Ceruletide acts in the abdomen, not in the brain, to produce satiety

Ceruletide (caerulein), a decapeptide extracted from the skin of the frog, Hyla caerulea, is very similar in structure to the C-terminal octapeptide of cholecystokinin (CCK-8). Although ceruletide and CCK-8 act through similar or identical receptors to produce the same visceral effects, previous studies in the rat suggested that peripherally administered ceruletide acted directly on the ventromedial hypothalamic (VMH) area to decrease food intake, but peripherally administered CCK-8 acted at a vagally innervated abdominal site to decrease food intake. Since it is unprecedented for these two peptides to produce the same effect by acting at different sites, we investigated the site of action of ceruletide's satiety effect in the rat and compared it to the site of action of CCK-8. The major results were: (1) intraperitoneal administration of ceruletide and CCK-8 inhibited food intake, but intraventricular administration did not; (2) the satiety effect of ceruletide and CCK-8 was not changed by bilateral lesions of the VMH; and (3) the satiety effect of ceruletide and CCK-8 was abolished or markedly reduced by bilateral abdominal vagotomy. We conclude that ceruletide acts at the same vagally innervated abdominal site to produce satiety as CCK-8 does and that neither peptide acts directly on the VMH area.     

Ineffectiveness of ceruletide to reduce food intake and body weight in obese women hospitalized for weight reduction and treated with a restricted diet. A double-blind study

Ceruletide is a synthetic decapeptide closely resembling the 8-carboxy-terminal peptide of cholecystokinin (CCK-8) with which it shares several biological properties. In a double-blind study versus placebo, we evaluated the effects of ceruletide on self-rated hunger and food intake at lunch time, as well as on body weight in 14 obese women hospitalized for weight reduction and on a restricted diet. During two 6-day courses of treatment with ceruletide or placebo, ceruletide 0.3 microgram/kg b.wt. or an equivalent volume of its diluent were injected IM at 11.30 a.m., i.e., 30 min before lunch. Feelings of hunger were quantitated, using a visual analogue self-rating scale, prior to the injection of ceruletide or placebo and 30 min thereafter, i.e., just prior to the start of meal ingestion, as well as 2 hr after the start of the meal. Duration and caloric content of food ingested at lunch, as well as morning body weight, were recorded daily. Ceruletide, compared to placebo, did not significantly influence any of the above variables. However, in the first three experimental days, the increase in self-rated hunger from values before the injection to 30 min thereafter was less marked, though not significantly so (0.05 less than p less than 0.1), with ceruletide than with placebo. Thus, it appears that ceruletide, under the experimental conditions of the present study, was not effective in enhancing the patients' motivation to lose weight and to further restrict their food intake at lunch time.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ceruletide increases threshold and tolerance to experimentally induced pain in healthy man

Previous studies suggested that ceruletide might be endowed with analgesic and sedative properties. To investigate the effects of ceruletide on experimentally induced pain and on central nervous functions, two studies, each involving 24 healthy subjects, were carried out in random double-blind fashion. Every subject participated in three experiments one week apart. In study 1, 120 and 60 ng/kg/hr ceruletide IV increased threshold and tolerance to electrically and threshold to thermally induced cutaneous pain significantly more than saline (p<0.001), the higher dose being slightly more active. Only mild sedative effects occurred. Study 2 compared the effects of 60 and 6 ng/kg/hr ceruletide IV to those of 0.4 mg/kg/hr pentazocine IV and investigated whether these effects were naloxone reversible. Both ceruletide doses, 60 ng/kg/hr slightly more than 6 ng/kg/hr, elevated threshold and tolerance to electrically induced and threshold to thermally induced pain markedly, pentazocine acted stronger and longer than ceruletide (p<0.001). Naloxone reversed the effects of pentazocine but not of ceruletide. Conclusion: ceruletide (1) exerts potent naloxone resistant analgesic effects, which, however, are inferior to those of pentazocine, and (2) produces only mild sedation.     

