Oestrogen and Progesterone Receptor Gene Expression in Canine Oocytes and Cumulus Cells Throughout the Oestrous Cycle

The aim of this research was to analyze oestrogen receptor-alpha (ER alpha), ER beta and progesterone receptor (PR) gene expression in the canine oocyte and cumulus cells throughout the oestrous cycle. Ovaries from 38 bitches were recovered after ovariohysterectomy and sliced. The phase of the oestrous cycle was determined by vaginal cytology, vaginoscopy and serum hormonal measurements. Oocytes were mechanically denuded by repeated pipetting. For each phase of the cycle, a sample was composed by a pool of 50 oocytes (sample number: prooestrus = 3, oestrus = 8, dioestrus = 5 and anoestrus = 5) or a pool of cumulus cells (prooestrus = 4, oestrus = 7, dioestrus = 4 and anoestrus = 6). Oocyte and cumulus cells` total RNA was isolated and reverse transcription was conducted to perform real-time PCR. Oestrogen receptor-alpha was expressed throughout the cycle in the oocyte (33.33%, 25.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively) and cumulus cells (50.0%, 47.14%, 25.0% and 66.67% for prooestrus, oestrus, dioestrus and anoestrus, respectively). In the oocyte, the ER beta was also expressed in all phases of the cycle (33.33%, 50.0%, 20.0% and 60.0% for prooestrus, oestrus, dioestrus and anoestrus, respectively), whereas in cumulus cells, ER beta was only expressed during prooestrus (50%) and oestrus (14.29%). Interestingly, while the oocyte PR was not detected in any phase of the cycle, this receptor was expressed during prooestrus (50%), oestrus (42.86%) and anoestrus (16.67%) in cumulus cells. In conclusion, canine oocytes express ER alpha and ER beta throughout the oestrous cycle, however, there is a lack of PR expression in all these phases. Moreover, in cumulus cells, only ER alpha was expressed throughout the oestrous cycle.

Regulation of Trypanosoma cruzi-Induced Myocarditis by Programmed Death Cell Receptor

Trypanosoma cruzi infection causes intense myocarditis, leading to cardiomyopathy and severe cardiac dysfunction. Protective adaptive immunity depends on balanced signaling through a T cell receptor and coreceptors expressed on the T cell surface. Such coreceptors can trigger stimulatory or inhibitory signals after binding to their ligands in antigen-presenting cells (APC). T. cruzi modulates the expression of coreceptors in lymphocytes after infection. Deregulated inflammation may be due to unbalanced expression of these molecules. Programmed death cell receptor 1 (PD-1) is a negative T cell coreceptor that has been associated with T cell anergy or exhaustion and persistent intracellular infections. We aimed to study the role of PD-1 during T. cruzi-induced acute myocarditis in mice. Cytometry assays showed that PD-1 and its ligands are strongly upregulated in lymphocytes and APC in response to T. cruzi infection in vivo and in vitro. Lymphocytes infiltrating the myocardium exhibited high levels of expression of these molecules. An increased cardiac inflammatory response was found in mice treated with blocking antibodies against PD-1, PD-L1, and to a lesser extent, PD-L2, compared to that found in mice treated with rat IgG. Similar results in PD-1(-/-) mice were obtained. Moreover, the PD-1 blockade/deficiency led to reduced parasitemia and tissue parasitism but increased mortality. These results suggest the participation of a PD-1 signaling pathway in the control of acute myocarditis induced by T. cruzi and provide additional insight into the regulatory mechanisms in the pathogenesis of Chagas` disease.

Expression of vascular endothelian growth factor-C does not predict occult lymphnode metastasis in early oral squamous cell carcinoma

Strong vascular endothelial growth factor-C (VEGF-C) expression has been correlated to occurrence of lymph-node metastases in patients with oral squamous cell carcinoma (OSCC). The incidence of occult lymph-node metastasis remains a decisive factor in the prognosis of patients with early OSCC. The aim of this study was to evaluate VEGF-C expression as a predictor of occult lymph-node metastasis in OSCC. Eighty-seven patients with primary OSCC arising in the tongue or floor of mouth, clinically T1N0M0 or T2N0M0, with (pN+) and without (pN0) occult lymph-node metastases were analyzed for VEGF-C expression by malignant cells. Occult lymph-node metastases (pN+) were detected in 22% of the 64 patients who were submitted to elective neck dissection. No statistically significant difference was found between OSCC with and without occult lymph-node metastasis in regard to VEGF-C immunoexpression by malignant cells and clinicopathologic features. Independently of VEGF-C expression, lymph-node metastasis (PN+) was the most significant prognostic factor for overall survival of patients with OSCC (p = 0.030). These findings indicate that isolated VEGF-C expression by malignant cells is not of predictive value for occult lymph-node metastasis in the early stages of OSCC.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

