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Design The design of the study was to investigate the prevalence of the following: 1) premature ovarian failure (POF) in patients with autoimmune Addison's disease (AD); 2) steroidproducing cells (StCA) and steroidogenic enzymes (17α-hydroxylase autoantibodies and P450 side-chain cleavage enzyme autoantibodies) in patients with or without POF; and 3) the value of these autoantibodies to predict POF. Patients The study included 258 women: 163 with autoimmune polyendocrine syndrome type 2 (APS-2), 49 with APS-1, 18 with APS-4, and 28 with isolated AD. Methods StCA were measured by an immunofluorescence technique and 17α-hydroxylase autoantibodies and P450 sidechain cleavage enzyme autoantibodies by immunoprecipitation assays. Results Fifty-two of 258 women with AD (20.2%) had POF. POF was diagnosed in 20 of 49 (40.8%) with APS-1, six of 18 (33.3%) with APS-4, 26 of 163 (16%) with APS-2, and none of 28 with isolated AD. In patients with APS-1 and APS-4, POF developed after AD, whereas it preceded AD in patients with APS-2. StCA were detected in 31 of 43 with POF (72%) and 51 of 198 without POF (25.7%). StCA were present in 22 of 38 with APS-1 (57.9%) (11 of 13 with POF); in five of 13 with APS-4 (38.5%) (three of four with POF); in 53 of 162 with APS-2 (32.7%) (17 of 26 with POF), and in one of 28 isolated AD patients (3.6%). Twelve of 13 patients with POF with a duration less than 5 yr (92.3%) and 18 of 25 with duration longer than 5 yr (72%) were StCA positive. Twenty-eight of 31 with POF (90.3%) were positive for at least one steroidogenic antibody. Forty-one women with AD less than 40 yr were followed up for a mean period of 9 yr. Eight of 21 women (38%) positive or seroconverted for steroidogenic autoantibodies developed POF at a mean age of 23 yr (six with APS-1, one with APS-2, and one with APS-4), and none of the 20 patients negative for steroidogenic autoantibodies developed POF. Conclusions This study indicates that AD is frequently associated with POF and that steroidogenic antibodies are markers of patients with POF. Steroidogenic autoantibodies are predictive markers of POF in patients with AD.

Measurements of TSH receptor autoantibodies (TRAb) using assays based on the human monoclonal TSH receptor autoantibody M22 or bovine TSH have been compared in 136 adult patients. They suffered from Graves' disease (GD, n=62), Hashimoto's thyroiditis (HT, n=26), or non-autoimmune hyperthyroidism (NAH, n=48) and were selected on the basis of undetectable, borderline or low TRAb levels (0.6-3 IU/l) as measured by TSH based TRAb assay (Dynotest TRAKhuman from BRAHMS). The time interval between initial diagnosis of GD and TRAb determination was high and ranged from 1 month to 3.5 years (median: 2.3 years). Using the kit manufacturer's cutoff values, 53/62 (85.5%) of the selected group of GD patients were TRAb positive (>0.4 IU/l) by M22 based TRAb ELISA (Medizym TRAb clone, Medipan) and 45/62 (72.6%) were TRAb positive (>1.5 IU/l) by TSH based TRAb assay. In the HT group, 9/26 (34.6%) sera were positive in the M22 based ELISA and all but one of these 9 were positive or borderline in the TSH based assay. ROC plot analysis of the GD group using the NAH group as reference showed that at 95% specificity, the bovine TSH based TRAb assay had a sensitivity of 62.9% (cutoff for positivity=1.64 IU/l) and the M22 based TRAb ELISA a sensitivity of 90.3% (cutoff for positivity=0.32 IU/l). Overall therefore, the M22 based Medizym TRAb clone assay is more sensitive than the bovine TSH based Dynotest TRAK human assay.

B-cells influence T-cell reactivity by facilitating antigen presentation, but the role of autoantibody-secreting B-cells in regulating T-cell responses in Type 1 diabetes is poorly defined. The aims of this study were to characterise epitopes on the IA-2 autoantigen for three monoclonal antibodies from diabetic patients by amino acid substitutions of selected residues of IA-2, establish contributions of these epitopes to binding of serum antibodies in Type 1 diabetes and relate B- and T-cell responses to overlapping determinants on IA-2. The monoclonal antibodies recognised overlapping epitopes, with residues within the 831-860 region of IA-2 contributing to binding; substitution of Glu836 inhibited binding of all three antibodies. Monoclonal antibody Fab fragments and substitution of residues within the 831-836 region blocked serum antibody binding to an IA-2 643-937 construct. IL-10-secreting T-cells responding to peptides within the 831-860 region were detected by cytokine-specific ELISPOT in diabetic patients and responses to 841-860 peptide were associated with antibodies to the region of IA-2 recognised by the monoclonal antibodies. The study identifies a region of IA-2 frequently recognised by antibodies in Type 1 diabetes and demonstrates that these responses are associated with T-cells secreting IL-10 in response to a neighbouring determinant.

F1 offspring of the African cichlid Pseudocrenilabrus multicolor victoriae Seegers, 1990 from swamp (low oxygen) and lake (high oxygen) origin were raised under normoxia and submitted to hypoxia acclimation (0.8 ± 0.4 mg·L–1) and normoxia acclimation (7.4 ± 0.3 mg·L–1) for 4 weeks. Haematocrit and lactate dehydrogenase (LDH) specific activities in the liver, white skeletal muscle, heart, and brain were measured. For haematocrit and LDH activities of liver, muscle, and heart, the response to acclimation depended upon population of origin. In general, fish from the swamp population showed a more “typical” hypoxic response (increased haematocrit and LDH activities), whereas fish from the lake population either did not respond or showed the opposite response. The results suggest that populations of P. m. victoriae sampled from habitats with diverse oxygen regimes differ in their physiological and biochemical responses to hypoxia.

