Enzyme activity and liver tissue PCB concentrations of chicks of northern fulmars (F. glacialis) and black-legged kittiwakes (R. tridactyla) from Kongsfjorden

Arctic seabirds are exposed to a wide range of halogenated organic contaminants (HOCs). Exposure occurs mainly through food intake, and many pollutants accumulate in lipid-rich tissues. Little is known about how HOCs are biotransformed in arctic seabirds. In this study, we characterized biotransformation enzymes in chicks of northern fulmars (Fulmarus glacialis) and black-legged kittiwakes (Rissa tridactyla) from Kongsfjorden (Svalbard, Norway). Phase I and II enzymes were analyzed at the transcriptional, translational and activity levels. For gene expression patterns, quantitative polymerase chain reactions (qPCR), using gene-sequence primers, were performed. Protein levels were analyzed using immunochemical assays of western blot with commercially available antibodies. Liver samples were analyzed for phase I and II enzyme activities using a variety of substrates including ethoxyresorufin (cytochrome (CYP)1A1/1A2), pentoxyresorufin (CYP2B), methoxyresorufin (CYP1A), benzyloxyresorufin (CYP3A), testosterone (CYP3A/CYP2B), 1-chloro-2,4-nitrobenzene (CDNB) (glutathione S-transferase (GST)) and 4-nitrophenol (uridine diphosphate glucuronyltransferase (UDPGT)). In addition, the hydroxylated (OH-) polychlorinated biphenyls (PCBs) were analyzed in the blood, liver and brain tissue, whereas the methylsulfone (MeSO2-) PCBs were analyzed in liver tissue. Results indicated the presence of phase I (CYP1A4/CYP1A5, CYP2B, and CYP3A) and phase II (GST and UDPGT) enzymes at the activity, protein and/or mRNA level in both species. Northern fulmar chicks had higher enzyme activity than black-legged kittiwake chicks. This in combination with the higher XOH-PCB to parent PCB ratios suggests that northern fulmar chicks have a different biotransformation capacity than black-legged kittiwake chicks.

(Tables 1,2) Polar bear (Ursus maritimus), beluga whale (Delphinapterus leucas) and ringed seal (Pusa hispida) liver microsomal protein content and EROD activity

The present study assessed and compared the oxidative and reductive biotransformation of brominated flame retardants, including established polybrominated diphenyl ethers (PBDEs) and emerging decabromodiphenyl ethane (DBDPE) using an in vitro system based on liver microsomes from various arctic marine-feeding mammals: polar bear (Ursus maritimus), beluga whale (Delphinapterus leucas), and ringed seal (Pusa hispida), and in laboratory rat as a mammalian model species. Greater depletion of fully brominated BDE209 (14-25% of 30pmol) and DBDPE (44-74% of 90pmol) occurred in individuals from all species relative to depletion of lower brominated PBDEs (BDEs 99,100, and 154; 0-3% of 30pmol). No evidence of simply debrominated metabolites was observed. Investigation of phenolic metabolites in rat and polar bear revealed formation of two phenolic, likely multiply debrominated, DBDPE metabolites in polar bear and one phenolic BDE154 metabolite in polar bear and rat microsomes. For BDE209 and DBDPE, observed metabolite concentrations were low to nondetectable, despite substantial parent depletion. These findings suggested possible underestimation of the ecosystem burden of total-BDE209, as well as its transformation products, and a need for research to identify and characterize the persistence and toxicity of major BDE209 metabolites. Similar cause for concern may exist regarding DBDPE, given similarities of physicochemical and environmental behavior to BDE209, current evidence of biotransformation, and increasing use of DBDPE as a replacement for BDE209.

