, a specific marker of androgen-responsiveness.

Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract.

Spinal and bulbar muscular atrophy (SBMA) is associated with an abnormal expansion of the (CAG)(n)repeat in the androgen receptor (AR) gene. Similar mutations have been reported in other proteins that cause neurodegenerative disorders. The CAG-coded elongated polyglutamine (polyGln) tracts induce the formation of neuronal intracellular aggregates. We have produced a model to study the effects of potentially 'neurotoxic' aggregates in SBMA using immortalized motoneuronal cells (NSC34) transfected with AR containing polyGln repeats of different sizes [(AR.Q(n = 0, 23 or 46)]. Using chimeras of AR.Q(n) and the green fluorescent protein (GFP), we have shown that aggregate formation occurs when the polyGln tract is elongated and AR is activated by androgens. In NSC34 cells co-expressing the AR with the polyGln of pathological length (AR.Q46) and the GFP we have noted the presence of several dystrophic neurites. Cell viability analyses have shown a reduced growth/survival rate in NSC34 expressing the AR.Q46, whereas testosterone treatment partially counteracted both cell death and the formation of dystrophic neurites. These observations indicate the lack of correlation between aggregate formation and cell survival, and suggest that neuronal degeneration in SBMA might be secondary to axonal/dendritic insults.

Mammography with synchrotron radiation.

PURPOSE: To determine the feasibility of using synchrotron radiation (SR) in diagnostic mammography. MATERIALS AND METHODS: Monochromatic SR of x-ray beams of selected energies of 14-20 keV were used to obtain mammograms of surgically removed breast specimens that contained tumor nodules. For comparison, conventional mammograms of the same specimens were also obtained. RESULTS: The mammograms obtained with SR had higher contrast and better resolution than did traditional mammograms and demonstrated excellent detail in all cases studied. The mean glandular doses at 17 and 18 keV were 1.56 and 0.83 mGy, respectively, which is compatible with the mean value of 1.41 mGy delivered with the conventional grid apparatus. CONCLUSION: SR mammography appears to be a promising diagnostic imaging technique.