Evaluation of three new strategies to fight obesity in families.

Aims. To evaluate 3 strategies to reduce weight in obese families. Research design and methods. 142 obese parents and 119 obese children kept a fat-calorie restriction diet. On top of this diet, the families were randomized in a three-factorial design to one or more of three weight-loss strategies: (1) an additional diet preferring carbohydrates having a low glycemic index (dual diet), (2) financial incentive, and (3) telemonitoring of weight and physical activity. Results. All children improved their BMI-SDS by 0.18 ± 0.25 (P < .001) independently of the weight-loss strategy. In parents, relative losses of weight (kg) were -6.4% versus -4.0% for dual diet versus calorie restriction (P = .029), -6.9% versus -3.4% for with or without financial incentive (P = .002), and -8.0% versus -4.8% for with or without telemonitoring (P = .033). The weight loss after financial incentive plus dual diet plus telemonitoring was -14.4%. Conclusions. All strategies were effective in adults, in particular when combined. Children improved their BMI-SDS regardless of the strategy.

Estimation of biogas produced by the landfill of Palermo, applying a Gaussian model.

In this work, a procedure is suggested to assess the rate of biogas emitted by the Bellolampo landfill (Palermo, Italy), starting from the data acquired by two of the stations for monitoring meteorological parameters and polluting gases. The data used refer to the period November 2005-July 2006. The methane concentration, measured in the CEP suburb of Palermo, has been analysed together with the meteorological data collected by the station situated inside the landfill area. In the present study, the methane has been chosen as a tracer of the atmospheric pollutants produced by the dump. The data used for assessing the biogas emission refer to night time periods characterized by weak wind blowing from the hill toward the city. The methane rate emitted by the Bellolampo dump has been evaluated using a Gaussian model and considering the landfill both as a single point source and as a multiple point one. The comparison of the results shows that for a first approximation it is sufficient to consider the landfill of Palermo as a single point source. Starting from the monthly percentage composition of the biogas, estimated for the study period, the rate of biogas produced by the dump was evaluated. The total biogas produced by the landfill, obtained as the sum of the emitted component and the recovered one, ranged from 7519.97 to 10,153.7m3/h. For the study period the average monthly estimations of biogas emissions into the atmosphere amount to about 60% of the total biogas produced by the landfill, a little higher than the one estimated by the company responsible for the biogas recovery plant at the landfill.

Estrogen disrupts chemokine-mediated chemokine release from mammary cells: implications for the interplay between estrogen and IP-10 in the regulation of mammary tumor formation.

Chemokines are pro-inflammatory cytokines that function to attract immune cells to the sites of tissue inflammation, injury or infection. We have formulated the hypothesis that release of one chemokine can serve, in a local paracrine or endocrine fashion, to induce the release of other chemokines from neighboring mammary cells. We set out to investigate whether specific chemokines could promote the release of other chemokine members from mammary cells, and whether estrogen could serve to disrupt the release of these chemokines from mammary cells. We found that treatment with the chemokine IP-10 resulted in significant increases in the amount of MIP-1alpha and MCP-1/JE released from murine mammary cells. Estrogen co-treatment significantly blocked the ability of IP-10 to trigger the release of MIP-1alpha and MCP-1/JE. Suppressive effects of estrogen were reversed upon co-treatment with 4-hydroxytamoxifen. Estrogen treatment significantly decreased expression of proteins corresponding to the chemokine receptors CXCR3 and CCR5 on mammary cells. Exposure of female mice to IP-10 in vivo significantly decreased the ability of estrogen to support the growth of CCL-51-based tumors in mammary tissue. Our results suggest that exposure of mammary tissue to estrogen may decrease the release of local chemokines from mammary cells, potentially increasing the risk of tumor growth through decreased immune surveillance. Ongoing studies are investigating the possible mechanisms through which IP-10 stimulates the release of chemokines from mammary cells, and how the action of IP-10 may serve to decrease mammary tumor formation.

Altered Bax expression and decreased apoptosis in bone marrow cells of lupus-susceptible NZB/W mice.

