Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows

The objective of this study was to evaluate the plasma concentrations of insulin-like growth factor-I (IGF-I), and the mRNA hepatic expression of IGF-I and of the growth hormone receptors GHR and GHR 1A, in postpartum beef cows. Four Angus and four crossbred (Angus x Nelore) postpartum suckled beef cows were used. Liver and blood samples were collected every 10 days, from calving to 40 days postpartum, for gene expression and for β-hydroxybutyrate and IGF-I assays, respectively. Samples for progesterone assay were collected every other day, from day 10 to 40 postpartum. Three cows ovulated before 40 days postpartum. IGF-I concentration was higher in Angus x Nelore than in Angus cows. There was no difference in the expression of GHR, GHR 1A and IGF-I according to breed or ovulatory status. IGF-I concentrations were higher in crossbred cows, but have not changed according to postpartum ovulatory status. Moreover, changes in postpartum IGF-I concentrations are not associated with changes in liver GHR, GHR 1A and IGF-I mRNA expression in either breed.

Participation of the endoplasmic reticulum protein chaperone thio-oxidoreductase in gonadotropin-releasing hormone receptor expression at the plasma membrane

Chaperone members of the protein disulfide isomerase family can catalyze the thiol-disulfide exchange reaction with pairs of cysteines. There are 14 protein disulfide isomerase family members, but the ability to catalyze a thiol disulfide exchange reaction has not been demonstrated for all of them. Human endoplasmic reticulum protein chaperone thio-oxidoreductase (ERp18) shows partial oxidative activity as a protein disulfide isomerase. The aim of the present study was to evaluate the participation of ERp18 in gonadotropin-releasing hormone receptor (GnRHR) expression at the plasma membrane. Cos-7 cells were cultured, plated, and transfected with 25 ng (unless indicated) wild-type human GnRHR (hGnRHR) or mutant GnRHR (Cys14Ala and Cys200Ala) and pcDNA3.1 without insert (empty vector) or ERp18 cDNA (75 ng/well), pre-loaded for 18 h with 1 µCi myo-[2-3H(N)]-inositol in 0.25 mL DMEM and treated for 2 h with buserelin. We observed a decrease in maximal inositol phosphate (IP) production in response to buserelin in the cells co-transfected with hGnRHR, and a decrease from 20 to 75 ng of ERp18 compared with cells co-transfected with hGnRHR and empty vector. The decrease in maximal IP was proportional to the amount of ERp18 DNA over the range examined. Mutants (Cys14Ala and Cys200Ala) that could not form the Cys14-Cys200 bridge essential for plasma membrane routing of the hGnRHR did not modify maximal IP production when they were co-transfected with ERp18. These results suggest that ERp18 has a reduction role on disulfide bonds in wild-type hGnRHR folding.

Prolactin inhibits auto- and cross-induction of thyroid hormone and estrogen receptor and vitellogenin genes in adult Xenopus (Amphibia) hepatocytes

It is well known that virtually every tissue of the amphibian larvae is highly sensitive to the mutually antagonistic actions of thyroid hormone (TH) and prolactin (PRL), but it is not known if adult amphibian tissues respond similarly to these two hormones. We have previously shown that very low doses of triiodothyronine (T3) rapidly and strongly potentiate the activation of silent vitellogenin (Vit) genes by estrogen (E2) and the autoinduction of estrogen receptor (ER) transcripts in primary cultures of adult Xenopus hepatocytes. This response to T3 is accompanied by the upregulation of thyroid hormone receptor b (TRb) mRNA. Using Northern blot and RNase protection assays, we now show that ovine PRL added for 12 h along with 2 x 10-9 M T3 will completely prevent potentiation of E2 induction of Vit mRNA in primary cultures of adult Xenopus hepatocytes. PRL also abolished the auto-upregulation of TRb mRNA and the cross-activation of autoinduction of ER mRNA. Thus, we show for the first time that the anti-TH action of PRL that is manifested in Xenopus tadpole tissues during metamorphosis is retained in adult liver, and suggest that the mutually antagonistic actions of the two hormones may be brought about by similar molecular mechanisms in larval and adult amphibian tissues

Immunohistochemical expression of oestrogen and progesterone receptors during experimental acute and chronic murine Schistosomiasis mansoni

