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Steel products are widely used in the construction industry and for the development of infrastructure projects, because they are versatile, durable, and affordable. Energy demand and ‘greenhouse gas’ (GHG) emissions associated with the United Kingdom(UK) Iron & Steelsector principally result from the large consumption of coal/coke used in conjunction with the blast furnace. Like other sectors of industry, efforts are being made to ensure that processing becomes ever more environmentally benign, or ‘green’. Thus, the notion of ‘green steel’ has entered into the industrial vocabulary over the last decade or so. It is a steel-making process designed principally to lower GHG emissions, as well as potentially cutting costs and improving the quality of steel, in comparison to conventional methods. The aim of this study was therefore to (i) elicit the various ways in which the term ‘green steel’ has recently been used in the literature; and (ii) compare and contrast different options for making UK steel production more environmentally benign, particularly in regard to its decarbonisation. Some key ‘deep decarbonisation’, or ‘disruptive’, options for producing green steelin the UK are evaluated drawing on the experience from other nation-states and regions. These include the prospects for carbon capture and storage(CCS), the use of bioenergy resources, hydrogen-based production, electrification, and the least desirable option of deindustrialisation(i.e., reducing or out-sourcing of UK steel production ‘offshore’). ‘Circular economy’ interventions or resource efficiency improvements (‘reduce, reuse, recycle’) are also discussed. The potential reductions in GHG emissions from the UK Iron & Steelsector overall out to 2050 are then illustrated by comparison with previous technology roadmaps or transition pathways. The lessons learned are applicable across much of the industrialised world.

Building ‘zero carbon’ homes will be essential for achieving the carbon reductions within industrialised countries required to meet their commitments under the 2015 Paris Agreementon climate change. Such high performance buildings may need a combination or ‘cluster’ of micro-generatorsto be installed, such as a heat pump to provide heating and a solar photovoltaic(PV) array to produce electricity. When sized and installed appropriately, these technologies have lower emissions than the conventional systems they displace (centralised grid electricity and gas-fired boilers). However, if the ‘embodied’ energy and carbon is not recouped from that saved during the lifetime of the micro-generator, then there is no net saving overall. This study therefore assesses a range of clustered micro-generators using an ‘integrated approach’ that combines energy analysis, environmental life-cycle assessment, and an indicative financial appraisal. Eight clusters of micro-generators were designated to meet the heat and electricity requirements of five different dwelling types, each one specified to two different UK performance standards (2006 building regulations and a zero-carbon specification). For these 80 scenarios, various combinations of heat pumps with solar hot waterand/or PV systems yield the most attractive performance metrics with all of the clusters having energy and carbon paybacks (4.5–5.5 and 5.0–7.0 years respectively) within their operational lifetimes, and would hence create net savings overall. But the clusters were generally found to have unattractive financial payback periods (50–80 years), although this result will be sensitive to the discount rate and prevailing energy prices and support mechanisms. The focus is on the use of clustered micro-generators in the context of UK transition pathways to a low-carbon economy out to 2050, but the lessons learned are applicable to many industrialised countries.

Produced by the liver, corticosteroid-binding globulin (CBG) regulates the plasma distribution and actions of glucocorticoids. A sex difference in pituitary growth hormone secretion patterns established during puberty in rats results in increased hepatic CBG production and 2-fold higher plasma corticosterone levels in females. Glucocorticoids control hepatic development and metabolic activities, and we have therefore examined how disrupting the SerpinA6gene encoding CBG influences plasma corticosterone dynamics, as well as liver gene expression in male and female rats before and after puberty. Comparisons of corticosterone plasma clearance and hepatic uptake in adult rats, with or without CBG, indicated that CBG limits corticosterone clearance by reducing its hepatic uptake. Hepatic transcriptomic profiling revealed minor sex differences (207 differentially expressed genes) and minimal effect of CBG deficiency in 30-day-old rats before puberty. While liver transcriptomes in 60-day-old males lacking CBG remained essentially unchanged, 2710 genes were differentially expressed in wild-type female vs male livers at this age. Importantly, ∼10% of these genes lost their sexually dimorphic expression in adult females lacking CBG, including those related to cholesterol biosynthesis, inflammation, and lipid and amino acid catabolism. Another 203 genes were altered by the loss of CBG specifically in adult females, including those related to xenobiotic metabolism, circadian rhythm, and gluconeogenesis. Our findings reveal that CBG consolidates the sexual dimorphism of the rat liver initiated by sex differences in growth hormone secretion patterns and provide insight into how CBG deficiencies are linked to glucocorticoid-dependent diseases.

Discrepancies in ELISA measurements of human corticosteroid-binding globulin (CBG) using detection monoclonal antibodies that recognize an epitope (9G12) within its reactive center loop (RCL), versus an epitope (12G2) in a different location, have suggested that CBG with a proteolytically cleaved RCL exists in blood samples. We have previously been unable to verify this biochemically, and sought to determine if N-glycosylation differences account for discrepancies in ELISA measurements of CBG.Molecular biological, biochemical and glycopeptide analyses were used to examine how N-glycosylation at specific sites, including at N347 within the RCL, affect CBG ELISA or steroid-binding capacity assay (BCA) measurements. Plasma from patients with congenital disorders of glycosylation (CDG) was also examined in these assays as examples of N-glycosylation defects.We demonstrate that an N-glycan at N347 within the CBG RCL limits the 9G12 antibody from recognizing its epitope, whereas the 12G2 antibody reactivity is unaffected, thereby contributing to discrepancies in ELISA measurements using these two antibodies. Qualitative differences in N-glycosylation at N238 also negatively affect the steroid-binding of CBG in the absence of an N-glycan at N347 caused by a T349A substitution. Desialylation increased both ELISA measurements relative to BCA values. Similarly, plasma CBG levels in both ELISAs were much higher than BCA values in several CDG patients.Plasma CBG measurements are influenced by variations in N-glycosylation. This is important given the increasing number of CDG defects identified recently and because N-glycosylation abnormalities are common in patients with metabolic and liver diseases.

Environmental or ‘ecological’ footprints have been widely used in recent years as indicators of resource consumption and waste absorption transformed on the basis of biologically productive land area [in global hectares (gha)] required per capitawith prevailing technology. It has been employed here to estimate the footprints associated with three low carbon, more electric transition pathways for the United Kingdom (UK): described as ‘Market Rules’ (MR), ‘Central Co-ordination’ (CC) and ‘Thousand Flowers’ (TF) respectively. These pathways focus on the power sector, including the potential for increasing use of low-carbon electricity for heating and transport, within the context of critical European Uniondevelopments and policies. Their overall environmental footprint has been disaggregated into various components: bioproductive and built land, carbon emissions, embodied energy, materials and waste, transport, and water consumption. This component-based approach provides, for example, a means for evaluating the implications for the so-called ‘energy-land-water nexus’. Electricity demand was projected to decrease significantly under the TF pathway by 2050, but its total environmental footprint (EF) was greater than either that under the MR or CC pathways. This is mainly due to the increase in the use of bioproductive land associated with solid biofuel production and that of the carbon footprint, which are both seen to be larger than under either the MR or CC cases. Water and waste footprint components made almost negligibly small contributions under all three transition pathways. Lessons can clearly be drawn for other industrialised nations attempting to decarbonise their electricity generation systems, although local circumstances will determine the country-specific findings.