When Hurricane Katrina struck the U.S. Gulf Coast, levees surrounding New Orleans, Louisiana, USA, were breached, leading to widespread flooding of the city and potential contamination from industrial spills, residential sources, and redistribution of pre-existing pollutants. We chemically characterized sediment samples from five New Orleans locations and used early development and mutagenesis in Japanese medaka (Oryzias latipes) as metrics of the toxic effects of these sediments. Sediment samples were analyzed for organohalogen pesticides, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and metals. One month after Hurricane Katrina, four of the five sites had unsafe concentrations of arsenic and one or more pesticides, pesticide metabolites, or polycyclic aromatic hydrocarbons. Medaka embryonic mortality and time to hatching both increased during exposure to aqueous extracts of sediments, with the greatest toxicity observed for the most heavily contaminated sediment. Exposure to sediment extracts did not, however, result in significantly elevated rates of mutagenesis. When the most contaminated site was resampled 4.5 months later, the sediment had lower contaminant concentrations and fewer deleterious effects on medaka development. Using the medaka bioassay, therefore, we demonstrate toxic effects of post-Hurricane Katrina sediments immediately following the storm, with some amelioration over time of contaminant concentrations and their negative biological effects.

This paper attempts to overcome the dichotomy between the broadly different and largely separate fisheries science and management (FSM) and ecosystem science and management (ESM) knowledge systems that characterise the international literature and are found in fisheries management practice in different countries. The paper argues that the construction of a heuristic we term the fisheries problematic, around issues and contexts, reveals the breadth of international fisheries management concerns and the variety of contexts in which these concerns are being faced. Adopting a political economy informed nature-society approach the paper considers ecological and socio-economic processes in their institutional settings in an attempt to shift from the either/or arguments around fish or ecosystems found in the FSM or ESM literatures to investigation that is grounded in understandings of the historically and geographically specific trajectories of fisheries related interactions and understandings of how knowledge about the trajectories and their interactions is fashioned. Drawing on recent conceptual innovations in the field, the paper develops a matrix-centred approach to explore ecological, industry, community and policy domains in New Zealand’s Quota Management System (QMS) and Individual Transferable Quota (ITQ) fisheries management regime. The extended framework prioritises scrutiny of the interaction amongst the four domains, as a strategy to help develop institutional frameworks that facilitate behaviours that are societally inclusive. The paper offers three conclusions. First, the landscape of New Zealand fisheries issues is very much a product of the contingent interaction of the QMS, a management regime designed around the principles of a FSM approach and laid down in a neo-liberal political environment and Maori aspirations encompassing the fisheries sector. Second, the conceptual mapping of FSM and ESM perspectives over New Zealand’s fisheries management experience highlights that a number of management issues have been down played by the commitment to FSM, a situation that has led to on-going tensions between commercial, recreational and customary stakeholders regarding fisheries management. Put another way, there is more to running a sustainable fishery (as defined in the Fisheries Act 1996) than QMS and other tools and dialogue about the development of these should be a priority. Third and more generally, improved dialogue on fisheries questions is likely to be most expeditiously advanced by studies that explicitly conceptualise and contextualise ecological and socio-economic processes and their institutional arrangements.

SPIRIT figure_HARTR1. (DOCX 16 kb)

The availability of human monoclonal antibodies (MAbs) to the TSHR has enabled major advances in our understanding of how TSHR autoantibodies interact with the receptor. These advances include determination of the crystal structures of the TSHR LRD in complex with a stimulating autoantibody (M22) and with a blocking type autoantibody (K1-70). The high affinity of MAbs for the TSHR makes them particularly suitable for use as ligands in assays for patient serum TSHR autoantibodies. Also, M22 and K1–70 are effective at low concentrations in vivo as TSHR agonists and antagonists respectively. K1-70 has important potential in the treatment of the hyperthyroidism of Graves’ disease and Graves’ ophthalmopathy. Small molecule TSHR antagonists described to date do not appear to have the potency and/or specificity shown by K1-70. New models of the TSHR ECD in complex with various ligands have been built. These models suggest that initial binding of TSH to the TSHR causes a conformational change in the hormone. This opens a positively charged pocket in receptor-bound TSH which attracts the negatively charged sulphated tyrosine 385 on the hinge region of the receptor. The ensuing movement of the receptor's hinge region may then cause activation. Similar activation mechanisms seem to take place in the case of FSH and the FSHR and LH and the LHR. However, stimulating TSHR autoantibodies do not appear to activate the TSHR in the same way as TSH.

The glycoprotein hormones luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyrotropin (TSH) show low-level cross-reactivity between their respective receptors (R). Patient serum autoantibodies to the thyrotropin receptor (TSHR) do not appear to cross-react with the luteinizing hormone receptor (LHR) or follicle-stimulating hormone receptor (FSHR), although the concentrations of autoantibody with which it is feasible to carry out experiments of this type are limited. Consequently, we have studied the effects of high doses of the thyroid-stimulating human monoclonal autoantibody (M22) on the LHR and FSHR.Chinese Hamster ovary (CHO) cells stably expressing the TSHR, LHR, and FSHR and purified M22 IgG preparations were used in the study.CHO-TSHR, CHO-LHR, and CHO-FSHR cells were incubated with bovine TSH (0.1-25mU/mL), human recombinant chorionic gonadotropin (hCG; 0.5-10mU/mL) or human recombinant FSH (100-5000mU/mL) or with M22 IgG (0.001-5.0 microg/mL), and the extracellular cyclic AMP was measured by radioimmunoassay.Cyclic AMP levels increased in a dose-dependent manner after incubation of CHO-TSHR cells with TSH or M22 IgG, and on a molar basis the effects of TSH and M22 were similar. Cyclic AMP stimulation was not detectable in CHO-LHR and CHO-FSHR cells after incubation with M22 IgG, whereas incubation with hCG or FSH, respectively, caused dose-dependent cyclic AMP stimulation. On a molar basis, concentrations of M22 IgG approximately 100x those of FSH causing clear stimulation were ineffective with CHO-FSHR cells. Similarly, molar concentration of M22 IgG 20,000x those of hCG causing clear stimulation had no effect on CHO-LHR cells.This study shows that at relatively high concentrations, M22 IgG is unable to stimulate cyclic AMP levels in CHO-LHR or CHO-FSHR cells, suggesting that TSHR autoantibodies have greater specificity for the TSHR than TSH itself.

