Production of macrocyclic ellagitannin oligomers by Oenothera laciniata callus cultures

Melanocyte-stimulating hormone (MSH) has been reported to enhance the experimental metastatic behaviour of melanoma cells in the mouse model. alpha-MSH production and MSH receptor (melanocortin 1 receptor gene) expression have been detected in cultured normal human melanocytes and metastasized melanomas. The exact role of MSH in the metastatic behaviour of human melanoma cells is, however, not yet known. To clarify a possible role of proopiomelanocortin (POMC)-derived peptides, including alpha-MSH, in melanoma development and progression, we analysed immunohistochemically the localization of alpha-MSH adrenocorticotrophic hormone (ACTH) and beta-endorphin in various kinds of benign pigmented naevocytic lesions and malignant melanomas. Three of 21 samples of common and dysplastic naevi showed detectable alpha-MSH staining in naevus cells, and five and six of 15 samples were weakly positive for ACTH and beta-endorphin staining, respectively. In melanoma samples, 24 of 45, 23 of 39 and 30 of 42 samples showed positive staining with alpha-MSH, ACTH and beta-endorphin antibodies, respectively. Furthermore, staining for all three antibodies was noted to be more intense and diffuse in samples of nodular melanoma, vertically growing acral lentiginous melanoma and superficial spreading melanoma as well as metastatic lesions compared with those of naevi. Although it is yet to be determined whether or not this strong staining for POMC-derived peptides in advanced melanoma cells indicates a role of autocrine or paracrine regulation, our results suggest a possible involvement of POMC gene products in melanoma progression.     

Antagonistic regulation of cell migration by epidermal growth factor and glucocorticoid in human gastric carcinoma cells

Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.     

Characterisation of ACTH peptides in human skin and their activation of the melanocortin-1 receptor

Alpha-Melanocyte-stimulating hormone (alpha-MSH) is a proopiomelanocortin (POMC)-derived peptide, which is produced in the pituitary and at other sites including the skin. It has numerous effects and in the skin has a pigmentary action through the activation of the melanocortin-1 (MC-1) receptor, which is expressed by melanocytes. Recent evidence suggests that the related POMC peptides such as adrenocorticotrophin (ACTH), which is the precursor of alpha-MSH, is also an agonist at the MC-1 receptor. By using immunocytochemistry, we confirmed the presence of alpha-MSH in human skin where staining was evident in keratinocytes and especially strong in melanocytes and possibly Langerhans cells. ACTH was also present and tended to show the strongest reaction in differentiated keratinocytes. Immunostaining was also observed for the prohormone convertases, PC1 and PC2, which are involved in the formation of ACTH and its cleavage to alpha-MSH, respectively. The amounts of immunoreactive ACTH exceeded those of alpha-MSH. Using HPLC we identified for the first time the presence of ACTH1-39, ACTH1-17, ACTH1-10, acetylated ACTH1-10, alpha-MSH, and desacetyl alpha-MSH in epidermis and in cultured keratinocytes. The ability of these peptides to activate the human MC-1 receptor was examined in HEK 293 cells that had been transfected with the receptor. All peptides increased adenylate cyclase in these cells with the following order of potency: ACTH1-17 > alpha-MSH > ACTH1-39 > desacetyl alpha-MSH > acetylated ACTH1-10 > ACTH1-10. ACTH1-17 also increased the dendricity and melanin content of cultured human melanocytes indicating that the peptide was able to activate MC-1 receptors when present in their normal location. However, as found with alpha-MSH, not all cultures were responsive and, as we have previously suggested, we suspect that this was the result of changes at the MC-1 receptor. Nevertheless, it would appear that ACTH peptides can serve as natural ligands of the MC-1 receptor on human melanocytes and their presence in the skin suggests that, together with alpha-MSH, they may have a role in the regulation of human melanocytes.     

