Future strategies for treating erectile dysfunction

Erectile dysfunction affects over half of all men between 50 and 70 years of age, and by the age of 40, about 40% of men may suffer from some form of erectile dysfunction. Many disease states, such as diabetes, hypertension, depression, and vascular disease, are associated with the condition, which may occur many years prior to the onset of these disorders. The phenomenal success of sildenafil in improving erections in men with erectile dysfunction is due to the fact that the drug, as a phosphodiesterase inhibitor, improves the relaxation of smooth muscle cells, which become dysfunctional with the aging process. However, not everyone responds to this medication, mainly because the efficacy of the drug is directly dependent on the release of nitric oxide from the nerve terminals of the cavernosal nerve, and this may become defective with aging/certain disease states. The goal of gene therapy for organic impotence is to allow the patient to sustain physiologically elicited erections without resorting to pharmacological treatment immediately prior to the sexual act. Experimental efforts in gene therapy for erectile dysfunction are likely to continue intensively in a series of directions, some specific to the nature of the selected gene to be manipulated or the physiology of the corpora cavernosa itself, and others extrapolatable from the advancement of gene therapy in general.     

Gene therapy of erectile dysfunction in the rat with penile neuronal nitric oxide synthase

Gene transfer to the penile corpora cavernosa of constructs of the inducible and endothelial nitric oxide synthase (NOS) cDNAs ameliorates erectile dysfunction in aged rats. In this study, we investigated whether the neuronal NOS (nNOS) variant responsible for erection, penile nNOS (PnNOS), can exert a similar effect, and whether the combination of electroporation with a helper-dependent adenovirus (AdV) improves gene transfer. PnNOS and beta-galactosidase cDNAs were cloned in plasmid (pCMV-PnNOS; pCMV-beta-gal) and "gutless" AdV (AdV-CMV-PnNOS; AdV-CMV-beta-gal) vectors, and injected into the penis of adult (beta-gal) or aged (PnNOS) rats, with or without electroporation. Penile erection was measured at different times after PnNOS cDNA injection, by electrical field stimulation of the cavernosal nerve. The expression of beta-galactosidase or PnNOS was estimated in penile tissue by either histochemistry and luminometry or Western blot, and the effects of AdV-CMV-PnNOS on mRNA expression were examined by a DNA microarray. We found that electroporation increased pCMV-beta-gal uptake, and its expression was detectable at 56 days. In the aged rats treated with pCMV-PnNOS and electroporation, the maximal intracavernosal:mean arterial pressure ratios were elevated for 11 and 18 days when compared with those in controls. Electroporation intensified penile uptake of as few as 10(6) viral particles (vp) of AdV-CMV-beta-gal, and with 10(7) vp beta-galactosidase was still detectable at 60 days. Electroporated AdV-CMV-PnNOS (10(7) vp) was effective at 18 days in stimulating the erection of aged rats, without inducing the expression of cytotoxic genes. In conclusion, intracavernosal gene therapy with PnNOS cDNA corrected the aging-related erectile dysfunction for at least 18 days when given by electroporation in a helper-dependent AdV at low viral loads.     

Structural basis for Chk1 inhibition by UCN-01

Chk1 is a serine-threonine kinase that plays an important role in the DNA damage response, including G(2)/M cell cycle control. UCN-01 (7-hydroxystaurosporine), currently in clinical trials, has recently been shown to be a potent Chk1 inhibitor that abrogates the G(2)/M checkpoint induced by DNA-damaging agents. To understand the structural basis of Chk1 inhibition by UCN-01, we determined the crystal structure of the Chk1 kinase domain in complex with UCN-01. Chk1 structures with staurosporine and its analog SB-218078 were also determined. All three compounds bind in the ATP-binding pocket of Chk1, producing only slight changes in the protein conformation. Selectivity of UCN-01 toward Chk1 over cyclin-dependent kinases can be explained by the presence of a hydroxyl group in the lactam moiety interacting with the ATP-binding pocket. Hydrophobic interactions and hydrogen-bonding interactions were observed in the structures between UCN-01 and the Chk1 kinase domain. The high structural complementarity of these interactions is consistent with the potency and selectivity of UCN-01.     

