Hormone replacement therapy and everyday memory in mid-aged New Zealand women

While empirical research to date has generally supported positive effects of estrogen on verbal memory performance in women, the literature examining specific effects of Hormone Replacement Therapy (HRT) on cognitive functioning in mid-life women is more equivocal. The Rivermead Behavioural Memory Test-Extended Version (RBMT-E), a measure of everyday memory functioning in adults within an average range of cognitive functioning, was administered to a sample of 104 New Zealand women aged 40 to 60 years who had self-selected to either use or not use HRT (53 HRT users and 51 non-users). Self-report. measures of mood, stress, general health and menopausal symptoms were also administered. These variables, along with age and education level, were used in analyses of group differences on the everyday memory measures. Results showed significant differences between the groups for three sub-tests of the RBMT-E:'Story Immediate', 'Story Delayed', and 'Message Delayed'. Women who use HRT scored higher on these subtests than those who do not use HRT. After calculation of a total profile score (adjusting for age and IQ), HRT users score higher than HRT non-users on the RBMT-E overall measure of Everyday Memory. These pilot results suggest that HRT use in this sample-is related to enhanced verbal memory in everyday memory tasks and that the RBMT-E may be a useful tool for further work in this area of research.

Growth hormone, insulin-like growth factor I and cell proliferation in the mouse blastocyst

The role of growth hormone (GH) in embryonic growth is controversial, yet preimplantation embryos express GH, insulin-like growth factor I (IGF-I) and their receptors. In this study, addition of bovine GH doubled the proportion of two-cell embryos forming blastocysts and increased by about 25% the number of cells in those blastocysts with a concentration-response curve showing maximal activity at 1 pg bovine GH ml(-1), with decreasing activity at higher and lower concentrations. GH increased the number of cells in the trophectoderm by 25%, but did not affect the inner cell mass of blastocysts. Inhibition of cell proliferation by anti-GH antiserum indicated that GH is a potent autocrine or paracrine regulator of the number of trophectoderm cells in vivo. Type 1 IGF receptors (IGF1R) were localized to cytoplasmic vesicles and plasma membrane in the apical domains of uncompacted and compacted eight-cell embryos, but were predominantly apparent in cytoplasmic vesicles of the trophectoderm cells of the blastocyst, similar to GH receptors. Studies using alphaIR3 antiserum which blocks ligand activation of IGF1R, showed that IGF1R participate in the autocrine or paracrine regulation of the number of cells in the inner cell mass by an endogenous IGF-I-IGF1R pathway. However, alphaIR3 did not affect GH stimulation of the number of trophectoderm cells. Therefore, CH does not use secondary actions via embryonic IGF-I to modify the number of blastocyst cells. This result indicates that GH and IGF-I act independently. GH may selectively regulate the number of trophectoderm cells and thus implantation and placental growth. Embryonic GH may act in concert with IGF-I, which stimulates proliferation in the inner cell mass, to optimize blastocyst development.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Growth hormone and epidermal growth factor in salivary glands of giant and dwarf transgenic mice

Epidermal growth factor (EGF) in rat salivary glands is regulated by testosterone, thyroxin, and growth hormone (GH). Salivary glands of 45-day-old giant and dwarf male and female transgenic mice were examined histologically and by immunohistochemistry (IHC) for EGF. Male giants showed no significant differences from wild-type (WT) parotid and submandibular glands. However, their sublingual glands expressed EGF diffusely and strongly in granular cells within the striated ducts, where they were not found in WT mice. Submandibular gland ducts of female WT were different, having individual granular cells strongly positive for EGF and distributed sporadically along the striated duct walls. Neither female GH-antagonist dwarf mice nor GH-receptor knockout mice had any granular cells expressing EGF in any gland. Obvious presence of granular duct cells in the sublingual glands of giant male mice suggests GH-upregulated granular cell EGF expression. Furthermore, absence of granular duct cells from all glands in female GH-antagonist and GH-receptor knockout transgenic mice suggests that GH is necessary for the differentiation of the granular cell phenotype in female salivary glands.

Interplay between plasma hormone profiles, sex and body condition in immature hawksbill turtles (Eretmochelys imbricata) subjected to a capture stress protocol

We investigated plasma hormone profiles of corticosterone and testosterone in immature hawksbill turtles (Eretmochelys imbricata) in response to a capture stress protocol. Further, we examined whether sex and body condition were covariates associated with variation in the adrenocortical response of immature turtles. Hawksbill turtles responded to the capture stress protocol by significantly increasing plasma levels of corticosterone over a 5 h period. There was no significant sex difference in the corticosterone stress response of immature turtles. Plasma testosterone profiles, while significantly different between the sexes, did not exhibit a significant change during the 5 h capture stress protocol. An index of body condition was not significantly associated with a turtle's capacity to produce plasma corticosterone both prior to and during exposure to the capture stress protocol. In summary, while immature hawksbill turtles exhibited an adrenocortical response to a capture stress protocol, neither their sex nor body condition was responsible for variation in endocrine responses. This lack of interaction between the adrenocortical response and these internal factors suggests that the inactive reproductive- and the current energetic- status of these immature turtles are important factors, that could influence plasma hormone profiles during stress. (C) 2003 Elsevier Inc. All rights reserved.

