Insulin-like growth factor 2 cDNA cloning and ontogeny of gene expression in the liver of the marsupial brushtail possum (Trichosurus vulpecula)

The cDNA sequence for insulin-like growth factor 2 (IGF-2) was determined from the liver of the marsupial brushtail possum (Trichosurus vulpecula) using reverse transcription followed by polymerase chain reaction (RT-PCR) with gene-specific primers. The 359 bp of possum sequence encompassed the mature peptide, 27 bp of the signal peptide, and 125 bp of the E-peptide. Alignment of the deduced amino acid sequence with those from other species indicated that the mature peptide was 71 amino acids in length, 4 amino acids longer than most other mammals. At both the nucleotide and amino acid levels there was a high degree of sequence identity with IGF-2 from other mammalian and nonmammalian species. Amino acid identity ranged from 94.4% with a variant form of human IGF-2 to 80.3% with zebrafinch IGF-2. Northern analysis revealed that radiolabeled possum IGF-2, cDNA hybridized to multiple transcripts in the liver of both adult possums and 150-day-old pouch young and that the overall level of expression was greater in pouch young. Semiquantitative RT-PCR with total RNA from liver samples of pouch young aged 12 to 150 days postpartum and adults confirmed that IGF-2 gene expression was two to three times more abundant in pouch young than in adults but there was no significant change in the level of expression during pouch life. Unlike other mammalian species, in which there is a decline in levels of liver IGF-2 gene expression around the time of birth, levels in the marsupial brushtail possum remain elevated for at least 150 days after birth. This suggests that the decline in liver IGF-2 expression in marsupials and eutherians occurs at a similar stage of development and may reflect a role for this growth factor during the postnatal growth and development of the marsupial, (C) 2001 Academic Press.

Growth hormone (GH)/GH receptor expression and GH-mediated effects during early bovine embryogenesis

Pituitary growth hormone (GH) stimulates postnatal growth and metabolism. The role of CH and its receptor (GHR) during prenatal development, however, is still controversial. As shown by reverse transcription polymerase chain reaction (RT-PCR), bovine in vitro fertilization embryos synthesized the transcript of GHR from Day 2 of embryonic life onwards. Real time RT-PCR revealed that synthesis of GHR mRNA was increased 5.9-fold in 6-day-old embryos compared with 2-day-old embryos. Using in situ hybridization, the mRNA encoding GHR was predominantly localized to the inner cell mass of blastocysts. The GHR protein was first visualized 3 days after fertilization. GH-specific transcripts were first detected in embryos on Day 8 of in vitro culture. As shown by transmission electron microscopy, GH treatment resulted in elimination of glycogen storage in 6- to 8-day-old embryos and an increase in exocytosis of lipid vesicles. These results suggest that a functional GHR able to modulate carbohydrate and lipid metabolism is synthesized during preimplantation development of the bovine embryo and that this GHR may be subject to activation by embryonic GH after Day 8.

Binding and functional studies with the growth hormone receptor antagonist, B2036-PEG (pegvisomant), reveal effects of pegylation and evidence that it binds to a receptor dimers

GH actions are dependent on receptor dimerization. The GH receptor antagonist, B2036-PEG, has been developed for treating acromegaly. B2036 has mutations in site 1 to enhance receptor binding and in site 2 to block receptor dimerization. Pegylation (B2036-PEG) increases half-life and lowers immunogenicity, but high concentrations are required to control insulin-like growth factor-I levels. We examined antagonist structure and function and the impact of pegylation on biological efficacy. Unpegylated B2036 had a 4.5-fold greater affinity for GH binding protein (GHBP) than GH but similar affinity for membrane receptor. Pegylation substantially reduced membrane binding affinity and receptor antagonism, as assessed by a transcription assay, by 39- and 20-fold, respectively. GHBP reduced antagonist activity of unpegylated B2036 but did not effect antagonism by B2036-PEG. B2036 down-regulated receptors, and membrane binding sites doubled in the presence of dimerization-blocking antibodies, suggesting that B2036 binds to a receptor dimer. It is concluded that the high concentration requirement of B2036-PEG for clinical efficacy relates to pegylation, which decreases binding to membrane receptor but has the advantages of reduced clearance, immunogenicity, and interactions with GHBP. Our studies suggest that B2036 binds to a receptor dimer and induces internalization but not signaling.

