Georges Hennen

Housekeeping genes as internal standards: use and limits

Quantitative studies are commonly realised in the biomedical research to compare RNA expression in different experimental or clinical conditions. These quantifications are performed through their comparison to the expression of the housekeeping gene transcripts like glyceraldehyde-3-phosphate dehydrogenase (G3PDH), albumin, actins, tubulins, cyclophilin, hypoxantine phsophoribosyltransferase (HRPT), L32. 28S, and 18S rRNAs are also used as internal standards. In this paper, it is recalled that the commonly used internal standards can quantitatively vary in response to various factors. Possible variations are illustrated using three experimental examples. Preferred types of internal standards are then proposed for each of these samples and thereafter the general procedure concerning the choice of an internal standard and the way to manage its uses are discussed.

Placental Growth Hormone Levels in Normal Pregnancy and in Pregnancies with Intrauterine Growth Retardation

ABSTRACT: To assess the possible role of placental growth hormone (GH) in fetoplacental growth, we measured placental and pituitary GH (GHN) in maternal plasma by means of two RIA using two MAb (5B4 recognizing both placental GH and GHN, and K24 recognizing only GHN) during pregnancy. IGF-I also was measured by RIA in the same samples after extraction. A transverse study of 186 samples obtained between 8 wk of amenorrhea (WA) and term confirmed the reported rise in GH immunoreactivity with 5B4 after 24 to 25 WA from 12.3 ± 2.0 mU/L (mean ± SEM) to a plateau of 27.5 ± 3.4 mU/L at 34 to 35 WA together with the decrease in GHN to undetectable levels by 24 to 25 WA. IGF-I levels increased from 164.0 ± 44.6 μUg/L at 24 to 25 WA to 331.6 ± 63.6 μUg/L at term. A longitudinal study of 31 normal pregnant women confirmed this hormonal pattern and the reported placental GH plateau after 35 WA. A drastic decrease in placental GH was observed with the onset of labor (from 26.9 ± 2.1 to 2.7 ± 1.1 mU/L), whereas the decrease in IGF-I was not significant (from 212.9 ± 26.5 to 162.4 ± 16.9 μUg/L). Interestingly, maternal plasma samples obtained after 31 WA until the initiation of labor in 22 cases of intrauterine growth retardation (six cases of toxemia, one chromosomal aberration, one maternofetal infection, 14 idiopathic) contained significantly lower amounts of placental GH (14.9 ± 1.6 mU/L versus 26.5 ± 1.2 mU/L in normal pregnancies; p < 0.001). Plasma IGF-I levels were also lower than normal (156.0 ± 25.5 μUg/L versus 285.1 ± 40.8 μUg/L; p < 0.001). These results suggest a relationship between placental GH levels in the maternal plasma and the development of the fetoplacental unit.

Effects of pituitary hormones on the prostate

Although essential, androgens alone are not sufficient to induce normal growth and functionality of the prostate. Nonandrogenic hormones must also be involved in the proliferation of the prostate cancer cells which do not respond to antiandrogenic therapy and which thus become androgen‐independent. Prolactin, but also growth hormone and luteinizing hormone, are potentially able to act on both normal and abnormal prostatic cells.

Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-I (IGF-I) and IGF-I receptor levels in the prostate of immature rats.

In this study we investigated the involvement of several different pituitary hormones on rat prostate development. 22-day-old Wistar rats, hypophysectomized (hypox) at 19 days of age were supplemented with highly purified human prolactin (hPRL), human luteinizing hormone (hLH), porcine follicle-stimulating hormone (pFSH), and bovine growth hormone (bGH) or with saline. Quantitative analysis of RNAs shows that treatment with either PRL or GH increases significantly steady-state mRNAs levels of the following genes in the prostate: androgen receptor (AR) (respectively 3.5- and 4.8-fold above hypox controls), IGF-I (5- and 2.7-fold), and IGF-I receptor (2.9- and 2.3-fold). LH and FSH, by contrast, have negative effects on these parameters. To test whether the enhancing effect of PRL and GH on AR-mRNA abundance was followed by increased content in the protein itself, binding assays were performed with the androgen agonist [3H]R1881 (131 and 153 fmol/mg protein while hypox controls contained 110 fmol/mg protein). In addition to the well-documented presence of prolactin receptors in prostatic tissues, we have further demonstrated, by means of nuclease S1 protection assays plus dot- and Northern-blot analyses, that a GH receptor mRNA is produced in the immature rat prostate. Moreover, we observed not only strong lactogenic but also purely somatogenic binding to be occurring in the immature prostates. Finally, we have studied IGF-I mRNA content in separated epithelial/stromal cell fractions and have concluded that IGF-I expression is principally located in the prostatic stroma. Taken together, these results suggest that PRL and GH are involved in regulating AR synthesis, at least partially by direct action on the organ. In this context IGF-I appears as a paracrine factor playing a role in epithelium/stroma interactions during prostatic development.

Porcine luteinizing hormone. The amino acid sequence of the β subunit

At the former International Symposium on Protein and Polypeptide Hormones, Ward et al. [l] presented a paper outlining slight modification in the complete primary structure of ovine luteinizing hormone subunit (0-LHP) as compared with their original results [2] . Simultaneously [3], we presented our data concerning the structure of the bovine chain (B-LH/3). A single point of discrepancy was apparent [3,4] in comparing both sequences which was later cleared on re-examination of the ovine structure [5]. In contrast to the complete homology between 0-LHfl and B-LHP, specific differences were expected for the corresponding porcine subunit (P-LH/3) in view of its composition [6]. In this report, the complete primary structure of the reduced and carboxymethylated P-LHP is presented with comparison to our results concerning the bovine chain (B-LH@.

