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.

Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line.

The expression of melatonin receptors (MR) of the Mel1a subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK-293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I-MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (Kd) value of 23.8 +/- 0.5 pM and a maximum number of binding sites (Bmax) value of 1.17 +/- 0.11 fmol/mg protein (n = 5), which are comparable with the reported Kd and Bmax values in human kidney cortex. Coincubation with GTPgammaS (10 microM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (Kd was increased by a factor of 1.5-2.0), with no significant difference in Bmax. Melatonin (1 microM) decreased the forskolin (10 microM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel1a receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel1a receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT-PCR, HEK-293 cells express both Mel1a and Mel1b messenger RNAs, but the messenger RNA level for Mel1b is several orders of magnitude lower than for Mel1a. We conclude that HEK-293 cells express MR predominantly of the Mel1a subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.

2-[125I]iodomelatonin-binding sites in the human kidney and the effect of guanosine 5'-O-(3-thiotriphosphate).

Putative melatonin receptors in normal kidney cortical tissues of patients with transitional cell carcinoma or renal cell carcinoma were characterized using a melatonin agonist, 2-[125I]iodomelatonin, as the radioligand. 2-[125I]Iodomelatonin-binding sites in the human kidney were stable, saturable, reversible, and of high affinity. The binding affinity was 15.2 +/- 2.5 pmol/L, and the binding density was 1.79 +/- 0.19 fmol/mg protein. The unity of the Hill coefficients and linearity of the Scatchard plots suggested that 2-[125I]iodomelatonin was bound to a single class of binding sites. Pharmacological characterization showed that these binding sites were highly specific to 2-iodomelatonin, melatonin, 6-hydroxymelatonin, and 6-chloromelatonin. Guanosine 5'-O-(3-thiotriphosphate) decreased the binding affinity and density of 2-[125I]iodomelatonin-binding sites in the kidney, suggesting that these binding sites are coupled to a G-protein. The characterization of 2-[125I]iodomelatonin-binding sites in normal kidney tissues taken from patients with transitional cell carcinoma or renal cell carcinoma suggests the existence of 2-[125I]iodomelatonin-binding sites in the human kidney cortex, which is in line with the findings of 2-[125I]iodomelatonin-binding sites in kidneys of other mammals and birds. The implication of a direct melatonin action on renal function in the human is proposed.

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.

Nocturnal elevation of plasma melatonin and urinary 5-hydroxyindoleacetic acid in young men: attempts at modification by brief changes in environmental lighting and sleep and by autonomic drugs.

In order to determine whether the human pattern of circulating melatonin resembles that previously described in lower animals, men 19-32 years old were exposed to a light-dark cycle with 14 hours of light per day (L:D 14:10). In whites and blacks, nocturnal (dark phase, sleeping) melatonin levels were almost always elevated to 0.05-0.1 ng/ml plasma compared with lower or undetectable levels during the day, measured by the tadpole bioassay. Thin-layer migration of bioactive material was identical to that for melatonin standard. A rhythm with nocturnal elevation of urinary 5-hydroxyindoleacetic acid (5-HIAA) was observed. Nocturnal (sleep phase) rise in blood melatonin (but not urinary 5-HIAA) continued during 21/2 day-night cycle lengths after the onset of constant light. Though the dark phase plasma melatonin rise was less marked after reversal of the sleep-wake cycle (no change in the light cycle), dark phase rise in urinary 5-HIAA continued. Though marked cardiovascular and other effects were produced by intravenous isoproterenol or scopolamine, no definite effect on melatonin levels was observed after either drug during the light phase in waking subjects.

Molecular and functional characterization of a partial cDNA encoding a novel chicken brain melatonin receptor.

