Cloning and nucleotide sequence of amidase gene from Pseudomonas putida

The peptidergic signal substance thyrotropin-releasing hormone (TRH) is inactivated by the TRH-degrading ectoenzyme (TRH-DE), a peptidase that exhibits an extraordinary high degree of substrate specificity and other unusual characteristics. There is no other ectopeptidase known capable of degrading this tripeptideamide, and vice versa, TRH is the only known substrate of this unique enzyme. Thus, studies on this enzyme may reveal new aspects on the function of the TRH signaling system. After succeeding in purifying this enzyme to homogeneity and cloning the cDNA encoding rat TRH-DE, molecular tools became available to study the expression of this enzyme by Northern blot analysis and in situ hybridization histochemistry. The stringent and tissue-specific regulation of the adenohypophyseal TRH-DE by estradiol and thyroid hormones strongly suggests that this enzyme may act as a regulatory element modulating pituitary hormone secretion. In brain, the expression of TRH-DE is not influenced by peripheral hormones but the distinct distribution pattern, and the high activities support the concept that in this tissue TRH-DE may act as a terminator of TRH signals.     

The effect of LHRH and TRH on human interferon-gamma production in vivo and in vitro

Accumulating evidence suggests that hypothalamic luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) are two hypophysiotropic factors which modulate the immune response. The aim of the present study was to determine the in vivo effects of an intravenous bolus of LHRH and TRH on plasma interferon (IFN)-gamma production in five normoprolactinemic women with irregular menstrual cycles. We also determined prolactin (PRL), thyrotropin (TSH), follicle stimulating hormone (FSH), and luteinizing hormone (LH) levels before and after intravenous administration of LHRH and TRH. The results demonstrate that intravenous bolus of LHRH/TRH increases plasma IFN-gamma levels, with the maximum response 45 min after in vivo administration of hypothalamic peptides and after peak levels of adenohypophyseal hormones (PRL: 15 min; TSH: 30 min; FSH: 30 min; LH: 30 min). In order to investigate a possible direct action of hypothalamic hormones on immune cells, we also evaluated, in the same subjects, the influence of LHRH and TRH on IFN-gamma production by human peripheral blood mononuclear cells (PBMCs), collected before the intravenous administration of the peptides and stimulated in vitro with bacterial superantigen staphylococcal enterotoxin A (SEA) and concanavalin A (Con A). LHRH and TRH, separately and together, significantly enhanced in vitro IFN-gamma production by SEA- and ConA-activated PBMCs. The present results suggest that hypothalamic peptides (LHRH and TRH) directly, and/or indirectly pituitary hormones (PRL, TSH, FSH, and LH) or IL-2, have stimulatory effect on IFN-gamma producing cells and are further evidence of interactions between the neuroendocrine and immune systems.     

Physiological and pathophysiological aspects of thyrotropin-releasing hormone gene expression in the human hypothalamus

Although the tripeptide thyrotropin-releasing hormone (TRH) was the first hypothalamic hormone to be isolated and characterized, only very few data were available on the central component of the hypothalamus-pituitary-thyroid (HPT) axis in the human brain until recently. We used immunocytochemistry to describe, for the first time, the distribution of TRH-containing cells and fibers in the human hypothalamus. Brain material was obtained with a short postmortem delay followed by fixation in paraformaldehyde, glutaraldehyde, and picric acid. Many TRH-containing cells were present in the paraventricular nucleus (PVN), especially in its dorsocaudal part. Some TRH cells were found in the suprachiasmatic nucleus (SCN), which is the circadian clock of the brain, and in the sexually dimorphic nucleus (SDN), which is in agreement with earlier observations in the rat hypothalamus. Dense TRH-containing fiber networks were present not only in the median eminence but also in a number of other hypothalamic areas, suggesting a physiological function of TRH as a neuromodulator or neurotransmitter in the human brain, in addition to its neuroendocrine role in pituitary secretion of thyroid-stimulating hormone (TSH). As a next step, we developed a technique for TRH mRNA in situ hybridization using a [35S] CTP-labeled TRH cRNA antisense probe in formalin-fixed paraffin-embedded sections. Numerous heavily labeled TRH mRNA-containing neurons were detected in the caudal part of the PVN, while some cells were present in the SCN and in the perifornical area. These results demonstrated the value of in situ hybridization for elucidating the chemoarchitecture of the human hypothalamus in routinely fixed autopsy tissue and enabled us to perform quantitative studies. As part of the neuroendocrine response to disease, serum concentrations of thyroid hormone decrease without giving rise to elevated concentrations of TSH, suggesting altered feedback control at the level of the hypothalamus and/or pituitary. In order to establish whether decreased activity of TRH cells in the PVN contributes to the persistence of low TSH levels in nonthyroidal illness (NTI), hypothalamic TRH gene expression was investigated in patients whose plasma concentrations of thyroid hormones had been measured just before death. Quantitative in situ hybridization showed a positive correlation of total TRH mRNA in the PVN and serum concentrations of TSH and triiodothyronine (T3) less than 24 hours before death, supporting our hypothesis. Current experiments aim at elucidating the mechanism by which hypothalamic thyroid hormone feedback control in TRH cells of patients with NTI is changed.     