Sex hormone-binding globulin (SHBG) is the main transporter of sex hormones in most vertebrates. Low SHBG levels have been linked to increased risk for diabetes and metabolic syndrome. Polymorphisms of the SHBGgene linked to low SHBG protein levels also strongly predicted increased risk of type 2 diabetes, thus raising the possibility that SHBG may play a role in the pathogenesis of insulin resistance and diabetes.To examine whether expression of human SHBGin mice may ameliorate the development of diabetes and metabolic syndrome in response to a high-fat diet (HFD).Transgene mice expressing a human SHBGtransgene (SHBG+) (N= 10/11; males/females) and their wild type littermates (N= 12/8; males/females) were fed HFD for 4.5 months.HFD induced comparable obesity in control and SHBG+mice. Male transgenes had higher muscle mass after 2–3.5 months HFD (0.43 ± 0.028 (n= 4) vs 0.38 ± 0.053 g (n= 7), P= 0.05). Fasting blood glucose, as well as insulin or HOMA-IR, was not different in transgenic vs wild-type males after 4–5 months HFD. Female transgenes had higher fasting glucose (152 ± 29 (n= 7) vs 115 ± 27 mg/dL, P= 0.01 (n= 8)), but mean insulin and HOMA-IR were not different. Likewise, insulin tolerance test and intra-peritoneal glucose tolerance test (GTT) were not different. Finally, SHBG+ mice were not different from controls in terms of liver enzymes, serum triglyceride levels and blood pressure.In mice with diet-induced obesity, human SHBGdid not protect against development of obesity or dysglycemia.

Progress in reducing industrial energy demand and carbon dioxide (CO2) emissions is evaluated with a focus is on the situation in the United Kingdom (UK), although the lessons learned are applicable across much of the industrialized world. The UKindustrial sector is complex, because it may be viewed as consisting of some 350 separate combinations of subsectors, devices and technologies. Various energy analysis and carbon accounting techniques applicable to industry are described and assessed. The contributions of the energy‐intensive (EI) and nonenergy‐intensive (NEI) industrial subsectors over recent decades are evaluated with the aid of decomposition analysis. An observed drop in aggregate energy intensity over this timescale was driven by different effects: energy efficiency improvements; structural change; and fuel switching. Finally, detailed case studies drawn from the Cementsubsector and that associated with Food and Drinkare examined; representing the EIand NEIsubsectors, respectively. Currently available technologies will lead to further, short‐term energy and CO2emissions savings in manufacturing, but the prospects for the commercial exploitation of innovative technologies by mid‐21st century are far more speculative. There are a number of nontechnological barriers to the take‐up of such technologies going forward. Consequently, the transition pathways to a low carbon future in UKindustry by 2050 will exhibit large uncertainties. The attainment of significant falls in carbon emissions over this period depends critically on the adoption of a limited number of key technologies [e.g., carbon capture and storage(CCS), energy efficiency techniques, and bioenergy], alongside a decarbonization of the electricity supply. WIREs Energy Environ2016, 5:684–714. doi: 10.1002/wene.212 This article is categorized under: Energy Efficiency > Science and MaterialsEnergy Efficiency > Economics and PolicyEnergy Efficiency > Climate and Environment

Sprague Dawley rats from different vendor colonies display divergent responses in a variety of experimental paradigms. An adjuvant-induced arthritis (AA) model of human rheumatoid arthritis was used to examine immune and endocrine responses to inflammatory challenge in Sprague Dawley rats from Charles River and Harlan colonies. Rats were injected with either complete Freund's adjuvant or physiological saline (control), weights, and paw volumes measured over 15 days, and blood and tissue were collected 16 days post-injection. Overall, Harlan rats developed more severe AA than Charles River rats. In addition, despite comparable corticosterone levels, corticosteroid binding globulin levels were lower in Harlan compared with Charles River rats in the absence of inflammation, suggesting that a lower corticosterone reservoir in Harlan rats may underlie their greater susceptibility to inflammation. With increasing AA severity, there was an increase in plasma corticosterone (total and free) and a decrease in corticosteroid binding globulin in both Charles River and Harlan rats. However, contrasting patterns of cytokine activation were observed in the hind paw, suggesting a reliance on different cytokine networks at different stages of inflammation, with Charles River rats exhibiting increased TNF-α, monocyte chemotactic protein-1 (MCP-1), keratinocyte chemoattractant/growth-regulated oncogene (KC/GRO), and IL-1β in the absence of clinical signs of arthritis, whereas Harlan had increased TNF-α, monocyte chemotactic protein-1, and IL-6 with mild to moderate arthritis. These colony-specific differences in endocrine and immune responses to AA in Sprague Dawley rats must be considered when comparing data from different laboratories and could be exploited to provide insight into physiological changes and therapeutic outcomes in arthritis and other inflammatory disorders.

Encoded by SerpinA6, plasma corticosteroid-binding globulin (CBG) transports glucocorticoids and regulates their access to cells. We determined how CBG influences plasma corticosterone and adrenal development in rats during the pubertal to adult transition using CRISPR/cas9 to disrupt SerpinA6gene expression. In the absence of CBG, total plasma corticosterone levels were ∼80% lower in adult rats of both sexes, with a greater absolute reduction in females than in males. Notably, free corticosterone and adrenocorticotropic hormone were comparable between all groups. Between 30 and 90 days of age, wild-type female rats showed increases in adrenal weight and the size of the corticosterone-producing region, the zona fasciculata (zf), in tandem with increases in plasma CBG and corticosterone concentrations, whereas no such changes were observed in males. This sex difference was lost in rats without CBG, such that adrenal growth and zf expansion were similar between sexes. The sex-specific effects of CBG on adrenal morphology were accompanied by remarkable changes in gene expression: ∼40% of the adrenal transcriptome was altered in females lacking CBG, whereas almost no effect was seen in males. Over half of the adrenal genes that normally exhibit sexually dimorphic expression after puberty were similarly expressed in males and females without CBG, including those responsible for cholesterol biosynthesis and mobilization, steroidogenesis, and growth. Rat adrenal SerpinA6transcript levels were very low or undetectable. Thus, sex differences in adrenal growth, morphology and gene expression profiles that emerge during puberty in rats are dependent on concomitant increases in plasma CBG produced by the liver.

