Hormonal effects on the secretion and glycoform profile of corticosteroid-binding globulin

Corticosteroid-binding globulin (CBG) is a plasma glycoprotein that is primarily synthesized in the liver and binds cortisol and progesterone with high affinity. In this study, a CBG secreting hepatocellular carcinoma derived cell line (HepG2) was used to investigate the hormonal regulation of hepatic CBG synthesis. HepG2 cells were grown for 72 h in 30, 300 and 3000 nM concentrations of estradiol (E₂), testosterone (T), insulin, thyroxin (T₄) and dexamethasone (DMZ) and the secreted CBG quantified by a novel enzyme-linked immunosorbent assay (ELISA). Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) was carried out to determine the effects of these hormones on the relative distribution of CBG glycoforms.

Insulin, T₄ and high concentrations of E₂ decreased the secretion of CBG by HepG2 cells (p < 0.05). Ethanol, the solvent used for E₂, T and DMZ, also significantly attenuated CBG secretion. 2D-PAGE resolved 13–14 glycoforms of CBG produced by HepG2 cells. Insulin caused a reduction in the synthesis of more acidic, while T₄ and DMZ decreased the production of more basic CBG glycoforms. Stimulation with E₂ resulted in the synthesis of additional isoforms of increased acidity, which may represent a type of CBG only seen during pregnancy in vivo. Possible physiological implications of these findings are discussed.     

Conversion of proteins from a non-polarized to an apical secretory pattern in MDCK cells

Previously it was shown that fusion proteins containing the amino terminus of an apical targeted member of the serpin family fused to the corresponding carboxyl terminus of the non-polarized secreted serpin, antithrombin, are secreted mainly to the apical side of MDCK cells. The present study shows that this is neither due to the transfer of an apical sorting signal from the apically expressed proteins, since a sequence of random amino acids acts the same, nor is it due to the deletion of a conserved signal for correct targeting from the non-polarized secreted protein. Our results suggest that the polarity of secretion is determined by conformational sensitive sorting signals.     

Characterization of the first adult de novo case of 46,X,der(Y)t(X;Y)(p22.3;q11.2)

Herein, we describe a case of an infertile man detected in postnatal diagnosis with FISH characterization and array-CGH used for genome-wide screening which allowed the identification of a complex rearrangement involving sex chromosomes, apparently without severe phenotypic consequences. The deletion detected in our patient has been compared with previously reported cases leading us to propose a hypothetical diagnostic algorithm that would be useful in similar clinical situations, with imperative multi disciplinary approach integrated with genetic counseling. Our patient, uniquely of reproductive age, is one of six reported cases of duplication of Xp22.3 (~ 8.4 Mb) segment and contemporary deletion of Yq (~ 42.9 Mb) with final karyotype as follows:

46,X,der(Y),t(X;Y)(Ypter → Yq11.221::Xp22.33 → Xpter).ish der(Y) (Yptel+,Ycen+,RP11-529I21+,RP11-506M9-Yqtel −,Xptel +). arrXp22.33p22.31(702–8,395,963, 8,408,289x1), Yq11.221q12 (14,569,317x1, 14,587,321–57,440,839x0)     

The cortisol response to ACTH in pigs,heritability and influence of corticosteroid-binding globulin

In the search for biological basis of robustness, this study aimed (i) at the determination of the heritability of the cortisol response to ACTH in juvenile pigs, using restricted maximum likelihood methodology applied to a multiple trait animal model, and (ii) at the study of the relationships between basal and stimulated cortisol levels with corticosteroid-binding globulin (CBG), IGF-I and haptoglobin, all important players in glucose metabolism and production traits. At 6 weeks of age, 298 intact male and female piglets from 30 litters (30 dams and 30 boars) were injected with 250 µg ACTH(1–24) (Synacthen). Blood was taken before ACTH injection to measure basal levels of cortisol, glucose, CBG, IGF-I and haptoglobin, and 60 min later to measure stimulated cortisol levels and glucose. Cortisol increased 2.8-fold after ACTH injection, with a high correlation between basal and stimulated levels (phenotypic correlation, r_p=0.539; genetic correlation, r_g=0.938). Post-ACTH cortisol levels were highly heritable (h²=0.684) and could therefore be used for genetic selection of animals with a more reactive hypothalamic–pituitary–adrenocortical axis. CBG binding capacity correlated with cortisol levels measured in basal conditions in males only. No correlation was found between CBG binding capacity and post-ACTH cortisol levels. Basal IGF-I concentration was positively correlated with BW at birth and weaning, and showed a high correlation with CBG binding capacity with a strong sexual dimorphism, the correlation being much higher in males than in females. Basal haptoglobin concentrations were negatively correlated with CBG binding capacity and IGF-I concentrations. Complex relationships were also found between circulating glucose levels and these different variables that have been shown to be related to glucose resistance in humans. These data are therefore valuable for the genetic selection of animals to explore the consequences on production and robustness traits, but also point at pigs as a relevant model to explore the underlying mechanisms of the metabolic syndrome including the contribution of genetic factors.     