Estrogen is believed to contribute to the development of the autoimmune disorder systemic lupus erythematosus (SLE) (lupus) in women. We hypothesized that estrogen might promote the development of lupus by altering apoptosis of bone marrow cells, perhaps through regulation of the apoptotic proteins Bax and Bcl-2. We compared the effects of estrogen (E2) and thrombopoietin (TPO) on the expression of Bax or Bcl-2 in bone marrow cells isolated from female non-lupus (NZW or NZB parental strains) or lupus-prone (NZB and NZW cross; NZB/W) mice. We report that the basal level of Bax in parental bone marrow cells was significantly greater than that of cells from NZB/W animals. Treatment of NZB or NZW marrow cells with E2 resulted in a significant decrease in Bax expression, which was completely reversed upon co-treatment with TPO. Bax expression was not significantly altered by E2 and/or TPO in NZB/W cells. Bcl-2 levels did not differ between murine strains under basal or hormone-treated conditions. Lower basal expression of Bax protein was associated with significantly less apoptosis for NZB/W marrow cells. In addition, there were significantly greater numbers of cells in bone marrow of lupus-susceptible animals. Our results indicate that bone marrow cells of NZB/W animals differ physiologically from those of NZW or NZB mice, and suggest that decreased expression of Bax in bone marrow precursors may lead to decreased apoptosis of mature blood cells in lupus-susceptible mice.

Altered bone marrow cell sensitivity in the lupus-prone NZB/W mouse: regulation of CFU-GM colony formation by estrogen, tamoxifen and thrombopoietin.

Estrogen is thought to contribute to the onset of systemic lupus erythematosus (SLE) in women through mechanisms that are not completely understood. Although estrogen serves as a negative regulator in normal hematopoietic development, little research has been conducted examining alteration in hematopoietic development triggered by estrogen in lupus-susceptible individuals. We examined whether estrogen and other factors could influence colony formation of bone marrow cells obtained from normal and lupus-susceptible mice. Bone marrow cells isolated from New Zealand Black (NZB) and lupus-prone New Zealand Black and New Zealand White cross (NZB/W) mice were cultured in the presence of granulocyte-macrophage colony stimulating factor (GM-CSF) alone or in combination with estrogen, thrombopoietin (TPO), tamoxifen, estrogen and TPO. or estrogen and tamoxifen, and plated in methylcellulose culture medium. Plates were scored for the number of CFU-GM (colony forming unit granulocyte-macrophage) colonies after 6d in culture. For females of both mouse strains, estrogen significantly decreased (P < 0.05) the number of GM colonies. Co-treatment of NZB/W cells, but not NZB cells, with TPO or tamoxifen reversed the suppressive action of estrogen (P < 0.05). In contrast, while estrogen did suppress colony formation from cells of NZB/W males (P < 0.05), neither TPO nor tamoxifen reversed this effect. Our results indicate that the sensitivity of bone marrow cells isolated from both female and male NZB/W lupus-prone mice to hormones/growth factors is qualitatively different from cells of NZB mice, and suggest that hematopoietic alterations at the level of the bone marrow may be related to the pathogenesis of SLE.

Macrophage inflammatory protein-1alpha and interferon-inducible protein 10 inhibit synergistically induced growth factor stimulation of MAP kinase activity and suppress phosphorylation of eukaryotic initiation factor 4E and 4E binding protein 1.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) and Steel factor (SLF) synergistically stimulate Raf-1 kinase activity, protein synthesis, and proliferation in hematopoietic MO7e cells; synergistic action of these factors is blocked by the suppressive chemokines macrophage inflammatory protein-1alpha (MIP-1alpha) and interferon-inducible protein 10 (IP-10; Aronica et al, J Biol Chem 270:21998, 1995). We assessed the potential for both stimulatory and inhibitory factors to act through the MAP kinase signaling pathway by studying the effects of growth factors and chemokines on MAP kinase activation. Also, because activation of kinase signaling pathways and stimulation of protein synthesis by peptide growth factors are associated with increased phosphorylation of eukaryotic initiation factor 4E (elF-4E) and the translational repressor 4E-binding protein 1 (4E-BP1) in some target cells, we investigated whether growth factor treatment could alter eIF-4E or 4E-BP1 phosphorylation state in MO7e cells. We report that treatment of MO7e cells with GM-CSF and SLF stimulated significant, greater-than-additive increases in MAP kinase activity and the phosphorylation of both eIF-4E and 4E-BP1. Increased 4E-BP1 phosphorylation correlated with a decrease in the association of 4E-BP1 with eIF-4E. Growth factor-induced phosphorylation of 4E-BP1 and dissociation of 4E-BP1 from eIF-4E was blocked in cells treated with rapamycin, wortmannin, or PD098059. Treatment of cells with IP-10 or MIP-1alpha blocked the stimulatory effects of GM-CSF and SLF, resulting in suppression of MAP kinase activity, eIF-4E and 4E-BP1 phosphorylation, and eIF-4E/4E-BP1 dissociation. Our results suggest that GM-CSF and SLF exert part of their combined growth-promoting effects on MO7e cells through activation of MAP kinase and enhancement of eIF-4E and 4E-BP1 phosphorylation and dissociation and that suppression of growth factor-induced protein synthesis by MIP-1alpha and IP-10 involves translational repression at the level of eIF-4E.