Heparan/chondroitin/dermatan sulfate primer 2-(6-hydroxynaphthyl)-O-beta-D-xylopyranoside preferentially inhibits growth of transformed cells

Melanocortins are proopiomelanocortin-derived peptides that include adrenocorticotropic hormone [ACTH (1-39)], alpha-melanocyte-stimulating hormone [alpha-MSH (1-13)], and related amino acid sequences. Melanocortin peptides have potent antiinflammatory/anticytokine activity. Because cytokines such as interleukin 1 (IL-1) and tumor necrosis factor (TNF) can be detrimental in HIV-infected patients, we investigated the effects of melanocortins on production of IL-1 and TNF alpha in the blood of HIV patients. Cytokine production was measured in whole blood samples stimulated with LPS in the presence or absence of alpha-MSH (1-13), alpha-MSH (11-13), ACTH (1-24), or ACTH (1-39). Melanocortins reduced production of both cytokines in a concentration-dependent fashion. In separate experiments on normal peripheral blood mononuclear cells (PBMC), alpha-MSH (1-13) inhibited production of IL-1 beta and TNF alpha induced by HIV envelope glycoprotein gp 120. These results suggest that stimulation of melanocortin receptors in inflammatory cells could be a novel way to reduce production of cytokines that promote HIV replication.     

Regulation of interleukin-6 (IL-6) secretion in primary cultured rat astrocytes: synergism of interleukin-1 (IL-1) and pituitary adenylate cyclase activating polypeptide (PACAP)

Adjuvant-induced arthritis (AA) in specific strains of rats is an immunologically mediated inflammatory disease which is also characterised by activation of the endocrine system. To further investigate the effects of AA on processing of the pro-opiomelanocortin (POMC) precursor in rat immune tissues, we utilised radioimmunoassays for adrenocorticotrophin (ACTH), beta-endorphin and alpha-melanocyte-stimulating hormone (alpha-MSH) to measure these peptides in the spleen and thymus. 14 days following adjuvant injection, spleen levels of ACTH were elevated in the AA group (4.47 +/- 1.04 ng/g tissue, n = 9) compared to controls (2.42 +/- 0.4 ng/g) and exacerbation of the disease by removal of circulating glucocorticoids through bilateral adrenalectomy (ADX) resulted in further elevation of spleen ACTH (5.11 +/- 1.22 ng/g). beta-Endorphin levels in both the AA (10.60 +/- 1.61 ng/g) and AA/ADX (13.37 +/- 2.36 ng/g) groups were higher than controls (5.57 +/- 0.65 ng/g). Conversely, alpha-MSH spleen levels were decreased in the AA (2.89 +/- 0.22 ng/g) and AA/ADX (2.22 +/- 0.33 ng/g) groups compared to controls (4.62 +/- 0.45 ng/g) and were also decreased following adrenalectomy. In the thymus, ACTH levels were elevated in the AA group (8.95 +/- 1.41 ng/g) compared to controls (5.79 +/- 0.63 ng/g), and the same pattern was evident for thymic alpha-MSH (0.64 +/- 0.08 ng/g in AA animals compared to control levels of 0.35 +/- 0.03 ng/g). Following G50 gel filtration, ACTH and beta-endorphin immunoreactivities (ir) were present in both spleen and thymus as two peaks, one which eluted near the void volume and one which eluted in a lower molecular mass position than the standards.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proopiomelanocorticotropin (POMC) peptides and lipoprotein lipase activity in vitro

Effects of alpha-melanocyte-stimulating hormone (alpha-MSH), beta-melanocyte-stimulating hormone (beta-MSH), beta-lipotropin (beta-LPH), and beta-endorphin (beta-EPH) at concentrations from 10(-9) M up to 10(-6) M on human adipose tissue lipoprotein lipase (LPL) were studied in a cell-free system. alpha-MSH and beta-MSH did not exert any effect on LPL; no degradation of these peptides in the incubation medium could be detected by HPLC analysis. beta-LPH and beta-EPH failed to alter enzyme activity. However, HPLC analysis revealed an unspecific rapid degradation of the peptides due to the activity of tissue proteases. Therefore, the protease inhibitors amastatin, antipain, APMSF, and TPCK were tested at concentrations of 10(-5), 10(-4), and 10(-3) M for their efficacy to inhibit degradation. None of the inhibitors was able to substantially reduce proteolysis of beta-LPH, as was the case with amastatin, APMSF, and TPCK for beta-EPH. However, antipain at 10(-4) M preserved at least 20% of the initial peptide concentration from proteolysis up to 150 min. Antipain caused a decrease in lipoprotein lipase activity (LPLA), which was dependent on concentration. The adverse effect of antipain at concentrations of 10(-4) M on LPL was completely abolished by beta-EPH at a concentration of 10(-6) M.     