Influence of growth hormone on the mandibular condylar cartilage of rats

Growth hormone (GH) stimulates mandibular growth but its effect on the mandibular condylar cartilage is not well. understood. Objective: This study was designed to understand the influence of GH on mitotic activity and on chondrocytes maturation. The effect of GH on cartilage thickness was also determined. Design: An animal model witt differences in GH status was determined by comparing mutant Lewis dwarf rats with reduced pituitary GH synthesis (dwarf), with normal rats and dwarf animals treated with GH. Six dwarf rats were injected with GH for 6 days, while other six normal rats and six dwarf rats composed other two groups. Mandibular condylar tissues were processed and stained for Herovici's stain and immunohistochemistry, for proliferating cell nuclear antigen (PCNA) and alkaline phosphatase (ALP). Measurements of cartilage thickness as well as the numbers of immunopositive cells for each antibody were analysed by one-way analysis of variance. Results: Cartilage thickness was significantly reduced in the dwarf animals treated with GH. PCNA expression was significant lower in the dwarf rats, but significantly increased when these animals were treated with GH. ALP expression was significant higher in the dwarf animals, while it was significantly reduced in the dwarf animals treated with GH. Conclusions: The results from this study showed that GH stimulates mitotic activity and delays cartilage cells maturation in the mandibular condyte. This effect at the cellular Level may produce changes in the cartilage thickness. (C) 2004 Elsevier Ltd. All rights reserved.

Human growth hormone presented by K14hGH-transgenic skin grafts induces a strong immune response but no graft rejection

Although immune responses leading to rejection of transplantable tumours have been well studied, requirements for epithelial tumour rejection are unclear. Here, we use human growth hormone (hGH) expressed in epithelial cells (skin keratinocytes) as a model neo-self antigen to investigate the consequences of antigen presentation from epithelial cells. Mice transgenic for hGH driven from the keratin 14 promoter express hGH in skin keratinocytes. This hGH-transgenic skin is not rejected by syngeneic non-transgenic recipients, although an antibody response to hGH develops in grafted animals. Systemic immunization of graft recipients with hGH peptides, or local administration of stimulatory anti-CD40 antibody, induces temporary macroscopic graft inflammation, and an obvious dermal infiltrate of inflammatory cells, but not graft rejection. These results suggest that a neo-self antigen expressed in somatic cells in skin can induce an immune response that can be enhanced further by induction of specific immunity systemically or non-specific immunity locally. However, immune responses do not always lead to rejection, despite induction of local inflammatory changes. Therefore, in vitro immune responses and in vivo delayed type hypersensitivity are not surrogate markers for immune responses effective against epithelial cells expressing neoantigens.

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

Crystallization and preliminary x-ray diffraction analysis of the unliganded human growth hormone receptor

The crystal structure of the extracellular domain of growth hormone receptor complexed to its ligand, growth hormone, has been known since 1992. However, no information exists for the unliganded form of the receptor. The human growth hormone receptor's extracellular ligand-binding domain, encompassing amino-acid residues 1 - 238, has been expressed in Escherichia coli, purified by anion ion-exchange chromatography and crystallized in its unliganded state by the hanging-drop vapour-diffusion method in 100 mM HEPES pH 7.0 containing 27.5%(w/v) PEG 5000 monomethyl ether and 200 mM ammonium sulfate as the co-precipitants. The crystals belong to the othorhombic space group C222(1), have unit-cell parameters a = 99.7, b = 112.2, c = 93.2 Angstrom and diffract to 2.5 Angstrom resolution using synchrotron radiation. The crystal structure will shed light on the nature of any conformation changes that occur upon ligand binding and will provide information to develop potential low-molecular-weight agonists/antagonists to treat clinical diseases in which the growth hormone receptor is implicated.

Mouse cellular cementum is highly dependent on growth hormone status

Cementum is known to be growth-hormone (GH)-responsive, but to what extent is unclear. This study examines the effects of extremes of GH status on cementogenesis in three lines of genetically modified mice; GH excess (giant), GH antagonist excess (dwarf), and GH receptor-deleted (GHR-KO) (dwarf). Age-matched mandibular molar tissues were processed for light microscope histology. Digital images of sections of first molar teeth were captured for morphometric analysis of lingual root cementum. Cross-sectional area of the cellular cementum was a sensitive guide to GH status, being reduced nearly 10-fold in GHR-KO mice, three-fold in GH antagonist mice, and increased almost two-fold in giant mice (p

Increased site 1 affinity improves biopotency of porcine growth hormone - Evidence against diffusion dependent receptor dimerization

Based on phage display optimization studies with human growth hormone (GH), it is thought that the biopotency of GH cannot be increased. This is proposed to be a result of the affinity of the first receptor for hormone far exceeding that which is required to trap the hormone long enough to allow diffusion of the second receptor to form the ternary complex, which initiates signaling. We report here that despite similar site 1 kinetics to the hGH/hGH receptor interaction, the potency of porcine GH for its receptor can be increased up to 5-fold by substituting hGH residues involved in site 1 binding into pGH. Based on extensive mutations and BIAcore studies, we show that the higher potency and site 1 affinity of hGH for the pGHR is primarily a result of a decreased off-rate associated with residues in the extended loop between helices 1 and 2 that interact with the two key tryptophans Trp(104) and Trp(169) in the receptor binding hot spot. Our mutagenic analysis has also identified a second determinant (Lys(165)), which in addition to His(169), restricts the ability of non-primate hormones to activate hGH receptor. The increased biopotency of GH that we observe can be explained by a model for GH receptor activation where subunit alignment is critical for effective signaling.