Cloning and characterization of exodus, a novel beta-chemokine.

Chemokines are a family of related proteins that regulate leukocyte infiltration into inflamed tissue. Some chemokines such as MIP-1 alpha also inhibit hematopoietic progenitor cell proliferation. Recently, three chemokines, MIP-1 alpha, MIP-1 beta, and RANTES, have been found to significantly decrease human immunodeficiency virus production from infected T cells. We report here the cloning and characterization of a novel human chemokine termed Exodus for its chemotactic properties. This novel chemokine is distantly related to other chemokines (28% homology with MIP-1 alpha) and shares several biological activities. Exodus is expressed preferentially in lymphocytes and monocytes, and its expression is markedly upregulated by mediators of inflammation such as tumor necrosis factor or lipopolysaccharide. Purified synthetic Exodus was found to inhibit proliferation of myeloid progenitors in colony formation assays. Exodus also stimulated chemotaxis of peripheral blood mononuclear cells. The sequence homology, expression, and biological activity indicate that Exodus represents a novel divergent beta-chemokine.

Altered hematopoiesis, behavior, and sexual function in mu opioid receptor-deficient mice.

The mu opioid receptor is thought to be the cellular target of opioid narcotics such as morphine and heroin, mediating their effects in both pain relief and euphoria. Its involvement is also implicated in a range of diverse biological processes. Using a mouse model in which the receptor gene was disrupted by targeted homologous recombination, we explored the involvement of this receptor in a number of physiological functions. Mice homozygous for the disrupted gene developed normally, but their motor function was altered. Drug-naive homozygotes displayed reduced locomotor activity, and morphine did not induce changes in locomotor activity observed in wild-type mice. Unexpectedly, lack of a functional receptor resulted in changes in both the host defense system and the reproductive system. We observed increased proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in both bone marrow and spleen, indicating a link between hematopoiesis and the opioid system, both of which are stress-responsive systems. Unexpected changes in sexual function in male homozygotes were also observed, as shown by reduced mating activity, a decrease in sperm count and motility, and smaller litter size. Taken together, these results suggest a novel role of the mu opioid receptor in hematopoiesis and reproductive physiology, in addition to its known involvement in pain relief.

Retroviral mediated gene transfer of Flt3 ligand enhances proliferation and MAP kinase activity of AML5 cells.

Flt3/flk-2 ligand (Flt3-L) co-stimulates and synergizes with cytokines such as granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor (G-CSF), interleukin-3 (IL-3), and erythropoietin in the proliferation of bone marrow and cord blood hematopoietic stem and progenitor cells. To study the biological effects of Flt3-L on the Flt3-L responsive AML5 cell line, the retroviral vector L(Flt3-L)SN was constructed based on the vector LXSN, but containing the human Flt3-L cDNA transcriptionally regulated by the Mo-MLV LTR. High-titer amphotropic producer cells that generated 10(6) cfu/mL after shuttle packaging through ecotropic packaging cells were isolated. AML5 cells were cultured overnight with L(Flt3-L)SN retroviral supernatant, 8 micrograms/mL polybrene, and 100 U/mL G-CSF, and expanded 1 week in medium with G-CSF. Transduced cells were selected in medium containing 0.4 mg/mL G418 and then in medium with 1.0 mg/mL G418. Retroviral mediated gene transfer in G418-resistant cells was confirmed after amplification by PCR of neo-specific sequences in genomic DNA. Northern blot analysis demonstrated L(Flt3-L)SN mRNA expression. Soluble Flt3-L was undetectable (< 100 pg/mL) by ELISA assay of conditioned medium from transduced cells, but Flt3-L was detected on the surface of AML5 cells by FACS analysis. Cells were plated in colony assay with and without 100 ng/mL Flt3-L, 100 U/mL G-CSF, and the combination. Gene transfer or growth factor treatment increased somewhat the clonogenicity of the nontransduced AML5 cells. More strikingly, L(Flt3-L)SN and each growth factor combination greatly increased the size of the resultant colonies such that the size of colonies from AML5/Flt3-L cells without added growth factor approximated that of the AML5 cells stimulated by exogenous soluble Flt3-L. Moreover, MAP kinase activity in L(Flt3-L)SN-transduced cells cultured without soluble Flt3-L was increased to the level induced in control cells by soluble Flt3-L. These results indicate that retroviral mediated gene transfer and autologous expression of the Flt3-L enhances growth and intracellular signaling of AML5 cells, information that should be of value for studying the effects of Flt3-L on immature subsets of primary hematopoietic stem and progenitor cells.