SUMMARYThe goal of the current study was to generate a comprehensive, multi-tissue perspective of the effects of chronic hypoxic exposure on carbohydrate metabolism in the Gulf killifish Fundulus grandis. Fish were held at approximately 1.3 mg l-1 dissolved oxygen (∼3.6 kPa) for 4 weeks, after which maximal activities were measured for all glycolytic enzymes in four tissues (white skeletal muscle, liver, heart and brain), as well as for enzymes of glycogen metabolism (in muscle and liver) and gluconeogenesis(in liver). The specific activities of enzymes of glycolysis and glycogen metabolism were strongly suppressed by hypoxia in white skeletal muscle, which may reflect decreased energy demand in this tissue during chronic hypoxia. In contrast, several enzyme specific activities were higher in liver tissue after hypoxic exposure, suggesting increased capacity for carbohydrate metabolism. Hypoxic exposure affected fewer enzymes in heart and brain than in skeletal muscle and liver, and the changes were smaller in magnitude, perhaps due to preferential perfusion of heart and brain during hypoxia. The specific activities of some gluconeogenic enzymes increased in liver during long-term hypoxic exposure, which may be coupled to increased protein catabolism in skeletal muscle. These results demonstrate that when intact fish are subjected to prolonged hypoxia, enzyme activities respond in a tissue-specific fashion reflecting the balance of energetic demands, metabolic role and oxygen supply of particular tissues. Furthermore, within glycolysis, the effects of hypoxia varied among enzymes, rather than being uniformly distributed among pathway enzymes.

Background Methylmercury (MeHg) is a known neurotoxic agent, but the mechanisms by which MeHg may act on reproductive pathways are relatively unknown. Several studies have indicated potential changes in hormone levels as well as declines in vertebrates with increasing dietary MeHg exposure. Objectives The purpose of this study was to identify alterations in gene expression associated with MeHg exposure, specifically those associated with previously observed changes in reproduction and reproductive biomarkers. Fathead minnows, Pimephales promelas, were fed one of three diets that were similar to documented concentrations of MeHg in the diets of wild invertivorous and piscivorous fish. We used a commercial macroarray in conjunction with quantitative polymerase chain reaction to examine gene expression in fish in relation to exposure to these environmentally relevant doses of MeHg. Results Expression of genes commonly associated with endocrine disruption was altered with Hg exposure. Specifically, we observed a marked up-regulation in vitellogenin mRNA in individual Hg-exposed males and a significant decline in vitellogenin gene expression in female fish with increasing Hg concentrations. Other genes identified by the macroarray experiment included those associated with egg fertilization and development, sugar metabolism, apoptosis, and electron transport. We also observed differences in expression patterns between male and female fish not related to genes specifically associated with reproduction, indicating a potential physiological difference in the reaction of males and females to MeHg. Conclusion Gene expression data may provide insight into the mechanisms by which MeHg affects reproduction in fish and indicate how MeHg differs in its effect from other heavy metals and endocrine-disrupting compounds.

This article presents a retention project in an associate degree nursing program. Initiatives were taken to retain students in the nursing program as well as to increase the pass rate on the National Council Licensure Examination (NCLEX)-RN. The first nursing course was primarily targeted for retention but initiatives were used with all students in the program. Preliminary results show a significant increase in retention, graduation, and increase in the scores on the NCLEX-RN.

Background:Autoantibodies to tryptophan hydroxylase (TPHAbs) directed against serotonin-producing enterochromaffin cells (EC) have been reported in autoimmune-polyendocrine-syndrome type 1 (APS-1) patients with gastrointestinal dysfunction (GID). Serotonin plays a critical role in enteric function and its peripheral blood levels reflect serotonin release from the gastrointestinal tract.Aims:We test the hypothesis that TPHAbs mark a distinct autoimmune component of APS-1 characterized by an autoimmune attack toward EC, which results in clinical GID.Methods:TPHAbs were measured in 64 APS-1 patients. Endoscopy with gastric (antrum/body) and duodenal biopsy was carried in 16 TPHAbs+ patients (8 with and 8 without GID) and in 2 TPHAbs- patients (without GID). Immunohistochemistry of biopsy specimens was carried out using antibodies to serotonin, chromogranin-A, CD3, CD4, CD8, and CD20. Serotonin serum levels were measured in TPHAbs+ and TPHAbs- patients who had endoscopy.Results:Thirty-seven of 64 patients were TPHAbs+ (11/12 with GID and 26/52 without GID; P < .001). Gastric and duodenal biopsies in all 8 TPHAb+ patients with GID showed lymphocytic infiltration with increased CD3+CD8+ intraepithelial lymphocytes and absence of EC. Furthermore, mean serotonin serum levels were below the normal range in TPHAb+ patients with GID (P < .01). In 8 TPHAb+ patients without GID gastric and duodenal biopsies showed different grades of inflammatory infiltration and reduced number of EC. Mean serotonin serum levels were near the lower limit of the normal range. In all TPHAbs+ patients the biopsies showed a reduced number of chromogranin-A positive cells consistent with enteroendocrine cells depletion. TPHAbs- patients without GID showed normal gastrointestinal mucosa and serotonin serum levels.Conclusions:TPHAbs appear to be markers of a distinct autoimmune component of APS-1. Progressive involvement of the gastrointestinal EC leads to the transition from preclinical to clinical disease, characterized by GID and reduced serotonin serum levels.

Expression of cytochrome P4501A (CYP1A) has been used as a biomarker for possible exposure to contaminants such as PCBs and dioxins in teleost fish. Using a quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) and a non-lethal gill biopsy, we estimated levels of CYP1A mRNA expression in Atlantic salmon ( Salmo salar ). Groups of ten Atlantic salmon juveniles (48–76 g) received an intraperitoneal injection of 50 μg g − 1 β-naphthoflavone (BNF) or vehicle. Their gill tissues were repeatedly sampled by non-lethal biopsies on day 0, 1, 2 and 7. Control fish expressed basal levels of CYP1A over the duration of sampling. BNF-treated salmon demonstrated similar levels of CYP1A to control fish at day 0 and higher levels over the course of each additional sampling point. Gill biopsies from wild salmon sampled from Millers River (South Royalston, Worcester County, MA, USA), known to contain PCBs, showed significantly higher CYP1A levels over an uncontaminated reference stream, Fourmile Brook (Northfield, Franklin County, MA, USA). We conclude that gill biopsies coupled with Q-RT-PCR analysis is a valuable tool in environmental assessment of wild Atlantic salmon populations and has the potential to be applied to other populations of fish as well.