INTRODUCTION: The responsibility of Schistosoma mansoni in female infertility is still controversial. This study was conducted to evaluate the effect of acute and chronic schistosomiasis mansoni infection on the endometrium using immunohistochemical analysis of uterine hormone receptor expression. METHODS: Twenty-four nonpregnant swiss albino mice were divided into three groups: control, noninfected; acute; and chronic Schistosoma mansoni infection. Histological sections of uterine specimens were examined by light microscope with an image analyzing system to detect structural histological, estrogen receptor (ER) and progesterone receptor (PR) expression in the endometrium. RESULTS: No secretory phase was detected in the endometrium in acute and chronic Schistosoma infection. Hormone receptor expression (ER and PR) showed statistically significant differences among the groups (p< 0.05), with significant low ER hormone expression in chronic infection, compared to control proliferative, control secretory and acute infection cases, and statistically significant high PR expression in both acute and chronic infection cases compared to the control secretory cases (p< 0.05). CONCLUSIONS: Schistosomiasis mansoni seems to have an important impact on the hormone expression of affected women. Further studies to explore the mechanism of such changes are recommended.

Expression of non-TLR pattern recognition receptors in the spleen of BALB/c mice infected with Plasmodium yoelii and Plasmodium chabaudi chabaudi AS

The spleen plays a crucial role in the development of immunity to malaria, but the role of pattern recognition receptors (PRRs) in splenic effector cells during malaria infection is poorly understood. In the present study, we analysed the expression of selected PRRs in splenic effector cells from BALB/c mice infected with the lethal and non-lethal Plasmodium yoelii strains 17XL and 17X, respectively, and the non-lethal Plasmodium chabaudi chabaudi AS strain. The results of these experiments showed fewer significant changes in the expression of PRRs in AS-infected mice than in 17X and 17XL-infected mice. Mannose receptor C type 2 (MRC2) expression increased with parasitemia, whereas Toll-like receptors and sialoadhesin (Sn) decreased in mice infected with P. chabaudi AS. In contrast, MRC type 1 (MRC1), MRC2 and EGF-like module containing mucin-like hormone receptor-like sequence 1 (F4/80) expression decreased with parasitemia in mice infected with 17X, whereas MRC1 an MRC2 increased and F4/80 decreased in mice infected with 17XL. Furthermore, macrophage receptor with collagenous structure and CD68 declined rapidly after initial parasitemia. SIGNR1 and Sn expression demonstrated minor variations in the spleens of mice infected with either strain. Notably, macrophage scavenger receptor (Msr1) and dendritic cell-associated C-type lectin 2 expression increased at both the transcript and protein levels in 17XL-infected mice with 50% parasitemia. Furthermore, the increased lethality of 17X infection in Msr1 -/- mice demonstrated a protective role for Msr1. Our results suggest a dual role for these receptors in parasite clearance and protection in 17X infection and lethality in 17XL infection.

AMPc - Sinalização intracelular: um software educacional

Chemicals binding to membrane receptors may induce events within the cell changing its behavior. Since these events are simultaneous and hard to be understood by students, we developed a computational model to dynamically and visually explore the cAMP signaling system to facilitate its understanding. The animation is shown in parts, from the hormone-receptor binding to the cellular response. There are some questions to be answered after using the model. The software was field-tested and an evaluation questionnaire (concerning usability, animations, models, and the software as an educational tool) was answered by the students, showing the software to be a valuable aid for content comprehension.

Loss of PTEN expression and AKT activation in HER2-positive breast carcinomas

PURPOSE: To examine the expression of AKT and PTEN in a series of HER2-positive primary invasive breast tumors using immunohistochemistry, and to associate these expression profiles with classic pathologic features such as tumor grade, hormone receptor expression, lymphatic vascular invasion, and proliferation.METHODS: A total of 104 HER2-positive breast carcinoma specimens were prepared in tissue microarrays blocks for immunohistochemical detection of PTEN and phosphorylated AKT (pAKT). Original histologic sections were reviewed to assess pathological features, including HER2 status and Ki-67 index values. The associations between categorical and numeric variables were identified using Pearson's chi-square test and the Mann-Whitney, respectively.RESULTS: Co-expression of pAKT and PTEN was presented in 59 (56.7%) cases. Reduced levels of PTEN expression were detected in 20 (19.2%) cases, and these 20 tumors had a lower Ki-67 index value. In contrast, tumors positive for pAKT expression [71 (68.3%)] were associated with a higher Ki-67 index value.CONCLUSION: A role for AKT in the proliferation of HER2-positive breast cancers was confirmed. However, immunohistochemical detection of PTEN expression did not correlate with an inhibition of cellular proliferation or control of AKT phosphorylation, suggesting other pathways in these mechanisms of control.

