Prolactin and growth hormone response to intracerebroventricular administration of the food opioid peptide gluten exorphin B5 in rats

Although it has long been known that opioid peptides cause marked changes of pituitary hormone secretion in both animals and humans, little is known about the possible effect(s) of food-derived opioids (exorphins) on pituitary function. In order to investigate the possible role of exorphins derived from wheat gluten on pituitary function, we gave the following treatments to four groups of male rats: intracerebroventricular (ICV) vehicle, Gluten Exorphin B5 (GE-B5) 200 microg ICV, naloxone intraperitoneally (IP) followed by vehicle ICV, naloxone IP followed by GE-B5 ICV. Blood samples for Prolactin (PRL) and Growth Hormone (GH) were taken at intervals for 90 minutes after vehicle or GE-B5 administration. GE-B5 strongly stimulated PRL secretion; its effect was completely abolished by naloxone administration. GH secretion was unaffected by GE-B5 under these experimental conditions. The present study shows for the first time that an opioid peptide derived from wheat gluten, GE-B5, has an effect on pituitary function when administered ICV; its mechanism of action appears to be mediated via classical opioid receptors.     

Evaluation of the circadian profile of peripheral plasma galanin concentrations in normal subjects

Galanin administration can influence pituitary function principally resulting in an increase in GH secretion. However, the role of circulating GAL levels in human endocrine function is still unknown. In the present study we simultaneously measured the circadian profiles of GAL, ACTH and GH in peripheral blood of ten adult subjects. Plasma samples were collected through an intravenous catheter at 0800, 1200, 1600, 2000, 2200, 2400, 0200, 0400 hours. The results were statistically evaluated by the cosinor analysis technique. A significant circadian rhythm of both plasma ACTH (p < 0.001) and GH levels (p < 0.03) was found with acrophases occurring at 0753 hrs and 0131 hrs for ACTH and GH, respectively. On the contrary, no significant rhythm was found in plasma GAL levels, indicating that no correlations exist between GAL and either GH or ACTH circadian profiles. Furthermore, the simultaneous assay of both GAL and GH plasma levels during a nocturnal frequent sampling performed in four volunteers showed the presence of peaks in GAL levels which, however, were not concomitant to the peaks in GH levels. These data demonstrate the lack of rhythmicity in the circadian profile of plasma GAL levels in healthy human subjects. The role of GAL in human endocrine function remains unknown and these results suggest that, in spite of the well documented increase in plasma GH concentrations following the intravenous administration of GAL, physiologically circulating levels of GAL are likely not involved in the regulation of GH secretion.     

Molecular characterization of p53 mutations in primary and secondary liver tumors: diagnostic and therapeutic perspectives

p53 is one of the most mutated genes in human cancer. We have performed the molecular characterization of p53 and have searched for correlations with etiological factors and clinical parameters in primary and secondary liver tumors. A systematic study was carried out, innovative in many respects, to determine the mutational pattern of all 11 exons of p53 and analysis was extended also to exons 1–4 and 9–11 and the exon/intron junctions. Our analyses were performed on case histories of 114 patients from the European area and highlighted p53 mutation patterns different from those reported in the literature for the same tumors. In our case history, different tumors of the same organ showed a different frequency and distribution of mutations. In analyzed tumor types, gene status was a prognostic indicator of survival because patients undergoing liver resection without mutated p53 had a more favorable prognosis than mutated patients. This suggests p53 molecular diagnosis could become a further criterion in the decision for surgery and possible therapies. We describe the ideal conditions for polymerase chain reaction (PCR), single-strand conformation polymorphism (SSCP), and direct sequencing, which we have set in order to optimize yields, sensitivity, and time of what might become a massive molecular screening.     

Possible involvement of Fas system in the induction of apoptosis in human astrocytic brain tumors

1. For a better understanding of the biological features of astrocytic tumors, we investigated apoptosis and its pathway, especially in the interaction between Fas and Fas ligand (FasL).2. We examined the presence of apoptosis in human astrocytic brain tumors by terminal deoxynucleotidyl transferase (TdT)-mediated d-UTP-biotin nick end labeling (TUNEL) and then apoptotic index (AI) was calculated. We also examined the distribution of Fas and FasL-positive tumor cells immunohistochemically. Labeling index (LI) for Fas and FasL was calculated as Fas-LI and FasL-LI, respectively, and compared to AI.3. Tumor cells expressing both Fas and FasL were TUNEL positive. Such cells were distributed sparsely in low-grade astrocytomas, but focally in glioblastomas. There was a close correlation among AI, Fas-LI, and FasL-LI, and astrocytic tumors with higher AI were associated with a longer survival time than that with lower AI.4. It was concluded that the Fas system may be involved in the apoptosis of astrocytic tumors, and AI can be a useful parameter for assessing prognosis of astrocytic tumors.     