Experimental treatment of brain tumor cells using CD suicide gene

Pro-opiomelanocortin (POMC) is a polyhormone precursor produced predominantly in the pars distalis and pars intermedia of the pituitary gland where it undergoes tissue specific processing to produce a whole array of peptides. We have shown previously that peptides derived from the N-terminal region of POMC are involved in adrenal growth in rats. Using specific two site immunoradiometric assays we have found that the plasma of 17 week old fetal sheep contain a 50 fold excess of pro-gamma-MSH over ACTH. As term approached, the levels of pro-gamma-MSH fell and ACTH rose with evidence of fragmentation of pro-gamma-MSH, suggesting that these peptides act in concert in the development of the fetal adrenal cortex and also provide the necessary drive to bring about parturition. In an attempt to explore the pathophysiology of adrenal function we have cloned human POMC cDNA which led to the discovery of a 9bp addition/deletion mutation in the C-terminus of gamma 3-MSH between positions 67-73. Chinese hamster ovary cells (CHO) cells stably transfected with constructs containing the variant POMC cDNAs have shown a degree of partial processing. Work is currently underway to further investigate the effects of these mutations on the processing by the prohormone converting enzymes PC1 and PC2.     

Dose-dependent doxycycline-mediated adrenocorticotropic hormone secretion from encapsulated Tet-on proopiomelanocortin Neuro2A cells in the subarachnoid space

We previously reported that polymer-encapsulated mouse neuroblastoma cells that are capable of secreting beta-endorphin may reduce pain sensitivity in rats after capsule implantation into the cerebrospinal fluid (CSF)-filled subarachnoid space of the spinal cord. The neuroblastoma cells carry the proopiomelanocortin (POMC) gene that encodes the precursor of adrenocorticotropic hormone (ACTH) and beta-endorphin. To control the expression of these hormones in the present study, a promoter that is inducible by administration of tetracycline derivatives such as doxycycline (Dox) was linked to the POMC gene. Encapsulated cells in the CSF space of rats stimulated by four intraperitoneal doses of Dox responded with ACTH expression as determined in a subsequence 36-hr in vitro incubation. The amount of ACTH released was dependent on the in vivo Dox dose. These findings indicate that gene expression in xenogeneic cells in the CSF space can be manipulated by injection of a relatively innocuous drug, and suggest that this system may be applicable to cell transplantation therapy in patients with central nervous system diseases that require temporary control of ligand delivery.     

Isolated congenital ACTH deficiency: a cleavage enzyme defect?

The pro-opiomelanocortin (POMC) gene encodes adrenocorticotrophin (ACTH) which is derived from precursors by proteolytic cleavage. Congenital, isolated ACTH deficiency is rare but may be familial and fatal. The aetiology is unknown though defects at both hypothalamus and adenohypophysis have been postulated. We have studied a female presenting with hypoglycaemia in the neonatal period. When studied at 6 weeks of age, ACTH was unmeasurable even after injection of corticotrophin releasing hormone (CRH1-41). ACTH precursors, quantitated by two-site immunoradiometric assay, were clearly measurable prior to treatment and were stimulated by CRH1-41 and suppressed by glucocorticoid administration. Concentrations of POMC, N-terminal pro-opiocortin (N-POC) and beta-endorphin (beta-EP) were within the normal adult range during glucocorticoid replacement therapy; ACTH and beta-lipotrophin remained undetectable. The secretion of glucagon, measured by radioimmunoassay, in response to hypoglycaemia was normal. By sequencing polymerase chain reaction products from the patient's genomic DNA, the entire coding region of the POMC gene was established to be normal. The results are compatible with a cleavage enzyme defect.     