An approach based on homology probing was used to clone a partial cDNA encoding a novel melatonin (ML) receptor (MLR) from chicken (Gallus domesticus) brain. Based on available deduced amino-acid sequence, the chicken MLR (cMLR) displayed greater sequence homology to the frog (Xenopus) MLR than cloned human/mammalian receptors, with overall identities of 73% and 66%, respectively. In order to gain functional expression, a chimeric frog/chicken (flc)MLR was constructed in which the 5' end of the cMLR, including the N-terminus, TM1 and part of the first intracellular loop was substituted by fMLR sequence. [125I]Iodo-ML bound with high affinity (Kd of approximately 35 pM) to COS-7 cells transiently expressing the flcMLR in a saturable and guanine nucleotide-sensitive manner with the following rank order of potency: 2-iodo-ML > ML > 6-Cl-ML > S20750 > 6-OH-ML > S20642 > S20753 > N-acetyl-5HT >> 5-HT. Estimated Ki values for these compounds at the flcMLR correlated well to those obtained in native chicken brain membranes. In line with the observed structural similarity to the fMLR, the flcMLR exhibited affinities for ML, 6-Cl-ML and 6-OH-ML approximately 10-fold lower than mammalian receptors. Functionally, opposing interactions between ML and dopamine receptor signal transduction pathways were observed with ML potently inhibiting dopamine D1A-receptor-mediated cAMP accumulation in cells (HEK-293) transiently co-expressing these receptors. cMLR mRNAs were found expressed in chicken brain and kidney with trace levels observed in the lung. The availability of cloned vertebrate MLRs distinct at both the amino acid and pharmacological level from their mammalian counterparts may now allow for the identification of those amino-acid residues and structural motifs that regulate ML-binding specificity and affinity.

Expression of mt1 melatonin receptor in rat retina: evidence for multiple cell targets for melatonin.

Melatonin is synthesized in the retina at night and acts as a local modulator within this tissue by mediating the effects of darkness. We investigated the expression and localization of the mt1 (Mel1a) melatonin receptor in rat retina in order to disclose the cellular and molecular bases of melatonin's action in the mammalian retina. Western blotting of the mt1 receptor in rat retina exhibited a single immunoreactive band of approximately 37,000 mol. wt, which corresponds to the predicted molecular size of the receptor. The mt1 receptor was immunocytochemically localized to both the inner and outer plexiform layers. During postnatal development, retina from two-week-old rats showed the highest mt1 immunoreactivity; the outer plexiform layer and horizontal cell bodies were strongly immunolabeled, with weaker labeling in the inner plexiform layer. Expression of mt1 receptor messenger RNA in the rat retina was demonstrated by reverse transcription-polymerase chain reaction and in situ hybridization. mt1 receptor transcripts were localized to ganglion cells, amacrine cells and horizontal cells. These results suggest that melatonin influences retinal physiology by acting on multiple retinal cell types, including ganglion, amacrine and horizontal cells, via the mt1 receptor expressed in their processes.

[125I]Iodomelatonin-binding sites in the pigeon brain: binding characteristics, regional distribution and diurnal variation.

The binding and pharmacological characteristics of melatonin-binding sites labelled by [125I]iodomelatonin in membrane preparations from the pigeon brain were determined. Specific binding of [125I]iodomelatonin in the membrane preparations of pigeon brain was rapid, stable, saturable and reversible. The [125I]iodomelatonin-binding sites had the following order of pharmacological affinities: melatonin greater than 6-chloromelatonin greater than N-acetylserotonin much greater than 5-hydroxytryptamine greater than tryptamine greater than 5-methoxytryptophol, greater than 1-acetylindole-3-carboxytryptamine, 5-hydroxyindole-3-acetic acid, L-tryptophan and 3-acetylindole. Compounds known to act on serotonin receptors, adrenoceptors or cholinoceptors were inactive compared with melatonin. Of the various brain regions studied, melatonin binding was greatest in the hypothalamus, intermediate in the mid-brain, pons-medulla and telencephalon, and low in the cerebellum. Subcellular fraction studies indicated that 39% of the binding was located in the mitochondrial fraction, 34% in the nuclear fraction, 21% in the microsomal fraction and 5.6% in the cytosol fraction. Scatchard analysis of the membrane preparations revealed a dissociation constant (Kd) of 206.3 +/- 57.9 pmol/l and a total number of binding sites (Bmax) of 26.7 +/- 1.9 fmol/mg protein in the middle of the light period (mid-light). In addition, saturation studies demonstrated that [125I]iodomelatonin-binding sites in pigeon brain membrane preparations were 36.2% higher at mid-light (26.7 +/- 1.9 fmol/mg protein) than in the middle of the dark period (19.6 +/- 1.25 fmol/mg protein), with no significant variation in their binding affinities.

[125I]iodomelatonin-binding sites in the bursa of Fabricius of birds: binding characteristics, subcellular distribution, diurnal variations and age studies.