Safety and tolerability of a new formulation (90 mg/kg/day divided every 12 h) of amoxicillin/clavulanate (Augmentin) in the empiric treatment of pediatric acute otitis media caused by drug-resistant Streptococcus pneumoniae

Ps4 (thyrotropin-releasing hormone [TRH]-enhancing peptide), one of the cryptic peptides resulting from the proteolytic processing of prepro-TRH to produce TRH, has a growing list of functions in addition to its well-established ability to enhance the TRH-induced release of thyrotropin (TSH) and prolactin from the pituitary. Intramedullary coadministration of Ps4 and TRH increased gastric acid secretion above the level produced by TRH alone and intracisternal infusion of Ps4 resulted in a substantial reduction in the levels of prepro-TRH-derived peptide levels in the rat pituitary, including Ps4. High-affinity receptors for Ps4 are widely distributed. In addition to the very high Ps4 binding capacity of the folliculo-stellate cells of the anterior pituitary, abundant Ps4 receptors are found in the urinary bladder, vas deferens, central nervous system, reproductive tissues, and pancreas. Targeted prepro-TRH gene disruption results in hyperglycemia as well as the expected hypothyroidism. The observed disregulation of thyroid and glucose homeostasis in the TRH "knockout" mouse clearly demonstrates that prepro-TRH-derived peptides and their cognate receptors within the pituitary, pancreas, and other neural and endocrine systems are of fundamental importance to a variety of physiological systems and merit structural and functional characterization.     

Cloning and characterization of a cDNA encoding a novel subtype of rat thyrotropin-releasing hormone receptor

A cDNA encoding a thyrotropin-releasing hormone (TRH) receptor expressed in the pituitary was previously cloned (De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) Biochem. J. 284, 891-899; De La Pena, P., Delgado, L. M., Del Camino, D., and Barros, F. (1992) J. Biol. Chem. 267, 25703-25708; Duthie, S. M., Taylor, P. L., Anderson, J., Cook, J., and Eidne, K. A. (1993) Mol. Cell Endocrinol. 95, R11-R15). We now describe the isolation of a rat cDNA encoding a novel subtype of TRH receptor (termed TRHR2) displaying an overall homology of 50% to the pituitary TRH receptor. Introduction of TRHR2 cDNA in HEK-293 cells resulted in expression of high affinity TRH binding with a different pharmacological profile than the pituitary TRH receptor. De novo expressed receptors were functional and resulted in stimulation of calcium transient as assessed by fluorometric imaging plate reader analysis. The message for TRHR2 was exclusive to central nervous system tissues as judged by Northern blot analysis. Studies of the expression of TRHR-2 message by in situ hybridization revealed a pattern of expression remarkably distinct (present in spinothalamic tract, spinal cord dorsal horn) from that of the pituitary TRH receptor (present in hypothalamus, and ventral horn of the spinal cord, anterior pituitary). Therefore, we have identified a novel, pharmacologically distinct receptor for thyrotropin-releasing hormone that appears to be more restricted to the central nervous system particularly to the sensory neurons of spinothalamic tract and spinal cord dorsal horn, which may account for the sensory antinociceptive actions of TRH.     

