Male Response to Female Ovulation in White-Faced Capuchins (Cebus capucinus): Variation in Fecal Testosterone,Dihydrotestosterone, and Glucocorticoids

Reproductive success is linked to dominance in male primates, reflecting the benefits of male competition. However, not all males compete successfully, suggesting that the costs of obtaining and maintaining high dominance status are significant. Here we examine the fecal metabolites of testosterone (fT) and dihydrotestosterone (fDHT) as bioactive androgens reflecting male reproductive effort, as well as fecal glucocorticoid (fGC) excretion as an index of stress in male white-faced capuchins (Cebus capucinus). We investigated the influence of female fertility (periovulatory vs. nonovulatory) on the hormonal responses of alpha and subordinate males. Over a 17-mo field season, we collected and analyzed weekly fecal samples (N = 992) from all 14 adult (> 10 yr) and subadult (≥ 6–10 yr) males residing in three study groups in the Santa Rosa Sector of the Área de Conservación Guanacaste, Costa Rica. Fecal samples (N > 2250) were also collected from group females (N = 28) to identify the fertile period using progesterone and estradiol assays. Alpha males had significantly higher fT, fDHT, and fGC levels than subordinate males independent of female reproductive state; further, adult subordinates had significantly higher fT, but not fDHT or fGC, than subadult subordinates. Male fT, fDHT, and fGC levels were significantly higher in the presence of fertile females, regardless of male dominance status and age. These findings indicate that the higher reproductive effort of alpha males comes with some costs (increased fGCs), and the presence of periovulatory females is associated with specific endocrine responses reflecting male reproductive effort and stress in white-faced capuchins.     

A structural modeling approach for the understanding of initiation and elongation of ALS-linked superoxide dismutase fibrils

Familial amyotrophic lateral sclerosis caused by mutations in copper-zinc superoxide dismutase (SOD1) is characterized by the presence of SOD1-rich inclusions in spinal cords. It has been shown that a reduced intra-subunit disulfide bridge apo-SOD1 can rapidly initiate fibrillation forming an inter-subunits disulfide under mild, physiologically accessible conditions. Once initiated, elongation can proceed via recruitment of either apo or partially metallated disulfide-intact SOD1 and the presence of copper, but not zinc, ions inhibit fibrillation. We propose a structural model, refined through molecular dynamics simulations, that, taking into account these experimental findings, provides a molecular explanation for the initiation and the elongation of SOD1 fibrils in physiological conditions. The model indicates the occurrence of a new dimeric unit, prone to interact one with the other due to the presence of a wide hydrophobic surface and specific electrostatic interactions. The model has dimensions consistent with the SOD1 fibril size observed through electron microscopy and provides a structural basis for the understanding of SOD1 fibrillation.

Two optimized combination assays to examine apoptosis pathways in clinical samples.

BACKGROUND: A consequence of a number of diseases is an alteration in apoptosis. Currently, there is no single assay that measures the main stages of apoptosis, requiring that multiple assays be performed. This hinders studies on clinical samples that have limited cell numbers. Our objective was to combine and optimize assays that target specific stages of apoptosis for use in a typical clinical blood sample. METHODS: Two flow cytometric assays were developed for use on peripheral blood mononuclear cells (PBMC) collected in two 8-ml tubes from a single draw. One measures caspase-12 activity, the level of active caspase-3 and DNA fragmentation. The second assesses depolarization of the mitochondria and phosphatidylserine externalization. Cell populations present within the samples were determined by flow cytometry. Apoptosis was validated by ELISA. RESULTS: Each assay was optimized for use with cell numbers and sample volumes typical of clinical blood samples. Each combination assay effectively distinguished apoptotic from nonapoptotic blood cells. CONCLUSIONS: This combined optimized method comprised of two independent assays makes it possible to assay the major pathways of apoptosis in addition to determining the blood cell subsets that are affected.     

Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.