Context:Plasma corticosteroid-binding globulin (CBG) transports cortisol but high progesterone levels at the maternal-fetal interface can displace cortisol from its steroid-binding site. A secretion-deficient CBG mutant (A51V) in ∼1 of 36 Chinese causes low circulating CBG levels.Objective:Assess the implications of a CBG deficiency on pregnancy outcomes.Participants and Design:From 1978 Chinese women screened at 12–16 weeks' gestation, 50 A51V carriers were identified and 46 were followed with 60 controls throughout pregnancy. Blood samples from another 2051 pregnant women were obtained at term to determine the secondary sex ratio (SSR) of newborns in an extended cohort (n = 101) of A51V mothers.Outcome Measures and Results:Among women recruited at 12–16 weeks' gestation, serum CBG increased progressively during pregnancy but was lower (P< .0001) in heterozygous A51V carriers than controls. Two women homozygous for A51V had very low serum CBG but their pregnancies progressed normally. The A51V mothers did not differ from controls in body mass index, gestational age at delivery, duration of parturition, blood pressure, gravidity, infant birth weight and size, or placental weights, and reported no unusual clinical symptoms. Peripheral CBG and progesterone levels correlated (r = 0.459) during first and second trimesters. Progesterone levels were much higher in intervillous blood and correlated (r = 0.637) with CBG levels. A female-skewed SSR in newborns of A51V mothers (0.77) differed (P< .05) from the SSR (1.17) in a reference cohort.Conclusions:CBG influences progesterone levels in peripheral blood and at the maternal-fetal interface. The female-skewed SSR suggests that male fetal survival is compromised in CBG-deficient mothers.

This review/research paper summarizes data on development of the external genitalia of the spotted hyena, a fascinating mammal noted for extreme masculinization of the female external genitalia. The female spotted hyena is the only extant mammal that mates and gives birth through a pendulous penis-like clitoris. Our studies indicate that early formation of the phallus in both males and females is independent of androgens; indeed the phallus forms before the fetal testes or ovaries are capable of synthesizing androgens. Likewise, pre- and postnatal growth in length of the penis and clitoris is minimally affected by “androgen status”. Nonetheless, several internal morphologies, as well as external surface features of the phallus, are androgen-dependent and thus account for dimorphism between the penis and clitoris. Finally, estrogens play a critical role in penile and clitoral development, specifying the position of the urethral orifice, determining elasticity of the urethral meatus, and facilitating epithelial–epithelial fusion events required for proper formation of the distal urethra/urogenital sinus and prepuce. Accordingly, prenatal inhibition of estrogen synthesis via administration of letrozole (an aromatase inhibitor) leads to malformations of the glans as well as the prepuce (hypospadias). The effects of prenatal androgens, anti-androgens and impaired estrogen synthesis correlated with the tissue expression of androgen and estrogen receptors.

A range of major risks associated with the production and use of biofuels in the rapidly changing United Kingdom (UK) energy and transport sectors have been identified and quantified. This was achieved with the aid of various stakeholder groups (academic researchers; industrialists; and a concatenated group of policy makers together with ‘green’ and international development groups), who completed an online internet questionnaire. Each stakeholder ranked 15 potential risks associated with the UK development and use of liquid biofuels according to their perceived ‘severity of impact’ and ‘likelihood of occurrence’ using a three-point scale. This data was then used to perform a ranking of the risks by multiplying scores for impact and occurrence. There was some variation between the different stakeholder groups, but the similar risks were ranked highly by each group. The overall ranking identified the main risks as being a lack of investor confidence in biofuel developments (the highest score); energy or fuel security issues; negative public perception of biofuels (equal second highest); increased food prices; high barriers to entry into the fuel market; and misdirected agricultural expansion or land use (equal fifth highest). Comments by the expert respondents also provide a qualitative evaluation of the present state of UK biofuel developments. The present trial illustrates the potential of using risk issues appraisal and ranking to evaluate developing risks to the UK biofuels landscape. Clearly such an exercise would need to be carried out periodically if it were to maintain its value to the biofuel-related industrial sector and other stakeholders, including policy makers.

CONTEXT: Cortisol is transported by corticosteroid-binding globulin (CBG) in blood. Single nucleotide polymorphisms (SNP) in the human CBG (SERPINA6) gene that disrupt CBG production or steroid binding are considered rare. OBJECTIVE: The objective of the study was to identify and determine the frequency of SNP in SERPINA6 that influence the production or cortisol-binding properties of CBG in Chinese subjects. PARTICIPANTS AND DESIGN: Blood samples from 2287 anonymous Chinese workers undergoing routine health tests were screened for the SERPINA6 coding sequence polymorphisms. MAIN OUTCOME MEASURES AND RESULTS: In a pilot study of 108 Chinese women, two nonsynonymous SNP were identified within SERPINA6 exon 2 encoding CBG A51V (n = 3) and CBG E102G (n = 1) variants. Sequence analysis of SERPINA6 exon 2 in a further 137 Chinese women revealed two other individuals with nonsynonymous SNP encoding CBGs R64Q and R64W as well as another CBG A51V carrier. The surprisingly high frequency of heterozygous CBG A51V carriers was confirmed in 1011 Chinese men (1:35) and 1031 other women (1:37). Individuals homozygous for these SNP were not identified. When expressed in Chinese hamster ovary cells, CBG A51V bound steroid normally, but its production/secretion was severely impaired; CBG E102G was produced normally, but its cortisol-binding capacity was abnormally low, whereas CBG R64Q and R64W were produced and bound cortisol normally. CONCLUSIONS: Defects in CBG A51V production explain why plasma CBG levels in individuals heterozygous for this variant are approximately 50% lower than normal. The high frequency of CBG A51V will allow clinical consequences of CBG deficiencies to be assessed for the first time in large patient populations.

Sex hormone-binding globulin (SHBG) transports androgens and estrogens in blood and regulates their access to target tissues. Hepatic production of SHBG fluctuates throughout the life cycle and is influenced primarily by metabolic and hormonal factors. Genetic differences also contribute to interindividual variations in plasma SHBG levels. In addition to controlling the plasma distribution, metabolic clearance, and bioavailability of sex steroids, SHBG accumulates in the extravascular compartments of some tissues and in the cytoplasm of specific epithelial cells, where it exerts novel effects on androgen and estrogen action. In mammals, the gene-encoding SHBG is expressed primarily in the liver but also at low levels in other tissues, including the testis. In subprimate species, Shbgexpression in Sertoli cells is under the control of follicle-stimulating hormone and produces the androgen-binding protein that influences androgen actions in the seminiferous tubules and epididymis. In humans, the SHBGgene is not expressed in Sertoli cells, but its expression in germ cells produces an SHBG isoform that accumulates in the acrosome. In fish, Shbg is produced by the liver but has a unique function in the gill as a portal for natural steroids and xenobiotics, including synthetic steroids. However, salmon have retained a second, poorly conserved Shbggene that is expressed only in ovary, muscle, and gill and that likely exerts specialized functions in these tissues. The present review compares the production and functions of SHBG in different species and its diverse effects on reproduction.