Sipaucins A-C,sesquiterpenoids from Siparuna pauciflora

The phytochemical investigation of the leaves of Siparuna pauciflora yielded three novel sesquiterpenoids: the germacrane sipaucin A, the elemane sipaucin B and sipaucin C, comprising a new type of carbon skeleton. In addition, four known aporphine alkaloids-nor-boldine, boldine, laurotetanine, and N-methyl-laurotetanine-were obtained. The evaluation of the antiplasmodial activity of the isolated compounds against two strains of Plasmodium falciparum (PoW, Dd2) showed a moderate activity of nor-boldine.     

Glucocorticoid-induced skeletal muscle atrophy is associated with upregulation of myostatin gene expression

The mechanisms by which excessive glucocorticoids cause muscular atrophy remain unclear. We previously demonstrated that dexamethasone increases the expression of myostatin, a negative regulator of skeletal muscle mass, in vitro. In the present study, we tested the hypothesis that dexamethasone-induced muscle loss is associated with increased myostatin expression in vivo. Daily administration (60, 600, 1,200 micro g/kg body wt) of dexamethasone for 5 days resulted in rapid, dose-dependent loss of body weight (-4.0, -13.4, -17.2%, respectively, P < 0.05 for each comparison), and muscle atrophy (6.3, 15.0, 16.6% below controls, respectively). These changes were associated with dose-dependent, marked induction of intramuscular myostatin mRNA (66.3, 450, 527.6% increase above controls, P < 0.05 for each comparison) and protein expression (0.0, 260.5, 318.4% increase above controls, P < 0.05). We found that the effect of dexamethasone on body weight and muscle loss and upregulation of intramuscular myostatin expression was time dependent. When dexamethasone treatment (600 micro g. kg-1. day-1) was extended from 5 to 10 days, the rate of body weight loss was markedly reduced to approximately 2% within this extended period. The concentrations of intramuscular myosin heavy chain type II in dexamethasone-treated rats were significantly lower (-43% after 5-day treatment, -14% after 10-day treatment) than their respective corresponding controls. The intramuscular myostatin concentration in rats treated with dexamethasone for 10 days returned to basal level. Concurrent treatment with RU-486 blocked dexamethasone-induced myostatin expression and significantly attenuated body loss and muscle atrophy. We propose that dexamethasone-induced muscle loss is mediated, at least in part, by the upregulation of myostatin expression through a glucocorticoid receptor-mediated pathway.     

Heterogeneity in growth and differentiation characteristics in male and female satellite cells isolated from turkey lines with different growth rates

The effects of growth- and gender-related differences on satellite cell proliferation and differentiation were investigated using satellite cells isolated from the pectoralis major muscle of a turkey line selected for increased 16-week body weight (F-line) and its unselected randombred control (RBC2-line). Proliferation rates within the F- and RBC2-lines did not differ between sexes. The F-line male and female satellite cells when compared to the RBC2-line male and female satellite cells proliferated at a faster rate. Differentiation rates were increased for the F-line male cells compared to both the F-line female and RBC2-line male satellite cells. No difference in differentiation rate was noted within the RBC2-line satellite cells. For satellite cells from females, the RBC2-line differentiated faster than the F-line. Morphological data on myotube length and the number of nuclei per myotube supported the differentiation data in that F-line male satellite cells had the longest myotubes with the most nuclei, there was no significant difference between myotubes within the RBC2-line, and female-derived myotubes from the RBC2-line were longer than those of the F-line by 96 h of fusion. These data are suggestive of both growth- and gender- related differences in satellite cell proliferation and differentiation.     