Changes in circulating levels of LH, FSH, LHbeta- and alpha-subunit after gonadotropin-releasing hormone, and of TSH, LHbeta- and alpha-subunit after thyrotropin-releasing hormone

The effect of the gonadotropin-releasing hormone (LRH) and thyrotropin-releasing hormone (TRH) on the blood levels of LH, FSH and TSH, and LHbeta- and alpha-subunit have been studied in 4 normal subjects during the first 20 min after administration of these releasing hormones. Increases in serum immunoreactive LH, LHbeta and alpha-subunit were seen in all subjects after LRH (100 mug iv) but in all subjects the rise in LH was preceded by a rise in alpha-subunit. All subjects showed an increase in TSH and 3 of the 4 subjects a rise in alpha-subunit after TRH (200 mug) but the alpha-subunit responses were smaller and less consistent than after LRH. Levels of LHbeta remained unchanged after TRH. The results demonstrate that the immunoreactive alpha-subunit of the pituitary glycoprotein hormones can be released independently of the intact hormones and that release occurs in response to the same releasing hormones, LRH and TRH, that release the intact hormones.     

The clinical impact of the thyrotropin-releasing hormone test

Because of its ability to cause the release of thyrotropin (TSH), prolactin (PRL), and, under particular circumstances, also of other adenohypopyseal hormones, from the pituitary, thyrotropin-releasing hormone (TRH) has been widely used as a diagnostic tool for about 30 years. The recent introduction of an ultrasensitive TSH assay, able to clearly distinguish suppressed from unsuppressed TSH levels, has rendered the use of the TRH test obsolete in the diagnosis of classic hyperthyroidism. On the contrary, the TRH test is still extremely useful in hyperthyroid patients with inappropriate secretion of thyrotropin, allowing the distinction between TSH-secreting pituitary tumors (usually unresponsive) and the pituitary variant of resistance to thyroid hormone (PRTH) syndrome (always responsive). In hypothyroidism, the TRH test is still of value in patients with preclinical primary hypothyroidism, as they show exaggerated TSH response, and in those with central hypothyroidism, allowing the differentiation between pituitary (secondary) and hypothalamic (tertiary) hypothyroidism. The availability of high-resolution imaging techniques such as magnetic resonance has rendered the use of the TRH test obsolete, to distinguish microprolactionomas from functional hyperprolactinemia. The TRH test still has great clinical value in the follow-up of patients with pituitary tumors (in particular somatotropinomas and clinically nonfunctioning pituitary adenomas) showing abnormal responses of anterior pituitary hormones other than TSH.     

Differential distribution of synaptotagmin I and rab3 in the anterior pituitary of four mammalian species

In the anterior pituitary of rat, gerbil, hamster and guinea pig, the presence and cellular distribution of the synaptic vesicle-associated proteins synaptotagmin I and rab3 were analyzed by immunoblotting and by immunocytochemical staining of serial semithin sections. Our results show that rab3 proteins are ubiquitously expressed in all endocrine cell types of both the anterior and intermediate lobe. In many cells, rab3 immunoreactivity was concentrated beneath the plasmalemma. This intracellular distribution coincided with the distribution of secretory granules, suggesting a possible association of rab3 proteins with the latter organelles. The staining patterns observed using two monoclonal rab3 antibodies with different isoform specificities are compatible with the recent suggestion that rab3B is the dominant rab3 isoform in anterior pituitary cells. However, we could demonstrate that also rab3A is present in endocrine adenohypophyseal cells, albeit at low levels. In contrast to rab3, synaptotagmin I immunoreactivity was only detected in a limited number of adenohypophyseal endocrine cells. Whereas the monoclonal synaptotagmin I antibody consistently failed to immunostain lactotrophs and endocrine cells of the intermediate lobe, other endocrine cell types displayed variable immunoreactivities towards this antibody. Although a low level of synaptotagmin I expression in the immunonegative cells cannot be excluded, the above observation may reflect a differential distribution of synaptotagmin isoforms in endocrine organs, as it has been described for the nervous system. Our study has established that endocrine cells of the anterior pituitary are endowed with proteins of the rab3 and synaptotagmin families which are generally thought to play important roles in the regulation of the trafficking and/or exocytosis of secretory organelles and, hence, probably fulfil similar functions in adenohypophyseal cells.     