Reversal of the order in which items are recalled reduces verbal span more than visuospatial span. We investigated behavioral performance and event-related potential components elicited during novel recognition span tasks with verbal (digits) and visuospatial (blocks) stimuli. Reversing item order reduced behavioral span and the amplitudes of the P3a and P3b event-related potential components in the verbal but not in the visuospatial tasks. P3a is thought to reflect detection of deviance from expectation, and hence the smaller P3a suggests expectancy of the stimulus sequence is attenuated in the digits backward task. P3b is thought to reflect allocation of attention to updating memory representations, hence the smaller P3b suggests attenuation of these attentional resources when the demanding operation of verbal order reversal is required in the digits backward span task.

Gonadotropin-releasing hormone (GNRH) activates the progesterone receptor (PGR) in pituitary cells and accentuates gonadotropin expression. We show that GNRH1 increases FshbmRNA levels in LbetaT2 mouse pituitary cells within 8 h and is three times more effective than GNRH2. By contrast, GNRH1 and GNRH2 do not affect Lhbgene expression in these cells. Within the same time frame, small interfering RNA (siRNA) knockdown of the PGR in LbetaT2 cells reduced GNRH1 activation of a PGR response element (PRE)-driven luciferase reporter gene and FshbmRNA levels by >50%. Chromatin immunoprecipitation (ChIP) assays also demonstrated that PGR loading on the PRE within the Fshbgene promoter in LbetaT2 cells occurred within 8 h after GNRH1 treatment and was lost by 24 h. While the GNRH1-induced upregulation of the PRE reporter gene and FshbmRNA levels was attenuated by cotreatment with protein kinase A (H-89) and protein kinase C (GF109203X) inhibitors, only GF109203X inhibited PGR phosphorylation at Ser249 in LbetaT2 cells. Immunoprecipitation assays also showed a progressive increase in the interaction between the PGR and its coactivator NCOA3 that peaked at 8 h coincident with the increase in FshbmRNA after GNRH1 treatment. The siRNA-mediated knockdown of NCOA3 in LbetaT2 cells also reduced FshbmRNA levels after GNRH1 treatment and loading of NCOA3 on the Fshbpromoter PRE in a ChIP assay. We conclude that the rapid effect of GNRH1 on Fshbexpression in LbetaT2 cells is mediated by PGR phosphorylation and loading at the PRE within the Fshbpromoter together with NCOA3.

Repeated stimulation of human motor cortex with pairs of magnetic pulses separated by 1.5 ms has been shown to increase the amplitude of the motor-evoked potentials in activated muscles, indicating a short-term plastic increase of corticospinal excitability. We show here that this increase is greater in the dominant than the nondominant motor cortex of right-handed participants. This finding is a direct demonstration that the potential for plastic synaptic change is greater in the dominant than the nondominant motor cortex and implies that a greater potential for reorganization within the dominant motor cortex is a mechanism of the greater dexterity of the dominant hand.

Event-related potentials were recorded during a warned choice reaction time task in which the warning signal was informative (it signaled the specific response to be required) or uninformative (it gave no response-specific information). The response signal specified a key press with either the middle or the ring finger of the left or the right hand. The informative warning signal facilitated reaction time in two separate protocols. The contingent negative variation elicited following the informative warning signal was larger than that following the uninformative warning signal, and this effect was larger over the left hemisphere than over the right hemisphere. This finding gives converging evidence that preparatory motor processes are predominantly lateralized to the dominant hemisphere.

Mammographic density is associated with increased breast cancer risk and is influenced by sex hormones. A T27Cpolymorphism (alleles A1and A2, respectively) in the 5′ promoter region of CYP17may be associated with elevated sex hormone levels. In a cross-sectional study of 181 pre- and 173 postmenopausal women, we examined the relationship of this polymorphism with mammographic density and other risk factors for breast cancer. Subjects were recruited across five categories of density. Risk factor and dietary information, anthropometric measures, and blood samples were obtained. Sex hormone, lipid, growth factor levels, and CYP17genotypes were determined. CYP17genotype was not associated with mammographic density levels before or after adjusting for risk factors for breast cancer. In premenopausal women, the A2allele was associated with higher levels of dehydroepiandrosterone sulfate, and in postmenopausal women, with higher levels of total estradiol and lower levels of follicle stimulating hormone. Among premenopausal women, interactions were observed between CYP17genotype and endogenous insulin levels as well as dietary variables associated with mammographic density. Our findings suggest that the CYP17 A2allele is associated with hormone levels, and interacts with insulin levels and diet to affect breast density levels and potentially breast cancer risk.

The liver X receptor/retinoid X receptor (LXR/RXR)-regulated gene ABCA1 effluxes cellular cholesterol and phospholipid to apolipoprotein A1 (apoA1), which is the rate-limiting step in high-density lipoprotein synthesis. The RXR pathway plays a critical role in testicular lipid trafficking, and RXRβ-deficient male mice are sterile and accumulate lipids in Sertoli cells. Here, we demonstrate that ABCA1 mRNA and protein are abundant in Sertoli cells, whereas germ cells express little ABCA1. LXR/RXR agonists stimulate ABCA1 expression in cultured Sertoli MSC1 and Leydig TM3 cell lines. However, Sertoli TM4 cells lack ABCA1, and TM4 cells or primary Sertoli cells cultured from ABCA1−/−mice both fail to efflux cholesterol to apoA1. Expression of exogenous ABCA1 restores apoA1-dependent cholesterol efflux in Sertoli TM4 cells. In vivo, ABCA1-deficient mice exhibit lipid accumulation in Sertoli cells and depletion of normal lipid droplets from Leydig cells by 2 months of age. By 6 months of age, intratesticular testosterone levels and sperm counts are significantly reduced in ABCA1−/−mice compared with wild-type (WT) controls. Finally, a 21% decrease (P= 0.01) in fertility was observed between ABCA1−/−males compared with WT controls across their reproductive lifespans.

In a crystal structure of the amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG), the biologically active estrogen metabolite, 2-methoxyestradiol (2-MeOE2), binds in the same orientation as estradiol. The high affinity of SHBG for 2-MeOE2 relies primarily on hydrogen bonding between the hydroxyl at C-3 of 2-MeOE2 and Asp(65) and an interaction between the methoxy group at C-2 and the amido group of Asn(82). Accommodation of the 2-MeOE2 methoxy group causes an outward displacement of residues Ser(128)-Pro(130), which appears to disorder and displace the loop region (Leu(131)-His(136)) that covers the steroid-binding site. This could influence the binding kinetics of 2-MeOE2 and/or facilitate ligand-dependent interactions between SHBG and other proteins. Occupancy of a zinc-binding site reduces the affinity of SHBG for 2-MeOE2 and estradiol in the same way. The higher affinity of SHBG for estradiol derivatives with a halogen atom at C-2 is due to either enhanced hydrogen bonding between the hydroxyl at C-3 and Asp(65) (2-fluoroestradiol) or accommodation of the functional group at C-2 (2-bromoestradiol), rather than an interaction with Asn(82). By contrast, the low affinity of SHBG for 2-hydroxyestradiol can be attributed to intra-molecular hydrogen bonding between the hydroxyls in the aromatic steroid ring A, which generates a steric clash with the amido group of Asn(82). Understanding how C-2 derivatives of estradiol interact with SHBG could facilitate the design of biologically active synthetic estrogens.