Measurement of cortisol,cortisone, prednisolone,dexamethasone and 11-deoxycortisol with ultra high performance liquid chromatography–tandem mass spectrometry: Application for plasma,plasma ultrafiltrate,urine and saliva in a routine laboratory

We describe an ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC MS/MS) method suitable for a routine laboratory to determine endogenous and exogenous glucocorticoids in plasma, plasma ultrafiltrate, urine and saliva in a single analytical run. After addition of a multi-analyte internal standard, a standardised sample preparation procedure with solid phase extraction followed, before injecting into a tandem mass spectrometer with positive mode electron spray ionisation and multiple reactant monitoring acquisition. The chromatography time was 3 min. The limit of quantitation for cortisol and cortisone in plasma was 3.75 nmol/L and linearity extended to 2000 nmol/L. The limit of quantitation for cortisol in plasma ultrafiltrate and saliva was 0.6 nmol/L. The limit of quantitation for 11-deoxycortisol and prednisolone was 5 nmol/L and for dexamethasone 1 nmol/L. The intra-assay CV was <5% and the inter-assay CV <10% for all analytes in all matrices. Comparison with an immuno-assay (IA) plasma cortisol method resulted in a regression equation of UHPLC = 0.79 × IA + 31.12 with R² = 0.960 (p < 0.0001). Comparison with a high performance liquid chromatography (HPLC) cortisol method yielded a regression equation of UHPLC = 1.06 × HPLC + 9.82, R² = 0.992 (p < 0.0001). The simultaneous measurement of endogenous and exogenous glucocorticoids contributed to patient care in cases with dexamethasone and metyrapone dynamic tests and unsuspected therapeutic glucocorticoid use.     

Temperature dependence of kinetic interactions between progesterone and the uterine cytoplasmic receptor

The temperature dependence of the rates of dissociation and association for progesterone-receptor interactions was measured over the temperature range of 0–20°C. The dissociation process is biphasic indicating that either two forms of receptor are present or that the binding of progesterone to the receptor is a concatenated reaction.The enthalpy of activation for the dissociation of progesterone from the receptor is about 26–28 kcal/mol and the entropic energy of activation is about ?5 kcal/mol. The enthalpy of activation for the association of these molecules is about 3 kcal/mol and the entropic energy of activation is about 6 kcal/mol. These data are consistent with a model of progesterone binding to the receptor that includes hydrogen bonds between each of the two ketone groups and hydrogen donors on the receptor protein and involves van der Waals' interactions, due to the close proximity of the receptor binding site to a large fraction of the progesterone surface.     

First cytogenetic information for Drymoreomys albimaculatus (Rodentia,Cricetidae), a recently described genus from Brazilian Atlantic Forest

The recently described taxon Drymoreomys albimaculatus is endemic to the Brazilian Atlantic Forest and its biology and genetics are still poorly known. Herein, we present, for the first time, the karyotype of the species using classical and molecular cytogenetics, which showed 2n=62, FN=62, and interstitial telomeric signals at the sex chromosomes. Nuclear and mitochondrial DNA sequences from the two karyotyped individuals verify the taxonomic identity as the recently described Drymoreomys albimaculatus and confirm the relationship of the species with other Oryzomyini. Additionally, external morphological information is provided.     

Paradoxical nature of estrogen agonist and antagonist binding in rat liver

Whereas Tamoxifen exerts potent antiestrogenic action in ER dependent breast cancer, it was largely without effect on rat liver gluconeogenesis which could be dramatically diminished by estrogens and androgens. Although estradiol was preferentially bound to an ER₄ component that coeluted with CBG from DE-52 columns, ³H-tamoxifen labelled the ER₃ moiety that was clearly distinct from transcortin. Similarly, testosterone was bound to the AR₄ entity but R-1881 was eluted in the AR₃ region. All these ER and AR populations were furthermore distinct from liver GR. These, for the first time, demonstrate polymorphic nature of AR and ER and suggest that agonist and antagonist actions may be expressed via separate populations of the receptor, contrary to the established, classical view that dictates competitive antagonism between them for the one and the same site.     

A requirement for ubiquinone in ATPase activity and oxidative phosphorylation.

The suggestion that a rapidly sedimenting rough endoplasmic reticulum fraction in close association with mitochondria, is the preferred site of cytochrome P-450 synthesis has been examined. The rate of cytochrome P-450 synthesis in the different subcellular fractions has been evaluated $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$, using the immunoprecipitation technique. The results indicate that the conventional microsomal fraction (100,000 X g sediment) is the major site of cytochrome P-450 synthesis and that the rapidly sedimenting rough endoplasmic reticulum fraction associated with mitochondria is not a preferred site for the hemoprotein synthesis.