(Table 1) Hepatic EROD activity and and retinoid concentrations in the liver, and plasma thyroid hormones of northern fulmars (Fulmarus glacialis) from Bjørnøya

We investigated whether the hepatic cytochrome P450 1A activity (measured as 7-ethoxyresorufin-O-deethylase (EROD)) and plasma thyroid hormone and liver retinoid concentrations were explained by liver and blood levels of halogenated organic contaminants (HOCs) in free-ranging breeding northern fulmars (Fulmarus glacialis) from Bjornoya in the Norwegian Arctic. Hepatic EROD activity and liver levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin toxic equivalents (TEQs) were positively correlated, suggesting that hepatic EROD activity is a good indicator for dioxin and dioxin-like HOC exposure in breeding northern fulmars. There were not found other strong relationships between HOC concentrations and hepatic EROD activity, plasma thyroid or liver retinoid concentrations in the breeding northern fulmars. It is suggested that the HOC levels found in the breeding northern fulmars sampled on Bjornoya were too low to affect plasma concentrations of thyroid hormones and liver levels of retinol and retinyl palmitate, and that hepatic EROD activity is a poor indicator of polychlorinated biphenyl (PCB) and pesticide exposure.

Radical scavenging activity of protein from tentacles of jellyfish Rhopilema esculentum

In this study, radical scavenging activity of protein from tentacles of jellyfish Rhopilema esculentum (R. esculentum) was assayed including superoxide anion radical and hydroxyl radical scavenging. The protein samples showed strong scavenging activity on superoxide anion radical and values EC50 of full protein (FP), first fraction (IFF), second fraction (SF), and 30% (NH4)(2)SO4 precipitate (Fr-1) were 2.65, 7.28, 1.10, and 22.51 mu g/mL, respectively, while values EC50 of BHA, BHT, and alpha-tocopherol were 31, 6 1, and 88 pg/mL, respectively. Also, the protein samples had strong scavenging effect on hydroxyl radical and the values EC50 of FP, FF, SF, Fr-1, and Fr-2 were 48.91, 27.72, 1.82, 16.36, and 160.93 mu g/mL, but values EC50 of Vc and mannitol were 1907 and 4536 mu g/mL, respectively. Of the five protein samples, SF had the strongest radical scavenging activity and may have a use as a possible supplement in the food and pharmaceutical industries. The radical scavenging activity was stable at high temperature so that R. esculentum may be used as a kind of natural functional food. (c) 2005 Elsevier Ltd. All rights reserved.

16 alpha-substituted analogs of the antiprogestin RU486 induce a unique conformation in the human progesterone receptor resulting in mixed agonist activity.

Previously, we have shown that agonists and antagonists interact with distinct, though overlapping regions within the human progesterone receptor (hPR) resulting in the formation of structurally different complexes. Thus, a link was established between the structure of a ligand-receptor complex and biological activity. In this study, we have utilized a series of in vitro assays with which to study hPR pharmacology and have identified a third class of hPR ligands that induce a receptor conformation which is distinct from that induced by agonists or antagonists. Importantly, when assayed on PR-responsive target genes these compounds were shown to exhibit partial agonist activity; an activity that was influenced by cell context. Thus, as has been shown previously for estrogen receptor, the overall structure of the ligand-receptor complex is influenced by the nature of the ligand. It appears, therefore, that the observed differences in the activity of some PR and estrogen receptor ligands reflect the ability of the cellular transcription machinery to discriminate between the structurally different complexes that result following ligand interaction. These data support the increasingly favored hypothesis that different ligands can interact with different regions within the hormone binding domains of steroid hormone receptors resulting in different biologies.

Nitrogen fertilizer and seed rate effects on Hagberg failing number of hybrid wheats and their parents are associated with alpha-amylase activity, grain cavity size and dormancy

Field experiments were carried out to assess the effects of nitrogen fertilization and seed rate on the Hagberg falling number (HFN) of commercial wheat hybrids and their parents. Applying nitrogen (200 kg N ha(-1)) increased HFN in two successive years. The HFN of the hybrid Hyno Esta was lower than either of its parents (Estica and Audace), particularly when nitrogen was not applied. Treatment effects on HFN were negatively associated with a-amylase activity. Phadebas grain blotting suggested two populations of grains with different types of a-amylase activity: Estica appeared to have a high proportion of grains with low levels of late maturity endosperm a-amylase activity (LMEA); Audace had a few grains showing high levels of germination amylase; and the hybrid, Hyno Esta, combined the sources from both parents to show heterosis for a-amylase activity. Applying nitrogen reduced both apparent LMEA and germination amylase. The effects on LMEA were associated with the size and disruption of the grain cavity, which was greater in Hyno Esta and Estica and in zero-nitrogen treatments. External grain morphology failed to explain much of the variation in LMEA and cavity size, but there was a close negative correlation between cavity size and protein content. Applying nitrogen increased post-harvest dormancy of the grain. Dormancy was greatest in Estica and least in Audace. It is proposed that effects of seed rate, genotype and nitrogen fertilizer on HFN are mediated through factors affecting the size and disruption of the grain cavity and therefore LMEA, and through factors affecting dormancy and therefore germination amylase. (c) 2004 Society of Chemical Industry.