Melanocortin and MCH precursor-derived NEI effects on striatum-midbrain co-cultures

The possibility of developmental effects of POMC-derived melanocortins and analogs on neurons of fetal rat brain regions exhibiting marked developmental melanocortin receptor expression, was studied in serum-free co-cultures of gestational day 18 striatal and mesencephalic cells, and compared with NEI and NGE. These two peptide fragments of the melanin concentrating hormone precursor, occurring in brain areas devoid of POMC terminals, cross-react with alpha-MSH antibodies; NEI elicits grooming similar to alpha-MSH. Neurofilament protein (NF), growth-associated protein (GAP-43) and synaptophysin of the synaptosomal fraction were determined by ELISA as markers for neuritogenesis, growth cones, and nerve terminal differentiation. Cell survival was analyzed by MTT assay, proportions of major cell types by immunocytochemistry. alpha-Melanocyte-stimulating hormone (alpha-MSH, effective concentration 250-2500 nM), the analog Nle4-, D-Phe7-alpha-MSH (NDP, 3.1-750 nM), and NEI (250 nM) increased NF in 3 day cultures by 11%, 17%, and 22%, respectively, whereas ACTH(1-24) and ACTH(1-39) (25 2500 nM) were ineffective. In 11 day cultures, alpha-MSH (250-750 nM), but not NDP, ACTH(1-24) or ACTH(1-39), increased synaptosomal synaptophysin by 11%. GAP-43 and cell survival remained unaffected. These data indicate that selected melanocortins as well as NEI can influence differentiation of neural processes in brain neurons.     

Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin

Reduction in the activity of the alpha-melanocyte-stimulating hormone (alpha-MSH) system causes obesity, and infusions of alpha-MSH can produce satiety, raising the possibility that alpha-MSH may mediate physiological satiety signals. Since alpha-MSH is coded for by the pro-opiomelanocortin (POMC) gene, we examined if POMC gene expression would be inhibited by fasting in normal mice or in models of obesity characterized by leptin insufficiency (ob/ob) or leptin insensitivity (db/db). In wild-type mice, hypothalamic POMC mRNA was decreased > 60% after a 2-day fast and was positively correlated with leptin mRNA. Similarly, compared with controls, POMC mRNA was decreased by at least 60% in both db/db and ob/ob mice. POMC mRNA was negatively correlated with both neuropeptide Y (NPY) and melanin-concentrating hormone (MCH) mRNA. Finally, treatment of both male and female ob/ob mice with leptin stimulated hypothalamic POMC mRNA by about threefold. These results suggest that impairment in production, processing, or responsiveness to alpha-MSH may be a common feature of obesity and that hypothalamic POMC neurons, stimulated by leptin, may constitute a link between leptin and the melanocortin system.     

Differential influence of a selective melanocortin MC4 receptor antagonist (HS014) on melanocortin-induced behavioral effects in rats

We injected i.c.v. the natural agonist alpha-MSH (melanocyte-stimulating hormone) and the first selective melanocortin MC4 receptor antagonist HS014 (cyclic [AcCys11, D-Nal14, Cys18, Asp-NH(2)22]-beta-MSH(11-22) in rats and scored a number of behavioral effects which have been related to the melanocortic peptides. The results showed that HS014 (5 microg/rat) completely blocked alpha-MSH (3 and 5 microg/rat)-induced grooming, yawning and stretching. Penile erections induced by alpha-MSH were, however, only partially blocked by HS014. Injections of alpha-MSH decreased food intake in food-deprived rats, whereas HS014 increased food intake. When the peptides were given together, the food intake was similar to that of saline treated controls. Locomotion/exploration and resting were not influenced by either peptide. Our data show that exogenous beta-MSH decreases food intake, and that an endogenous central melanocortinergic inhibitory tone on feeding prevails which can be blocked with HS014, leading to an increase in food intake. Our data also provide evidence that grooming, stretching and yawning in rats may be mediated by the melanocortin MC4 receptor, whereas penile erections might perhaps be mediated by some other melanocortin receptor.     