The melatonin-binding sites in membrane preparations of the bursa of Fabricius of birds were studied using [125I]iodomelatonin as a radioligand. The binding sites were stable, saturable, reversible and of high affinity. Scatchard analysis of specific binding revealed equilibrium binding constants (Kd) of (means +/- S.E.M.) 43.1 +/- 5.9, 73.3 +/- 7.6 and 35.3 +/- 4.8 pmol/l respectively at the mid-point of the light period (mid-light) in chickens, pigeons and quail, with a total number of binding sites (Bmax) of 1.23 +/- 0.15, 1.33 +/- 0.18 and 0.94 +/- 0.07 fmol/mg protein. The diurnal variation in [125I]iodomelatonin binding showed that the Bmax was 45, 115 and 70% higher at mid-light than at mid-dark in the bursae of chickens, pigeons and quail respectively. The Kd value determined from kinetic analysis was 49.0 +/- 6.4 pmol/l at mid-light in the chicken bursa. The [125I]iodomelatonin-binding sites of chicken bursal membranes had the following order of pharmacological affinities: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetyl-serotonin > 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-carboxaldehyde, 3-acetylindole, L-tryptophan, 5-hydroxyindole-3-acetic acid, 5-hydroxytryptophan suggesting that the [125I]iodomelatonin-binding sites were highly specific for melatonin. The subcellular distribution of binding sites in the chicken bursa was in the following descending order: nuclear > mitochondrial > microsomal > cytosolic fraction. There was an age-related decrease in [125I]iodomelatonin-binding in chicken bursal membranes, with higher densities in the neonate.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in the serum concentrations of testosterone in mice and hamsters during their critical periods of neural sexual differentiation.

Male and female mice and hamsters were decapitated 1-5 days after birth and serum concentrations of testosterone determined by radioimmunoassay. In the two species studied, serum levels of testosterone in male pups were significantly (P less than 0.05) higher than those obtained in female neonates. This lends support to the hypothesis that circulating levels of testosterone play an important role in the process of neural sexual differentiation in rodents. Moreover, the sex differences in serum concentrations of testosterone in neonatal rodents together with the detectable levels of testosterone in female neonates may suggest that androgenization is a dose-dependent phenomenon. Alternatively, they may indicate that a minimum concentration of the steroid must be present for androgenization to occur during the critical period of neural sexual differentiation and that this 'threshold' is exceeded in male but not in female rodents.

The identification of 125I-labelled iodomelatonin-binding sites in the testes and ovaries of the chicken (Gallus domesticus).

The existence of 125I-labelled iodomelatonin-binding sites in chicken ovaries and testes was investigated. The specific binding of 125I-labelled iodomelatonin to chicken ovarian and testicular tissue satisfies all the criteria for a binding site. It was rapid, stable, saturable, reversible, specific and of high affinity. Equilibrium studies showed that total and non-specific binding increased over a range of 5-150 pmol 125I-labelled iodomelatonin/l tested, with specific binding reaching saturation towards the middle range of radioligand concentrations. Scatchard analyses indicated a dissociation constant (Kd) of 36.5 +/- 5.3 pmol/l (means +/- S.E.M.) in the membrane preparations of chicken testes at the middle point in the period of light and a maximum number of binding sites (Bmax) of 0.93 +/- 0.40 fmol/mg protein (n = 6). In membrane preparations of chicken ovaries, the Kd was 102.2 +/- 27.3 pmol/l and the Bmax was 2.77 +/- 0.38 fmol/mg protein (n = 6). Equilibrium and kinetic dissociation constants in the picomolar range indicate high-affinity and physiologically relevant 125I-labelled iodomelatonin-binding sites. Competitive inhibition studies determined the following order of relative potency for inhibition of 125I-labelled iodomelatonin-binding to chicken gonadal membranes: 6-chloromelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-acetic acid, 5-hydroxyindole-3-acetic acid and L-tryptophan. The presence of 125I-labelled iodomelatonin-binding sites suggests a direct pineal-gonadal connection in the chicken.

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney.

The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44.6 +/- 4.4 pmol/l (n = 6) and a total number of binding sites (Bmax) of 6.43 +/- 0.60 fmol/mg protein (n = 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1.0, suggesting a single class of 125I-labelled iodomelatonin-binding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53.4% higher at mid-light than at mid-point of the dark period (P < 0.05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22.5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin >> 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function.(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in the level of retinal melatonin and persistence of its diurnal rhythm in rats after pinealectomy.