The effect of dopamine on neurohypophysial hormone release in vivo and from the rat neural lobe and hypothalamus in vitro

1. The rat hypothalamus (containing the supra-optic nuclei, paraventricular nuclei, median eminence and proximal pituitary stalk) has been incubated in vitro and shown to be capable of releasing the neurohypophysial hormones, oxytocin and arginine vasopressin, at a steady basal rate about one twentieth that of the rat neural lobe superfused in vitro. 2. The hypothalamus and neural lobe in vitro released both hormones in a similar arginine vasopressin/oxytocin ratio of about 1-2:1. However, when release was expressed relative to tissue hormone content, the hypothalamus was shown to release about three times as much arginine vasopressin and six times as much oxytocin as the neural lobe. 3. Dopamine in a concentration range of 10(-3)-10(-9)M caused graded increases in hormone release from the hypothalamus in vitro to a maximum fivefold increase over preceding basal levels. The demonstration that apomorphine also stimulated hormone release whereas noradrenaline was relatively ineffective suggested that a specific dopamine receptor was involved. A separate cholinergic component in the release process was indicated by the finding that acetylcholine stimulated release to a maximum fivefold increase in concentrations of 10(-3)-10(-9)M. 4. The fact that the isolated hypothalamus can be stimulated by dopamine and acetylcholine to release increased amount of oxytocin and arginine vasopressin raises the question of the origin and fate of the hormones released in this way. The possibility that they could be released into the hypophysial portal circulation from median eminence to affect the anterior lobe of the pituitary is discussed. 5. In similar doses, both dopamine and noradrenaline injected into the lateral cerebral ventricles of the brain of the anaesthetized, hydrated, lactating rat caused the release of arginine vasopressin and oxytocin. Apomorphine release both hormones but at a higher dose level and to less effect than the catecholamines. 6. The hormone release induced in vivo by dopamine could be prevented by the prior administration of haloperidol or phentolamine and these antagonists were equally effective in blocking the hormone release due to noradrenaline. The involvement of a specific dopamine receptor was more clearly implicated by the use of pimozide which completely inhibited the hormone release due to dopamine and apomorphine but not that due to noradrenaline. 7. It is suggested that the release of neurohypophysial hormones can be stimulated via a dopaminergic nervous pathway in addition to a cholinergic one. The possibility that the osmoreceptor mechanism for the release of antidiuretic hormone from the neural lobe of the pituitary may involve such a dopaminergic pathway is discussed.     

Timing of completed suicides among residents of Olmsted County, Minnesota, 1951-1985

In man, GHRH has been shown to potentiate the TSH-releasing activity of TRH. To study the way by which GHRH affects TRH-stimulated TSH release, we examined the effect of GHRH (1-29)NH2 on basal and stimulated TSH secretion in intact male rats and superfused dispersed rat pituitary cells. In the intact rats, GHRH(1-29)NH2 potentiated TRH-stimulated TSH release in the evening, but potentiation was not observed in the morning and in dispersed pituitary cells. Basal TSH levels were not changed by GHRH(1-29)NH2. It is concluded that GHRH(1-29)NH2 potentiates the TSH-releasing activity of TRH in the evening in rats possibly through suprahypophyseal disinhibition.     

