Pregnenolone-binding components in the porcine adrenal gland

The object of this study was to determine whether binding components for pregnenolone, analogous to those described in the adrenal cortex of guinea pigs and rats, were present in the porcine adrenal. A binding component for pregnenolone in the cytosolic fraction of porcine adrenal was demonstrated by sucrose density gradient centrifugation. It banded maximally at 9.6% sucrose (w/w) compared to 12.2% and 12.4% sucrose (w/w) for the plasma-binding component and serum albumin, respectively. At a pregnenolone concentration of 1 X 10(-5) M, specific cytosolic binding of 1 X 10(-8) M [3H]pregnenolone was decreased by 42%. The fractions from sucrose gradients which bound pregnenolone maximally contained 3 beta-hydroxysteroid dehydrogenase-isomerase. The cytosolic supernatant of porcine adrenal gland was resolved by chromatography on hydroxyapatite into eleven fractions, four of which bound added pregnenolone and three of which displayed enzymatic activity. Electrophoretic analysis of the enzymatically active fractions in polyacrylamide gel showed that two of them were of heterogeneous composition, whereas the third, most enzymatically active, fraction consisted principally of one band of high molecular weight.     

EST-based analysis of gene expression in the porcine brain

Since pig is an important livestock species worldwide, its gene expression has been investigated intensively, but rarely in brain. In order to study gene expression profiles in the pig central nervous system, we sequenced and analyzed 43,122 highquality 5’ end expressed sequence tags (ESTs) from porcine cerebellum, cortex cerebrum, and brain stem cDNA libraries, involving several different prenatal and postnatal developmental stages. The initial ESTs were assembled into 16,101 clusters and compared to protein and nucleic acid databases in GenBank. Of these sequences, 30.6% clusters matched protein databases and represented function known sequences; 75.1% had significant hits to nucleic acid databases and partial represented known function; 73.3% matched known porcine ESTs; and 21.5% had no matches to any known sequences in GenBank. We used the categories defined by the Gene Ontology to survey gene expression in the porcine brain.     

Nestin expression in rat adrenal gland

The constituents of the intermediate filament network of adrenal gland cells have not been deeply investigated in vivo. Adrenocortical cells have been reported to express cytokeratins and vimentin, but the intermediate filament components of the adrenomedullary cells are still unknown. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous and muscle systems. It has been reported to be unable to form filaments by itself and it co-assembles with vimentin. Using immunocytochemical and biochemical approaches, the present study demonstrates that nestin is expressed in situ either in the cortex or in the medulla of adult rat adrenal glands. Nestin-negative cells prevalently form the zona glomerulosa whereas the zona fasciculata and the zona reticularis are mainly nestin-immunoreactive. Nestin-positive cells always express vimentin-like immunoreactivity but several cells apparently expressing only vimentin are detectable too. Nestin is also expressed by adrenomedullary cells that also display a faint vimentin-like immunoreactivity. We hypothesise that the inconstant detection of nestin in adrenocortical cells depends on their different functional moments. Moreover, even though our data do not allow to confirm vimentin in adrenomedullary cells, in situ detection of nestin in the adrenal medulla indirectly supports in vivo expression of vimentin in chromaffin cells.     

Light activates the adrenal gland: timing of gene expression and glucocorticoid release

Light is a powerful synchronizer of the circadian rhythms, and bright light therapy is known to improve metabolic and hormonal status of circadian rhythm sleep disorders, although its mechanism is poorly understood. In the present study, we revealed that light induces gene expression in the adrenal gland via the suprachiasmatic nucleus (SCN)-sympathetic nervous system. Moreover, this gene expression accompanies the surge of plasma and brain corticosterone levels without accompanying activation of the hypothalamo-adenohypophysial axis. The abolishment after SCN lesioning, and the day-night difference of light-induced adrenal gene expression and corticosterone release, clearly indicate that this phenomenon is closely linked to the circadian clock. The magnitude of corticostereone response is dose dependently correlated with the light intensity. The light-induced clock-dependent secretion of glucocorticoids adjusts cellular metabolisms to the new light-on environment.     