Analysis of nine mouse monoclonal antibodies (mAbs) to the thyrotropin receptor (TSHR) with TSH antagonist activity showed that only one of the mAbs (RSR B2) was an effective antagonist of the human thyroid stimulating autoantibody M22. Crystals of B2 Fab were analyzed by x-ray diffraction and a crystal structure at 3.3 A resolution was obtained. The surface charge and topography of the B2 antigen binding site were markedly different from those of the thyroid-stimulating mAb M22 and these differences might contribute to the different properties of the two mAbs. B2 (but not other mouse TSHR-specific mAbs) was also an effective antagonist of thyroid stimulating autoantibody activity in 14 of 14 different sera from patients with Graves' disease. 125I-labeled B2 bound to the TSHR with high affinity (2 x 10(10) L/mol) and patient serum TSHR autoantibodies inhibited labeled B2 binding to the receptor in a similar way to inhibition of labeled TSH binding (r = 0.75; n = 20). Furthermore, labeled B2 binding was inhibited by patient serum TSHR autoantibodies with TSH antagonist activity and also by mouse and human thyroid stimulating mAbs. Overall, mAb B2 is a powerful antagonist of thyroid stimulating autoantibodies (and TSH) thus resembling closely patient serum TSH antagonist TSHR autoantibodies. Furthermore, B2 might have potentially important in vivo applications when tissues containing the TSHR (including those in the orbit) need to be made unresponsive to stimulating autoantibodies.

The role of oxygen in regulating patterns of gene expression in mammalian development, physiology, and pathology has received increasing attention, especially after the discovery of the hypoxia-inducible factor (HIF), a transcription factor that has been likened to a “master switch” in the transcriptional response of mammalian cells and tissues to low oxygen. At present, considerably less is known about the molecular responses of nonmammalian vertebrates and invertebrates to hypoxic exposure. Because many animals live in aquatic habitats that are variable in oxygen tension, it is relevant to study oxygen-dependent gene expression in these animals. The purpose of this review is to discuss hypoxia-induced gene expression in fishes from an evolutionary and ecological context. Recent studies have described homologs of HIF in fish and have begun to evaluate their function. A number of physiological processes are known to be altered by hypoxic exposure of fish, although the evidence linking them to HIF is less well developed. The diversity of fish presents many opportunities to evaluate if inter- and intraspecific variation in HIF structure and function correlate with hypoxia tolerance. Furthermore, as an aquatic group, fish offer the opportunity to examine the interactions between hypoxia and other stressors, including pollutants, common in aquatic environments. It is possible, if not likely, that results obtained by studying the molecular responses of fish to hypoxia will find parallels in the oxygen-dependent responses of mammals, including humans. Moreover, novel responses to hypoxia could be discovered through studies of this diverse and species-rich group.

To better understand the molecular relationship between contaminant exposure in Atlantic salmon and the cytochrome P450 system, we have cloned a CYP1A gene using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The cloned cDNA possesses all characteristic motifs of teleost CYP1A genes including the start codon, heme-binding region, stop codon, and poly-adenylation signal. The cloned gene is also characterized by an extended 3′ untranslated region (UTR; 1025bp) containing three AUUUA sequences that are likely to be a target for rapid degradation of its mRNA. Its coding region (1569 base pairs) shares 97.4% and 96.7% sequence identity with the corresponding regions of rainbow trout CYP1A1 and CYP1A3 genes, respectively. In addition, the deduced amino acid sequence of salmon CYP1A shows 96.9% and 96.2% identity with rainbow trout P450 1A1 and P450 1A3 enzymes, respectively. Phylogenetic analysis of a sample of CYP genes confirmed that Atlantic salmon CYP1A is closely related to rainbow trout CYP1A genes as well. Northern analysis showed that CYP1A mRNA is at a significantly higher level in the liver of Atlantic salmon injected intraperitoneally with β-Naphthoflavone (BNF), suggesting that this gene is inducible. Southern analysis verified the presence of at least one copy of the CYP1A gene in the genomic DNA of Atlantic salmon. These results reveal the transcriptional and inductive properties of an Atlantic salmon CYP1A gene with an extended 3′ UTR, and provide molecular tools for studying the effects of xenobiotics on Atlantic salmon populations.

Objective: To study possible mechanisms for the inhibition of cytochrome P450 C21 (steroid 21-hydroxylase) enzyme activity by P450 C21 autoantibodies (Abs)in vitro.Design: Two possible mechanisms for the inhibition of P450 C21 enzyme activity by P450 C21 Abs were studied: (a) conformational changes in the P450 C21 molecule induced by Ab binding and (b) the effects of Ab binding to P450 C21 on the electron transfer from the nicotinamide adenine dinucleotide phosphate reduced (NADPH) cytochrome P450 reductase (CPR) to P450 C21.Methods: The effect of P450 C21 Ab binding on the conformation of recombinant P450 C21 in yeast microsomes was studied using an analysis of the dithionite-reduced CO difference spectra. The effect of P450 C21 Abs on electron transfer was assessed by analysis of reduction of P450 C21 in the microsomes in the presence of CO after addition of NADPH.Results: Our studies confirmed the inhibiting effect of P450 C21 Abs on P450 C21 enzyme activity. Binding of the Abs did not induce significant change in the P450 C21 peak at 450 nm (native form) and did not produce a detectable peak at 420 nm (denatured form) in the dithionite-reduced CO difference spectra. This indicated that conformation of P450 C21 around the heme was not altered compared with the native structure. However, incubation of the P450 C21 in yeast microsomes with P450 C21 Ab inhibited the fast phase electron transfer from the CPR to P450 C21.Conclusions: Our observations suggested that the mechanism by which P450 C21 Abs inhibit P450 C21 enzyme activity most likely involves inhibition of the interaction between the CPR and P450 C21.

