Development of human single-chain antibodies against SARS-associated coronavirus.

The outbreak of severe acute respiratory syndrome (SARS), caused by a distinct coronavirus, in 2003 greatly threatened public health in China, Southeast Asia as well as North America. Over 1,000 patients died of the SARS virus, representing 10% of infected people. Like other coronaviruses, the SARS virus also utilizes a surface glycoprotein, namely the spike protein, to infect host cells. The spike protein of SARS virus consists of 1,255 amino acid residues and can be divided into two sub-domains, S1 and S2. The S1 domain mediates the binding of the virus to its receptor angiotensin-converting enzyme 2, which is abundantly distributed on the surface of human lung cells. The S2 domain mediates membrane fusion between the virus and the host cell. Hence two strategies can be used to block the infection of the SARS virus, either by interfering with the binding of the S1 domain to the receptor or by blocking the fusion of the virus with the cell membrane mediated by the S2 domain. Several antibodies against the S1 domain have been generated and all of them are able to neutralize the virus in vitro and in vivo using animal models. Unfortunately, point mutations have been identified in the S1 domain, so that the virus isolated in the future may not be recognized by these antibodies. As no mutation has been found in the S2 domain indicating that this region is more conserved than the S1 domain, it may be a better target for antibody binding. After predicting the immunogenicity of the epitopes of the S2 domain, we chemically synthesized two peptides and also expressed one of them using a recombinant DNA method. We screened a phage displaying library of human single-chain antibodies (ScFv) against the predicted epitopes and obtained a human ScFv which can recognize the SARS virus in vitro.

Immunomodulating effects of CKBM on the cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy volunteers.

The current study investigated the immunomodulating effect of CKBM on cytokine induction in peripheral blood mononuclear cells (PBMCs) isolated from 20 healthy volunteers. Cytometric Bead Analysis (CBA) was used to study IL-2, IL-4, IL-6, IL-10, TNF-alpha and IFN-gamma. TNF-alpha and IL-6 were significantly increased in a CKBM dose- and time-dependent manner. Flow cytometry analysis showed an increased intracellular staining of IL-6 but not of TNF-alpha in CKBM treated PBMCs. In addition, MTT cell cytotoxicity assay showed that CKBM concentrations below 5% did not significantly affect the metabolic activities of PBMCs. The current study indicated that CKBM may modulate the immune response by inducing the secretions of TNF-alpha and IL-6, which are cytokine mediators of innate immunity and inflammation preparing or "priming" the body to combat diseases.

Anti-cancer and pro-apoptotic effects of an herbal medicine and Saccharomyces cerevisiae product (CKBM) on human hepatocellular carcinoma HepG2 cells in vitro and in vivo.

Hepatocellular carcinoma is a major health problem worldwide. Different treatment strategies have been developed to cope with this problem. Herbal medicine is now widely studied in both Eastern and Western countries. In this study, we used both in vitro and in vivo model to illustrate the anti-tumor effect of a product, CKBM, consisting of herbal medicine and specially processed Saccharomyces cerevisiae. Dose-dependent anti-proliferation effect was observed on in vitro growth of human hepatoma HepG2 cells after 48 hours incubation with CKBM. At the 50% inhibitory concentration (IC50) no significant toxic effect was observed on normal human fibroblasts Hs68 and human liver WRL-68 cells. The results of morphological changes, detection of DNA fragmentation, flow cytometric analysis and Western blot analysis indicated that this anti-tumor effect of CKBM was mediated via the process of apoptosis. In addition, HepG2 cells- bearing nude mice model was used for in vivo anti-tumor study. Our results showed that 14-day treatment with 0.8 ml daily dosage of CKBM could inhibit 54.1% of tumor growth. The plasma activities of enzymes specific for heart and liver, namely creatine kinase, lactate dehydrogenase, aspartate transaminase and alanine transaminase, remained at normal levels, indicated that CKBM did not produce toxicity to the host.