Vitamin D receptor haplotypes affect lead levels during pregnancy

Pregnant women are particularly susceptible to toxic effects associated with lead (Pb) exposure. Pb accumulates in bone tissue and is rapidly mobilized from bones during pregnancy, thus resulting in fetal contamination. While vitamin D receptor (VDR) polymorphisms modify bone mineralization and affect Pb biomarkers including blood (Pb-B) and serum (Pb-S) Pb concentrations, and %Pb-S/Pb-B ratio, the effects of these polymorphisms on Pb levels in pregnant women are unknown. This study aimed at examining the effects of three (Fokl, Bsml and Apal) VDR polymorphisms (and VDR haplotypes) on Pb levels in pregnant women. Pb-B and Pb-S were determined by inductively coupled plasma mass spectrometry in samples from 256 healthy pregnant women and their respective umbilical cords. Genotypes for the VDR polymorphisms were determined by PCR and restriction fragment length digestion. While the three VDR polymorphisms had no significant effects on Pb-B, Pb-S or %Pb-S/Pb-B ratio, the haplotype combining the f, a, and b alleles for the Fokl, Apal and Bsml polymorphisms, respectively, was associated with significantly lower Pb-S and %Pb-S/Pb-B (P<0.05). However, maternal VDR haplotypes had no effects on Pb levels in the umbilical cords. To our knowledge, this is the first study showing that a combination of genetic polymorphisms (haplotype) commonly found in the VDR gene affects Pb-S and %Pb-S/Pb-B ratios in pregnant women. These findings may have major implications for Pb toxicity because they may help to predict the existence of a group of subjects that is genetically less prone to Pb toxicity during pregnancy. (C) 2010 Elsevier B.V. All rights reserved.

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.

Arachidonic acid stimulates glucose uptake in 3T3-L1 adipocytes by increasing GLUT1 and GLUT4 levels at the plasma membrane: Evidence for involvement of lipoxygenase metabolites and peroxisome proliferator-activated receptor

Exposure of insulin-sensitive tissues to free fatty acids can impair glucose disposal through inhibition of carbohydrate oxidation and glucose transport. However, certain fatty acids and their derivatives can also act as endogenous ligands for peroxisome proliferator-activated receptor gamma (PPARgamma ), a nuclear receptor that positively modulates insulin sensitivity. To clarify the effects of externally delivered fatty acids on glucose uptake in an insulin-responsive cell type, we systematically examined the effects of a range of fatty acids on glucose uptake in 3T3-L1 adipocytes. Of the fatty acids examined, arachidonic acid (AA) had the greatest positive effects, significantly increasing basal and insulin-stimulated glucose uptake by 1.8- and 2-fold, respectively, with effects being maximal at 4 h at which time membrane phospholipid content of AA was markedly increased. The effects of AA were sensitive to the inhibition of protein synthesis but were unrelated to changes in membrane fluidity. AA had no effect on total cellular levels of glucose transporters, but significantly increased levels of GLUT1 and GLUT4 at the plasma membrane. While the effects of AA were insensitive to cyclooxygenase inhibition, the lipoxygenase inhibitor, nordihydroguaiaretic acid, substantially blocked the AA effect on basal glucose uptake. Furthermore, adenoviral expression of a dominant-negative PPARgamma mutant attenuated the AA potentiation of basal glucose uptake. Thus, AA potentiates basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes by a cyclooxygenase-independent mechanism that increases the levels of both GLUT1 and GLUT4 at the plasma membrane. These effects are at least partly dependent on de novo protein synthesis, an intact lipoxygenase pathway and the activation of PPARgamma with these pathways having a greater role in the absence than in the presence of insulin.

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.

Dengue virus binding to human leukocyte cell lines: receptor usage differs between cell types and virus strains

Monocyte macrophages (M phi) are thought to be the principal target cells for the dengue viruses (DV), the cause of dengue fever and hemorrhagic fever. Cell attachment is mediated by the virus envelope (E) protein, but the host-cell receptors remain elusive. Currently, candidate receptor molecules include proteins, Fc receptors, glycosaminoglycans (GAGs) and lipopolysaccharide binding CD14-associated molecules. Here, we show that in addition to M phi, cells of the T- and B-cell lineages, and including cells lacking GAGs, can bind and become infected with DV. The level of virus binding varied widely between cell lines and, notably, between virus strains within a DV serotype. The latter difference may be ascribable to one or more amino acid differences in domain II of the E protein. Heparin had no significant effect on DV binding, while heparinase treatment of cells in all cases increased DV binding, further supporting the contention that GAGs are not required for DV binding and infection of human cells. In contrast to a recent report, we found that lipopolysaccharide (LPS) had either no effect or enhanced DV binding to, and infection of various human leukocyte cell lines, while in all virus-cell combinations, depletion of Ca2+/Mg2+ enhanced DV binding. This argues against involvement of beta (2) integrins in virus-host cell interactions, a conclusion in accord with the demonstration of three virus binding membrane proteins of < 75 kDa. Collectively, the results of this study question the purported exclusive importance of the E protein domain III in DV binding to host cells and point to a far more complex interaction between various target cells and, notably, individual DV strains. (C) 2001 Elsevier Science B.V. All rights reserved.