The fidelity of aminoacylation of tRNA Thr by the threonyl-tRNA synthetase (ThrRS) requires the discrimination of the cognate substrate threonine from the noncognate serine. Misacylation by serine is corrected in a proofreading or editing step. An editing site has been located 39 A away from the aminoacylation site. We report the crystal structures of this editing domain in its apo form and in complex with the serine product, and with two nonhydrolyzable analogs of potential substrates: the terminal tRNA adenosine charged with serine, and seryl adenylate. The structures show how serine is recognized, and threonine rejected, and provide the structural basis for the editing mechanism, a water-mediated hydrolysis of the mischarged tRNA. When the adenylate analog binds in the editing site, a phosphate oxygen takes the place of one of the catalytic water molecules, thereby blocking the reaction. This rules out a correction mechanism that would occur before the binding of the amino acid on the tRNA.

This study evaluated whether the African cyprinid Barbus neumayeri from Rwembaita Swamp (low-oxygen) and Njuguta River (high-oxygen) in the Kibale National Park, Uganda differed in traits related to aerobic and anaerobic metabolic potential. Haematocrit was measured as an index of blood oxygen-carrying capacity, and tissue activities and isozyme composition of lactate dehydrogenase (LDH) were measured as indices of tissue anaerobic capacity. To address whether site-dependent differences were acute responses v. longer-term adjustments to environmental conditions, these variables were measured in fish sampled shortly after collection and after laboratory maintenance under well-aerated conditions. In fish sampled in the field, those from the low-oxygen site had significantly higher haematocrit, but this difference disappeared after long-term laboratory maintenance. In contrast, fish from the low-oxygen site had higher liver LDH activities than fish from the high-oxygen site, and this difference persisted during laboratory maintenance. Polymorphism was detected at both the LDH-A and LDH-B loci, and genotype frequencies for LDH-B differed significantly between collection sites. These results demonstrate physiological, biochemical and genetic differences in B. neumayeri from habitats differing in dissolved oxygen availability and suggest both acute and long-term responses to local environmental conditions.

The properties of a human monoclonal antibody to the thyrotropin receptor (TSHR) (M22) with the characteristics of patient sera thyroid stimulating autoantibodies is described. Similar concentrations (pmol/L) of M22 Fab and porcine TSH had similar stimulating effects on cyclic adenosine monophosphate (cAMP) production in TSHR-transfected Chinese hamster ovary cells whereas higher doses of intact M22 immunoglobulin G (IgG) were required to cause the same level of stimulation. Patient sera containing TSHR autoantibodies with TSH antagonist (blocking) activity inhibited M22 Fab and IgG stimulation in a similar way to their ability to block TSH stimulation. Thyroid-stimulating monoclonal antibodies (TSmAbs) produced in mice inhibited 125I-TSH binding and 125I-M22 Fab binding to the TSHR but the mouse TSmAbs were less effective inhibitors than M22. These competition studies emphasized the close relationship between the binding sites on the TSHR for TSH, TSHR autoantibodies with TSH agonist activity, and TSHR autoantibodies with TSH antagonist activity. Recombinant M22 Fab could be produced in Escherichia coli and the recombinant and hybridoma produced Fabs were similarly active in terms of inhibition of TSH binding and cAMP stimulation. The crystal structure of M22 Fab was determined to 1.65 A resolution and is that of a standard Fab although the hypervariable region of the heavy chain protrudes further from the framework than the hypervariable region of the light chain. The M22 antigen binding site is rich in aromatic residues and its surface is dominated by acidic patches on one side and basic patches on the other in agreement with an important role for charge-charge interactions in the TSHR-autoantibody interaction.

We describe the case of a baby born to a mother with Addison's disease in the context of Autoimmune Polyendocrine Syndrome Type 2. Adrenal cortex autoantibodies and steroid 21-hydroxylase autoantibodies were detectable in the sera of both mother and baby, suggesting the transplacental passage of these autoantibodies. Adrenal autoantibodies were present in the baby's serum at delivery, at 3, 6 and till 34 months of age but no signs of clinical or subclinical adrenal insufficiency were found in the baby during the observation period. These data suggest that the presence of adrenal autoantibodies in serum alone is not a sufficient cause for the development of autoimmune adrenalitis.

This paper examines the constitution of ‘sustainable management’ within the context of the New Zealand marine environment. Firstly this paper examines the difference between ‘sustainable ecosystems’ and ‘sustainable utilisation’. The distinction is important if we are to make sense of the different ways in which various stakeholders (fishers, fisheries companies, scientists, fisheries managers and environmental groups) use ‘sustainability’ in the management of New Zealand's marine environment. We then examine how contestation results in sustainable management becoming a governmentality. We propose that such contestation transforms stakeholders into subjects whose everyday practices and relationships are influenced by seemingly incommensurable understandings of the lexicon that surrounds sustainable management. We conclude try considering how the potential revelation of new unknowns may be needed in order for disparate stakeholders to forge a unified approach to New Zealand's marine management.

SUMMARY Many environmental pollutants induce expression of the cytochrome P450(CYP) 1A subfamily of genes. We integrated cellular and molecular biological techniques to examine the effects of β-naphthoflavone (BNF) exposure in lake trout brain CYP1A distribution and dynamics. Over a 32-day time-course,real time quantitative reverse transcription polymerase chain reaction(Q-RT-PCR) results showed that CYP1A mRNA induction in response to BNF exposure occurred rapidly and continued to rise in the BNF-treated lake trout after 4 h, with a peak at or after 2 days. Messenger RNA levels fell after 4 days, and this trend continued after 16 days of exposure. In situhybridization indicated that CYP1A mRNA was universally elevated in the brain of BNF-exposed fish and was mainly expressed in the endothelia and occasionally in the glial cells. CYP1A immunoreactivity was induced in the olfactory bulb and valvula cerebelli of BNF-treated fish. Other brain areas showed constitutive CYP1A immunoreactivity in both control and BNF-treated fish. Some BNF-treated fish contained multifocal hemorrhages in the brain tissue, and these fish had overall depressed CYP1A immunoreactivity in the brain. The relationship between transcriptional and translational effects of BNF exposure in the brain of juvenile lake trout is discussed.