Ultracytochemical localization of particulate guanylate cyclase in rat adrenal gland exposed to stimulation by porcine brain natriuretic peptide

Summary We studied the cytochemical localization of particulate guanylate cyclase (GC) in rat adrenal gland after stimulation with porcine brain natriuretic peptide (pBNP) by electron microscopy. In the adrenal cortex, GC activity, as demonstrated by the presence of reaction product, was prevalently localized to the zona glomerulosa and zona fasciculata, while the zona reticularis showed little GC reaction product. In the adrenal medulla, GC reaction product was present only in adrenalin-containing cells. All GC positivity was associated with intracellular membranes. No GC reaction product was detected in specimens incubated in media devoid of pBNP. In parallel samples incubated in the presence of rat atrial natriuretic factor (rANF), the distribution of rANF-stimulated GC activity was similar to that of pBNP-stimulated GC activity.     

Differential expression of a stress-modulating gene, BRE, in the adrenal gland, in adrenal neoplasia, and in abnormal adrenal tissues.

Genes that modulate the action of hormones and cytokines play a critical role in stress response, survival, and in growth and differentiation of cells. Many of these biological response modifiers are responsible for various pathological conditions, including inflammation, infection, cachexia, aging, genetic disorders, and cancer. We have previously identified a new gene, BRE, that is responsive to DNA damage and retinoic acid. Using multiple-tissue dot-blotting and Northern blotting, BRE was recently found to be strongly expressed in adrenal cortex and medulla, in testis, and in pancreas, whereas low expression was found in the thyroid, thymus, small intestine and stomach. In situ hybridization and immunohistochemical staining indicated that BRE was strongly expressed in the zona glomerulosa of the adrenal cortex, which synthesizes and secretes the mineralocorticoid hormones. It is also highly expressed in the glial and neuronal cells of the brain and in the round spermatids, Sertoli cells, and Leydig cells of the testis, all of which are associated with steroid hormones and/or TNF synthesis. However, BRE expression was downregulated in human adrenal adenoma and pheochromocytoma, whereas its expression was enhanced in abnormal adrenal tissues of rats chronically treated with nitrate or nitrite. These data, taken together, indicate that the expression of BRE is apparently associated with steroids and/or TNF production and the regulation of endocrine functions. BRE may play an important role in the endocrine and immune system, such as the cytokine-endocrine interaction of the adrenal gland.     

Nestin expression in normal adrenal gland and adrenocortical tumors

Human adrenocortical cells have been shown to express cytokeratins and vimentin. Nestin is an intermediate filament protein that is mainly expressed in the developing nervous system and that has been recently reported in rat adrenal gland as well. Using immunohistochemical and biochemical approaches, the present study demonstrates that nestin is constantly expressed in situ in the cortex of normal human adrenal glands. Nestin expressing cells were prevalently located in the zona reticularis but some positive cells could be spotted in the zona fasciculata as well. Moreover, patches of nestin-positive cells have been constantly detected on sections of cortical adenomas. In contrast, adrenal carcinomas displayed a variable number of nestin-immunoreactive cells that in some cases were virtually absent. Samples of renal clear cell carcinoma metastasis in the adrenals were also examined which did not show nestin-immunoreactivity. We propose that a positive nestin-immunoreaction could be useful in differential diagnosis of clear cell tumors in adrenal glands.     

Tissue-specific hypomethylation and expression of rat phosphoenolpyruvate carboxykinase gene induced by in vivo treatment of fetuses and neonates with 5-azacytidine

Rat fetuses of 17-19-day gestation were injected in utero with 5-azacytidine (two to three daily injections of 40 micrograms/fetus). Neonates were injected with seven daily injections (1 mg/kg). DNA samples were isolated from the fetal and neonatal livers and neonatal spleen and subjected to analysis of their methylation status. Overall methylation was analyzed by the nearest-neighbor analysis (at CpG sites) and the pattern of methylation at CCGG sites by Southern blot analysis using phosphoenolpyruvate carboxykinase (PEPCK) sequences as probes. While DNAs from the liver and spleen undergo hypomethylation to the same extent in response to the 5-azacytidine treatment, the changes in the methylation patterns of the PEPCK gene in the two tissues are strikingly different. The changes observed indicate that a decrease in the methylase activity (inhibition by 5-azacytidine) results in site- and tissue-specific hypomethylation. The tissue-specific changes in the methylation pattern are associated with a tissue-specific expression of the PEPCK gene. Although the gene is hypomethylated by azacytidine in both liver and spleen, it is expressed only in the liver. The expression of already active genes (PEPCK in the kidney and albumin in the liver) is not further enhanced by the drug.     