Increase of fat oxidation and weight loss in obese mice caused by chronic treatment with human growth hormone or a modified C-terminal fragment

OBJECTIVE: To observe the chronic effects of human growth hormone (hGH) and AOD9604 (a C-terminal fragment of hGH) on body weight, energy balance, and substrate oxidation rates in obese (ob/ob) and lean C57BL/6Jmice. In vitro assays were used to confirm whether the effects of AOD9604 are mediated through the hGH receptor, and if this peptide is capable of cell proliferation via the hGH receptor. METHOD: Obese and lean mice were treated with hGH, AOD or saline for 14 days using mini-osmotic pumps. Body weight, caloric intake, resting energy expenditure, fat oxidation, glucose oxidation, and plasma glucose, insulin and glycerol were measured before and after treatment. BaF-BO3 cells transfected with the hGH receptor were used to measure in Vitro I-125-hGH receptor binding and cell proliferation. RESULTS: Both hGH and AOD significantly reduced body weight gain in obese mice. This was associated with increased in vivo fat oxidation and increased plasma glycerol levels (an index of lipolysis). Unlike hGH, however, AOD9604 did not induce hyperglycaemia or reduce insulin secretion. AOD9604 does not compete for the hGH receptor and nor does it induce cell proliferation, unlike hGH. CONCLUSIONS: Both hGH and its C-terminal fragment reduce body weight gain, increase fat oxidation, and stimulate lipolysis in obese mice, yet AOD9604 does not interact with the hGH receptor. Thus, the concept of hGH behaving as a pro-hormone is further confirmed. This data shows that fragments of hGH can act in a manner novel to traditional hGH-stimulated pathways.

Genetic and environmental contributions to educational attainment in Australia

The genetic and environmental contributions to educational attainment in Australia are examined using a multiple regression model drawn from the medical research literature. Data from a large sample of Australian twins are analysed. The findings indicate that at least as much as 50 percent and perhaps as much as 65 percent of the variance in educational attainments can be attributed to genetic endowments. It is suggested that only around 25 percent of the variance in educational attainments may be due to environmental factors, though this contribution is shown to be around 40 percent when adjustments for measurement error and assortative mating are made. The high fraction of the observed variation in educational attainments due to genetic differences is consistent with results reported by Heath et al. (Heath, A.C., Berg, K., Eaves, L.J., Solaas, M.H., Corey, L.A., Sundet, J., Magnus, P., Nance, W.E., 1985. Education policy and the heritability of educational attainment. Nature 314(6013), 734-736.), Tambs et al. (Tambs, K., Sundet, J.M., Magnus, P., Berg, K., 1989. Genetic and environmental contributions to the covariance between occupational status, educational attainment and IQ: a study of twins. Behavior Genetics 19(2), 209-222.), Vogler and Fulker (Vogler, G.P., Fulker, D.W., 1983. Familial resemblance for educational attainment. Behavior Generics 13(4), 341-354.) and Behrman and Taubman (Behrman, J., Taubman, P., 1989. Is schooling mostly in the genes? Nature-nurture decomposition using data on relatives. Journal of Political Economy 97(6), 1425-1446.), suggesting that the finding is robust. (C) 2001 Elsevier Science Ltd. All rights reserved.