Optimizing recovery of peptides from an octadecylsilyl (ODS) cartridge

An analytical procedure has been studied in detail to optimize the recovery of the radiolabeled and unlabeled synthetic peptides substance P and methionine-enkephalin from a reversed phase octadecylsilyl disposable cartridge, which is one of the first steps to preferentially enrich peptides from a biologic matrix. Our previous research on biologic peptides indicated the need for the present study. The column flow-rate was found to be one of the most important experimental parameters; and its effect on the recovery of those two synthetic peptides was determined by the measurement of recovered radioactivity and by high performance liquid chromatography u.v. detection. The accuracy and reproducibility of the flow-rate affected recovery, and it was necessary to regulate the flow-rate with a syringe infusion pump. Recovery was optimum using a slow rate for both sample application and elution. Furthermore, a slow elution was more important than a slow rate of sample application. Such basic recovery studies on individual synthetic peptides are required before undertaking peptide extractions from biologic matrices.     

Activation of CCK-A receptors induces elevation of plasma corticosterone in rats.

Cholecystokinin octapeptide (CCK-8) and ceruletide (1 microgram/kg) produced a pronounced increment of plasma corticosterone levels at 30 min after intraperitoneal administration. The response to these peptides was suppressed by pretreatment with a selective antagonist for CCK-A receptors, (-)L-364,718, in a dose-related manner, but not with an antagonist for CCK-B receptors, (+)L-365,260. However, (-)L-364,718 itself had no effect on basal levels of plasma corticosterone. These results indicate that peripheral administration of CCK-8 and ceruletide stimulates the hypothalamo-pituitary adrenal axis through the activation of CCK-A receptors, but not CCK-B receptors.     

Influence of Potassium Concentration in Microdialysis Perfusate on Basal and Stimulated Striatal Dopamine Release: Effect of Ceruletide, a Cholecystokinin-Related Peptide

Abstract: The in vivo microdialysis method was used to study the effect of the cholecystokinin-related peptide, ceruletide, on extracellular levels of dopamine (DA) in the striatum following perfusion with various K⁺ concentrations. Increasing the K⁺ concentration in the perfusate from 4 to 15 or 17.5 m M did not change basal DA release or release evoked by electrical stimulation of the medial forebrain bundle (MFB). However, when the perfusing solution contained 20 or 30 m M K⁺, dose-dependent reductions of both basal and MFB-stimulated DA release occurred. Subcutaneous administration of ceruletide at 160 μg/kg had no influence on the basal or MFB-stimulated DA release with 4 or 15 m M K⁺ in the perfusate. However, after perfusion with 17.5 m M K⁺, ceruletide significantly attenuated the basal and MFB-stimulated DA release. Carbachol (10 μ M ) locally applied via the dialysis probe also attenuated MFB-stimulated DA release after perfusion with 17.5 m M K⁺. From these results, we conclude that under appropriate depolarization of striatal DA terminals, ceruletide induces further depolarization and inactivation of nigrostriatal DA terminals. The present data suggest that this effect may be mediated via intrinsic cholinergic neurons in the striatum.     

Peptidome analysis of human skim milk in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. Due to the difficulties associated with detection technology of the peptides, the expression of the peptides present in human milk is not known widely. In recent years, peptidome analysis has received increasing attention. In this report, the analysis of endogenous peptides in human milk was done by mass spectrometry. A method was also developed by our researchers, which can be used in the extraction of peptide from human milk. Analysis of the extracts by LC–MS/MS resulted in the detection of 1000–3000 Da peptide-like features. Out of these, 419 peptides were identified by MS/MS. The identified peptides were found to originate from 34 proteins, of which several have been reported. Analysis of the peptides’ cleavage sites showed that the peptides are cleaved with regulations. This may reflect the protease activity and distribution in human body, and also represent the biological state of the tissue and provide a fresh source for biomarker discovery. Isotope dimethyl labeling analysis was also used to test the effects of premature delivery on milk protein composition in this study. Differences in peptides expression between breast milk in term milk (38–41 weeks gestation) and preterm milk (28–32 weeks gestation) were investigated in this study. 41 Peptides in these two groups were found expressed differently. 23 Peptides were present at higher levels in preterm milk, and 18 were present at higher levels in term milk.     