Evidence for the expression of growth hormone receptors in human placenta

Since human placenta produces a growth hormone variant, it seemed important to search for evidence of GH receptors in that organ. Evidence for the expression of the GH receptor (GHR) gene was obtained by northern blot analysis. In addition, GHR poly A+ RNA was detected in RNA from cultured trophoblastic cells, but not from placenta fibroblasts. There was a low but significant specific binding of pituitary GH-N and placental GH-V to placenta plasma membranes. Both variants apparently bound to the same receptor, which is present in the first trimester as well as in the term placenta. These results suggest that placental GH may have paracrine or autocrine functions in the placenta.

Thyroid-stimulating hormone binding to cultured thyroid cells

The suggestion that thyroid stimulating hormone (TSH) interacts with its target tissue at a superficial cell site was made by Pastan et al. [I] on the basis of experiments showing the persistent effect of TSH on [ l-14C]glucose oxidation in thyroid slices exposed to the hormone and washed thoroughly in hormone-free medium. Additional support to this idea was given by Yamashita and Field [2] and Wolff and Jones [3] who showed that plasma membranes purified from thyroid gland contained a TSH-sensitive adenylcyclase. Previous investigations demonstrated that TSH induced the reorganization into follicles of cultured isolated thyroid cells (Fayet and Tixier [4], Fayet et al. [5] ), via the adenylcyclase-cyclic AMP system (Fayet and Lissitzky [6], Lissitzky et al. [7]) whereas monolayer cultures were obtained in its absence. TSHor dibutyryl cyclic AMP-stimulated cells showed the organizational [8] and the specific metabolic properties of the gland follicular cells [7]. This communication reports the direct measurement of specific TSH-receptor interaction in this system and some of the properties of the binding sites. 2. Materials and methods

Surgical Management of Amiodarone-associated Thyrotoxicosis: Too Risky or Too Effective?

Abstract. Amiodarone-associated thyrotoxicosis, often clinically mild and resolutive after amiodarone discontinuation or under medical therapy, is sometimes drug unresponsive and not uncommonly follows a dramatic, even fatal course. Therefore, we considered a surgical solution in 15 severely amiodarone-associated thyrotoxic patients. Twelve men and three women (mean age 68 years, range 50–84 years) underwent radical thyroidectomy for clinical and biologically proved amiodarone-associated thyrotoxicosis. In six surgery was the first-line therapeutic option. In the other nine thyroidectomy seemed unavoidable considering the unresponsiveness to medical therapy and rapid deterioration of the patients’ clinical condition, with life-threatening cardiac failure in three. In every patient surgery was conducted without immediate or delayed complications. Total thyroidectomy proved uniformly, definitively, and rapidly effective in controlling thyrotoxicosis in all patients, with a spectacular reversal of cardiac failure in the three most critical cases. Surgery was beneficial to our 15 patients and undoubtedly life-saving in the three most worrying cases. These results suggest that thyroidectomy should be more liberally regarded as an interesting alternative to conventional, but unpredictably effective, medical therapies.

Placental growth hormone and IGF-I in a pregnant woman with Pit-1 deficiency

The respective contributions of pituitary and placental GH to circulating IGF‐I in pregnant women have not been well established. We measured the serum concentrations of placental growth hormone (PGH) and IGF‐I in a woman with pit‐1 deficiency before, during and after pregnancy, resulting in the birth of a healthy child (not pit‐1 deficient). Both PGH and IGF‐I concentrations were below the assay detection limit before and after pregnancy. During pregnancy, PGH and IGF‐I levels increased steadily; the concentrations of PGH and IGF‐I in late pregnancy were comparable with levels previously measured in normal pregnancies. PGH and IGF‐I concentrations were strongly correlated throughout pregnancy (r = 0.90; P = 0.002). PGH was undetectable in cord serum, whilst the IGF‐I concentration was within the normal range. The findings of this case study corroborate the notion that PGH is the prime regulator of maternal serum IGF‐I during pregnancy.

Cyclical Cushing's disease and its successful control under sodium valproate.

Several subgroups of Cushing’s disease were recently described (anterior or intermediate lobe origin, hyper-or hypo-pulsatility of Cortisol, presence or absence of response after GRH or TRH, cyclical Cushing’s disease). We present here a detailed case report on a patient suffering from Cushing’s disease whose endocrine functions were extensively investigated. Treatment with bromocriptine, as well;as subsequent transsphenoidal surgery, were followed by rapid but transient reversal of symptoms. When clinical manifestations reoccurred, daily measurements of free urinary Cortisol revealed a cyclic pattern of Cortisol hyperexcretion. A study of ultradian rhythm revealed hyperpulsatility of Cortisol secretion. More interestingly, a treatment with sodium valproate, a drug known to inhibit CRH production, was followed by a rapid and longstanding normalization of clinical and biological data for 2 years. Based on these data, and on information from the literature, the present case of Cushing’s disease exhibits characteristics suggesting a possible hypothalamic origin.