The influence of androgen administration on the structure and function of the brain-pituitary-gonad axis of sexually immature platyfish,Xiphophorus maculatus

Summary This report demonstrates that the administration of testosterone (T) or 11-ketotestosterone (11-KT) to sexually immature (8 wks old) male platyfish (Xiphophorus maculatus) of early-and late-maturing genotypes affects the synthesis and/or release of luteinizing hormone-releasing hormone (LHRH), as assessed by immunocytochemical evaluation, increases the number and activity of pituitary gonadotropes, stimulates the production of sperm and, thus, advances the age of sexual maturation over that dictated by the genome. We also show that 11-KT and T affect different LHRH-containing centers in the brain and have differential effects on rate and degree of sexual maturation, regardless of whether the hormones are administered to early or late-maturing genotypes.     

Piping plover chick ecology following landscape-level disturbance

Population declines of disturbance-dependent species due to suppression of natural disturbances are realized across ecosystems. The piping plover (Charadrius melodus; plover), a disturbance-dependent and conservation-reliant shorebird that nests on sandy beaches and barrier islands on the Atlantic Coast, was listed under the United States Endangered Species Act in 1986. In 2012, Hurricane Sandy landed on Fire and Westhampton islands, barrier island nesting sites for plovers in New York, USA. Hurricane Sandy was a natural disturbance in this system, creating abundant nesting habitat. The number of chicks produced by a pair, or a population, is a direct measure of reproductive output, and gaining a better understanding of productivity and chick behavior following large-scale habitat creation may improve plover habitat management and potentially species persistence. We evaluated the effects of landscape features on habitat selection, behavior, and survival of plover broods using logistic regression, generalized linear mixed effects models, and survival models. Plover broods selected flatter sites with less dense vegetation than available at random. Chick foraging rates were highest in moist substrates and were lower in areas of higher nesting plover density. Chick survival was greater for broods that hatched earlier in the breeding season and increased as chicks aged. Generally, providing access to sites with flatter, moist substrates will likely result in higher quality brood rearing habitat on the landscape. Ultimately, vegetation removal and habitat management may be needed to reduce plover nesting density and ensure sufficient habitat, which may in turn improve plover chick survival. Moreover, sustaining natural landscape disturbances such as those resulting from storms, and not taking actions to prevent hurricane-created overwash, will allow these landscape features to persist.     

Disruption of the Cytokeratin Cytoskeleton and Inhibition of Hepatocytic Autophagy by Okadaic Acid

To learn whether autophagy might be dependent on any of the major cytoskeletal elements, the effect of various cytoskeleton inhibitors on autophagy and cytoskeletal organization was studied in isolated rat hepatocytes. Autophagy, measured as the sequestration of endogenous lactate dehydrogenase, was completely inhibited in isolated rat hepatocytes by the protein phosphatase inhibitor okadaic acid (30 nM). Only small effects were seen with vinblastine (10 μM) or cytochalasin D (10 μM). Indirect immunofluorescence microscopy with antibody to a 55-kDa cytokeratin, corresponding to human cytokeratin 8 (CK8), revealed that whereas control cells contained a well-organized network of cytokeratin intermediate filaments, okadaic acid disrupted this network into small spherical aggregates. Treatment with cytochalasin D or vinblastine, which disrupt microfilaments and microtubules, respectively, had no detectable effect on the cytokeratin filament distribution. Neither the microtubule network (detected by indirect immunofluorescence with antibodies against α- and β-tubulin) nor the actin microfilament network (detected by rhodamine-palloidin) was disrupted by okadaic acid. Naringin (100 μM), a putative protein kinase-inhibitory flavonoid, offered complete protection against the autophagy-inhibitory and cytokeratin-disruptive effects of okadaic acid. Two other flavonoids, genistein (100 μM) and prunin (100 μM) as well as KN-62 (10 μM), a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II), likewise displayed a good ability to protect against the effect of okadaic acid upon cytokeratin organization, while no such protection was seen with H-89 (20 μM), an inhibitor of the cyclic nucleotide-dependent protein kinases, or with H-7 (100 μM), which in addition inhibits protein kinase C. The results suggest that the cytokeratin cytoskeleton of hepatocytes is subject to rapid control by phosphorylation and dephosphorylation and that cytokeratin filaments may somehow be involved in the autophagic process.     

Impact of Submarine Groundwater Discharge on Marine Water Quality and Reef Biota of Maui

Generally unseen and infrequently measured, submarine groundwater discharge (SGD) can transport potentially large loads of nutrients and other land-based contaminants to coastal ecosystems. To examine this linkage we employed algal bioassays, benthic community analysis, and geochemical methods to examine water quality and community parameters of nearshore reefs adjacent to a variety of potential, land-based nutrient sources on Maui. Three common reef algae, Acanthophora spicifera, Hypnea musciformis, and Ulva spp. were collected and/or deployed at six locations with SGD. Algal tissue nitrogen (N) parameters (δ¹⁵N, N %, and C:N) were compared with nutrient and δ¹⁵N-nitrate values of coastal groundwater and nearshore surface water at all locations. Benthic community composition was estimated for ten 10-m transects per location. Reefs adjacent to sugarcane farms had the greatest abundance of macroalgae, low species diversity, and the highest concentrations of N in algal tissues, coastal groundwater, and marine surface waters compared to locations with low anthropogenic impact. Based on δ¹⁵N values of algal tissues, we estimate ca. 0.31 km² of Kahului Bay is impacted by effluent injected underground at the Kahului Wastewater Reclamation Facility (WRF); this region is barren of corals and almost entirely dominated by colonial zoanthids. Significant correlations among parameters of algal tissue N with adjacent surface and coastal groundwater N indicate that these bioassays provided a useful measure of nutrient source and loading. A conceptual model that uses Ulva spp. tissue δ¹⁵N and N % to identify potential N source(s) and relative N loading is proposed for Hawaiʻi. These results indicate that SGD can be a significant transport pathway for land-based nutrients with important biogeochemical and ecological implications in tropical, oceanic islands.     