Analysis of the Primary Sequence of Human Myelin Basic Protein Peptides 1–44 and 90–170 by Fast Atom Bombardment Mass Spectrometry

The originally described sequence of human myelin basic protein peptide 45-89 has recently been shown to contain two errors which have now been resolved. In the present study fast atom bombardment mass spectrometry was utilized to analyze the primary sequence of the other portions, peptides 1-44 and 90-170 of human myelin basic protein. The results obtained confirm the accuracy of the primary sequence published for both of these terminal peptides.     

Demonstration of two distinct tachykinin receptors in rat brain cortex

Eledoisin and substance P are members of a class of peptides termed tachykinins. They share a similar spectrum of biological activities but their relative potencies in various pharmacological assays differ. We have investigated whether there is more than one receptor for these tachykinins in rat brain cortex membranes. 125I-Bolton Hunter-conjugated eledoisin specifically binds to rat brain cortex membranes with high affinity. The binding is inhibited over 95% by unlabeled eledoisin (6.6 microM). Scatchard analysis of the binding of this ligand is curvilinear suggesting that there are two binding sites with KD values of 0.9 +/- 0.7 nM and 20 +/- 10 nM. We tested various analogs and fragments of substance P and eledoisin for their ability to inhibit the binding of 125I-Bolton Hunter-conjugated eledoisin and 125I-Bolton Hunter-conjugated substance P to these membranes. The following peptides are more potent as inhibitors of the 125I-Bolton Hunter-conjugated eledoisin binding site than of the 125I-Bolton Hunter-conjugated substance P binding site: nonradioactive Bolton Hunter-conjugated eledoisin (greater than 100-fold), eledoisin (12-fold), kassinin (22-fold), neuromedin K (greater than 58-fold), and pyroglutamyl substance P(6-11)hexapeptide (4-fold). In contrast, substance P (21-fold), physalaemin (8-fold), and substance P methyl ester (1200-fold) were more potent as inhibitors of 125I-Bolton Hunter-conjugated substance P binding. These results suggest that these two ligands may bind to distinct receptors. 125I-Bolton Hunter-conjugated substance P binds specifically to rat parotid cell receptors, but 125I-Bolton Hunter-conjugated eledoisin does not, indicating that parotid cells contain only one of the receptor subtypes. The cortex membrane binding of both ligands is stimulated by low concentrations of MnCl2 (ED50 = 0.05 mM) and is inhibited by guanylyl-5'-(beta, gamma-imido)diphosphate (IC50 = 0.5 microM).     

Structure and functions of the novel hypothalamic RFamide neuropeptides R-RFa and 26RFa in vertebrates

A number of RFamide peptides have been characterized in invertebrate species and these peptides have been found to exert a broad spectrum of biological activities. In contrast, in vertebrates, our knowledge on RFamide peptides is far more limited and only a few members of the RFamide peptide family have been identified in various vertebrate classes during the last years. The present review focuses on two novel RFamide peptides, Rana RFamide (R-RFa) and 26RFa, that have been recently isolated from the amphibian brain. R-RFa shares the C-terminal LPLRFamide motif with other RFamide peptides previously identified in mammals, birds and fish. The distribution of R-RFa in the frog brain exhibits strong similarities with those of other LPLRFamide peptides, notably in the periventricular region of the hypothalamus. There is also evidence that the physiological functions of R-RFa and other LPLRFamide peptides have been conserved from fish to mammals; in particular, all these peptides appear to be involved in the control of pituitary hormone secretion. 26RFa does not exhibit any significant structural identity with other RFamide peptides and this peptide is the only member of the family that possesses an FRFamide motif at its C-terminus. The strong conservation of the primary structure of 26RFa from amphibians to mammals suggests that this RFamide peptide is involved in important biological functions in vertebrates. As for several other RFamide peptides, 26RFa-containing neurons are present in the hypothalamus, notably in two nuclei involved in the control of feeding behavior. Indeed, 26RFa is a potent stimulator of appetite in mammals. Concurrently, recent data suggest that 26RFa exerts various neuroendocrine regulatory activities at the pituitary and adrenal level.     