The effects of melatonin and Ginkgo biloba extract on memory loss and choline acetyltransferase activities in the brain of rats infused intracerebroventricularly with beta-amyloid 1-40.

Intraventricular infusion of rats with beta-amyloid for 14 days resulted in memory deficit in the water maze as well as decreases in choline acetyltransferase activities and somatostatin levels in the cerebral cortex and hippocampus. These changes were not altered by daily intraperitoneal injection of 20 mg/Kg melatonin. Orally administered Ginkgo biloba extract, however, partially reversed the memory deficit and the decrease in choline actyltransferase activities in the hippocampus. The latter treatment failed to reverse the decrease in somatostatin levels. The results indicate that orally administered Ginkgo biloba extract can protect the brain against beta-amyloid from changes leading to memory deficit through its effect on the cholinergic system.

Variations of mt1 melatonin receptor density in the rat uterus during decidualization, the estrous cycle and in response to exogenous steroid treatment.

The expression of mt1 receptor protein in the rat uterus was investigated using an anti-mt1 polyclonal antibody against the rat mt1 receptor. A melatonin receptor protein of 37 kDa was detectable by Western blotting in the rat uterine membrane preparations. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, was used to localize melatonin receptors in the uterus of the estrous rats and to study the changes of melatonin receptors in pregnancy. Melatonin receptors were found to be localized in the estrous rat uterine antimesometrial stroma. As decidualization of the uterine stroma progressed during pregnancy, the melatonin binding sites were progressively reduced and became confined to the antimesometrial non-decidualized outer stroma. 2-[125I]Iodomelatonin binding sites were not seen in the mesometrial stromal cells during pregnancy. The role of ovarian hormones in the regulation of uterine melatonin receptors was examined by studying the binding at various phases of the estrous cycle, after ovariectomy with and without follow-on treatment of estradiol (E2), progesterone (P4) or both. 2-[125I]Iodomelatonin binding in the rat uterus fluctuated during the estrous cycle, being lowest during metestrus. Ovariectomy caused an almost 70% reduction of 2-[125I]iodomelatonin binding compared with the control. Injections of ovariectomized (OVX) rats with E2 or P4 alone or in combination for 11 days induced a partial restoration of 2-[125I]iodomelatonin binding in the OVX rats. The results show that mt1 melatonin receptors in the rat antimesometrial stroma are regulated by ovarian hormones.

Pretreatment with melatonin reduces volume of cerebral infarction in a rat middle cerebral artery occlusion stroke model.

Melatonin is a potent scavenger of free radicals and an indirect antioxidant. Recent studies have shown that melatonin possesses beneficial effects in experimental models of brain trauma and global cerebral ischemia. The effects of pretreatment with melatonin on volume of cerebral infarction were investigated in the present study. Adult male Sprague-Dawley rats were anesthetized with sodium pentobarbital to undergo right-sided endovascular middle cerebral artery occlusion (MCAO) for 3 hr. A single dose of melatonin (1.5, 5, 15, or 50 mg/kg in 1 mL normal saline) or its vehicle was given via an intraperitoneal injection at 0.5 hr before MCAO. Relative infarction volumes on day 3 after MCAO were significantly reduced in the groups treated with melatonin at 5 (mean +/- S.E.M., 15.7 +/- 2.5%) or 15 (21.4 +/- 3.1 %) mg/kg but not at 1.5 (30.6 +/- 3.5%) or 50 (26.7 +/- 2.8%) mg/ kg when compared with the vehicle group (33.9 +/- 3.5%). There was no significant difference in the arterial blood pressure (BP), heart rate (HR) and relative cerebral blood flow among the experimental groups. These results indicate that pretreatment with melatonin at a dose between 5 and 15 mg/kg protects against focal cerebral ischemia.

Receptor-mediated modulation of avian caecal muscle contraction by melatonin: role of tyrosine protein kinase.