Human sex hormone-binding globulin (SHBG) binds estradiol and testosterone with high affinity. Plasma SHBG is produced by hepatocytes, but the human SHBG gene is also expressed in the testis. Little is known about SHBG gene expression in the human testis, but human SHBG transcripts accumulate in a spermatogenic stage-dependent manner in the testes of mice containing an 11-kb human SHBG transgene. We have now found that human SHBG transcripts containing an alternative exon 1 sequence are located specifically in the testicular germ cells of these transgenic mice, whereas murine SHBG transcripts are confined to Sertoli cells. In addition, we have detected immunoreactive human SHBG in the acrosome during all stages of spermiogenesis in mice containing an 11-kb human SHBG transgene. Western blots of germ cell extracts from these transgenic mice and from human sperm indicate that the immunoreactive human SHBG in the acrosome composes electrophoretic variants, which are 3-5 kDa smaller than the major electrophoretic isoforms of human SHBG in the blood. This apparent size difference is due in part to differences in glycosylation of plasma and acrosomal SHBG isoforms. The function of the human SHBG isoform in the acrosome is unknown, but it binds steroid ligands with high affinity. This is the first demonstration that human SHBG transcripts encode an SHBG isoform that remains within a cellular compartment.

The amino-terminal laminin G-like domain of human sex hormone-binding globulin (SHBG) contains a single high affinity steroid-binding site. Crystal structures of this domain in complex with several different steroid ligands have revealed that estradiol occupies the SHBG steroid-binding site in an opposite orientation when compared with 5 alpha-dihydrotestosterone or C19 androgen metabolites (5 alpha-androstan-3 beta,17 beta-diol and 5 alpha-androstan-3 beta,17 alpha-diol) or the synthetic progestin levonorgestrel. Substitution of specific residues within the SHBG steroid-binding site confirmed that Ser(42) plays a key role in determining high affinity interactions by hydrogen bonding to functional groups at C3 of the androstanediols and levonorgestrel and the hydroxyl at C17 of estradiol. Among residues participating in the hydrogen bond network with hydroxy groups at C17 of C19 steroids or C3 of estradiol, Asp(65) appears to be the most important. The different binding mode of estradiol is associated with a difference in the position/orientation of residues (Leu(131) and Lys(134)) in the loop segment (Leu(131)-His(136)) that covers the steroid-binding site as well as others (Leu(171)-Lys(173) and Trp(84)) on the surface of human SHBG and may provide a basis for ligand-dependent interactions between SHBG and other macromolecules. These new crystal structures have also enabled us to construct a simple space-filling model that can be used to predict the characteristics of novel SHBG ligands.

Sex hormone-binding globulin (SHBG) is the major sex steroid-binding protein in human plasma and is produced by the liver. Plasma SHBG levels vary considerably between individuals and are influenced by hormonal, metabolic, and nutritional factors. We have now found that a (TAAAA)npentanucleotide repeat, located within an alusequence at the 5′ boundary of the human SHBGpromoter, influences its transcriptional activity in association with downstream elements, including an SP1-binding site. Furthermore, SHBGalleles within the general population contain at least 6–10 TAAAA repeats, and the transcriptional activity of a human SHBGpromoter-luciferase reporter construct containing 6 TAAAA repeats was significantly lower than for similar reporter constructs containing 7–10 TAAAA repeats when tested in human HepG2 hepatoblastoma cells. This difference in transcriptional activity reflected the preferential binding of a 46-kDa liver-enriched nuclear factor to an oligonucleotide containing 6 rather than 7–10 TAAAA repeats. Thus, a (TAAAA)nelement within the humanSHBGpromoter influences transcriptional activity in HepG2 cells and may contribute to differences in plasma SHBG levels between individuals.

Human sex hormone-binding globulin (SHBG) transports sex steroids in the blood. It functions as a homodimer, but there is little information about the topography of its dimerization domain, and its steroid binding stoichiometry is controversial. The prevailing assumption is that each homodimeric SHBG molecule contains a single steroid-binding site at the dimer interface. However, crystallographic analysis of the amino-terminal laminin G-like domain of human SHBG has shown that the dimerization and steroid-binding sites are distinct and that both monomers within a homodimeric complex are capable of binding steroid. To validate our crystallographic model of the SHBG homodimer, we have used site-directed mutagenesis to create SHBG variants in which single amino acid substitutions (V89E and L122E) were introduced to produce steric clashes at critical positions within the proposed dimerization domain. The resulting dimerization-deficient SHBG variants contain a steroid-binding site with an affinity and specificity indistinguishable from wild-type SHBG. Moreover, when equalized in terms of their monomeric subunit content, dimerization-deficient and wild-type SHBGs have essentially identical steroid binding capacities. These data indicate that both subunits of the SHBG homodimer bind steroid and that measurements of the molar concentration of SHBG homodimer in serum samples have been overestimated by 2-fold.

Abstract: The early (R1) and late (R2) components of the cutaneous blink reflex in right-handed humans were recorded in three experiments to examine the lateral symmetry of a simple excitatory process in their brainstem pathways and of the control of the excitability of the pathways by gaps in acoustic noise. Experiment 1 showed that a stimulus below R1-elicitation threshold increased the excitability of the right R1 pathway more than the left but that the rate of decay was similar on both sides. Experiment 2 showed that a brief unilateral gap in noise affected the R1 and R2 reflex pathways bilaterally. Experiment 3 showed that R2 varied with gap duration and that gaps to the left and right ears had indistinguishable effects. The finer temporal resolution of events in the right sensory field of right-handers seen in psychophysical judgments is not seen in the descending control of brainstem excitability.

•Energy use and decarbonisation options for the UK Glasssector have been evaluated.•The glass sector was examined in its historical context and industrial structure.•Glass is largely recyclable, and can contribute to ‘circular economy’ aspirations.•The techno-economic improvement potential of glass-making processes was assessed.•‘Technology roadmaps’ were developed for various alternative scenarios out to 2050.

Administration of recombinant murine tumor necrosis factor (TNF) to mice results in lethal shock, characterized by hypotension, hypothermia, and dramatic induction of cytokines released in the circulation, such as interleukin-6 (IL-6). The sensitivity of mice to the effects of murine TNF varies from strain to strain. DBA/2 mice were found to be considerably more resistant to TNF than C57BL/6 mice. The resistance proved to be dominant since (C57BL/6 × DBA/2)F1 mice were also resistant. Using BXD recombinant inbred mice and a dose of TNF lethal for C57BL/6 but not for DBA/2 mice, we found that the resistance to TNF links to loci coding for corticosteroid-binding globulin (Cbg), α1-protease inhibitor (Spi1), contrapsin (Spi2) and the contrapsin-regulating geneSpi2rthat form a gene cluster on chromosome 12. Quantitative trait-loci analysis of TNF-induced induction of IL-6 and of hypothermia also points to the importance of this locus (P< 0.0002 andP= 0.017, respectively), more particularly theCbgandSpi2loci, in the resistance to TNF. We propose to name the locus “TNF protection locus.” The data suggest that endogenous protease inhibitors and/or glucocorticoids play a significant role in the attenuation of TNF-induced lethal shock. This study also demonstrates that loci affecting important biological responses can be identified with very high resolution using recombinant inbred mice.