降糖肽的发展现状及研究进展

糖尿病是一组以高血糖为特征的代谢性疾病,是由于胰岛素分泌缺陷或其生物功能受损,或两者兼有引起。糖尿病可能导致各种组织,特别是眼、肾、心脏、血管、神经的慢性损害、功能障碍。糖尿病主要分为Ⅰ型糖尿病和Ⅱ型糖尿病,这两类糖尿病均存在明显的遗传异质性。目前用于降低或控制血糖的药物主要有硫脉类、双肌类、苯甲酸衍生物类、糖苷酶抑制剂类和噻唑烷二酮类。研发出安全有效的降糖药物已经成为全世界关注的焦点,其中,生物活性肽是目前最广泛研究的潜在治疗剂之一,其最佳用途正在开发中。迄今为止,已经发现了许多天然肽和合成肽,其具有通过不同机制介导的优异的抗糖尿病效果。新兴技术和药物输送系统的应用进一步促进了预期目标成果的达成。临床前和临床研究中一些具有更好效力和安全性的优秀肽已经被确定。因此,对这些肽的进一步详细研究可能会筛选出临床上有用的抗糖尿病药物。     

Solid Phase Synthesis and Application of Labeled Peptide Derivatives: Probes of Receptor-Opioid Peptide Interactions

Solid phase synthetic methodology has been developed in our laboratory to incorporate an affinity label (a reactive functionality such as isothiocyanate or bromoacetamide) into peptides (Leelasvatanakij and Aldrich J Peptide Res 56, 80, 2000), and we have used this synthetic strategy to prepare affinity label derivatives of a variety of opioid peptides. To date side reactions have been detected only in two cases, both involving intramolecular cyclization. We have identified several peptide-based affinity labels for δ opioid receptors that exhibit wash-resistant inhibition of binding to these receptors and are valuable pharmacological tools to study opioid receptors. Even in cases where the peptide derivatives do not bind covalently to their target receptor, studying their binding has revealed subtle differences in receptor interactions with particular opioid peptide residues, especially Phe residues in the N-terminal “message” sequences. Solid phase synthetic methodology for the incorporation of other labels (e.g. biotin) into the C-terminus of peptides has also been developed in our laboratory (Kumar and Aldrich Org Lett 5, 613, 2003). These two synthetic approaches have been combined to prepare peptides containing multiple labels that can be used as tools to study peptide ligand-receptor interactions. These solid phase synthetic methodologies are versatile strategies that are applicable to the preparation of labeled peptides for a variety of targets in addition to opioid receptors.     

Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry

The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.     

The effect of acidic residues and amphipathicity on the lytic activities of mastoparan peptides studied by fluorescence and CD spectroscopy

Some mastoparan peptides extracted from social wasps display antimicrobial activity and some are hemolytic and cytotoxic. Although the cell specificity of these peptides is complex and poorly understood, it is believed that their net charges and their hydrophobicity contribute to modulate their biological activities. We report a study, using fluorescence and circular dichroism spectroscopies, evaluating the influence of these two parameters on the lytic activities of five mastoparans in zwitterionic and anionic phospholipid vesicles. Four of these peptides, extracted from the venom of the social wasp Polybia paulista, present both acidic and basic residues with net charges ranging from +1 to +3 which were compared to Mastoparan-X with three basic residues and net charge +4. Previous studies revealed that these peptides have moderate-to-strong antibacterial activity against Gram-positive and Gram-negative microorganisms and some of them are hemolytic. Their affinity and lytic activity in zwitterionic vesicles decrease with the net electrical charges and the dose response curves are more cooperative for the less charged peptides. Higher charged peptides display higher affinity and lytic activity in anionic vesicles. The present study shows that the acidic residues play an important role in modulating the peptides’ lytic and biological activities and influence differently when the peptide is hydrophobic or when the acidic residue is in a hydrophilic peptide.     