Determinants of general practitioner use among women in Australia

This study investigates the use of general practitioner services by women in Australia. Although there is a universal health insurance system (Medicare) in Australia, there are variations in access to services and out of pocket costs for services. Survey data from 2350 mid-age (45-50 years) and 2102 older (70-75 years) women participating in the Australian Longitudinal Study on Women's Health were linked with Medicare data to provide a range of individual and contextual variables hypothesised to explain general practitioner use. Structural equation modelling showed that physical health was the most powerful explanatory factor of general practitioner use. However, after adjusting for self-reported health, out of pocket cost per consultation was inversely associated with use of services. The out of pocket cost was generally lower for women with low socioeconomic status but cost was also directly related to geographical remoteness. Women living in more remote areas had higher out of pocket costs and poorer access to services. Women who reported better access to care were more likely to be satisfied with their most recent general practice consultation and less likely to be sceptical of the value of medical care. These results show the need for health policies that improve the equitable use of general practitioner services in Australia. (C) 2001 Elsevier Science Ltd. All rights reserved.

Industrial relations decentralisation and the growth of 12-hour shifts in Australia

In the last two decades, increasing numbers of workplaces in Australia have introduced 12-hour shifts. This increase is due, in part, to government policies aimed at promoting labour flexibility. The purpose of this paper is to examine the cover afforded by the Workplace Relations Act 1996 and other industrial relations legislation in terms of shift-workers’ health and safety. Particular reference is made to the broader social, economic and political context surrounding the introduction and use of 12-hour shifts, as it is this context that shapes the constraints and opportunities facing employers and employees in the work arrangements they choose and how they are negotiated. We conclude that the current system of regulating industrial relations in Australia is largely outcome-focused and inadequate. The bargaining process receives little regulation in terms of considering how changes could affect health and safety in the workplace or how changes might affect individual workers. As a result, the increased introduction of unsafe shiftworking arrangements is a worrying, and likely, prospect.

NMR structure and backbone dynamics of a concatemer of epidermal growth factor homology modules of the human low-density lipoprotein receptor

The ligand-binding region of the low-density lipoprotein (LDL) receptor is formed by seven N-terminal, imperfect, cysteine-rich (LB) modules. This segment is followed by an epidermal growth factor precursor homology domain with two N-terminal, tandem, EGF-like modules that are thought to participate in LDL binding and recycling of the endocytosed receptor to the cell surface. EGF-A and the concatemer, EGF-AB, of these modules were expressed in Escherichia coli. Correct protein folding of EGF-A and the concatemer EGF-AB was achieved in the presence or absence of calcium ions, in contrast to the LB modules, which require them for correct folding. Homonuclear and heteronuclear H-1-N-15 NMR spectroscopy at 17.6 T was used to determine the three-dimensional structure of the concatemer. Both modules are formed by two pairs of short, anti-parallel beta -strands. In the concatemer, these modules have a fixed relative orientation, stabilized by calcium ion-binding and hydrophobic interactions at the interface. N-15 longitudinal and transverse relaxation rates, and {H-1}-N-15 heteronuclear NOEs were used to derive a model-free description of the backbone dynamics of the molecule. The concatemer appears relatively rigid, particularly near the calcium ion-binding site at the module interface, with an average generalized order parameter of 0.85 +/- 0.11. Some mutations causing familial hypercholesterolemia may now be rationalized. Mutations of D41, D43 and E44 in the EGF-B calcium ion-binding region may affect the stability of the linker and thus the orientation of the tandem modules. The diminutive core also provides little structural stabilization, necessitating the presence of disulfide bonds. The structure and dynamics of EGF-AB contrast with the N-terminal LB modules, which require calcium ions both for folding to form the correct disulfide connectivities and for maintenance of the folded structure, and are connected by highly mobile linking peptides. (C) 2001 Academic Press.