It has been suggested that N‐glycans act as a general sorting signal for secretory proteins in MDCK cells [Scheiffele et al. (1995) Nature 378, 96–98]. Human corticosteroid binding globulin contains six consensus sites for N‐glycosylation and is known to be secreted to the apical side of MDCK cells. Our results show that wild‐type corticosteroid binding globulin is N‐glycosylated when it is recombinantly expressed in MDCK cells. Six mutants, each lacking one of the N‐glycosylation sites, and a mutant lacking all six N‐glycosylation sites were also secreted to the apical side of MDCK cells in a polarized manner. Thus, the N‐glycans on corticosteroid binding globulin do not act as an apical sorting signal in MDCK cells.

The oral administration of 150 μg desogestrel and 30 μg ethinyl estradiol (EE2) increases (P< 0.001) serum concentrations of sex-hormone-binding globulin (SHBG) and corticosteroid-binding globulin (CBG), whereas treatment with 150 μg levonorgestrel and 30 μg EE2only increases serum CBG concentrations. No changes in serum albumin concentrations occurred during or after treatment with either preparation, and increases in SHBG and CBG returned to the pretreatment values 1month after treatment ceased. The serum distribution of levonorgestrel was unchanged during treatment, whereas the increase in serum SHBG concentrations after treatment with the preparation containing desogestrel decreased (P< 0.001) the percentage of non-protein-bound 3-keto-desogestrel and the percentage of albumin-bound 3-keto-desogestrel but increased (P< 0.001) the SHBG-bound fraction. Oral contraceptives containing either progestogen decrease the mean serum non-protein-bound testosterone concentrations, especially during treatment with desogestrel (P< 0.001), and desogestrel may therefore be the more appropriate progestogen for the treatment of women prone to androgenic side effects.

Objective: We tested the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep. Methods: Twenty-one sheep were instrumented under general anesthesia with maternal and fetal arterial and venous catheters and myometrial electromyogram electrodes at 117 days’ gestation (dGA). At 125 dGA at 12:00 PM, after 2 days of baseline recording, either saline (n= 7, control group), betamethasone (n= 7), or dexamethasone (n= 7) was administered into the fetal jugular vein at a rate of 10 µg/hour. A total dose of 0.48 mg was given over the next 48 hours. The animals underwent autopsy 3 days after the end of the infusion period (130 dGA), or earlier if labor resulted from the glucocorticoid administration. Daily maternal and fetal arterial blood samples (4 mL) for hormone measurement were taken at 10:00 amthroughout the study period. Additional arterial blood samples were taken if the animal developed labor. Maternal plasma progesterone and fetal ACTH and Cortisol concentrations were measured by radioimmunoassay, and corticosteroid-binding globulin (CBG) binding capacity was determined by saturation analysis. Myometrial activity was monitored continuously throughout the experimental protocol. Results: All seven betamethasone-treated animals developed labor after the glucocorticoid infusion regimen. In contrast, only two of seven dexamethasone-treated animals developed labor. Fetal treatment with betamethasone produced a greater and earlier fall in maternal plasma progesterone than fetal treatment with dexamethasone. Both betamethasone and dexamethasone treatments elevated fetal plasma CBG to similar binding capacities. Elevated fetal plasma ACTH and Cortisol concentrations at the end of the infusion period in both betamethasone- and dexamethasone-treated animals were not related to the development of labor-type contractions. Conclusion: These data support the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep.

α1-Proteinase inhibitor (α1-PI) controls several proteinases that play important roles during inflammation and tissue remodeling, but its function during mammalian development remains obscure. We have therefore studied the ontogeny of α1-PI mRNA in selected fetal and newborn sheep tissues by Northern blotting. Furthermore, because glucocorticoids play important roles in fetal maturation and parturition in sheep, we measured the relative amounts of glucocorticoid receptor (GR) mRNA in the same tissues and compared the effect of dexamethasone (DEX) administration on α1-PI mRNA levels in liver and kidney of fetal and adult sheep. In the ontogeny study, a 1.8-kb α1-PI mRNA was found in fetal adrenal gland and lung at Days 60–70 but was low or undetectable in these tissues at later gestational ages. By contrast, the abundance of hepatic α1-PI mRNA did not vary between Days 60 and 130 of gestation, but decreased significantly (p< 0.05) at term (Days 145–147) and in newborn lambs. In the fetal kidney, it was also present between Days 60 and 130, and its relative abundance increased significantly (p< 0.05) in term and newborn lambs. These changes did not correlate with GR mRNA abundance, which was relatively stable in all tissues except the kidney, where a significant (p< 0.05) decrease was observed in neonates. Compared to saline treatment, DEX infusion (2 μg/min for 15 min every 2 h for 96 h) to fetal sheep beginning on Day 130 of gestation resulted in a significant decrease in fetal liver α1-PI mRNA abundance in the fetal liver, but no corresponding change in the fetal kidney. In adult sheep liver, a similar effect was seen after administration of 10 mg DEX per day in comparison to saline controls. We therefore conclude that 1) the widespread tissue distribution of α1-PI mRNA during mid-gestation becomes more restricted to the liver and kidney during late gestation; 2) glucocorticoids decrease the relative amount of α1-PI mRNA in the liver but not in the kidney. These results suggest that the decrease in liver α1-PI mRNA abundance in term fetuses and neonates may be induced by increases in fetal plasma cortisol concentrations during the last few days of gestation.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis of fetal sheep during late gestation is associated with increases in plasma concentrations of adrenocorticotropic hormone (ACTH) and cortisol, and ultimately results in parturition. However, the mechanisms contributing to the concurrent increases in ACTH and cortisol are unclear. Plasma estradiol-17β (E2) concentrations increase progressively in the prepartum ovine fetus, and we hypothesized that E2may influence HPA activity by affecting either basal and/or hypoxemia-stimulated ACTH release. We examined potential mechanisms, including altered expression of pro-opiomelanocortin (POMC) in fetal pituitary corticotrophs, and changes in corticosteroid binding globulin (CBG) and/or the enzymes 11β hydroxy steroid dehydrogenase (11β HSD)-1 or 11β HSD-2 in liver and placenta, that could alter negative feedback control. We infused fetal sheep at 127 d of gestation with either E2(100 μg/24 h) or saline for 100 h. Fetal arterial blood samples were collected at 8 h intervals during the infusion of E2or saline (n=4), for measurement of basal plasma ACTH and cortisol concentrations, as well as plasma corticosteroid binding capacity (CBC). Placenta and fetal liver samples were collected at 100 h for measurement of placental 11 β HSD-1 and 11β HSD-2 mRNA and hepatic CBG and 11β HSD-1 mRNA, by Northern blotting. Fetal pituitary samples were collected for measurement of POMC mRNA byin situhybridization. In a separate experiment, fetuses were exposed to 2 h of hypoxemia at 75 h of E2or saline infusion (n=4), and fetal arterial blood samples were collected during the period of hypoxemia for measurement of plasma ACTH and cortisol concentrations. E2infusion had no effect on basal plasma concentrations of ACTH or total cortisol, or on the stimulated levels of ACTH or total cortisol achieved in response to hypoxemia. Basal fetal pituitary POMC mRNA also did not change significantly with E2infusion. No significant increases were observed in plasma CBC during E2administration. However, hepatic CBG and 11β HSD-1 mRNA were significantly elevated in the livers of E2-treated fetuses. Placental 11β HSD-1 mRNA; but not 11β HSD-2 mRNA was increased by E2treatment. These data do not support a direct effect of exogenous E2at the level of basal or hypoxemia-stimulated ACTH output, but suggest that elevated E2concentrations may alter the expression of genes encoding proteins implicated in tonic regulation of fetal HPA function.