To study the short- and long-term effects of pinealectomy on the level of retinal melatonin, male rats adapted to a photoperiod of 12 h light: 12 h darkness (with lights on at 06.00 h) were pinealectomy. In the short-term experiment, the rats were decapitated 1 week after pinealectomized. In the long-term experiment, 1 month was allowed for recovery. Melatonin was extracted from retinae and quantified by radioimmunoassay. A diurnal rhythm of retinal melatonin was found to persist after pinealectomy in both experiments. An increase in retinal melatonin was demonstrated 1 month after pinealectomy, indicating a compensatory effect on melatonin in the retinae of pinealectomized rats. Thus, biosynthesis of melatonin in the retina may be modulated through a negative feedback system.

Melatonin in the retina of rats: a diurnal rhythm.

Melatonin was extracted by chloroform from the retina of the rat and measured by radioimmunoassay. In rats housed under a régime of 12 h light: 12 h darkness, melatonin content in the retina, like that in the pineal, serum and brain tissue, was high from midway through the dark to early in the period of light, and low from half-way through the light period to early in the period of darkness. A similar result was found in a second experiment. The existence of a diurnal rhythm of melatonin in the retina of rats as observed in this study is consistent with the suggestion that melatonin may regulate the diurnal rhythm of eye pigmentation in vertebrates.

Serum concentrations of testosterone, oestrogens, luteinizing hormone and follicle-stimulating hormone in male and female rats during the critical period of neural sexual differentiation.

Untreated male and female rat pups were killed 1--5 days post partum and the serum concentrations of testosterone, oestrogens, LH and FSH were determined by radioimmunoassay. At all five sampling times, the serum concentrations of testosterone in male rats were about three times higher than those in female rats, but serum levels of oestrogens did not differ between the sexes. Serum concentrations of LH and FSH were lower in male than in female pups. In another study, rats were decapitated 1--10 days after birth and serum concentrations of testosterone were determined with a different radioimmunoassay. Again, at all four sampling times, the concentration of testosterone was significantly higher in the male than in the female pups.

Cortisol decreases 2[125I]iodomelatonin binding sites in the duck thymus.

The immunosuppressive effect of chronic glucocorticoid treatment on 2[125I]iodomelatonin binding in the duck thymus was studied. Two-week-old ducks were injected intraperitoneally with either 1 mg of cortisol per day (experimental group) or an equivalent volume of vehicle (control group) in the middle of the light period for 7 days. 2[125I]Iodomelatonin binding assays were performed on thymic membranes. Cortisol injection reduced the body weight gain, size of the bursa of Fabricius and absolute weights of the primary lymphoid organs but had no effect on the spleen weights. The relative weights of the spleen were increased while those of the primary lymphoid organs were unchanged. The density of the thymus 2[125I]iodomelatonin binding sites was decreased while the affinity was not affected. The modulation of the thymic 2[125I]iodomelatonin binding sites by changes in the immune status of the duck suggests that these binding sites represent physiologically relevant melatonin receptors and that melatonin exerts its action on the lymphoid tissues directly. Our findings support the hypothesis that the thymus is the target site for the immunomodulatory interactions between the pineal melatonin and the adrenal steroids. A possible inhibitory influence of adrenal steroids on the immuno-enhancing effect of melatonin is also suggested.

Melatonin receptors in the chicken kidney are up-regulated by pinealectomy and linked to adenylate cyclase.

The effect of pinealectomy on the characteristics of melatonin receptors in the chicken kidney was studied. One-day-old chicks were operated and kept under a 12 h/12 h light/dark photoperiod. Six weeks after operation, the animals were sacrificed at mid-light and mid-dark. Serum melatonin was determined by radioimmunoassay and kidney melatonin receptors were studied by radioreceptor assay using the melatonin agonist 2-[125I]iodomelatonin as the radioligand. Pinealectomy significantly reduced the mid-dark serum melatonin level and abolished the diurnal rhythm of 2-[125I]-iodomelatonin binding in the kidney. The density of 2-[125I]-Iodomelatonin binding sites in the kidney at mid-dark was increased significantly to a value comparable to the mid-light density after pineal ablation. Our results suggest that melatonin receptors in the chicken kidney are directly regulated by melatonin in the circulation. The coupling of kidney melatonin receptors to adenylate cyclase was investigated. The basal and forskolin-stimulated cAMP production in chicken kidney explants was studied following melatonin or melatonin plus pertussis toxin treatment. Levels of cAMP in chicken kidney explants were extracted and determined by radioimmunoassay. Melatonin had no effect on basal cAMP levels. However, melatonin significantly inhibited the forskolin-stimulated cAMP accumulation at a concentration of 10 pmol/l. Inhibitory effects of melatonin on the forskolin-stimulated cAMP increase in the chicken kidney were totally blocked by preincubating the kidney tissue with 1.0 micrograms/ml pertussis toxin. Our results suggest that kidney melatonin receptors may modulate the adenylate cyclase leading to biological responses in the renal system.