Carnitine biosynthesis: identification of the cDNA encoding human gamma-butyrobetaine hydroxylase

gamma-Butyrobetaine hydroxylase (EC 1.14.11.1) is the last enzyme in the biosynthetic pathway of L-carnitine and catalyzes the formation of L-carnitine from gamma-butyrobetaine, a reaction dependent on alpha-ketoglutarate, Fe2+, and oxygen. We report the purification of the protein from rat liver to apparent homogeneity, which allowed N-terminal sequencing using Edman degradation. The obtained amino acid sequence was used to screen the expressed sequence tag database and led to the identification of a human cDNA containing an open reading frame of 1161 base pairs encoding a polypeptide of 387 amino acids with a predicted molecular weight of 44.7 kDa. Heterologous expression of the open reading frame in the yeast Saccharomyces cerevisiae confirmed that the cDNA encodes the human gamma-butyrobetaine hydroxylase. Northern blot analysis showed gamma-butyrobetaine hydroxylase expression in kidney (high), liver (moderate), and brain (very low), while no expression could be detected in the other investigated tissues.     

The Italian Registry for Adrenal Cortical Carcinoma: analysis of a multiinstitutional series of 129 patients. The ACC Italian Registry Study Group

A combined anterior pituitary (CAP) function test was assessed in eight healthy male beagle dogs. The CAP test consisted of sequential 30-second intravenous administrations of four hypothalamic releasing hormones in the following order and doses: 1 microgram of corticotropin-releasing hormone (CRH)/kg, 1 microgram of growth hormone-releasing hormone (GHRH)/kg, 10 micrograms of gonadotropin-releasing hormone (GnRH)/kg, and 10 micrograms of thyrotropin-releasing hormone (TRH)/kg. Plasma samples were assayed for adrenocorticotropin, cortisol, GH, luteinizing hormone (LH), and prolactin (PRL) at multiple times for 120 min after injection. Each releasing hormone was also administered separately in the same dose to the same eight dogs in order to investigate any interactions between the releasing hormones in the combined function test. Compared with separate administration, the combined administration of these four hypothalamic releasing hormones caused no apparent inhibition or synergism with respect to the responses to CRH, GHRH, and TRH. The combined administration of these four hypothalamic releasing hormones caused a 50% attenuation in LH response compared with the LH response to single GnRH administration. The side effects of the combined test were confined to restlessness and nausea in three dogs, which disappeared within minutes after the administration of the releasing hormones. It is concluded that with the rapid sequential administration of four hypothalamic releasing hormones (CRH, GHRH, GnRH, and TRH), the adenohypophyseal responses are similar to those occurring with the single administration of these secretagogues, with the exception of the LH response, which is lower in the CAP test than after single GnRH administration.     

A low-Km 5'-nucleotidase from rat brain cytosolic fraction: purification, kinetic properties, and description of regulation by a novel factor that increases sensitivity to inhibition by ATP and ADP

A readily soluble 5'-nucleotidase was purified 1,800-fold from rat brain 105,000-g supernatant. The enzyme showed similarity to the 5'-nucleotidase ectoenzyme of plasma membranes. It exhibited a low Km for AMP, which was preferred over IMP as substrate. It was inhibited by free ATP and ADP and by alpha,beta-methylene ADP. The enzyme appeared to be a glycoprotein on the basis of its interaction with concanavalin A. It contained a phosphatidylinositol moiety because treatment with phosphatidylinositol-specific phospholipase C increased its hydrophilicity. A single subunit of Mr = 54,300 +/- 800 was observed, which is appreciably smaller than published values for the 5'-nucleotidase ectoenzyme or for other low- Km "soluble" 5'-nucleotidases. The soluble 5'-nucleotidase showed an elution profile on AMP-Sepharose affinity chromatography or on Mono Q ion-exchange chromatography different from that of the brain ectoenzyme. Forty-two percent of the soluble 5'-nucleotidase in brain 105,000-g supernatant did not bind to a Mono Q ion-exchange column because of its interaction with a soluble factor. This factor could be removed by chromatography on concanavalin A-Sepharose. The factor had the novel property of increasing the sensitivity of the purified soluble 5'-nucleotidase toward the inhibitor ATP by 20-fold. This factor was also able to increase the inhibition of brain 5'-nucleotidase ectoenzyme by ATP.     