Characterization of the MTF-1 transcription factor from zebrafish and trout cells

Two Euphausia superba Dana endo-1.4-beta-xylanases (A, and B), hydrolysing xylan in the same manner as the enzyme classified as EC 3.2.1.8, were isolated and purified. (2) The enzymes were distinguished by their molecular mass and charge, affinities towards the oat xylan (Km of 4.1 and 7.7 mg ml(-1), respectively), values of activation energy in oat xylan hydrolysis (35.5 and 42.5 kJ mol(-1), respectively), as well as the way in which they split the substrate. (3) In vitro they showed the same optimal temperature (37-40 degrees C), optimal pH (5.7-6.0), very low thermostability, and were stabilized and activated by Ca2+ and Mg2+ ions, as well as by some unidentified substances with molecular mass less than 17 kDa, present in crude extracts of krill.     

Distribution, abundance, and conservation of Vinaceous Amazons (Amazona vinacea) in Argentina and Paraguay

ABSTRACT.   Vinaceous Amazons ( Amazona vinacea ) are endemic to the Atlantic forest of southeastern Brazil, eastern Paraguay, and the province of Misiones in Argentina. We searched for Vinaceous Amazons throughout the western part of its range in Argentina and Paraguay during 1639 days of fieldwork from 1997 to 2006. These parrots have disappeared from most areas where they were historically recorded in these countries, and are now limited to a few sites in northeastern Paraguay and central Misiones (Argentina). We estimate the minimum remaining populations at 220 individuals in Paraguay and 203 individuals in Argentina. Important sites for the species are (1) the farming area from San Pedro to Tobuna (Misiones, Argentina) and (2) the Itaipú reserves complex and Reserva Natural Privada Itabó (Paraguay). In our surveys, Vinaceous Amazons were absent from the largest tracts of forest in Misiones, and were most often observed feeding, roosting, and nesting in small forest remnants and in agricultural areas that included forest fragments and isolated trees. Threats to amazons in these areas include nest poaching, forest clearing, and being shot as a crop pest. We confirmed 40 Vinaceous Amazons kept as pets in 35 homes between San Pedro and Tobuna. Environmental education and law enforcement are urgently needed to reduce threats in populated areas, and subsistence farmers need technical and logistical support to slow or stop the conversion of forest into cropland. Finally, additional study is needed to determine this amazon's habitat preferences, nest site requirements, and demography in different habitats.     

Effects of Beta-Alanine Supplementation on Brain Homocarnosine/Carnosine Signal and Cognitive Function: An Exploratory Study

Objectives

Two independent studies were conducted to examine the effects of 28 d of beta-alanine supplementation at 6.4 g d^-1 on brain homocarnosine/carnosine signal in omnivores and vegetarians (Study 1) and on cognitive function before and after exercise in trained cyclists (Study 2).

Methods

In Study 1, seven healthy vegetarians (3 women and 4 men) and seven age- and sex-matched omnivores undertook a brain 1H-MRS exam at baseline and after beta-alanine supplementation. In study 2, nineteen trained male cyclists completed four 20-Km cycling time trials (two pre supplementation and two post supplementation), with a battery of cognitive function tests (Stroop test, Sternberg paradigm, Rapid Visual Information Processing task) being performed before and after exercise on each occasion.

Results

In Study 1, there were no within-group effects of beta-alanine supplementation on brain homocarnosine/carnosine signal in either vegetarians (p = 0.99) or omnivores (p = 0.27); nor was there any effect when data from both groups were pooled (p = 0.19). Similarly, there was no group by time interaction for brain homocarnosine/carnosine signal (p = 0.27). In study 2, exercise improved cognitive function across all tests (P<0.05), although there was no effect (P>0.05) of beta-alanine supplementation on response times or accuracy for the Stroop test, Sternberg paradigm or RVIP task at rest or after exercise.

Conclusion

28 d of beta-alanine supplementation at 6.4g d^-1 appeared not to influence brain homocarnosine/carnosine signal in either omnivores or vegetarians; nor did it influence cognitive function before or after exercise in trained cyclists.