Neuroendocrinology of melatonin in reproduction: recent developments.

The circadian melatonin rhythm with high levels in the dark period is important for the synchronization of reproductive response to appropriate environmental conditions in animals. The target sites of melatonin action on reproductive functions remain to be clarified. Using autoradiography (ARG) and radioreceptor binding assays with 2[125I]iodomelatonin, a melatonin agonist, as the radioligand, studies on the sites of melatonin action have increased significantly in the last ten years. The recent cloning of melatonin receptor subtypes also allowed the characterization of receptor(s) to the molecular level. Earlier reports have documented that the hypothalamic-pituitary axis plays a vital role in the regulation of reproduction by melatonin. This is supported in part by the demonstration of melatonin receptors in the suprachiasmatic nuclei (SCN) in the brain and pars tuberalis (PT) in the pituitary. However, the nature of SCN and PT involvement in the reproductive action of melatonin remains unknown. In addition to the hypothalamus and pituitary, the two classical sites of melatonin action, other targets have been identified. The recent demonstration of 2[125I]iodomelatonin binding sites or melatonin receptors in the testis, epididymis, vas deferens, prostate, ovary and mammary gland suggest the concept of multiple sites of melatonin action on the reproductive system. The presence of melatonin receptors in the said tissues is consistent with earlier reports of direct melatonin actions on different levels of the reproductive system. This multiple levels of melatonin action, from the hypothalamus, pituitary, gonads to other reproductive tissues form a robust system of photoperiodic control in animal reproduction. This would guarantee successful gestation and delivery of the offspring at a time with optimum food availability and ultimately favourable for the survival of species. Molecular and cellular studies of melatonin signaling system(s), its regulation and effects on downstream functional events in the future may provide new insights and directions for the study of the physiology and pharmacology of fertility and contraception in animals and humans.

Evaluation of fluorinated indole derivatives for electron-capture negative ionization mass spectrometry: application to the measurement of endogenous 5-methoxyindole-3-acetic acid in rat.

A series of N-trifluoroacetyl/pentafluoropropionyl-O-trifluoroethyl/ pentafluoropropyl/heptafluorobutyl ester derivatives of 5-methoxyindole-3-acetic acid (5MIAA) were synthesized. Under electron-capture negative ionization conditions, the N-trifluoroacetyl derivatives were found to yield relatively abundant, analyte-specific M-. molecular ions and [M-HF]-., [M-HF-CF2CO]-. and [M-CF3CO]- fragment ions, while the N-pentafluoropropionyl derivatives yielded predominantly the reagent-specific pentafluoroacylium C2F5CO- ion. 5-[2H3]Methoxyindole-3-acetic acid was prepared in high yield by a new synthetic procedure and used as the internal standard in subsequent gas chromatographic/mass spectrometric analysis. Using the N-trifluoroacetyl-O-pentafluoropropyl ester derivative, femtomole to low picomole per gland/organ per g ml-1 levels of endogenous 5MIAA were identified and determined in the rat pineal gland, retina, whole brain and serum.

[125I]iodomelatonin binding sites in the chicken spinal cord: binding characteristics and diurnal variation.

[125I]Iodomelatonin ([125I]MEL) binding in chicken spinal cords was characterized. The specific binding of [125I]MEL to the membrane preparation of spinal cord was rapid, stable, saturable, reversible, specific and of high affinity. Scatchard analysis of the specific binding data indicated an equilibrium dissociation constant (Kd) of 30.7 +/- 3.43 pM and a maximum number of binding sites (Bmax) of 1.69 +/- 0.14 fmol/mg protein in spinal cords collected at mid-light. The diurnal variation study showed that the Bmax was 32% higher (P < 0.05) at mid-light compared to mid-dark. The pharmacological characteristics demonstrated that [125I]MEL binding sites was highly specific for melatonin. Our results suggest that melatonin may exert a direct action on the spinal cord.