Heat-shock protein 90 complexes in resting and thrombin-activated platelets

The orexins are peptides which were recently isolated from the rat hypothlamus. They play a role in energy homeostasis and regulation of feeding as well as in other functions such as the sleep-wake cycle. The involvement of glucocorticoids in stress processes as well as in body weight regulation is well known. In the present paper, we investigated the role of glucocorticoids on hypocretin (Hcrt)/orexin (OX) pathway in Sprague-Dawley rats. We confirmed by in situ hybridization that prepro-Hcrt/OX mRNA expression is restricted to the lateral hypothalamus area with extension to the perifornical nucleus and the posterior hypothalamic area. Lateral hypothalamic prepro-Hcrt/OX mRNA expression was decreased by 50% after adrenalectomy (99.8+/-5.0 vs 49.2+/-4.4 nCi/g, p<0.01). Peripheral glucocorticoid treatment (dexamethasone) restored its expression to normal levels (105.4+/-6.1 nCi/g). The present data provide direct evidence that Hcrt/OX expression in the lateral hypothalamus is modulated by the glucocorticoids status. As the Hcrt/Ox system is closely interactive with the corticotropin-releasing hormone and neuropeptide Y systems, we propose that hypocretin/orexins peptides constitute a very sensitive key relay for mediating both stress and feeding behavior.     

Immunocytochemistry of myoepithelial cells in the salivary glands

MECs are distributed on the basal aspect of the intercalated duct and acinus of human and rat salivary glands. However, they do not occur in the acinus of rat parotid glands, and sometimes occur in the striated duct of human salivary glands. MECs, as the name implies, have structural features of both epithelial and smooth muscle cells. They contract by autonomic nervous stimulation, and are thought to assist the secretion by compressing and/or reinforcing the underlying parenchyma. MECs can be best observed by immunocytochemistry. There are three types of immunocytochemical markers of MECs in salivary glands. The first type includes smooth muscle protein markers such as -SMA, SMMHC, h-caldesmon and basic calponin, and these are expressed by MECs and the mesenchymal vasculature. The second type is expressed by MECs and the duct cells and includes keratins 14, 5 and 17, 1β1 integrin, and metallothionein. Vimentin is the third type and, in addition to MECs, is expressed by the mesenchymal cells and some duct cells. The same three types of markers are used for studying the developing gland.

Development of MECs starts after the establishment of an extensively branched system of cellular cords each of which terminates as a spherical cell mass, a terminal bud. The pluripotent stem cell generates the acinar progenitor in the terminal bud and the ductal progenitor in the cellular cord. The acinar progenitor differentiates into MECs, acinar cells and intercalated duct cells, whereas the ductal progenitor differentiates into the striated and excretory duct cells. Both in the terminal bud and in the cellular cord, the immediate precursors of all types of the epithelial cells appear to express vimentin. The first identifiable MECs are seen at the periphery of the terminal bud or the immature acinus (the direct progeny of the terminal bud) as somewhat flattened cells with a single cilium projecting toward them. They express vimentin and later -SMA and basic calponin. At the next developmental stage, MECs acquire cytoplasmic microfilaments and plasmalemmal caveolae but not as much as in the mature cell. They express SMMHC and, inconsistently, K14. This protein is consistently expressed in the mature cell. K14 is expressed by duct cells, and vimentin is expressed by both mesenchymal and epithelial cells.

After development, the acinar progenitor and the ductal progenitor appear to reside in the acinus/intercalated duct and the larger ducts, respectively, and to contribute to the tissue homeostasis. Under unusual conditions such as massive parenchymal destruction, the acinar progenitor contributes to the maintenance of the larger ducts that result in the occurrence of striated ducts with MECs. The acinar progenitor is the origin of salivary gland tumors containing MECs. MECs in salivary gland tumors are best identified by immunocytochemistry for -SMA. There are significant numbers of cells related to luminal tumor cells in the non-luminal tumor cells that have been believed to be neoplastic MECs.     