The crystal structures of threonyl-tRNA synthetase (ThrRS) from Staphylococcus aureus , with ATP and an analogue of threonyl adenylate, are described. Together with the previously determined structures of Escherichia coli ThrRS with different substrates, they allow a comprehensive analysis of the effect of binding of all the substrates: threonine, ATP and tRNA. The tRNA, by inserting its acceptor arm between the N-terminal domain and the catalytic domain, causes a large rotation of the former. Within the catalytic domain, four regions surrounding the active site display significant conformational changes upon binding of the different substrates. The binding of threonine induces the movement of as much as 50 consecutive amino acid residues. The binding of ATP triggers a displacement, as large as 8 A at some C α positions, of a strand-loop-strand region of the core β-sheet. Two other regions move in a cooperative way upon binding of threonine or ATP: the motif 2 loop, which plays an essential role in the first step of the aminoacylation reaction, and the ordering loop, which closes on the active site cavity when the substrates are in place. The tRNA interacts with all four mobile regions, several residues initially bound to threonine or ATP switching to a position in which they can contact the tRNA. Three such conformational switches could be identified, each of them in a different mobile region. The structural analysis suggests that, while the small substrates can bind in any order, they must be in place before productive tRNA binding can occur.

14C/C ratios in samples from radial transects across individual thalli of Caloplaca trachyphylla collected at two sites were measured and the results used to investigate whether 14C/C data might provide some insight into the magnitude of carbon turnover in this lichen species. The 14C/C data suggest that significant internal recycling/translocation of carbon is unlikely in the sampled thalli. However, converting the 14C/C data for the larger intact thalli sampled at each site to calendar years, using the atmospheric ⊃14C record, does not yield constant or even monotonically varying growth rates. Since crustose lichen growth rates are constant or decrease with thallus size, and since the 14C/C data from these larger thalli show a relatively small spread in 14C/C data values compared to the Northern Hemisphere atmospheric ⊃14C record over the past 50 years, the 14C/C data suggest that carbon turnover may be occurring. Carbon turnover was modelled starting with the atmospheric ⊃14C record. Turnover was incorporated so that for each year in the record a constant percentage of the total carbon was lost annually and replaced by new photosynthetically fixed carbon with a 14C/C ratio equal to that of the contemporary atmosphere. The 14C/C data from the radial samples were then converted to a calendar year using the model record. Constant annual carbon turnover values of 0, 0·5, 1, 1·5, 2, 2·5, 3, 3·5, 4, 4·5, 5, 5·5, 6, 7, 8, 9, 10, 15, 20, 25 and 50% were modelled. Carbon turnover values between 3 and 6% created ⊃14C model records that when applied to 14C/C data from the thalli produced constant radial growth rates that were: (1) identical for all lichens at a given site, and (2) independent of lichen size at a given site. The 14C/C data further indicate that annual carbon turnover in this species of lichen is 14C/C data from the thalli and might explain the discrepancies between the standard atmospheric ⊃14C record and the 14C/C ratios observed in C. trachyphylla.

Lichen samples were collected and observations about lichen communities were recorded at sites in the intermountain western United States. Specifically the states of Idaho, Montana, Wyoming, Nevada, Utah, Colorado, Arizona, and New Mexico were included in this study. The minor and trace-element concentrations in many of the lichens collected were determined by proton induced X-ray emission (PIXE). These data are part of a base line assessment of current air pollution conditions in this region. These data also provide an opportunity to study some aspects of lichen physiology. Using the data from 508 foliose and fruticose lichens, frequency distributions for concentrations of phosphorus and calcium are considered. Phosphorus has a closely-spaced, bimodal distribution: one mode for foliose lichens and one mode for fruticose lichens. This suggests that all lichen genera in this study have similar requirements and absorption mechanisms for phosphorus. Calcium has a complex frequency distribution and concentrations that range from 450 mg/kg to 14 % dry weight. Contributions to this complex distribution pattern can be understood if the data are resolved into growth form, genera within each growth form, and in some cases species within a given genus. This complex dependence on calcium is strong evidence that lichens develop specific calcium-related adaptations in order to accommodate various habitat conditions.

Many estuarine habitats are characterized by episodes of hypoxia, the frequency and severity of which may vary seasonally. Accordingly, resident fish species may show seasonal differences in their capacity to tolerate hypoxia. We have tested this hypothesis in the gulf killifish, Fundulus grandis, sampled from the Lake Pontchartrain estuary (Louisiana) at different times of the year. We measured 2 indicators of hypoxia tolerance, the frequency of aquatic surface respiration (ASR) during gradual reduction in dissolved oxygen (D.O.) and survival time during severe hypoxic stress, and found both to be significantly affected by season. Fish collected during the summer did not engage in ASR until the D.O. concentration dropped to values lower than that required to elicit ASR by fish collected during other seasons. Laboratory acclimation of fish to low oxygen did not change the relationship between ASR behavior and D.O., suggesting that the observed seasonal effect on ASR was not simply due to previous exposure of summer fish to environmental hypoxia. Furthermore, fish collected during the summer and winter had significantly longer survival times during exposure to severe hypoxia than fish collected during the fall. Survival analysis indicated that the condition of fish was positively associated with survival time, and seasonal variation in condition accounted for about half of the observed difference between survival times of fish collected during the summer and fall. Seasonal variation in ASR and survival, when taken together, demonstrate that hypoxia tolerance in F. grandis may be subject to acclimatization. An increase in hypoxia tolerance during the summer could increase survivorship of fish when exposed to elevated temperatures and low oxygen concentrations which prevail during the summer months.

Microplastics are known to be an increasing component found within both marine sediments and the water column. This study carried out an initial assessment of the levels of microplastics present within the Solent estuarine complex, focusing specifically on the water column. A plankton net trawl survey was carried out, with samples analysed using visual observation and Fourier Transform Infrared Spectroscopy (FT-IR). The study identified significant quantities of plastics, ranging in shape, with hot spots found at confluence points within the estuary. Though the FT-IR analysis was inconclusive, the nature of the samples indicates the effect of oceanographic conditions on the prevalent types of microplastics found, which in turn identifies key local sources such as wastewater treatment plants and the plastics industry as being the dominant inputs.