Biology and mechanisms of action of synergistically stimulated myeloid progenitor cell proliferation and suppression by chemokines.

A number of cytokines can act together to stimulate/enhance the proliferation of hematopoietic stem and progenitor cells in a greater than additive fashion. An example of this is the combination of a colony-stimulating factor with a potent costimulating molecule such as steel factor. Certain members of the chemokine family of cytokines can suppress this synergistically enhanced proliferation. This review focuses on cytokines involved in these stimulating/enhancing/suppressing effects with regard to biological activity and what is beginning to be learned about the intracellular signal transduction events that may be mediating these effects. Examples of intracellular mediators involved include, but are not limited to, the Raf-1/ MAP kinase pathway and cyclin-dependent kinase inhibitors p21cip-1 and p27kip-1 for cell proliferation, and eukaryotic initiation factor-4E and 4E binding protein 1 for protein synthesis.

Advances in understanding the postreceptor mechanisms of action of GM-CSF, G-CSF, and Steel factor.

Intracellular signaling events occurring downstream of receptor activation for the colony-stimulating factors GM-CSF and G-CSF and Steel factor the latter a member of the tyrosine kinase receptor family of hematopoietic growth factors, are discussed. Hematopoietic signaling pathways, including the Ras/Raf-1/MAP kinase cascade and the Jak-STAT pathway are defined and links existing between separate signaling pathways are discussed. Emphasis is given to exploring the relationships that exist between activation of receptor-associated proteins and signal transduction pathways, and the regulation of gene transcription, translation, and hematopoietic cell proliferation. A model system exploring the synergistic interaction between GM-CSF and Steel factor in the regulation of hematopoietic cell proliferation is presented.

Interferon-inducible protein 10 and macrophage inflammatory protein-1 alpha inhibit growth factor stimulation of Raf-1 kinase activity and protein synthesis in a human growth factor-dependent hematopoietic cell line.

Stimulatory cytokines, including granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), act in a synergistic manner to stimulate the growth of hematopoietic progenitor cells, an effect also demonstrated for the growth factor-dependent human hematopoietic cell line MO7e. While little is known about the mechanisms responsible for mediating synergistic interactions of cytokines, Raf-1, a component of the MAP kinase signaling pathway, is thought to play a role in the stimulatory response evoked by several cytokines, including SLF and GM-CSF. Interferon-inducible protein-10 (IP-10) and macrophage inflammatory protein-1 alpha (MIP-1 alpha) are members of the chemokine family of suppressive cytokines. Prior exposure of hematopoietic cells to chemokines, including IP-10 and MIP-1 alpha, inhibits the synergistic action of growth factors on stimulating cell proliferation. We report that treatment of MO7e cells with the combination of GM-CSF and SLF directly stimulates statistically significant synergistic increases in the phosphorylation and activation of Raf-1 kinase, and in cellular protein synthesis levels. Pretreatment of MO7e cells with IP-10 or MIP-1 alpha blocked synergistic growth factor action, resulting in statistically significant suppression of cell proliferation, protein synthesis, and Raf-1 phosphorylation and activation. IP-10 and MIP-1 alpha treatment also evoked significant increases in intracellular cAMP levels. Pretreatment of cells with agents which serve to raise intracellular cAMP levels, or with cAMP analogs inhibited the synergistic actions of GM-CSF and SLF in a manner similar to IP-10 and MIP-1 alpha. In addition, treatment of cells with a potent inhibitor of cAMP-dependent protein kinase A blocked the suppressive action of MIP-1 alpha and IP-10 on Raf-1 kinase activity and on MO7e cell proliferation. The ability of IP-10 and MIP-1 alpha to antagonize the synergistic action of GM-CSF and SLF appears to involve inactivation of Raf-1 and the down-regulation of protein synthesis. Our findings suggest that both MIP-1 alpha and IP-10 mediate their suppressive effects in MO7e cells by stimulating increases in cellular cAMP levels and activating protein kinase A, a mechanism we believe to be unique to these chemokines and not one applied to all growth suppressive members of the chemokine superfamily (for example, interleukin 8 and platelet factor 4).