Abstract: Melatonin receptors in the quail caecum were studied by 2[125I]iodomelatonin binding assay and the involvement of tyrosine protein kinase in the melatonin-induced contraction was explored. The binding of 2[125I]iodomelatonin in the quail caecum membrane preparations was saturable, reversible and of high affinity with an equilibrium dissociation constant (Kd) of 24.6 +/- 1.1 pm (n = 7) and a maximum number of binding sites (Bmax) of 1.95 +/- 0.09 fmol (mg/protein) (n = 7). The relative order of potency of indoles in competing for 2[125I]iodomelatonin binding was: 2-iodomelatonin > melatonin > 2-phenylmelatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that ML(1) receptors are involved. The binding was inhibited by Mel1b melatonin receptor antagonists, luzindole and 4-phenyl-2-propionamidotetralin (4-P-PDOT) as well as by non-hydrolyzable analogs of GTP like GTPgammaS and Gpp(NH)p but not by adenosine nucleotides. The latter suggests that the action of melatonin on the caecum is G-protein linked. Cumulative addition of melatonin (1-300 nM) potentiated both the amplitude and frequency of spontaneous contractions in the quail caecum. The potentiation of rhythmic contractions was blocked by both luzindole and 4-P-PDOT. Antagonists of tyrosine kinase, genistein(2 microM) and erbstatin(4 microM) suppressed the modulation of spontaneous contractions by melatonin, but not inhibitors of protein kinase C (PKC) or protein kinase A (PKA). Melatonin-induced increment in spontaneous contraction was blocked by nifedipine (0.4 nM). Thus, we suggest that melatonin potentiates spontaneous contraction in the quail caecum via interacting with G-protein-coupled Mel(1b) receptor which may activate L-type Ca2+ channels by mobilizing tyrosine kinases.

Modulation of blood glucose by melatonin: a direct action on melatonin receptors in mouse hepatocytes.

Melatonin receptors were studied in isolated mouse hepatocytes using the 2[(125)I]iodomelatonin binding assay. The binding of 2[(125)I]iodomelatonin to hepatocytes isolated from the mouse using collagenase was stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (K(d)) obtained from saturation studies was 10.0 +/- 0.4 pmol/l (n = 16), which was comparable to the K(d) obtained from kinetics studies (6.9 +/- 1.2 pmol/l, n = 3), and the maximum number of binding sites (B(max)) was 2.9 +/- 0.4 fmol/mg protein (n = 16). The relative order of potency of indoles in competing for 2[(125)I]iodomelatonin binding was 2-iodomelatonin > 2-phenylmelatonin > 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that the binding was mediated by the ML(1) receptor subtype. The linear Rosenthal plots, the close proximity of the Hill coefficient to unity and the monophasic competition curves suggest that a single class of 2[(125)I]iodomelatonin binding sites is present in the mouse hepatocytes. Guanosine 5'-O-(3-thiotriphosphate) dose-dependently inhibited 2[(125)I]iodomelatonin by lowering the affinity of binding, while no inhibitory effects of adenosine nucleotides were observed, suggesting that the binding sites are G-protein linked. Western immunoblotting was used to identify the melatonin receptor subtype in mouse hepatocytes using anti-Mel(1a) and anti-Mel(1b). Hepatocyte membrane extract reacted with anti-Mel(1b) but not anti-Mel(1a) giving a peptide-blockable band of 36 kD, supporting the hypothesis that the melatonin receptors in mouse hepatocytes are of the Mel(1b) subtype. Melatonin injection and a high plasma glucose level affected 2[(125)I]iodomelatonin binding in the whole mouse liver homogenates. Plasma glucose was elevated by mid-light intraperitoneal injection of melatonin (4 and 40 mg/kg body weight) in a dose-dependent manner with maximum elevation achieved 1 h after injection. 2[(125)I]Iodomelatonin binding at this time showed increased K(d) with no changes in B(max). When the plasma glucose returned to normal within 2 h, the binding remained lowered with increased K(d) but no changes in B(max). Elevation of plasma glucose by 2-deoxyglucose injection (500 mg/kg), on the other hand, decreased the binding by decreasing the B(max) without affecting the K(d). Suppression of plasma glucose by insulin injection (3 IU/kg) did not change the binding. Thus, melatonin may act directly on the liver to elevate the plasma glucose level, and changes in plasma glucose level itself may in turn affect hepatic melatonin binding.