Identifying trajectories of adolescent smoking: An application of latent growth mixture modeling

The goal of the current study was to identify discrete longitudinal patterns of change in adolescent smoking using latent growth mixture modeling. Five distinct longitudinal patterns were identified. A group of early rapid escalators was characterized by early escalation (at age 13) that rapidly increased to heavy smoking. A pattern characterized by occasional puffing up until age 15, at which time smoking escalated to moderate levels was also identified (late moderate escalators). Another group included adolescents who, after age 15, began to escalate slowly in their smoking to light (0.5 cigarettes per month) levels (late slow escalators). Finally, a group of stable light smokers (those who smoked 1-2 cigarettes per month) and a group of stable puffers (those. who smoked only a few puffs per month) were also identified. The stable puffer group was the largest group and represented 25% of smokers.

Changes in non-22-kilodalton (kDa) isoforms of growth hormone (GH) after administration of 22-kDa recombinant human GH in trained adult males

GH is being used by elite athletes to enhance sporting performance. To examine the hypothesis that exogenous 22-kDa recombinant human GH (rhGH) administration could be detected through suppression of non-22-kDa isoforms of GH, we studied seventeen aerobically trained males (age, 26.9 +/- 1.5 yr) randomized to rhGH or placebo treatment (0.15 IU/kg/day for 1 week). Subjects were studied at rest and in response to exercise (cycle-ergometry at 65% of maximal work capacity for 20 min). Serum was assayed for total GH (Pharmacia IRMA and pituitary GH), 22-kDa GH (2 different 2-site monoclonal immunoassays), non-22-kDa GH (22-kDa GH-exclusion assay), 20-kDa GH, and immunofunctional GH. In the study, 3 h after the last dose of rhGH, total and 22-kDa GH concentrations were elevated, reflecting exogenous 22-kDa GH. Non-22-kDa and 20-kDa GH levels were suppressed. Regression of non-22-kDa or 20-kDa GH against total or 22-kDa GH produced clear separation of treatment groups. In identical exercise studies repeated between 24 and 96 h after cessation of treatment, the magnitude of the responses of all GH isoforms was suppressed (P < 0.01), but the relative proportions were similar to those before treatment. We conclude: 1) supraphysiological doses of rhGH in trained adult males suppressed exercise-stimulated endogenous circulating isoforms of GH for up to 4 days; 2) the dearest separation of treatment groups required the simultaneous presence of high exogenous 22-kDa GH and suppressed 20-kDa or non-22-kDa GH concentrations; and 3) these methods may prove useful in detecting rhGH abuse in athletes.

The response of molecular isoforms of growth hormone to acute exercise in trained adult males

Circulating GH consists of multiple molecular isoforms, all derived from the one gene in nonpregnant humans. To assess the effect of a potent stimulus to pituitary secretion on GH isoforms, we studied 17 aerobically trained males (age, 26.9 +/- 1.5 yr) in a randomized, repeat measures study of rest vs. exercise. Exercise consisted of continuous cycle ergometry at approximately 80% of predetermined maximal oxygen uptake for 20 min. Serum was assayed for total, pituitary, 22-kDa, recombinant, non-22-kDa, 20-kDa, and immunofunctional GH. All isoforms increased during, peaked at the end, and declined after exercise. At peak exercise, 22-kDa GH was the predominant isoform. After exercise, the ratios of non-22 kDa/total GH and 20-kDa GH/total GH increased and those of recombinant/pituitary GH decreased. The disappearance half-times for pituitary GH and 20-kDa GH were significantly longer than those for all other isoforms. We conclude that 1) all molecular isoforms of GH measured increased with and peaked at the end of acute exercise, with 22-kBa GH constituting the major isoform in serum during exercise; and 2) the proportion of non-22-kDa isoforms increased after exercise due in part to slower disappearance rates of 20-kDa and perhaps other non-22-kDa GH isoforms. It remains to be determined whether the various biological actions of different GH isoforms impact on postexercise homeostasis.