Chronic prenatal exposure to cocaine alters cerebrovascular responses in newborn pigs

Maternal cocaine abuse may increase the incidence of perinatal asphyxia. In nonexposed asphyxiated neonates, decreased cerebrospinal fluid (CSF) cAMP concentrations are associated with poor neurological outcome. On the other hand, cocaine increases central nervous system (CNS) cAMP. Therefore, we hypothesized that in utero cocaine exposure may increase brain cAMP and thereby preserve cerebrovascular responses to cAMP-dependent stimuli following asphyxia. Pregnant pigs received either cocaine (1 mg/kg, i.v.) twice weekly during the last trimester or normal saline vehicle (sham-control) and were allowed to deliver vaginally at term. Cranial windows were implanted in the newborn pigs within the first week of life and used to collect CSF for cAMP determinations and to assess changes in pial arteriolar diameters (PAD). In the first part of the study, pial arteriolar responses to different vasodilator and vasoconstrictor stimuli were evaluated in piglets prior to asphyxia (n = 20). In newborn pigs exposed to cocaine, cerebrovascular responses to hypercapnia and norepinephrine were significantly exaggerated compared to controls. Then, piglets were randomly selected for the second part of the study that involved prolonged asphyxia (n = 12). In cocaine-exposed but not sham-control piglets, CSF cAMP increased markedly during asphyxia. In the sham piglets, but not the cocaine-exposed piglets, CSF cAMP fell progressively below the baseline during recovery. Cerebrovascular reactivity to cAMP-dependent stimuli (hypercapnia and isoproterenol) was preserved during recovery from asphyxia in the cocaine-exposed piglets but significantly attenuated in the sham controls. We conclude that piglets with chronic prenatal exposure to cocaine show exaggerated cerebrovascular responses to vasogenic stimuli and preserved cAMP-dependent cerebral vasoreactivity following asphyxia.     

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

The ATP binding cassette multidrug transporter LmrA and lipid transporter MsbA have overlapping substrate specificities

LmrA is an ATP binding cassette (ABC) multidrug transporter in Lactococcus lactis that is a structural and functional homologue of the human multidrug resistance P-glycoprotein MDR1 (ABCB1). LmrA is also homologous to MsbA, an essential ABC transporter in Escherichia coli involved in the trafficking of lipids, including Lipid A. We have compared the substrate specificities of LmrA and MsbA in detail. Surprisingly, LmrA was able to functionally substitute for a temperature-sensitive mutant MsbA in E. coli WD2 at non-permissive temperatures, suggesting that LmrA could transport Lipid A. LmrA also exhibited a Lipid A-stimulated, vanadate-sensitive ATPase activity. Reciprocally, the expression of MsbA conferred multidrug resistance on E. coli. Similar to LmrA, MsbA interacted with photoactivatable substrate [3H]azidopine, displayed a daunomycin, vinblastine, and Hoechst 33342-stimulated vanadate-sensitive ATPase activity, and mediated the transport of ethidium from cells and Hoechst 33342 in proteoliposomes containing purified and functionally reconstituted protein. Taken together, these data demonstrate that MsbA and LmrA have overlapping substrate specificities. Our observations imply the presence of structural elements in the recently published crystal structures of MsbA in E. coli and Vibrio cholera (Chang, G., and Roth, C. B. (2001) Science 293, 1793-1800; Chang, G. (2003) J. Mol. Biol. 330, 419-430) that support drug-protein interactions and suggest a possible role for LmrA in lipid trafficking in L. lactis.     

Beta-scission of side-chain alkoxyl radicals on peptides and proteins results in the loss of side-chains as aldehydes and ketones

Exposure of proteins to radicals in the presence of O(2) results in side-chain oxidation and backbone fragmentation; the interrelationship between these processes is not fully understood. Recently, initial attack on Ala side-chains was shown to give alpha-carbon radicals (and hence backbone cleavage) and formaldehyde, via the formation and subsequent beta-scission, of C-3 alkoxyl radicals. We now show that this side-chain to backbone damage transfer, is a general mechanism for aliphatic side-chains. Oxidation of Val, Leu, and Asp residues by HO(*)/O(2) results in the release of a family of carbonyls (including formaldehyde, acetone, isobutyraldehyde, and glyoxylic acid) via the formation, and subsequent beta-scission of alkoxyl radicals. The concentration of these products increases with the HO(*) flux. The release of multiple carbonyls confirms the occurrence of oxidation at C-3 and C-4 for Val, and these sites, plus C-5, for Leu. The detection of glyoxylic acid and CO(2)(-*) from Asp demonstrates the occurrence of competing beta-scission processes for the Asp C-3 alkoxyl radical. The yield of hydroperoxides and released carbonyls account for 10-145% of the initial HO(*). The greater than 100% yields confirm the occurrence of chain reactions in peptide/protein oxidation, with more than one residue being damaged per initiating radical.