The non-protein bound percentage of ST-1435 was measured by centrifugal ultrafiltration-dialysis in undiluted female serum at 37°C. It was found that as much as 13% of ST-1435 in serum is not bound to proteins. The results also show that the affinities of SHBG and CBG of ST-1435 are very low and that SHBG and CBG do not bind ST-1435 under physiological conditions in serum. We suggest that ST-1435 is bound mainly to serum albumin, and that the binding to that accounts for more than 87% of total serum ST-1435 concentrations. During the treatment period of 3 months, no change in SHBG or CBG binding levels was observed when ST-1435 was administered parenterally.The lack of any interaction between ST-1435 and high affinity serum steroid binding proteins, and the very high percentage of non-protein bound ST-1435 in serum, probably explain its extremely high biological potency at the hypothalamic-pituitary level, when compared for example with d-norgestrel. For the same reason, practically all the ST-1435 in hepatic portal blood is probably taken-up and very rapidly metabolised by the liver. This may explain why oral administration of ST-1435 results in low and inadequate plasma concentrations for contraceptive purposes, while alternative parenteral routes of administration result in relatively much higher serum concentrations of the steroid. Finally, because ST-1435 does not bind to SHBG and CBG under physiological conditions, and does not change plasma SHBG and CBG binding capacities, ST-1435 treatment will not indirectly alter the amounts of endogeneous sex steroid hormones or their distribution in plasma. Therefore, side effects such as acne or hirsutism are unlikely to develop as a result of sustained administration of this potent progestin.

Objective:We tested the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep.Methods:Twenty-one sheep were instrumented under general anesthesia with maternal and fetal arterial and venous catheters and myometrial electromyogram electrodes at 117 days' gestation (dGA). At 125 dGA at 12:00 PM,after 2 days of baseline recording, either saline (n = 7, control group), betamethasone (n = 7), or dexamethasone (n = 7) was administered into the fetal jugular vein at a rate of 10 μg/hour. A total dose of 0.48 mg was given over the next 48 hours. The animals underwent autopsy 3 days after the end of the infusion period (130 dGA), or earlier if labor resulted from the glucocorticoid administration. Daily maternal and fetal arterial blood samples (4 mL) for hormone measurement were taken at 10:00 AMthroughout the study period. Additional arterial blood samples were taken if the animal developed labor. Maternal plasma progesterone and fetal ACTH and cortisol concentrations were measured by radioimmunoassay, and corticosteroid-binding globulin (CBG) binding capacity was determined by saturation analysis. Myometrial activity was monitored continuously throughout the experimental protocol.Results:All seven betamethasone-treated animals developed labor after the glucocorticoid infusion regimen. In contrast, only two of seven dexamethasone-treated animals developed labor. Fetal treatment with betamethasone produced a greater and earlier fall in maternal plasma progesterone than fetal treatment with dexamethasone. Both betamethasone and dexamethasone treatments elevated fetal plasma CBG to similar binding capacities. Elevated fetal plasma ACTH and cortisol concentrations at the end of the infusion period in both betamethasone- and dexamethasone-treated animals were not related to the development of labor-type contractions.Conclusion:These data support the hypothesis that betamethasone is more potent than dexamethasone in inducing the essential mechanisms of parturition in sheep.

Orienting to one of two levels of stimulus significance (a distress squeal or a simulated mimic squeal) in female hooded rats was measured by suppression of ongoing drinking. Subsequent generalization tests with the same stimulus presented in different contexts showed that generalization of habituation across contexts was a function of stimulus significance: Habituation to the distress squeal was restricted to the context in which it first had been presented, whereas habituation to the mimic squeal generalized across the different contexts.

Three experiments measured orienting of rats to stimuli varying in intrinsic significance. In the first, rats showed greater reactivity to a recording of a conspecific’s distress squeal than to a simulated mimic squeal when the stimuli were presented at 100 dB but not at 80 dB, and orienting to all stimuli habituated rapidly with repeated stimulus exposures. Experiment 2 showed that, following several stimulus exposures, recovery of orienting after rest periods of 1 and 7 days was a function of the stimulus: Orienting to the distress squeal presented at 100 dB recovered more rapidly than did orienting to the mimic squeal at 100 dB or to either the mimic or the distress squeal presented at 80 dB. Experiment 3 showed that habituation to the most significant stimulus, the distress squeal presented at 100 dB, was retarded at long (24-h) interstimulus intervals, presumably a consequence of less stimulus-to-stimulus transfer of habituation with this stimulus. The results are discussed in terms of biological constraints on habituation of orienting and on recovery of orienting following habituation.

Use-dependent facilitation of motor-evoked potentials evoked by transcranial magnetic stimulation with repetition of simple movements has been well established. Motor-evoked potentials were recorded from two intrinsic hand muscles before and after blocks of motor practice in which study participants made repeated ballistic pinch responses with either their left or their right hand. Despite similar increases in behavioral performance by each hand (measured by the peak acceleration of the force generated by the index finger), practice-related increases in the amplitude of the motor-evoked potentials were greater in the left than in the right motor cortex of right-handed participants. This finding supports the hypothesis that the dominant motor cortex has a greater ability to reorganize with experience than the non-dominant motor cortex.

The amino‐teminal laminin G‐like domain of human sex hormone‐binding globulin (SHBG), which contains the steroid‐binding site and the dimerization domain, has been produced in Escherichia coli, purified to homogeneity and crystallized in complex with 5α‐­dihydrotestosterone (DHT) in two different crystal forms. Native data sets have been collected for tetragonal crystals (space group P4122 or P4322; unit‐cell parameters a= 52.2, c= 148.4 Å) diffracting to 3.3 Å and trigonal crystals (R32; a= 104.0, c= 84.4 Å) diffracting to better than 1.6 Å. Since both crystal forms can only accommodate a single monomer in the asymmetric unit and share twofold rotational symmetry, it is proposed that the homodimer of this truncated form of SHBG, as observed in ultracentrifugation experiments, displays C2point‐group symmetry.