Growth hormone binding protein in normal and aneuploid pregnancy: a paradoxical decrease in trisomy 18

Objective To explore whether abnormalities in growth hormone binding protein (GHBP) may underlie the growth restriction associated with fetal aneuploidy. Design A retrospective casecontrol study. Setting Monash Medical Centre, Clayton, Victoria, Australia. Population Twenty-one trisomy 18, and 30 trisomy 21 pregnancies, and 170 chromosomally normal pregnancies at 15-18 weeks of gestation representing three to five controls per case matched for source, gestation and duration of storage. Methods GHBP was measured using a ligand immunofunctional assay. Results In the chromosomally normal pregnancies GHBP levels decreased slightly but significantly across the narrow gestational window studied. Compared with controls, levels of GHBP, expressed as median (95% CI) multiples of the median (MoM), in the trisomy 21 pregnancies were similar, 1.0 (0.92-1.39) MoM and 1.27 (1.04-1.50) MoM, respectively; P = 0.061 (Mann-Whitney CI test) but were significantly reduced in the trisomy 18 pregnancies, 0.68 (0.51-0.84) MoM; P = 0.0014 (Mann-Whitney U test). Conclusions These data suggest that decreased levels of maternal growth hormone binding protein, and by implication growth hormone receptor complement, may underlie the early severe growth restriction that is characteristic of trisomy 18.

"They're scared of it": Intergroup determinants of attitudes toward children with HIV

The study used an intergroup perspective to explore teachers' willingness to teach children with HIV within mainstream education. One hundred thirty-eight teachers from 13 high schools across the United Kingdom participated in a survey. The results suggest that previous contact with an individual who was HIV positive (whether an adult or a child) was strongly associated with greater willingness to teach a child with HIV. However, this effect disappeared when intergroup anxiety, positive affect, and positive beliefs were included as covariates. There was little evidence that instrumental variables (i.e., relatively pragmatic concerns about infection) were associated with willingness to teach. Participants who had some contact reported lower fear of contagion from a child with HIV, but there was no evidence that this was associated with overall attitudes. The results are interpreted to suggest that interventions to reduce prejudice should not simply concentrate on improving knowledge about HIV transmission.

Suppression of keratinocyte growth and differentiation by transforming growth factor beta 1 involves multiple signaling pathways

Transforming growth factor beta1 treatment of keratinocytes results in a suppression of differentiation, an induction of extracellular matrix production, and a suppression of growth. In this study we utilized markers specific for each of these functions to explore the signaling pathways involved in mediating these transforming-growth-factor-beta1-induced activities. In the first instance, we found that the induction of extracellular matrix production (characterized by 3TP-Lux reporter activity) was induced in both keratinocytes and a keratinocyte-derived carcinoma cell line, SCC25, in a dose-dependent manner. Furthermore, transforming growth factor beta1 also suppressed the differentiation-specific marker gene, transglutaminase type 1, in both keratinocytes and SCC25 cells. In contrast, transforming growth factor beta1 inhibited proliferation of keratinocytes but did not cause growth inhibition in the SCC25 cells. Transforming-growth-factor-beta1-induced growth inhibition of keratinocytes was characterized by decreases in DNA synthesis, accumulation of hypophosphorylated Rb, and the inhibition of the E2F:Rb-responsive promoter, cdc2, and an induction of the p21 promoter. When the negative regulator of transforming growth factor beta1 signaling, SMAD7, was overexpressed in keratinocytes it could prevent transforming-growth-factor-beta1-induced activation of the 3TP-Lux and the p21 promoter. SMAD7 could also prevent the suppression of the transglutaminase type 1 by transforming growth factor beta1 but it could not inhibit the repression of the cdc2 promoter. These data indicate that the induction of 3TP-Lux and p21 and the suppression of transglutaminase type 1 are mediated by a different proximate signaling pathway to that regulating the suppression of the cdc2 gene. Combined, these data indicate that the regulation of transforming growth factor beta1 actions are complex and involve multiple signaling pathways.