Post-endocytic sorting of calcitonin receptor-like receptor and receptor activity-modifying protein 1

Calcitonin receptor-like receptor (CLR) and the receptor activity-modifying protein 1 (RAMP1) comprise a receptor for calcitonin gene-related peptide (CGRP). Although CGRP induces endocytosis of CLR/RAMP1, little is known about post-endocytic sorting of these proteins. We observed that the duration of stimulation with CGRP markedly affected post-endocytic sorting of CLR/RAMP1. In HEK and SK-N-MC cells, transient stimulation (10(-7) M CGRP, 1 h), induced CLR/RAMP1 recycling with similar kinetics (2-6 h), demonstrated by labeling receptors in living cells with antibodies to extracellular epitopes. Recycling of CLR/RAMP1 correlated with resensitization of CGRP-induced increases in [Ca(2+)](i). Cycloheximide did not affect resensitization, but bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPases, abolished resensitization. Recycling CLR and RAMP1 were detected in endosomes containing Rab4a and Rab11a, and expression of GTPase-defective Rab4aS22N and Rab11aS25N inhibited resensitization. After sustained stimulation (10(-7) M CGRP, >2 h), CLR/RAMP1 trafficked to lysosomes. RAMP1 was degraded approximately 4-fold more rapidly than CLR (RAMP1, 45% degradation, 5 h; CLR, 54% degradation, 16 h), determined by Western blotting. Inhibitors of lysosomal, but not proteasomal, proteases prevented degradation. Sustained stimulation did not induce detectable mono- or polyubiquitination of CLR or RAMP1, determined by immunoprecipitation and Western blotting. Moreover, a RAMP1 mutant lacking the only intracellular lysine (RAMP1K142R) internalized and was degraded normally. Thus, after transient stimulation with CGRP, CLR and RAMP1 traffic from endosomes to the plasma membrane, which mediates resensitization. After sustained stimulation, CLR and RAMP1 traffic from endosomes to lysosomes by ubiquitin-independent mechanisms, where they are degraded at different rates.

Increased salivary immunoglobulin A and reduced alpha-amylase activity in whole saliva from spastic cerebral palsy individuals

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expression of a gene encoding a 16.9-kDa heat-shock protein, Oshsp16.9, in Escherichia coli enhances thermotolerance

A gene encoding the rice 16.9-kDa class I low-molecular-mass (LMM) heat-shock protein (HSP), Oshsp16.9, was introduced into Escherichia coli using the pGEX-2T expression vector to analyze the possible function of this LMM HSP under heat stress. It is known that E. coli does not normally produce class I LMM HSPs. We compared the survivability of E. coli XL1-Blue cells transformed with a recombinant plasmid containing a glutathione S-transferase (GST)–Oshsp16.9 fusion protein (pGST-FL cells) with the control E. coli cells transformed with the pGEX-2T vector (pGST cells) under heat-shock (HS) after isopropyl β-d-thiogalactopyranoside induction. The pGST-FL cells demonstrated thermotolerance at 47.5°C, a treatment that was lethal to the pGST cells. When the cell lysates from these two E. coli transformants were heated at 55°C, the amount of protein denatured in the pGST-FL cells was 50% less than that of the pGST cells. Similar results as pGST-FL cells were obtained in pGST-N78 cells (cells produced a fusion protein with only the N-terminal 78 aa in the Oshsp16.9 portion) but not in pGST-C108 cells (cells produced a fusion protein with C-terminal 108 aa in the Oshsp16.9 portion). The acquired thermotolerant pGST-FL cells synthesized three types of HSPs, including the 76-, 73-, and 64-kDa proteins according to their abundance at a lethal temperature of 47.5°C. This finding indicates that a plant class I LMM HSP, when effectively expressed in transformed prokaryotic cells that do not normally synthesize this class of LMM HSPs, may directly or indirectly increase thermotolerance.