Macrophage inflammatory protein-1 alpha enhances growth factor-stimulated phosphatidylcholine metabolism and increases cAMP levels in the human growth factor-dependent cell line M07e, events associated with growth suppression.

The immunoregulatory C-C chemokine, macrophage inflammatory protein-1 alpha (MIP-1 alpha) has suppressive activity on proliferation of stem cells and early subsets of myeloid progenitor cells. A receptor for C-C chemokines that binds MIP-1 alpha has been characterized, cloned, and shown to be related structurally to neuropeptide receptors that couple through G-proteins to phospholipase-C and adenyl cyclase. Yet, very little information on the intracellular mechanisms of action of MIP-1 alpha is available. We show here that the human factor-dependent cell line M07e is responsive to the cell cycle-suppressive effects of MIP-1 alpha, has specific membrane-binding sites for MIP-1 alpha, and that treatment of these cells with this chemokine increases the phosphatidylcholine (PC) and phosphocholine turnover rates in cells that are synergistically stimulated by the combination of granulocyte-macrophage colony-stimulating factor and steel factor but not these factors acting singly. Additional, MIP-1 alpha treatment induces a dose- and time-dependent increase in intracellular cAMP levels in M07e cells. Both exogenous PC and dibutyryl cAMP were found to suppress the proliferation of M07e colony-forming cells to a level similar to that of MIP-1 alpha, further implicating cAMP and PC metabolism in MIP-1 alpha-induced M07e suppression. RANTES, a related chemokine, with weak or incomplete binding to the cloned MIP-1 alpha receptor, did not suppress M07e colony-forming cells, nor did it increase intracellular cAMP levels, but it did enhance growth factor-induced PC turnover, further supporting the involvement of cAMP in MIP-1 alpha suppression while demonstrating that increased PC turnover alone is not sufficient for suppression. These findings support the idea that the human MIP-1 alpha receptor is coupled to phospholipid and cAMP metabolism in a manner similar to other 7-transmembrane, G-protein-linked receptors and suggest that a phosphatidylcholine hydrolytic cycle and an associated increase in cAMP are part of the mechanisms of action of MIP-1 alpha.

Estrogen action via the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene transcription.

Estrogenic hormones, believed to exert most of their effects via the direct interaction of their receptors with chromatin, are found to increase cAMP in target breast cancer and uterine cells in culture and in the intact uterus in vivo. Increases in intracellular cAMP are evoked by very low concentrations of estradiol (half maximal at 10 pM) and by other physiologically active estrogens and antiestrogens, but not by an inactive estrogen stereoisomer. These increases in cAMP result from enhanced membrane adenylate cyclase activity by a mechanism that does not involve genomic actions of the hormones (are not blocked by inhibitors of RNA and protein synthesis). The estrogen-stimulated levels of cAMP are sufficient to activate transcription from cAMP response element-containing genes and reporter plasmid constructs. Our findings document a nongenomic action of estrogenic hormones that involves the activation of an important second-messenger signaling system and suggest that estrogen regulation of cAMP may provide an additional mechanism by which this steroid hormone can alter the expression of genes.

Regulation of progesterone receptor gene expression in MCF-7 breast cancer cells: a comparison of the effects of cyclic adenosine 3',5'-monophosphate, estradiol, insulin-like growth factor-I, and serum factors.