Radial 14C/C profiles across three individuals of the crustose lichen Rhizocarpon geographicum (L.) DC. have been determined using accelerator mass spectrometry. These data were used to assess whether lifespan estimates can be determined in this species using 14C/C isotope ratio measurements. 14C/C profiles are relatively flat with Δ14C values (deviations from the modern radiocarbon standard) for the radial samples displaying a small spread ranging from 130 to 200 per mil. The data are consistent with carbon cycling based on growth patterns involving replacement and fusion of areoles within the thallus as well as or instead of cellular or molecular replacement. Consequently, lifespan estimates cannot be obtained from 14C/C measurements of this species and the Δ14C profiles provide no insights into whether the relationship between size and age is linear or curvilinear in this species.

There are, and have been, many school-based sex education projects in this country which have used peer leaders (students delivering an educational programme who are of similar, or slightly older, age than the students receiving the programme). Rigorous evaluation of the methodology remains scant. This paper describes a comparative investigation of peer-led and adult-led sex education in National Curriculum Year 9 (aged 13/14 years). The results from this study suggest that peer leaders appear to be more effective in establishing conservative norms and attitudes related to sexual behaviour than the adults. Peer leaders were less effective than adults in imparting factual information and getting students involved in classroom activities. These findings suggest that both adult-led and peer-led methods may have a place in effective sex education--the challenge being to determine which areas are best dealt with by whom.

Survivorship of zebrafish, Danio rerio, was measured during lethal hypoxic stress after pretreatment in water at either ambient oxygen or at a lowered, but nonlethal, level of oxygen. Acclimation to nonlethal hypoxia (pO(2) congruent with 15 Torr; ca. 10% air-saturation) for 48 hr significantly extended survival time during more severe hypoxia (pO(2) congruent with 8 Torr; ca. 5% air-saturation) compared to survival of individuals with no prior hypoxic exposure. The magnitude of the acclimation effect depended upon the sex of the fish: hypoxia pretreatment increased the survival times of males by a factor of approximately 9 and that of females by a factor of 3 relative to controls. In addition, survival time of control and hypoxia acclimated fish depended upon when in the year experiments were conducted. Survival times were 2-3 times longer when measured in the late fall or winter compared to survival times measured during the spring or summer. These results demonstrate a direct survival benefit of short-term acclimation to hypoxia in this genetically tractable fish. The fact that the acclimation effect depended upon the sex of the fish and the season during which experiments were conducted demonstrates that other genetic and/or environmental factors affect hypoxia tolerance in this species. J. Exp. Zool. 289:266-272, 2001.

Threonyl-tRNA synthetase, a class II synthetase, uses a unique zinc ion to discriminate against the isosteric valine at the activation step. The crystal structure of the enzyme with an analog of seryl adenylate shows that the noncognate serine cannot be fully discriminated at that step. We show that hydrolysis of the incorrectly formed ser-tRNA(Thr) is performed at a specific site in the N-terminal domain of the enzyme. The present study suggests that both classes of synthetases use effectively the ability of the CCA end of tRNA to switch between a hairpin and a helical conformation for aminoacylation and editing. As a consequence, the editing mechanism of both classes of synthetases can be described as mirror images, as already seen for tRNA binding and amino acid activation.

Peer-led health education in school is widely used. Advocates suggest it is an effective method based on the belief that information, particularly sensitive information, is more easily shared between people of a similar age. Critics suggest that this is a method not based on sound theory or evidence of effectiveness. This review evaluates school-based health education programmes which have set out to compare the effects of peers or adults delivering the same material. The identified studies indicated that peer leaders were at least as, or more, effective than adults. Although this suggests that peer-led programmes can be effective, methodological difficulties and analytical problems indicate that this is not an easy area to investigate, and research so far has not provided a definitive answer.

Sections of the crustose saxicolous lichen, Caloplaca trachyphylla, were dated using 14C accelerator mass spectrometry (AMS). The data show a stron linear dependence of radial position on time (r=0.993), suggesting a constant radial growth rate. This specimen had averaged a marginal growth rate of 1.48 mm/year. Extrapolation of the growth curve yields a thallus age of 20 years. These data demonstrate the feasibility of using AMS technology to precisely date lichen tissues and determine growth rates of lichen thalli.

Purified native human thyroid peroxidase (nTPO) isolated from thyroid tissue and recombinant (r)TPO produced in High Five insect cells have been compared. nTPO and rTPO were purified to about 95% homogeneity and showed similar UV and visual spectra and similar 412 nm per 280 nm absorbance ratios (0.4 for nTPO and 0.4 for rTPO). The nTPO and rTPO guaiacol oxidation enzyme activities were about 1,000 guaiacol units per milligram of protein. TPO autoantibody binding characteristics of nTPO and rTPO were analyzed in an assay based on 125I-labeled nTPO and precipitation with protein A. In the assay, the effect of unlabeled nTPO or rTPO on TPO autoantibody binding from 25 patients sera was studied. Unlabeled nTPO or rTPO (from 0 to 160 ng/mL) inhibited the binding of TPO autoantibodies in a dose-dependent manner in the case of each serum studied (from 100% in the absence of unlabeled TPO to 5%-10% in the presence of 160 ng/mL of TPO). The inhibition profile for each serum was essentially identical in the case of both TPO preparations. The effect of TPO autoantibodies on enzyme activity of rTPO was analyzed after incubation of rTPO with TPO autoantibody-positive serum immunoglobulin G (IgG) (n = 12), TPO monoclonal antibodies reactive with two different epitopes on the TPO, IgG (n = 3) from glutamic acid decarboxylase autoantibody positive patient sera, and IgG (n = 3) from healthy blood donors. Effective complexing of TPO by TPO autoantibodies was tested by precipitating the complexes with solid phase protein A and measuring the TPO enzyme activity in the resulting supernatants. These studies showed that the TPO enzyme activity was not affected by incubation with TPO autoantibody-positive IgG or monoclonal antibodies despite effective complexing of the autoantibodies with TPO. Overall, our studies demonstrate that nTPO and rTPO produced in insect cells are very similar in terms of enzyme activity, UV and visible spectra, and reactivity with autoantibodies. Furthermore, in our study, TPO autoantibodies did not appear to inhibit TPO enzyme activity.