Activated Ras prevents downregulation of Bcl-X(L) triggered by detachment from the extracellular matrix. A mechanism of Ras-induced resistance to anoikis in intestinal epithelial cells

Detachment of epithelial cells from the extracellular matrix (ECM) results in a form of apoptosis often referred to as anoikis. Transformation of intestinal epithelial cells by oncogenic ras leads to resistance to anoikis, and this resistance is required for the full manifestation of the malignant phenotype. Previously, we demonstrated that ras-induced inhibition of anoikis in intestinal epithelial cells results, in part, from the ras-induced constitutive downregulation of Bak, a pro-apoptotic member of the Bcl-2 family. Since exogenous Bak could only partially restore susceptibility to anoikis in the ras-transformed cells, the existence of at least another component of the apoptotic machinery mediating the effect of activated ras on anoikis was suggested. Indeed, here we show that, in nonmalignant rat and human intestinal epithelial cells, detachment from the ECM or disruption of the cytoskeleton results in a significant downregulation of the antiapoptotic effector Bcl-X(L), and that activated H- or K-ras oncogenes completely abrogate this downregulation. In addition, we found that enforced downregulation of Bcl-X(L) in the ras-transformed cells promotes anoikis and significantly inhibits tumorigenicity, indicating that disruption of the adhesion-dependent regulation of Bcl-X(L) is an essential part of the molecular changes associated with transformation by ras. While the ras-induced downregulation of Bak could be reversed by pharmacological inhibition of phosphatidylinositol 3 kinase (PI 3-kinase), the effect of ras on Bcl-X(L) was PI 3-kinase- and mitogen-activated protein kinase (MAP kinase)-independent. We conclude that ras-induced resistance to anoikis in intestinal epithelial cells is mediated by at least two distinct mechanisms: one that triggers downregulation of Bak and another that stabilizes Bcl-X(L) expression in the absence of the ECM.     

Estrogen mitogenic action. I. Demonstration of estrogen-dependent MTW9/PL2 carcinogen-induced rat mammary tumor cell growth in serum-supplemented culture and technical implications

Summary The MTW9/PL cell line was established by our laboratory in culture from the carcinogen-induced hormone-responsive MT-W9A rat mammary tumor of a Wistar-Furth (W/Fu) rat. This tumor formed estrogen, androgen, and progesterone responsive tumors in W/Fu rats (Sirbasku, D. A., Cancer Res. 38:1154–1165; 1978). It was later used to derive the MTW9/PL2 cell population which was also estrogen-responsive in vivo (Danielpour, D., et al., In Vitro Cell. Dev. Biol. 24∶42–52; 1988). In the study presented here, we describe serum-supplemented culture conditions in which the MTW9/PL2 cells demonstrate≥80-fold steroid hormone growth responses. All sera used were steroid hormone-depleted by charcoal-dextran treatment at 34°C. The studies were done with horse serum as well as serum from other mammalian species. The growth of the MTW9/PL2 cells was biphasic in response to hormone-depleted serum. Concentrations of ≤5% (v/v) promoted optimum growth. Above this concentration, serum was inhibitory. Concentrations ≥40% (v/v) inhibited growth altogether. Addition of 1.0×10⁻¹³−1.0×10⁻⁸ M 17β-estradiol (E₂) reversed the inhibition completely. At 1.0×10⁻⁸ M, estrone, estriol and diethylstilbestrol promoted growth as well as E₂. Testosterone and dihydrotestosterone promoted growth only at ≥10⁻⁷ M. Progesterone was effective only at≥10⁻⁶ M. Cortisol was ineffective. Labeled-hormone-binding analysis and Western immunoblotting documented that MTW9/PL2 cells had estrogen and progesterone receptors but not androgen or cortisol receptors. Estrogen treatment of MTW9/PL2 cells induced a concentration and time dependent increase in progesterone receptors. We conclude (1) the MTW9/PL2 population is the first highly steroid hormone-responsive rat mammary tumor cell line to be established in culture from a carcinogen-induced tumor, and (2) sera from a number of species including horse, rat and human contain an inhibitor which mediates estrogen sensitive MTW9/PL2 cell growth in culture.