We have compared regulation of progesterone receptor (PR) gene expression in MCF-7 human breast cancer cells by cAMP and that by estradiol (E2) and insulin-like growth factor-I (IGF-I). Treatment of cells with 8-bromo-cAMP or agents known to activate protein kinase-A, namely cholera toxin plus 3-isobutyl-1-methylxanthine (CT plus IBMX; which increased intracellular cAMP > 10-fold) evoked an increase in PR protein levels as did treatment with IGF-I or E2. Increases in PR caused by IGF-I were not accompanied by increases in PR mRNA, whereas PR mRNA levels were markedly induced by E2 and CT plus IBMX, showing regulation at different levels by these agents. The increases in PR mRNA evoked by E2 or CT plus IBMX were almost completely abolished by treatment with antiestrogen. Treatment with cycloheximide inhibited CT- plus IBMX-mediated PR mRNA stimulation, but not the induction of E2, indicating that the PR mRNA response to cAMP is not a primary one and probably requires de novo protein synthesis. Distinct effects of serum were observed on the expression of PR in MCF-7 cells. PR mRNA and protein were consistently elevated when cells were cultured under low serum conditions (0-0.5% charcoal dextran-treated calf serum) and were reduced as the serum concentration was increased, suggesting that a serum factor(s) repress constitutive PR levels. Also, the ability of CT plus IBMX to stimulate PR declined markedly for cells grown in medium containing higher (5%) serum levels; by contrast, the ability of E2 to induce PR was increased at the higher serum concentration. Thus, unknown serum factors interfere with the action of cAMP in up-regulating PR, whereas serum factors are important for the effectiveness of E2 in stimulating PR. These observations indicate that regulation of PR occurs at different levels by several factors (cAMP, E2, and IGF-I) and imply that cAMP, serum factors, and growth factors, such as IGF-I, in addition to E2 will be of importance in determining PR levels and, hence, cell sensitivity to progestins.

Stimulation of estrogen receptor-mediated transcription and alteration in the phosphorylation state of the rat uterine estrogen receptor by estrogen, cyclic adenosine monophosphate, and insulin-like growth factor-I.

We have shown previously that exposure of rat uterine cells in primary culture to estradiol (E2), insulin-like growth factor-I (IGF-I), or agents which alter intracellular cAMP levels, such as cholera toxin plus isobutylmethylxanthine (CT + IBMX) and 8-Br-cAMP, results in the up-regulation of cellular levels of the progesterone receptor, an effect believed to be mediated through the activation of estrogen receptor (ER) and phosphorylation pathways. We have therefore undertaken studies using transient transfection of these uterine cell cultures with a simple estrogen-responsive reporter gene in order to determine the ability of these agents to stimulate ER-mediated gene transcription directly. We also compared the ability of these same agents to alter the phosphorylation state of the endogenous uterine ER protein. Plasmid DNA containing two tandem estrogen responsive elements and a TATA box linked to the chloramphenicol acetyl transferase (CAT) gene was introduced into immature rat uterine cells grown in primary culture. Treatment of transfected cells with 10(-9) M E2, CT (1 micrograms/ml) + IBMX (10(-4) M), 8-Br-cAMP (10(-4) M), or IGF-I (20 ng/ml) resulted in an 8- to 10-fold induction of CAT activity. CAT activity stimulated by all agents was nearly completely suppressed by coincubation with the antiestrogen ICI 164,384 (ICI) or the protein kinase (PK) inhibitor H8. CAT activity induced by 8-Br-cAMP was more readily suppressed by ICI than that induced by E2, indicating that ER in cells exposed to 8-Br-cAMP is either unoccupied or minimally occupied by ligand. The level of ER phosphorylation in uterine cells was increased 3- to 5-fold upon exposure to E2, CT + IBMX, 8-Br-cAMP, or IGF-I. Of interest, the antiestrogen ICI also elicited a similar increase in overall ER phosphorylation. The PK inhibitors H8 and PKI suppressed the increase in overall ER phosphorylation stimulated by these agents by 50-75%. The results of our study indicate that E2, IGF-I, and agents which raise intracellular cAMP are able to stimulate ER-mediated trans-activation and ER phosphorylation. The fact that antiestrogen (ICI) evokes a similar increase in ER phosphorylation without a similar increase in transcription activation indicates that an increase in overall ER phosphorylation does not necessarily result in increased transcriptional activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Progesterone receptor regulation in uterine cells: stimulation by estrogen, cyclic adenosine 3',5'-monophosphate, and insulin-like growth factor I and suppression by antiestrogens and protein kinase inhibitors.