High plasma proopiomelanocortin in aggressive adrenocorticotropin-secreting tumors

A specific propiomelanocortin (POMC) immunoradiometric assay was developed using antibodies directed against ACTH and beta-endorphin (beta end). Partially purified standard POMC was prepared from the human small cell lung carcinoma cell line DMS-79 culture medium. Ten units (U) POMC had the same displacement ability as one pg beta end in a C-terminal beta end radioimmunoassay and thus were close if not equal to 10 pg POMC. This POMC assay was used to investigate patients with ACTH-dependent Cushing's syndrome. Plasma POMC was undetectable (< 60 U/mL) in 17 normal controls and in 4 patients with Addison's disease (concomitant ACTH plasma levels between 362 and 1058 pg/mL). Forty-two patients with Cushing's disease were studied, either before (n = 25) or after (n = 17) bilateral adrenalectomy: 7 patients with highly invasive macroadenomas had high POMC plasma levels, between 240 and 4200 U/ml (concomitant ACTH plasma levels between 77 and 5730 pg/mL); 35 patients, including one with an invasive macroadenoma, had undetectable POMC plasma levels (concomitant ACTH plasma levels between 31 and 2820 pg/mL). Among 20 patients with histologically proven ectopic ACTH syndrome, 16 had high POMC plasma levels, between 80 and 8000 U/mL (concomitant ACTH plasma levels between 45 and 9265 pg/mL); all those tumors were malignant, and the highest POMC/ACTH plasma levels ratios (taken as an index of altered POMC processing) were observed in the 3 patients with small cell carcinomas of the lung; in one of these patients, ACTH and POMC plasma levels both decreased during the course of chemotherapy, in parallel with the reduction of the tumoral mass. Four patients with ectopic ACTH syndrome had undetectable POMC plasma levels (concomitant ACTH plasma levels between 78 and 335 pg/mL): they were all typical bronchial carcinoids. These data show that high POMC plasma level is neither specific for nor constant in ectopic ACTH syndrome. Rather it should be considered as a marker of tumor aggressivity, in pituitary- and non-pituitary tumors. Its diagnostic help appears limited for the most frequent cause of occult ectopic ACTH syndrome, the typical bronchial carcinoids.     

Neuropeptides are potent modulators of human in vitro immunoglobulin E synthesis

We determined the effect of adrenocorticotropin hormone (ACTH) on the regulation of IgE synthesis. Depending on the concentration, ACTH enhanced or inhibited IgE synthesis in a culture system where IgE synthesis was induced with interleukin-4 (IL-4) and anti-CD40 monoclonal antibody in peripheral blood mononuclear cells. Similar effects on IgE synthesis were observed by adding ACTH-related peptides, e.g. corticotropin-releasing factor (CRF), the inducer of ACTH, or alpha-melanocyte stimulating hormone (alpha-MSH), a cleavage product of ACTH. However, ACTH had no effect on IgG or IgM synthesis in this culture system. ACTH did not act directly on either B or T cells as there was no influence on IgE synthesis in a system using purified B cells alone or co-cultured with T cells. The effect of ACTH on IgE synthesis was mediated by accessory cells. This was shown by priming purified CD14-positive monocytes with ACTH and reconstitution experiments. Therefore, these findings suggest that ACTH and the related peptides CRF and alpha-MSH can influence the microenvironment modulating an IL-4 and anti-CD40 monoclonal antibody driven class switching to IgE via accessory cells.     

Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels

Two full-length cDNAs, gbr-2A and gbr-2B, encoding inhibitory amino acid receptor subunits have been amplified and cloned from Caenorhabditis elegans mRNA. The 5' 732 bp of the two cDNAs, encoding 237 amino acids, are identical. The 3' 758 bp of the gbr-2B cDNA are present within the 3' untranslated region of the gbr-2A clone. As a result, the two cDNAs are predicted to encode subunits which share a common extracellular N-terminal sequence of 237 amino acids, but different, though closely related, C-terminal sequences which include four predicted membrane-spanning regions. A search of the EMBL database revealed that the sequences of the two subunits are most closely related to the alpha-subunit of the C. elegans avermectin receptor. Northern blot analysis showed the presence of two related mRNAs of approximately 2.2 and 1.5 kb in a developmentally mixed population of C. elegans. The genomic DNA sequence confirms that both mRNAs were transcribed from the same gene, gbr-2, suggesting that the closely related 3' sequences have arisen as a result of a partial gene duplication event. We propose that C. elegans is utilising alternative splicing to generate receptor subunits with identical extracellular, ligand-binding domains but different transmembrane, channel forming domains.