Endogenous testosterone increases L-type Ca2+ channel expression in porcine coronary smooth muscle

Evidence indicates that gender and sex hormonal status influence cardiovascular physiology and pathophysiology. We recently demonstrated increased L-type voltage-gated Ca2+ current (ICa,L) in coronary arterial smooth muscle (CASM) of male compared with female swine. The promoter region of the L-type voltage-gated Ca2+ channel (VGCC) (Cav1.2) gene contains a hormone response element that is activated by testosterone. Thus the purpose of the present study was to determine whether endogenous testosterone regulates CASM ICa,L through regulation of VGCC expression and activity. Sexually mature male and female Yucatan swine (7-8 mo; 35-45 kg) were obtained from the breeder. Males were left intact (IM, n=8), castrated (CM, n=8), or castrated with testosterone replacement (CMT, n=8; 10 mg/day Androgel). Females remained gonad intact (n=8). In right coronary arteries, both Cav1.2 mRNA and protein were greater in IM compared with intact females. Cav1.2 mRNA and protein were reduced in CM compared with IM and restored in CMT. In isolated CASM, both peak and steady-state ICa were reduced in CM compared with IM and restored in CMT. In males, a linear relationship was found between serum testosterone levels and ICa. In vitro, both testosterone and the nonaromatizable androgen, dihydrotestosterone, increased Cav1.2 expression. Furthermore, this effect was blocked by the androgen receptor antagonist cyproterone. We conclude that endogenous testosterone is a primary regulator of Cav1.2 expression and activity in coronary arteries of males.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

The effects of stanozolol and boldenone undecylenate on scrotal width, testis weight, and sperm production in pony stallions

Fifty mature pony stallions were randomly assigned to one of five treatment groups: Group 1- controls (no treatment), Group 2 - 0.55 mg/kg stanozolol weekly for 13 treatments, Group 3 - 1.1 mg/kg stanozolol every 3 weeks for 5 treatments, Group 4 - 1.1 mg/kg boldenone undecylenate every 3 weeks for 5 treatments, and Group 5 - 0.55 mg/kg boldenone undecylenate weekly for 13 treatments. Scrotal widths (SW), combined testis weights (CTW), and daily sperm productions (DSP) were not different between Groups 1 and 2. Ponies in Group 5 had smaller SW (P<0.01), smaller CTW and decreased DSP compared to controls (P < 0.05). Although SW for ponies in Groups 3 and 4 were less than for controls (P < 0.01), CTW and DSP were not different. The only treatment regime that did not alter SW, CTW, and DSP was Group 2 (0.55 mg/kg stanozolol weekly for 13 treatments).     

Unique regional distribution of delta 4-3-ketosteroid-5 alpha-oxidoreductase and associated epididymal morphology in the marsupial, Didelphis virginiana

The epididymal epithelial ultrastructure has been described in the adult male North American opossum, Didelphis virginiana. Morphological results have suggested that absorptive activity is prominent in the proximal epididymal region by virtue of numerous microvilli, an endocytotic complex, dense granules, and multivesicular bodies in the apical cytoplasm. In contrast, the middle and distal epididymal regions exhibit ultrastructural features indicative of protein synthesis such as large invaginated euchromatic nuclei, large nucleoli, and increased amounts of granular endoplasmic reticulum. It is in the middle and distal epididymal regions where sperm head rotation and sperm pairing take place. Epididymal delta 4-3-ketosteroid-5 alpha-oxidoreductase (5 alpha-reductase) activity also has been measured. It has been found that the level of enzyme activity differs significantly (p less than 0.01) between the proximal, middle, and distal epididymal regions. Enzyme-specific activity has been found to be highest in the middle region (47.6 +/- 5.4 picomoles 5 alpha-reduced androgens formed/b/mg protein), lower in the distal region (18.3 +/- 0.7 picomoles 5 alpha-reduced androgens formed/b/mg protein), with little activity (2.4 +/- 1.2 picomoles 5 alpha-reduced androgens formed/h/mg protein) found in the proximal epididymal region. This regional distribution of enzyme activity differs markedly from that reported for eutherian mammals. Both the suggested epididymal protein synthetic and secretory activity and the level of epididymal 5 alpha-reductase activity appear to correlate regionally with the morphological changes that occur in the opossum spermatozoa as they transit the epididymis.     

Hypothalamic glycogen synthase kinase 3β has a central role in the regulation of food intake and glucose metabolism

GSK3β (glycogen synthase kinase 3β) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3β activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3β in leptin-deficient Lepob/ob mice and show that intracerebroventricular injection of a GSK3β inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3β inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3β in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3β signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.     