Primary uterine cell cultures were used to study multifactor regulation of progesterone receptor (PR) and the signal transduction pathways which may serve to mediate that regulation. Increases in intracellular cAMP, brought about by treatment with cholera toxin plus isobutyl methyl xanthine or by addition of 8-bromo-cAMP, result in 6- to 7-fold increases in the intracellular content of PR as monitored by [3H]R5020 binding and by Western immunoblot using anti-PR antibodies. In these primary cultures of uterine cells isolated from 19-day-old immature rats, 8-bromo-cAMP evokes significant increases in PR by 8 h with maximal increases by 24 h. This time course and magnitude of PR stimulation are similar to those evoked by maximally effective concentrations of estradiol (3 x 10(-9) M) or IGF-I (20 ng/ml). Dose-response studies reveal that 10(-6) to 10(-4) M concentrations of 8-bromo-cAMP (8-Br-cAMP) elicit a maximal response. In contrast, 8-bromo-cGMP over a wide concentration range was unable to elevate cellular PR levels. Under these culture conditions, cell proliferation was not altered by treatment with any of these agents. Although estrogen, cAMP, and insulin-like growth factor I (IGF-I) may act via different pathways to increase PR, the effects evoked by maximally effective concentrations of these agents are not additive implying involvement of a common component. The increases in PR evoked by estradiol, cAMP, or IGF-I are markedly suppressed by treatment with antiestrogen (ICI 164,384) or the cyclic nucleotide-dependent protein kinase inhibitor H8 or the protein kinase A inhibitor PKI, indicating the involvement of the estrogen receptor and phosphorylation pathways in PR regulation by these three agents. The present studies identify cAMP, as well as estrogen and IGF-I, as important regulators of the level of PR in uterine cells and suggest that multiple factors, including those affecting intracellular cAMP levels, might influence responsiveness to progestins via regulation of the intracellular PR content.

Exogenous human growth hormone-releasing factor and ovine somatotropin improve growth performance and composition of gain in lambs.

The objectives of this study were 1) to compare intermittent subcutaneous administration of human growth hormone-releasing factor (hGRF) at two doses with a similar regimen of ovine somatotropin (oST) for effects on growth and composition of gain in growing lambs and 2) to determine whether increasing the dietary amino acid availability enhances response to oST or hGRF. Eighty crossbred ewe and wether lambs (25.5 kg live weight) were assigned randomly in pairs to receive four daily injections of excipient, 40 micrograms oST/kg BW, 5 micrograms hGRF/kg BW or 10 micrograms hGRF/kg BW for 42 (n = 80) or 56 (n = 40) d. Doses were adjusted weekly for BW. Mean plasma oST concentrations increased from 2.03 ng/ml prior to treatment to 20.64, 4.80 and 5.45 ng/ml with oST, 5 and 10 micrograms/kg hGRF doses, respectively. Lambs did not become refractory to hGRF. Cumulative gain increased approximately 18% with 7 wk of treatment with oST and the low dose of hGRF (both P less than .01), and feed efficiency improved 21% with oST and 18% with both doses of hGRF (both P less than .05). Carcass lipid accretion rate decreased 22% to 30% (P less than .001), and carcass protein accretion rate increased 30% to 36% (P less than .001) with hGRF and oST treatment, respectively. Addition of fishmeal to the diet at 4% to replace an equal amount of soy protein improved gain 8.5%; it improved efficiency 14.2% (P less than .05) across all treatments, and it significantly enhanced the effects of oST on feed efficiency (interaction P less than .12) and hind leg muscle weights.

Management of male pseudohermaphroditism: a case report spanning twenty-one years.

Management of infants born with ambiguous sex organs requires the close collaboration of a team of professionals: pediatric endocrinologist, pediatric psychologist, gynecologist, and surgeon. The interdisciplinary teamwork of a group of professionals is described in the case of a patient announced at birth as a male but within days reannounced and reared as a female. Over the next 21 years, she became completely aware of her endocrine condition, including its medical classification (male pseudohermaphroditism), her chromosomal (46 XY) and gonadal (testes) status, and her early history of male sex assignment. The clinical management described herein helped this person deal effectively with her condition despite unsuccessful reconstructive vagal surgery. Substantiation of this is provided by the patient's personal comments.