Cloning and expression of the porcine myogenin gene

Abstract

Myogenin is a member of the myoD family of myogenic regulatory factors which direct the initiation of myogenesis during skeletal muscle development. Myogenin is responsible for the differentiation of proliferating myoblasts into myotubes and the absence of this factor results in severe muscular deficiency. Using PCR primers designed from mammalian myogenin sequences, a 215 bp PCR product was amplified from porcine genomic DNA and found to be 89.7% homologous to mouse myogenin. A porcine cosmid library was screened with the myogenin PCR probe and a 6.5 kb portion of a cosmid containing the pig myogenin gene was sequenced. The sequence includes 5´ flanking region, the entire protein coding region composed of three exons and two introns and 3´ flanking region. The protein coding region was highly conserved (≥92.5%) with mouse and human myogenin coding regions. Transfection of pig myogenin into C3H10T1/2 cells, a multipotential fibroblast cell line, resulted in 28% myogenic conversion assayed by cell fusion to form multinucleated cells and synthesis of sarcomeric myosin. These results indicate that the cloned myogenin gene was capable of initiating myogenesis in a fibroblast cell line.     

Somatostatin gene expression in the thymus gland

A complex pattern of interactions appears to exist between the immune and neuroendocrine systems. Recently, vasopressin, oxytocin and vasoactive intestinal peptide have been isolated from the thymus. Using a rat somatostatin antisense RNA probe we have demonstrated expression of the somatostatin gene in the rat thymus. Furthermore, we have shown that the levels of thymic somatostatin mRNA exhibit a bell-shaped response to dexamethasone administration. Lipocortin I and II antisense RNA probes have been used as a positive control for the effects of the dexamethasone. We would suggest that somatostatin acts in the thymus in a paracrine mode to modulate T lymphocyte development.     

Extraction and immunochemical characterization of delta sleep-inducing peptide-like material from the porcine pituitary and adrenal gland

The naturally occurring forms of delta sleep-inducing peptide (DSIP) are not fully identified. In the present study, porcine pituitaries and adrenal glands were extracted in water, saline or acid under various conditions and immunoreactive DSIP (IR-DSIP) quantified by radioimmunoassay. The highest concentrations were measured in anterior pituitary extracts (40.8 +/- 2.6 ng/g tissue weight) recovered using water with aprotinin. However, high performance liquid chromatography (HPLC) indicated degradation of hydrophobic forms of IR-DSIP in water extracts. Extraction in acetic acid including C18 Sep-Pak purification resulted in an elution profile of IR-DSIP in adrenal extracts with a major peak coeluting with synthetic DSIP [DSIP(1-9)], whereas anterior pituitary extract showed material of higher hydrophobicity. Approximately 30% of IR-DSIP in anterior pituitary as well as in adrenal gland extracts seemed to be glucosylated, as based on concanavalin A chromatography. One of the DSIP-immunoreactive components by immunoblotting (molecular mass 25 kDa) was identified in both pituitary and adrenal gland extracts. In conclusion, several chromatographically distinct forms of IR-DSIP are present in the porcine pituitary and adrenal gland. IR material eluting as DSIP(1-9) is present in adrenal gland extract. The procedure and solution used for tissue extraction seem to be essential in order to obtain reliable elution positions on HPLC.     

Opioid peptides in adrenal gland

Enkephalin-like immunoreactive peptides have been observed in adrenal glands of all species studied with the highest contents found in dogs and cows, and the lowest in rats. These peptides are located both in gland cells and in afferent nerve terminals. Bovine adrenal glands contain opioid peptides in many molecular forms. The peptides include a group of low molecular weight forms (M.W. <1000) which are capable of binding to the opiate receptor, and a group of high molecular weight forms (M.W. >1000) which contain enkephalin within their peptide sequence, but are devoid of opioid activity unless treated with trypsin. The physiological role(s) of the adrenal enkephalin-like material is not clear at present. However, it has been observed that nicotine-stimulated release of catecholamine from isolated chromaffin cells can be reduced by opiate agonists, suggesting that enkephalin-like peptide in nerve terminals may act on chromaffin cells. Several lines of evidence suggest that enkephalin-like peptides in gland cells can be released into the bloodstream.