Direct action of melatonin in human granulosa-luteal cells.

The direct involvement of melatonin in modulation of ovarian steroidogenesis, the high levels of melatonin found in human follicular fluid, and the presence of melatonin binding sites in the ovary led us to hypothesize that melatonin acts as a modulator of ovarian function. In contrast to the hypothalamus and pituitary, the mechanism of melatonin action at the level of the ovary is still poorly understood. In the present study, we investigated the gene expression of the two different forms of melatonin receptors in human granulosa-luteal cells, using RT-PCR. PCR products corresponding to the expected sizes of the melatonin receptor subtypes, mt(1)-R and MT(2)-R, were obtained from granulosa-luteal cells, and the authenticity of the PCR products was confirmed by Southern blot hybridization with cDNA probes. Subsequent cloning and sequence analysis revealed that the ovarian mt(1)-R and MT(2)-R cDNAs are identical to their brain counterparts. Because gonadotropins and GnRH acting through specific receptors in the human ovary regulate cellular functions, we investigated the role of melatonin in the regulation of FSH receptor, LH receptor, GnRH, and GnRH receptor levels. Treatment with melatonin (10 pM-100 nM) significantly increased LH receptor mRNA levels without altering the expression of the FSH receptor gene. Both GnRH and GnRH receptor mRNA levels were significantly decreased, to 61% and 45% of control levels, respectively, after melatonin treatment. Melatonin treatment alone had no effect on basal progesterone production but enhanced the effects of human CG-stimulated progesterone production. Because MAPKs are activated in response to a diverse array of extracellular stimuli leading to the regulation of cell growth, division, and differentiation, and because melatonin has been shown to modulate cellular proliferation and differentiation, in this study, we demonstrated that melatonin activated MAPK in a dose- and time-dependent manner. In summary, our studies demonstrate, for the first time, that melatonin can regulate progesterone production, LH receptor, GnRH, and GnRH receptor gene expression through melatonin receptors in human granulosa-luteal cells, which may be mediated via the MAPK pathway and activation of Elk-1. Our results support the notion that melatonin plays a direct role in regulating ovarian function.

Effects of halothane, pentobarbital and ketamine on serum melatonin levels in the early scotophase in New Zealand white rabbits.

In mammals, the nocturnal rise in pineal melatonin is regulated by signals from the endogenous clock, the hypothalamic suprachiasmatic nuclei. There have been few reports on whether anaesthetics which modulate multisynaptic neuronal functions affect melatonin secretion. We studied the effects of three commonly used anaesthetics, halothane, pentobarbital and ketamine, on serum melatonin levels in male New Zealand white rabbits. Seven blood samples were collected, 30-60 min apart, before, during and after anaesthesia. Experiments were performed in the late light and early dark period, so that changes in melatonin secretion would be reflected in the onset and/or level of nocturnal serum melatonin. Serum melatonin levels were determined by radioimmunoassay. Our results indicated that halothane attenuated the release of melatonin and pentobarbital had no apparent effect, whereas ketamine potentiated the release of melatonin. These findings suggest that melatonin levels may be affected in patients anaesthetized with halothane or ketamine, resulting in disturbed biological rhythms, especially the sleep-wake cycle following recovery.

Effect of light on 5-methoxyindole-3-acetic acid in the rat.

This study aims to determine the effects of light on the levels of 5-MIAA to provide further information on this indoleamine, using a sensitive and specific radioimmunoassay (RIA) for immunoreactive 5-methoxyindole-3-acetic acid (5-MIAA) developed in our laboratories using a specific antibody and tritiated label. Significant differences were found in the immunoreactive 5-MIAA levels between mid-light and mid-dark pineal glands in rats adapted to 12/12 hrs light/dark and in constant darkness. Under constant light, the circadian rhythm was abolished. The rat serum displayed no diurnal variations in 5-MIAA levels under aux photic conditions. The persistence of rhythms found in constant darkness but abolished in constant light suggests that the pineal 5-MIAA is endogenous and uses light as an environmental cue. In addition to melatonin, 5-MIAA could possibly be another pineal photoperiodic signal in animals.