Multimerization and DNA Binding Properties of INI1/hSNF5 and Its Functional Significance

INI1/hSNF5/BAF47/SMARCB1 is an HIV-1 integrase (IN)-binding protein that modulates viral replication in multiple ways. A minimal IN-binding domain of INI1, S6 (amino acids 183–294), transdominantly inhibits late events, and down-modulation of INI1 stimulates early events of HIV-1 replication. INI1 both stimulates and inhibits in vitro integration depending on IN concentration. To gain further insight into its role in HIV-1 replication, we purified and biochemically characterized INI1. We found that INI1 forms multimeric structures. Deletion analysis indicated that the Rpt1 and Rpt2 motifs form the minimal multimerization domain. We isolated mutants of INI1 that are defective for multimerization using a reverse yeast two-hybrid system. Our results revealed that INI1 residues involved in multimerization overlap with IN-binding and nuclear export domains and are required for nuclear retention and co-localization with IN. Multimerization-defective mutants are also defective for mediating the transdominant effect of INI1-S6-(183–294). Furthermore, we found that INI1 is a minor groove DNA-binding protein. Although IN binding and multimerization are required for INI1-mediated inhibition, the acceptor DNA binding property of INI1 may be required for stimulation of in vitro strand transfer activities of IN. Binding of INI1 to IN results in the formation of presumably inactive high molecular weight IN-INI1 complexes, and the multimerization-defective mutant was unable to form these complexes. These results indicate that the multimerization and IN binding properties of INI1 are necessary for its ability to both inhibit integration and influence assembly and particle production, providing insights into the mechanism of INI1-mediated effects in HIV-1 replication.HIV-1³ replication is a dynamic process that is modulated by the interaction of several host cellular proteins (1). A genome-wide siRNA-mediated knockdown indicated that hundreds of host factors are involved in the stimulation or inhibition of HIV-1 replication (2). Understanding the interplay between the host proteins and the HIV-1 viral proteins is essential to fully comprehend the dynamic relationship between the virus and the host.INI1/hSNF5/BAF47/SMARCB1 is a core component of the SWI/SNF chromatin-remodeling complex. It interacts directly with the HIV-1-encoded integrase (IN) required for the integration of the viral DNA into the host chromosome (3, 4). IN mediates the insertion of viral cDNA into host chromosomal DNA by sequential steps of 3′ processing and strand transfer (or joining) (4, 5). INI1 binds directly to HIV-1 IN in vitro and in vivo and modulates several steps of HIV-1 replication (3, 6–8). The ectopically expressed minimal IN-binding domain of INI1 transdominantly and potently inhibits HIV-1 assembly and particle production (8). The inhibitory effect is dependent on IN-INI1 interaction and is abrogated when an IN mutant defective for interaction with INI1 is used (8). Furthermore, particle production is minimal in cells lacking INI1, and reintroduction of INI1 into these cells can partially correct the defect (6). These results indicate that INI1 is required for HIV-1 late events. Additional studies have indicated that INI1 is selectively incorporated into HIV-1 but not other retroviral and lentiviral particles (9). Virally encapsidated INI1 is required for post-entry early events of HIV-1 replication prior to integration (6). These studies indicate that producer cell-associated as well as virion-associated INI1 is required for HIV-1 replication. Contrary to these proviral functions of INI1, siRNA-mediated knockdown studies indicate that INI1 in the target cells inhibits early events of HIV-1 replication (7). These studies indicate that whereas INI1 in the target cells may act as an antiviral host protein, HIV-1 may subvert the INI1 antiviral effect, and HIV-1 may utilize this host factor for late events in the producer cells and for early preintegration events in the target cells. Interestingly, in an earlier study, we demonstrated that partially purified INI1 both inhibits and stimulates in vitro integration in a manner dependent on IN concentration (3). Although INI1 stimulates in vitro strand transfer reactions at low IN concentrations, it inhibits the reaction at high concentrations (3). Further structure-function analysis of INI1 is required to understand this complex and dual role of INI1 during HIV-1 replication.INI1 gene is also a tumor suppressor that is biallelically deleted in aggressive pediatric cancers known as rhabdoid tumors (10). INI1 mutations have been found in other soft tissue cancers (11–13). The mechanism of INI1-mediated tumor suppression is not fully understood. INI1 protein has two highly conserved domains that are imperfect direct repeats (termed Rpt1 and Rpt2) of each other and a third conserved coiled coil domain (termed homology region 3 or HR3) at the C terminus. The Rpt1 and Rpt2 domains appear to be involved in protein-protein interaction with various cellular and viral proteins (3, 14–18). Additionally, the Rpt2 domain harbors a masked nuclear export signal, and the C-terminal domain is involved in inhibiting the nuclear export of the protein in the steady state. INI1 exhibits nonspecific DNA binding activity (18). The cancer-associated mutations occur throughout the open reading frame of the INI1 gene, suggesting that mutation in any one of the INI1 domains may inactivate the protein and that multiple domains are required for its function (19–21).To gain further insight into the mechanism of its action, we purified INI1 protein to homogeneity and characterized it biochemically. Here we report, for the first time, that INI1 forms dimeric and higher order multimeric structures. We have characterized the multimerization domain of INI1 and found that multimerization and IN binding activities of INI1 are required for inhibition of in vitro integration. Furthermore, we found that the multimerization, IN binding, and nuclear export properties of INI1 are important for transdominant effects. In addition, we found that INI1 possesses a minor groove DNA binding activity and that the nonspecific acceptor DNA binding activity of INI1 may be required for stimulation of in vitro integration. Finally, we found that multimerization of the full-length protein is necessary for its ability to be retained in the nucleus and to co-localize with HIV-1 IN in the nucleus. Thus, our studies provide novel insights into the mechanism by which INI1 regulates HIV-1 replication.