In order to better understand the distribution pattern of mineral elements in lichen tissues, thin sections (15 μm) of the foliose, vagrant soil lichen Xanthoparmelia chlorochroa were examined using proton microprobe Particle induced X-ray emission (PIXE). This technique was used to make two-dimensional scans, with 5 μm resolution, across tissue cross sections of the test species. Element maps for Si, P, S, Cl, K, Ca, Ti, Mn, Fe, Cu, Zn, and As have been prepared. Several elements are strongly localized in the element maps. PIXE data are complimented with STIM, light micrographs, and SEM images. Preliminary data suggest that nuclear microprobe techniques may be useful in elucidating element absorption and transport mechanisms in lichens.

Once the decision was made to catalog electronic serials (electronic journals or e-journals) at the University of Pennsylvania, it was up to the serials catalogers to decide how much information would be provided in the record, as well as to develop a “standard” form. Even though the cataloging would come from two separate units (the main University Library and the Biomedical Library), the records would be part of the same OPAC. As very few other libraries were cataloging Internet resources at the time and national standards were still under discussion, it was necessary to “learn by doing.” The authors describe the process of creating cataloging records for the more than 1550 Internet serial resources and the progression from brief, basic records on the library's gopher to full-level cataloging of Internet titles for these items. Examples of problems unique to this format are supplied; discussion of foreign and domestic online newspapers and established publisher-and/or vendor-generated scholarly...

Summary Steroidogenic enzyme autoantibodies (SEAbs) are frequently present and are markers of autoimmune premature ovarian failure (POF) in females with autoimmune Addison's disease (AAD). The prevalence and significance of SEAbs in males with AAD have not yet been defined. We studied the prevalence of SEAbs in a large cohort of males with AAD and assessed the relationship between SEAbs positivity and testicular function. A total of 154 males with AAD (mean age 34 years) were studied. SEAbs included autoantibodies to steroid-producing cells (StCA), detected by immunofluorescence, and steroid 17α-hydroxylase (17α-OHAbs) and side chain cleavage enzyme (SCCAbs) measured by immunoprecipitation assays. Gonadal function was evaluated by measuring follicle-stimulating hormone (FSH), luteinizing hormone (LH), total testosterone (TT), sex hormone-binding globulin (SHGB), anti-müllerian hormone (AMH) and inhibin-B (I-B). Twenty-six males, 10 SEAbs(+) and 16 SEAbs(–), were followed-up for a mean period of 7·6 years to assess the behaviour of SEAbs and testicular function. SEAbs were found in 24·7% of males with AAD, with the highest frequency in patients with autoimmune polyendocrine syndrome type 1 (APS-1). The levels of reproductive hormones in 30 SEAbs(+) males were in the normal range according to age and were not significantly different compared to 55 SEAbs(–) males (P > 0·05). During follow-up, both SEAbs(+) and SEAbs(–) patients maintained normal testicular function. SEAbs were found with high frequency in males with AAD; however, they were not associated with testicular failure. This study suggests that the diagnostic value of SEAbs in males with AAD differs compared to females, and this may be related to the immunoprivileged status of the testis.

Air pollutants were collected in Logan, Cache County, UT, in February 1993 during two periods of atmospheric inversion accompanied by fog. The following atmospheric species were determined: (1) gaseous SO2, NO2 (semi-quantitatively),HNO3, NH3, and HF; (2) fine particulate SO4 =, NO3 -, NH4 +, F–, H+, C, Si, S, K, Ca, Ti, Mn, Fe, Ni, Cu, Zn, Pb, Se, Br, and Sr, and; (3) fine particulate mass, which was calculated. The major components of fine particulate matter were carbonaceous material, ammonium nitrate, and ammonium sulfate, while the soil component was small. Calculated, fine particulate mass averaged 80 μg/m3 and reached concentrations as high as 120 μg/m3. SO2/Sox and NO2/NOy mole ratios generally varied between 0.2 and 0.1 during inversions. These ratios also showed moderate but consistent diurnal patterns. The emission inventory for Cache County indicates sources of SO2 and NOx but not significant amounts of primary sulfate and nitrate. The observations reported here indicate there is sign...

Total fluoride (gas plus fine particle), spherical aluminosilicate particles, particulate selenium, arsenic, lead, bromine, and absorption of light by fine particles have been used to characterize chemical profiles for sources of sulfur oxides impacting the Grand Canyon National Park Class I Visibility Region. During the Project MOHAVE (Measurement of Haze and Visual Effects) winter and summer intensive studies in 1992, these various species were determined at seven sampling sites in and around the Grand Canyon. Extensive upper air and surface-based meteorological measurements were examined to determine probable geographical origins of the air mass present during a given sampling period for each sampling site. Samples corresponding to air masses dominated by transport from a single major source region were used to determine a source profile for each region. Source regions which have been characterized by this analysis include the San Joaquin Valley area, the southern California coastal urban area, the Baja, California-Imperial Valley area, the Arizona and Mexico area (including major smelters) south of the Grand Canyon, the area southeast of the Grand Canyon, the Colorado Plateau area, the Wasatch Front in Utah, and the area in Nevada to the west and northwest of the Grand Canyon. Source profiles giving the ratio of each endemic marker to SO x for each identified regional source in these areas have been determined. The source profiles for the various regional sources are all statistically different and distinguishable from those for other geographically adjoining sources.

International audience; The estimation of the bulk-solvent contribution to the diffraction of a macromolecular crystal makes use of a solvent mask which delimits the bulk-solvent regions in the crystal. It is shown that the way this mask is usually defined in CNS contains a bias which can lead to absurd results in the case of very large structures, where the calculations can only be made on relatively coarse grids. A modified procedure is described and applied to 70S ribosome data at 5.5 A resolution. The B factor affecting the bulk solvent is also discussed. Even in this case of very high and widely variable atomic B factors, it seems sufficient to consider a constant and isotropic B factor for the bulk solvent. This is initially set to the average value of the atomic B factor, but can be refined.