Characterization of GH3 cells overexpressing basic fibroblast growth factor (FGF-2)

Basic fibroblast growth factor (FGF-2) is not only a potent mitogen for various cells but also a multifunctional factor with angiogenic and chemotactic activity, and the capacity to induce the synthesis of various proteinases and to modulate endocrine function. To clarify the role played by FGF-2 in the progression of pituitary tumor, we fused rat FGF-2 cDNA to the promoter SR alpha, consisting of the early promoter of SV40 and HTLV(I)-LTR, and we cotransfected GH3 cells with pSV2-neo by an electroporation method. After selection by G418, we obtained 7 neomycin-resistant clones. Southern blot analysis of genomic DNA revealed the presence of transfected rat FGF-2 cDNA in 4 of the 7 clones. To measure FGF-2 molecules, we established a new immuno-fluorometric assay system, using 3 monoclonal antibodies against different portions of human FGF-2. This assay had a minimum sensitivity of 10 pg/ml and cross-reacted neither with acidic fibroblast growth factor (FGF-1) nor insulin-like growth factor 1 (IGF-1), even at a concentration of 100 ng/ml. Although FGF-2 was undetectable in the culture medium of any of the clones, the cell homogenate contained a significant amount of FGF-2 (7.2 ng/mg protein) in 1 of the 4 FGF-2-transfected clones (GH3FGF(+)), whereas FGF-2 was not detected (< 5.2 pg/mg protein) in the cell homogenates of either the parent GH3 cells or the control cells transfected with pSV2-neo alone (GH3FGF(-)), GH3FGF(+) grew as adherent cells and formed epithelial sheets with a growth rate similar to that of control cells. The amount of prolactin(PRL) released by TRH was greater in GH3FGF(+) than that in GH3 or GH3FGF(-). On the other hand, the sensitivity to SRIF was increased in GH3FGF(+) compared with that in other clones. The findings of these in vitro studies indicate that FGF-2, if it is expressed in pituitary tumor cells, plays little if any role in cell growth but may modulate certain cell functions such as responsiveness to hormones.     

Is HIV infection a risk factor for advanced cervical cancer?

TNF-alpha plays a critical role in the cascade of neuroendocrine events during inflammation and septic shock. It also affects the release of pituitary hormones and acts as a growth factor in immune and nonimmune cells. The aim of the present study was to investigate the release of TNF-alpha from rat anterior pituitary cells and the effect of the steroid medium on its release. Cultured anterior pituitary cells from lactating rats spontaneously released TNF-alpha. The presence of lipopolysaccharide (LPS, 0.1 microg/mL) in the culture medium significantly increased TNF-alpha release and inhibited prolactin release. Chronic estrogenization of ovariectomized rats or the presence of 17 beta-estradiol in the culture medium also increased TNF-alpha release. LPS significantly stimulated TNF-alpha release in all groups and abrogated the estrogen-induced prolactin release. We also investigated the effect of TNF-alpha on prolactin release. The presence of TNF-alpha (50 ng/mL) in the culture medium inhibited prolactin release from anterior pituitary cells. These data show that anterior pituitary cells in culture release TNF-alpha and that this release is stimulated by estrogens. Our results also indicate that LPS inhibits prolactin release in an estrogenic environment, suggesting that TNF-alpha could affect pituitary hormone release during endotoxemia.     

Signalling properties of glycosylphosphatidylinositols and their regulated release from membranes in the turnover of glycosylphosphatidylinositol-anchored proteins

Glycosylphosphatidylinositols are involved in transmembrane signalling, and their signal mediated release from the cell membrane has been demonstrated for several hormones and growth factors. Presently, however, only indirect evidence exists for the enzyme responsible for the signal mediated release of glycosylphosphatidylinositols. Indirect evidence from product identification led to the conclusion that in mammals the hormone sensitive activity is that of a glycosylphosphatidylinositol anchor hydrolyzing phospholipase C. On the other hand a mammalian glycosylphosphatidylinositol anchor hydrolyzing phospholipase D is well-established. This enzyme most likely functions in the intracellular turnover of glycosylphosphatidylinositols, however, its possible relation to the signalling properties of glycosylphosphatidylinositols remains unclear.     

Purification of calcitonin-like peptides from rat brain and pituitary

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.