Human corticosteroid binding globulin (CBG) cDNA fragments were radiolabeled and hybridized in situ to metaphase chromosome preparations. The results localized the CBG gene to the q31–q32.1 region of human chromosome 14. This location also contains the genes for two closely related serine protease inhibitors: alpha1-proteinase inhibitor and alpha1-antichymotrypsin. It is therefore likely that these genes evolved by duplication events, and it would appear that this region contains a series of functionally related genes.

About 1 in 100 women enter menopause before age 40; they are generally diagnosed as having premature ovarian failure (POF). The only gene that has consistently been associated with POF is FMR1 (Fragile X Mental Retardation 1), but the finding that only about 14% of women with familial POF carry an FMR1 premutation suggests that other genes may be associated with POF.

When salmonid (rainbow trout and coho salmon) sex hormone-binding globulin (SHBG-alpha) cDNAs produced from liver RNA extracts were expressed in Chinese hamster ovary (CHO) cells, the steroid-binding characteristics of the recombinant proteins were found to differ substantially from the SHBG present in the blood of these species. Like SHBG in salmonid blood, the recombinant SHBG-alpha bound testosterone and ethinylestradiol (EE) with very high affinity but bound estradiol (E) with low affinity when analyzed in a competitive steroid-binding assay using [3H]5α-dihydrotestosterone ([3H]DHT) as labeled ligand. However, when the blood was analyzed using [3H]E as the labeled ligand a high affinity interaction was observed which produced a unique binding specificity profile in a competitive steroid-binding assay, in which DHT and EE displayed very poor affinity. This suggested that two forms of SHBG exist in salmonid blood which may be attributed to an early genome duplication event in salmonids, as evidenced by the recent discovery of an SHBG related gene sequence (SHBG-beta) in the rainbow trout genome (NCBI EF577269). We therefore cloned and expressed salmonid SHBG-beta forms in CHO cells and compared their steroid-binding properties with SHBG in blood samples using [3H]DHT and [3H]E. Importantly, recombinant salmonid SHBG-beta does not bind DHT or EE but exhibits a high affinity for testosterone and E, and its steroid-binding profile indicates that it represents a unique form of SHBG in salmonids. Moreover, the affinity of SHBG-beta for E is substantially higher than the affinity of SHBG-alpha for its preferred ligands (androstenedione and testosterone). This difference in steroid-binding specificity can be exploited for the determination of SHBG-alpha and -beta forms in a blood samples. Unlike SHBG-alpha which is expressed primarily in the liver, salmonid SHBG-beta is expressed in the ovary, muscle and stomach, and not in the liver. However, the plasma levels of SHBG-beta are at least as high as those of SHBG-alpha in the blood of mature animals. It therefore remains to be determined what tissue represents the main source of plasma SHBG-beta, and how its plasma concentrations vary during the life cycle of salmonids. Supported by the National Science and Engineering Council of Canada.

The fathead minnow (fhm) is a forage fish endemic to North America (NA) and an important link in the food chain. It is used increasingly as a sentinel organism in the assessment of environmental health. Previous studies in zebrafish indicate that sex hormone-binding globulin (SHBG) in the gills serves as a portal for the uptake of chemicals, including xenobiotics, from the aquatic environment. We have now cloned the full-length cDNA for fhmSHBG and expressed the recombinant protein in Chinese hamster ovary cells. Compared to zebrafish SHBG, fhmSHBG is 85% and 81% identical at the nucleotide and amino acid sequence levels, respectively. Biochemical characterization of recombinant fhmSHBG revealed that it has similar steroid-binding properties as zebrafish SHBG. In a competitive ligand binding assay using [3H]-androstenedione as labeled ligand, we have found that a herbicide (linuron), which is widely used in NA and shown to have anti-androgenic activity in fish and rats, binds to fhmSHBG in the micromolar range, suggesting that this anthropogenic compound with endocrine disrupting potential may "gain access" to fish through binding to SHBG in their gills. We then proceeded to develop a bioassay-guided fractionation procedure to identify novel natural compounds that act as ligands of fhmSHBG. Extracts from coniferous trees widely used in the pulp and paper industry were initially tested against fhmSHBG using radio-ligand binding assay. Some extracts (e.g. from Jack Pine) contained materials that competed strongly with [3H]-androstenedione for binding to fhmSHBG, whereas other plant extracts (e.g. Eastern hemlock) failed to compete at all. Subsequent fractionation of constituent compounds in the plant extracts using high-performance liquid chromatography, followed by radio-ligand binding assays, allowed us to isolate a single compound (mol wt 288 Da) that readily displaces endogenous steroids from fhmSHBG. This compound also binds the coho salmon homolog of fhmSHBG (i.e., coho SHBGα), albeit with less potency, but it does not bind to coho salmon SHBGβ. Identification and assessments of the binding affinity of this and other natural compounds for fhmSHBG are in progress. These studies indicate that both synthetic compounds and natural compounds in plant extracts bind to fish SHBGα homologs, and can be identified using the strategies we have developed. This work was supported by an NSERC Discovery grant (GLH) and by an NSERC Industrial Research and Development Fellowship (VLM).(poster)

Plasma levels of sex hormone-binding globulin (SHBG) are linked to infertility and risk for the metabolic syndrome. Previously, we found that increased exposures to monosaccharide induces hepatic palmitate accumulations and reduces human SHBGgene expression by down-regulating hepatocyte nuclear factor 4 alpha (HNF-4A) in transgenic mice and HepG2 hepatoblastoma cells. Here, we show that forkhead box A2 (FoxA2) participates in this regulatory pathway. Knock-down of Foxa2 by siRNA increased SHBGgene transcription and SHBG secretion, as tested by luciferase reporter assay and immunofluorometric assay, respectively, in HepG2 cells, while over-expression of FoxA2 had the opposite effect. To test if the effect of FoxA2 on SHBGexpression in these cells is direct or indirect, we measured HNF-4A, a key regulator of SHBGtranscription, levels in FoxA2 knocked-down or over-expressing cells, and found that FoxA2 is negatively associated with HNF-4A levels, which suggests that FoxA2 regulates SHBG production indirectly through HNF-4A. Since FoxA2 is a key regulator of hepatic lipid homeostasis, we further tested whether FoxA2 could participate in this regulatory pathway. When incubating HepG2 cells in high (10 mM) glucose, knock-down of FoxA2 attenuated glucose-induced repression of HNF-4A and SHBG levels by changing intra-cellular lipid accumulations. In summary, our results indicate that FoxA2 regulates hepatocyte lipid homeostasis, and in turn alters SHBG production at a transcriptional level through HNF-4A. Further studies of FoxA2-regulated alterations in lipid profiles within HepG2 cells are underway. These observations provide further insight the molecular mechanism responsible for altered hepatic SHBG expression in response to changes in nutritional status, and during different physiological conditions. Supported by an operating grant from the Canadian Institutes of Health Research.(poster)