Genetic analysis and cAMP measurement: comparison between lean and obese anovulating mice

PURPOSE: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups.METHODS: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method.RESULTS: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229±52 fMol) compared to the Control Group (1,814±45 fMol).CONCLUSIONS: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Prevalence of ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms in Brazilian breast cancer-unaffected women

Polymorphisms of hormone receptor genes have been linked to modifications in reproductive factors and to an increased risk of breast cancer (BC). In the present study, we have determined the allelic and genotypic frequencies of the ERα-397 PvuII C/T, ERα-351 XbaI A/G and PGR PROGINS polymorphisms and investigated their relationship with mammographic density, body mass index (BMI) and other risk factors for BC. A consecutive and unselected sample of 750 Brazilian BC-unaffected women enrolled in a mammography screening program was recruited. The distribution of PGR PROGINS genotypic frequencies was 72.5, 25.5 and 2.0% for A1A1, A1A2 and A2A2, respectively, which was equivalent to that encountered in other studies with healthy women. The distribution of ERα genotypes was: ERα-397 PvuII C/T: 32.3% TT, 47.5% TC, and 20.2% CC; ERα-351 XbaI A/G: 46.3% AA, 41.7% AG and 12.0% GG. ERα haplotypes were 53.5% PX, 14.3% Px, 0.3% pX, and 32.0% px. These were significantly different from most previously published reports worldwide (P < 0.05). Overall, the PGR PROGINS genotypes A2A2 and A1A2 were associated with fatty and moderately fatty breast tissue. The same genotypes were also associated with a high BMI in postmenopausal women. In addition, the ERα-351 XbaI GG genotype was associated with menarche ≥12 years (P = 0.02). ERα and PGR polymorphisms have a phenotypic effect and may play an important role in BC risk determination. Finally, if confirmed in BC patients, these associations could have important implications for mammographic screening and strategies and may be helpful to identify women at higher risk for the disease.

Single-nucleotide polymorphism analysis of GH, GHR, and IGF-1 genes in minipigs

Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits - body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC) - three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.

Metronomic chemotherapy in the neoadjuvant setting: results of two parallel feasibility trials (TraQme and TAME) in patients with HER2+ and HER2− locally advanced breast cancer

Neoadjuvant chemotherapy has practical and theoretical advantages over adjuvant chemotherapy strategy in breast cancer (BC) management. Moreover, metronomic delivery has a more favorable toxicity profile. The present study examined the feasibility of neoadjuvant metronomic chemotherapy in two cohorts [HER2+ (TraQme) and HER2− (TAME)] of locally advanced BC. Twenty patients were prospectively enrolled (TraQme, n=9; TAME, n=11). Both cohorts received weekly paclitaxel at 100 mg/m2 during 8 weeks followed by weekly doxorubicin at 24 mg/m2 for 9 weeks in combination with oral cyclophosphamide at 100 mg/day (fixed dose). The HER2+ cohort received weekly trastuzumab. The study was interrupted because of safety issues. Thirty-six percent of patients in the TAME cohort and all patients from the TraQme cohort had stage III BC. Of note, 33% from the TraQme cohort and 66% from the TAME cohort displayed hormone receptor positivity in tumor tissue. The pathological complete response rates were 55% and 18% among patients enrolled in the TraQme and TAME cohorts, respectively. Patients in the TraQme cohort had more advanced BC stages at diagnosis, higher-grade pathological classification, and more tumors lacking hormone receptor expression, compared to the TAME cohort. The toxicity profile was also different. Two patients in the TraQme cohort developed pneumonitis, and in the TAME cohort we observed more hematological toxicity and hand-foot syndrome. The neoadjuvant metronomic chemotherapy regimen evaluated in this trial was highly effective in achieving a tumor response, especially in the HER2+ cohort. Pneumonitis was a serious, unexpected adverse event observed in this group. Further larger and randomized trials are warranted to evaluate the association between metronomic chemotherapy and trastuzumab treatment.

Effect of estrogen receptor-alpha (ESR1) gene polymorphism on high density lipoprotein levels in response to hormone replacement therapy

Studies have shown that estrogen replacement therapy and estrogen plus progestin replacement therapy alter serum levels of total, LDL and HDL cholesterol levels. However, HDL cholesterol levels in women vary considerably in response to hormone replacement therapy (HRT). A significant portion of the variability of these levels has been attributed to genetic factors. Therefore, we investigated the influence of estrogen receptor-alpha (ESR1) gene polymorphisms on HDL levels in response to postmenopausal HRT. We performed a prospective cohort study on 54 postmenopausal women who had not used HRT before the study and had no significant general medical illness. HRT consisted of conjugated equine estrogen and medroxyprogesterone acetate continuously for 1 year. The lipoprotein levels were measured from blood samples taken before the start of therapy and after 1 year of HRT. ESR1 polymorphism (MspI C>T, HaeIII C>T, PvuII C>T, and XbaI A>G) frequencies were assayed by restriction fragment length polymorphism. A general linear model was used to describe the relationships between HDL levels and genotypes after adjusting for age. A significant increase in HDL levels was observed after HRT (P = 0.029). Women with the ESR1 PvuII TT genotype showed a statistically significant increase in HDL levels after HRT (P = 0.032). No association was found between other ESR1 polymorphisms and HDL levels. According to our results, the ESR1 PvuII TT genotype was associated with increased levels of HDL after 1 year of HRT.