Chimeric Galphaq subunits can distinguish the long form of the Xenopus Mel1c melatonin receptor from the mammalian mt1 and MT2 melatonin receptors.

The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.

Melatonin and its analogs potentiate the nifedipine-sensitive high-voltage-activated calcium current in the chick embryonic heart cells.

Effects of melatonin and its analogs on the voltage-activated calcium current of embryonic chick ventricular cardiomyocytes were investigated. Myocytes were dissociated from 14- to 16-day-old chicks (yellow Red Rob) embryonic hearts and cultured for 2 3 days. Calcium currents were studied by the patch-clamp technique. Whole-cell current recording showed nifedipine-sensitive, high-voltage-activated L-type calcium current inactivated in 70-100 ms during the voltage step period of 200 ms. There was no evidence of low-voltage-activated T-type calcium channels. Melatonin (ejected solution: 50 micromol/L melatonin; concentration at the vicinity of recording cell: about 1-5 micromol/L melatonin) and its analogs, 2-iodomelatonin and 2-iodo-n-butanol-5-methoxytryptamine, significantly increased the amplitude of the calcium current by 42-62%. The effect of melatonin on the L-type calcium current was not desensitised by repeated melatonin treatment. Our results suggest a specific melatonin receptor-mediated action on the calcium channel of the embryonic chick myocyte. The melatonin-induced increase in high-voltage calcium current may increase myocyte contractility and enhance cardiac output. A regulatory role of melatonin on the chick cardiac function should be further considered.

[Inhibitory effect of melatonin on the development of pituitary prolactin-producing tumors induced by 17-beta-estradiol].

OBJECTIVE: To examine the inhibitory effect of melatonin (MLT) on the development of pituitary prolactin-producing tumors (prolactinoma) induced by 17-beta-estradiol (E2), in vivo, and explore MLT's oncostatic mechanisms. METHODS: The prolactinomas were established by implanting E2-laden silastic capsules subcutaneously in Sprague-Dawley male rats. MLT doses 0.05, 0.25, 0.50, 1.00, and 2.00 mg/rat were administrated separately to 5 groups subcutaneously starting seven days prior to tumor induction for 97 days. The matched controls were given equal volumes of 4% alcohol in saline. RESULTS: (1) The prolactinoma weights in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 25.91% (P > 0.05), 48.78% (P < 0.01), 36.78% (P < 0.05), 31.04% (P > 0.05) and 35.22% (P > 0.05) respectively which were lower than that of control group; (2) The PRL mRNA levels of prolactinoma in 0.05, 0.25, and 0.50 mg MLT dose groups were 33.67% (P < 0.05), 25.51% (P < 0.05) and 41.84% (P < 0.01) respectively which were lower than that of control group as estimated by Northern Blot, and the in situ hybridization studies; (3) The DNA contents of prolactinoma in 0.05, 0.25 and 0.50 mg MLT dose groups were 40.73% (P < 0.001), 51.15% (P < 0.001) and 60.23% (P < 0.001) respectively which were lower than that of control group by laser scanning confocal microscopy; (4) Plasma peroxidative lipid contents in 0.05, 0.25, 0.50, 1.00 and 2.00 mg MLT dose groups were 26.45% (P < 0.05), 23.97% (P < 0.05), 47.11% (P < 0.001), 66.12%(P < 0.001) and 64.46% (P < 0.001) respectively which were lower than that of control group. The correlation coefficient between MLT doses and plasma peroxidative lipid contents was -0.8257 (P < 0.05). CONCLUSIONS: MLT in suitable doses is able to inhibit the development of E2-induced prolactinoma by inhibiting the expression of PRL gene and the DNA synthesis. The link between MLT antioxidative action and its inhibitory effect on development of prolactinoma should be further investigated.