Melanocortin-1 receptor genotype is a risk factor for basal and squamous cell carcinoma

MC1R gene variants have previously been associated with red hair and fair skin color, moreover skin ultraviolet sensitivity and a strong association with melanoma has been demonstrated for three variant alleles that are active in influencing pigmentation: Arg151Cys, Arg160Trp, and Asp294His. This study has confirmed these pigmentary associations with MC1R genotype in a collection of 220 individuals drawn from the Nambour community in Queensland, Australia, 111 of whom were at high risk and 109 at low risk of basal cell squamous cell carcinoma. Comparative allele frequencies for nine MC1R variants that have been reported in the Caucasian population were determined for these two groups, and an association between prevalence of basal cell carcinoma, squamous cell carcinoma, solar keratosis and the same three active MC1R variant alleles was demonstrated [odds ratio=3.15 95% CI (1.7, 5.82)]. Three other commonly occurring variant alleles: Val60Leu, Val92Met, and Arg163Gln were identified as having a minimal impact on pigmentation phenotype as well as basal cell carcinoma and squamous cell carcinoma risk. A significant heterozygote effect was demonstrated where individuals carrying a single MC1R variant allele were more likely to have fair and sun sensitive skin as well as carriage of a solar lesion when compared with those individuals with a consensus MC1R genotype. After adjusting for the effects of pigmentation on the association between MC1R variant alleles and basal cell carcinoma and squamous cell carcinoma risk, the association persisted, confirming that presence of at least one variant allele remains informative in terms of predicting risk for developing a solar-induced skin lesion beyond that information gained through observation of pigmentation phenotype.

A titrimetric respirometer measuring the nitrifiable nitrogen in wastewater using in-sensor-experiment

Measurement of nitrifiable nitrogen contained in wastewater by combining the existing respirometric and titrimetric principles is reported. During an in-sensor-experiment using nitrifying activated sludge. both the dissolved oxygen (DO) and pH in the mixed liquor were measured, and the FH was controlled at a set-point through titration of base or acid. A combination of the oxygen uptake rate (OUR), which was obtained from the measured DO signal, and the titration data allowed calculation of the nitrifiable nitrogen and the short-term biological oxygen demand (BOD) of the wastewater sample that was initially added to the sludge. The calculation was based solely on stoichiometric relationships. The approach was preliminarily tested with two types of wastewaters using a prototype sensor. Good correlation was obtained. (C) 2000 Elsevier Science Ltd. All rights reserved.

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-P belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/ AIB-1. In this study we demonstrate that SRC-P is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-P to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-P, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-P and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

Activation of the peroxisome proliferator-activated receptor-alpha enhances cell death in cultured cerebellar granule cells

Peroxisome proliferator-activated receptor-alpha (PPAR alpha) is a member of the steroid hormone receptor superfamily. In rodents, PPAR alpha. alters genes involved in cell cycle regulation in hepatocytes. Some of these genes are implicated in neuronal cell death. Therefore, in this study, we examined the toxicological consequence of PPAR alpha activation in rat primary cultures of cerebellar granule neurons. Our studies demonstrated the presence of PPAR alpha mRNA in cultures by reverse transcriptase-polymerase chain reaction. After 10 days in vitro, cerebellar granule neuron cultures were incubated with the selective PPAR alpha activator 4-chloro-6-(2,3-xylidino)2-pyrimidinylthioacetic acid (Wy-14,643). The inherent toxicity of Wy-14,643 and the effect of PPAR alpha activation following toxic stimuli were assessed. In these studies, neurotoxicity was induced through reduction of extracellular [KCl] from 25 mM to 5.36 mM. We observed no inherent toxicity of Wy-1 4,643 (24 hr) in cultured cerebellar granule cells. However, after reduction of [KCl], cerebellar granule cell cultures incubated with Wy-14,643 showed significantly greater toxicity than controls. These results suggest a posssible role for PPAR(x in augmentation of cerebellar granule neuronal death after toxic stimuli. (C) 2001 Wiley-Liss, Inc.