The insulin receptor substrate 1 associates with phosphotyrosine phosphatase SHPTP2 in liver and muscle of rats

Insulin stimulates the tyrosine kinase activity of its receptor resulting in the phosphorylation of its cytosolic substrate, insulin receptor substrate-1 (IRS-1) which, in turn, associates with proteins containing SH2 domains. It has been shown that IRS-1 associates with the tyrosine phosphatase SHPTP2 in cell cultures. While the effect of the IRS-1/SHPTP2 association on insulin signal transduction is not completely known, this association may dephosphorylate IRS-1 and may play a critical role in the mitogenic actions of insulin. However, there is no physiological demonstration of this pathway of insulin action in animal tissues. In the present study we investigated the ability of insulin to induce association between IRS-1 and SHPTP2 in liver and muscle of intact rats, by co-immunoprecipitation with anti-IRS-1 antibody and anti-SHPTP2 antibody. In both tissues there was an increase in IRS-1 association with SHPTP2 after insulin stimulation. This association occurred when IRS-1 had the highest level of tyrosine phosphorylation and the decrease in this association was more rapid than the decrease in IRS-1 phosphorylation levels. The data provide evidence against the participation of SHPTP2 in IRS-1 dephosphorylation in rat tissues, and suggest that the insulin signal transduction pathway in rat tissues is related mainly to the mitogenic effects of the hormone.

Antisense mRNA for NPY-Y1 receptor in the medial preoptic area increases prolactin secretion

We investigated the participation of neuropeptide Y-Y1 receptors within the medial preoptic area in luteinizing hormone, follicle-stimulating hormone and prolactin release. Four bilateral microinjections of sense (control) or antisense 18-base oligonucleotides of messenger ribonucleic acid (mRNA) (250 ng) corresponding to the NH2-terminus of the neuropeptide Y1 receptor were performed at 12-h intervals for two days into the medial preoptic area of ovariectomized Wistar rats (N = 16), weighing 180 to 200 g, treated with estrogen (50 µg) and progesterone (25 mg) two days before the experiments between 8.00 and 10:00 a.m. Blockade of Y1 receptor synthesis in the medial preoptic area by the antisense mRNA did not change plasma luteinizing hormone or follicle-stimulating hormone but did increase prolactin from 19.6 ± 5.9 ng/ml in the sense group to 52.9 ± 9.6 ng/ml in the antisense group. The plasma hormones were measured by radioimmunoassay and the values are reported as mean ± SEM. These data suggest that endogenous neuropeptide Y in the medial preoptic area has an inhibitory action on prolactin secretion through Y1 receptors.

The diversity of abnormal hormone receptors in adrenal Cushing's syndrome allows novel pharmacological therapies

Recent studies from several groups have indicated that abnormal or ectopic expression and function of adrenal receptors for various hormones may regulate cortisol production in ACTH-independent hypercortisolism. Gastric inhibitory polypeptide (GIP)-dependent Cushing's syndrome has been described in patients with either unilateral adenoma or bilateral macronodular adrenal hyperplasia; this syndrome results from the large adrenal overexpression of the GIP receptor without any activating mutation. We have conducted a systematic in vivo evaluation of patients with adrenal Cushing's syndrome in order to identify the presence of abnormal hormone receptors. In macronodular adrenal hyperplasia, we have identified, in addition to GIP-dependent Cushing's syndrome, other patients in whom cortisol production was regulated abnormally by vasopressin, ß-adrenergic receptor agonists, hCG/LH, or serotonin 5HT-4 receptor agonists. In patients with unilateral adrenal adenoma, the abnormal expression or function of GIP or vasopressin receptor has been found, but the presence of ectopic or abnormal hormone receptors appears to be less prevalent than in macronodular adrenal hyperplasia. The identification of the presence of an abnormal adrenal receptor offers the possibility of a new pharmacological approach to control hypercortisolism by suppressing the endogenous ligands or by using specific antagonists for the abnormal receptors.