Inhibition of malignant trophoblastic cell proliferation in vitro and in vivo by melatonin.

Melatonin inhibited thymidine incorporation into human choriocarcinoma JEG-3 cells at physiological and pharmacological concentrations in the present study. Gene expression of MT2 receptor, but not that of mt1 receptor, was detected in JEG-3 cells by reverse transcription-polymerase chain reaction (RT-PCR). The gene expression profile of the two human melatonin receptor subtypes in JEG-3 cells was identical to that previously reported for JAr cells, whose proliferation had also been shown to be similarly inhibited by physiological and pharmacological concentrations of melatonin. In contrast, melatonin had no effect on thymidine incorporation into 3A-Sub-E cells (a transformed trophoblast cell line), in which gene expression of both receptor subtypes could not be detected. The data suggest that in human placental trophoblasts, a correlation may exist between MT2 receptor gene expression and the direct anti-proliferative action of melatonin. Although melatonin has been reported to induce G1/S delay in cell cycle progression of JAr cells, no significant changes in the percentages of JEG-3 cells in different cell cycle phases upon melatonin treatment was recorded by flow cytometric analysis. This indicates that G1/S transition delay is probably not an important cellular mechanism in the direct anti-proliferative action of melatonin on human JEG-3 cells in vitro. Furthermore, melatonin inhibited the growth of both JAr and JEG-3 xenograft tumors in athymic nude mice, and prolonged the survival of those animals that developed choriocarcinoma. While the number of apoptotic tumor cells was not increased by melatonin, the pineal hormone induced significant decreases in the numbers of JAr and JEG-3 cells expressing proliferating cell nuclear antigen (PCNA) and cyclin A in the tumors. Taking into account both the in vitro and in vivo findings, it is likely that the inhibitory effect of melatonin on choriocarcinoma JAr and JEG-3 cell proliferation in vivo is largely a direct action of the hormone on the tumor cells.

Potential involvement of mt1 receptor and attenuated sex steroid-induced calcium influx in the direct anti-proliferative action of melatonin on androgen-responsive LNCaP human prostate cancer cells.

Melatonin, a pineal secretory product, has been shown to exert a direct anti-proliferative action on the androgen-sensitive LNCaP prostate cancer cell line through hitherto undefined mechanisms. In this communication, expression of mt1 melatonin receptor protein in human prostate cancer tissues and LNCaP cells was demonstrated by immunohisto(cyto)chemistry and western blotting, hence supporting the use of LNCaP cell line as a model for the study of melatonin signaling in prostate cancer cell growth. Using 3H-thymidine incorporation assay, LNCaP cell proliferation was inhibited by 2-iodomelatonin, a high-affinity melatonin receptor agonist. Furthermore, melatonin inhibited 3H-thymidine incorporation into LNCaP cells and attenuated 5alpha-dihydrotestosterone (DHT) or 17beta-estradiol (E2)-induced stimulation of LNCaP cell proliferation at physiological and pharmacological concentrations. Similar concentration-dependent inhibition of sex steroid-induced stimulation of thymidine incorporation into LNCaP cells by 2-iodomelatonin was also observed. Interestingly, attenuation of sex steroid-stimulated calcium influx into LNCaP cells by pharmacological concentrations of melatonin was recorded, whereas 2-iodomelatonin had no effect on cytosolic calcium changes induced by sex steroids. In addition, proliferative and cytosolic calcium changes were associated with inhibition of total prostate-specific antigen (PSA) production by LNCaP cells at high physiological and pharmacological concentrations of melatonin. Our data suggest that activated mt1 receptor and attenuated sex steroid-induced calcium influx are two important mechanisms mediating the direct anti-proliferative action of melatonin on androgen-responsive human prostate cancer cells.

Biological basis and possible physiological implications of melatonin receptor-mediated signaling in the rat epididymis.

The mammalian epididymis plays an important role in sperm maturation, an important process of male reproduction. Specific high-affinity 2-[(125)I]iodomelatonin binding sites, satisfying the pharmacokinetic properties of specific receptors, have been found in the rat corpus epididymis, suggesting a direct melatonin action on epididymal physiology. Subsequent molecular and cell biology studies have identified these 2-[(125)I]iodomelatonin binding sites to be mt(1) (MEL(1A)) and MT(2) (MEL(1B)) melatonin receptor subtypes. Changes in the binding characteristics of these receptors in the rat corpus epididymis in response to castration and steroid hormones like testosterone and hydrocortisone indicated that these membrane melatonin receptors are biologically functional receptors, whose activities are differentially regulated by testosterone and hydrocortisone. These melatonin receptors are coupled to pertussis toxin (PTX)-sensitive G(i) protein and probably participate in androgenic and adrenergic regulation of rat corpus epididymal epithelial cell functions. Furthermore, rat corpus epididymal epithelial cell proliferation was stimulated by melatonin, whose action was dependent on the concentration and duration of exposure to the hormone. Interestingly, an MT(2) receptor ligand (4-phenyl-2-propionamidotetraline, 4-P-PDOT) induced a stimulatory effect on epididymal epithelial cell proliferation similar to that produced by melatonin. In contrast, a nuclear melatonin receptor agonist (1-[3-allyl-4-oxo-thiazolidine-2-ylidene]-4-methyl-thiosemi-car bazone , CGP52608) and 8-bromo-cAMP inhibited epididymal epithelial cell proliferation. Taken together, our data lead us to postulate that one of the possible physiological functions of melatonin on the rat epididymis is the stimulation of mt(1) and MT(2) melatonin receptors resulting in the inhibition of cAMP signaling and an increase in epithelial cell proliferation.

Dopaminergic and GABAergic amacrine cells are direct targets of melatonin: immunocytochemical study of mt1 melatonin receptor in guinea pig retina.

Distribution of the mt1 melatonin receptor in the guinea pig retina was immunocytochemically investigated using peptide-specific anti-mt1 receptor antibody. Western blots of the guinea pig retina showed a single band at approximately 37 kilodalton (kD) immunoreactive to the anti-mt1 antibody. The most intense immunoreactivity for the mt1 receptor was detected in the cell bodies of ganglion cells. Their dendrites and axons were also immunolabeled. Subpopulations of amacrine cells, the inner plexiform layer, and the outer plexiform layer also exhibited moderate to weak immunolabeling. The mt1-positive amacrine cells were located either at the vitreal border of the inner nuclear layer or displaced in the ganglion cell layer. Double immunolabeling using antibodies to the mt1 receptor and tyrosine hydroxylase revealed that the majority of dopaminergic amacrine cells showed mt1 immunoreactivity. Almost all the ICA type dopaminergic cells were mt1 positive while the 2CA type cells less frequently exhibited mt1 immunoreaction. By double immunolabeling for the mt1 receptor and GABA, more than 50% of the mt1-immunoreactive amacrine cells were shown to be GABAergic neurons. Approximately one-third of the GABAergic amacrine cells were immunolabeled for the mt1 receptor. The present results demonstrate expression of the mt1 receptor in diverse neuronal cell types in the guinea pig retina and provide the first evidence for the direct effect of melatonin on dopaminergic and GABAergic amacrine cells via the mt1 receptor.

Pituitary prolactin-secreting tumor formation: recent developments.

Prolactinoma is the most common type of primary pituitary tumors. It occurs more frequently in women than in men. Dopaminergic agonists are effective in the shrinkage of prolactin-secreting pituitary tumor and are preferred in some patients. However, pituitary radiotherapy may enable the long-term removal of prolactin-secreting tumor cells. Recent evidence suggests that prolactinoma is a heterogeneous disorder with complicated and multifactorial etiology and pathogenesis. Apparently, a thorough understanding of prolactinoma tumorigenesis would be important. To facilitate investigations on tumorigenesis of prolactinoma, animal models for prolactinomas have been developed. These models have expedited our progress in the recent years. Many researchers consider the F(344) rat to be the most sensitive strain of rats to estrogen (E(2))-induced prolactinoma formation. Nonetheless, E(2) treatment for 60 days also induces the formation of pituitary prolactin-secreting adenoma in male Sprague-Dawley (SD) rats. Evidently, the SD rat is also a good animal for prolactinoma investigations. Following E(2) implantation, prolactinomas developed in the eutopic adenohypophysis in situ and/or ectopic pituitary grafted under the renal capsule in SD rats. These observations favor the hypothesis that prolactinoma growth is the result of pathological changes in the adenohypophysis and/or hypothalamus. In the latter case, abnormal release of hypothalamic dopamine, GABA, or brain-gut peptides (such as cholecystokinin, vasoactive intestinal polypeptide, galanin, angiotensin, opioid peptide, gastrin, gastrin-releasing peptide, pancreatic polypeptide, and adrenocorticotropic hormone) results in some of the pathological changes that may lead to hyperprolactinemia and/or prolactinoma development. Dysregulation of prolactin synthesis and secretion may be the result of prolactin gene modulation. In E(2)-induced rat prolactinomas, prolactin mRNA contents and the expression of some proto-oncogenes, e.g. c-myc and c-ras, TGFalpha and TGFbeta1 mRNA were significantly changed. The above findings are consistent with results in human prolactinoma development. In addition, in rats abnormal expression of the prolactin gene was correlated with hypomethylated status of CpG sites in exons 1, 2 and 4 of the prolactin gene, as well as the increase in hypersensitive sites to DNase 1 in the encoding region of the prolactin gene. In E(2)-treated rats, a point mutation with a base substitution from cytidine (C) to adenine (A) was found at the -36-bp site of the proximal promoter of the prolactin gene in eutopic pituitary prolactinomas, but no change was observed in the same sequence of the prolactin gene in ectopic prolactinoma. The association of a base substitution with the hyperexpression of the prolactin gene in eutopic prolactinomas suggests that different mechanisms may mediate the formation of eutopic and ectopic prolactin-secreting tumors. Melatonin decreases the expression of the prolactin gene in vitro suggesting that this pineal hormone may be a potential anticarcinogen in vivo. It has also been shown that MT(2) (Mel(1b)) melatonin receptors are expressed in anterior pituitary cells. The use of melatonin as a preventive or therapeutic drug for prolactinomas should be further investigated. In summary, improved knowledge on tumorigenesis of prolactinomas, especially in the rat model, was noted. These E(2)-induced rat prolactinoma models would facilitate future investigations, and expected results shall be fruitful and exciting for the development of future drug designs for the prevention and/or treatment of prolactin-secreting pituitary tumors.

Circadian variation of portal, arterial and venous blood levels of melatonin in pigs and its relationship to food intake and sleep.

Circadian levels of melatonin were determined in the hepatic portal vein, cranial vena cava, and the lower aorta of ten juvenile pigs. Blood was sampled every hour for a total of 24 hr via temporary cannulas introduced into blood vessels under anesthesia. No peak levels of melatonin were found in the mid-scotophase, but hepatic portal concentrations peaked at 06.00 hr. Overall levels of melatonin were highest in the hepatic portal vein (range 35-65 pg/mL), followed by an artery (range 30-55 pg/mL) and the vena cava (range 25-35 pg/mL). Levels of melatonin exhibit strong variation between individual pigs, but generally the average levels from all three sources follow each other's time course. However, on occasion, melatonin levels in the hepatic portal vein varied independently from the levels in the vena cava. Large portal peaks were usually preceded by a feeding period and were associated with a subsequent period of sleep. The data indicate that: 1) there is no clear circadian rhythm of melatonin in the peripheral blood of pigs, 2) relatively little melatonin is metabolized during the first liver passage, 3) food intake may elevate melatonin levels in the hepatic portal vein, and 4) increased levels of melatonin originated in the gastrointestinal tract may induce sleep.