Localization and characterization of evolutionarily conserved chromogranin A-derived peptides in the rat and human pituitary and adrenal glands

Chromogranin A (CgA) is a neuroendocrine protein that undergoes proteolytic cleavage in secretory granules. The aim of the present study was to characterize the peptides WE14 and EL35 that are derived from evolutionarily conserved regions of CgA in rat and human endocrine tissues. In the rat pituitary, HPLC analysis revealed that WE14 is present as a single immunoreactive peak, whereas EL35 elutes in two molecular forms. Authentic WE14 is also produced in both rat and human adrenal glands, while EL35 displays a variable elution profile depending on the tissue extract, indicating the existence of different forms of EL35 in these tissues. Immunohistochemical labeling of the rat pituitary showed that WE14 and EL35 occur in gonadotropes and melanotropes, but not in corticotropes. A strong immunoreaction for both peptides was also observed in rat adrenochromaffin cells. In the human adrenal gland, the WE14 and EL35 antisera revealed intense labeling of adrenomedullary cells in adult and nests of chromaffin progenitor cells in fetal adrenal. Finally, WE14 and EL35 immunoreactivity was detected in pheochromocytoma tissue where WE14 occurred as a single immunoreactive form, while EL35 displayed different forms. The observations that WE14 and EL35: (1). have been preserved during vertebrate evolution, (2). are processed in a cell-specific manner, and (3). occur during ontogenesis of the adrenal gland strongly suggest that these peptides play a role in endocrine tissues. In addition, the existence of differentially processed CgA-derived peptides in normal and tumorous tissues may provide new tools for the diagnosis and prognosis of neuroendocrine tumors.     

Ceruletide, ceruletide analogues and cholecystokinin octapeptide (CCK-8): effects on motor behaviour, hexobarbital-induced sleep and harman-induced convulsions

Ten ceruletide analogues and cholecystokinin octapeptide (CCK-8) were compared with ceruletide regarding neuropharmacological effects in mice after subcutaneous administration. The effects under study were catalepsy, prolongation of hexobarbital-induced sleep and delay in onset of harman-induced convulsions. Ceruletide and several analogues were more potent than the reference drugs, diazepam and haloperidol. Desulfation, deamidation and shortening of the peptide chain by five amino acids strongly reduced or abolished the pharmacological activities of ceruletide. Other chemical modulations mostly weakened the efficacy, but to an unequal extent for the three effects, which altered the pharmacological selectivity.     

Central nervous system effects of peptides, 1980-1985: a cross-listing of peptides and their central actions from the first six years of the journal Peptides

A tabular synopsis is presented for articles concerned with the effects of peptides on the central nervous system that appeared in the journal Peptides from 1980-1985. A table arranged alphabetically by peptide and one arranged by effects, both listing routes of injection, species, direction of change, and qualifying notes, provides easy cross-referencing of peptides and their effects. Over 80 peptides and over 135 effects are listed. The list of peptides includes, but is not limited to: ACTH, angiotensin, bombesin, bradykinin, calcitonin, casomorphin, CCK, ceruletide, CGRP, CRF, dermorphin, DSIP, dynorphin, endorphins, enkephalins, GRF, gastrin, LHRH, litorin, metkephamid, MIF-l, motilin, MSH, NPY, NT, oxytocin, ranatensin, sauvagine, substances P and K, somatostatin, TRH, VIP, vasopressin, and vasotocin. The list of effects includes, but is not limited to: aggression, alcohol, analgesia, attention, avoidance, behavior, cardiovascular regulation, catalepsy, conditioned behavior, convulsions, dopamine binding and metabolism, discrimination, drinking, EEG, exploration, feeding, fever, gastric secretion, GI motility, grooming, learning, locomotor behavior, mating, memory, neuronal activity, open field, operant behavior, rearing, respiration, satiety, scratching, seizure, sleep, stereotypy, temperature, thermoregulation and tolerance.     

Isolation and characterization of corticotropin- and melanotropin-related peptides from the neurointermediary lobe of the rat pituitary by reversed-phase liquid chromatography

A novel procedure utilizing reversed-phase high-performance liquid chromatography for the extraction and purification of peptides from biological tissues has been applied to the isolation of corticotropin-like intermediary lobe peptide (CLIP) and alpha-melanocyte-stimulating hormone (alpha-MSH) from the neurointermediary lobe of the rat pituitary. The isolation and characterization of two major forms of CLIP and two major forms of alpha-MSH are described. The isolated peptides have been identified by using enzymatic digestions and peptide mapping. The main form of ClIP is a peptide which has been modified by phosphorylation of the serine residue at position 31. This is the first peptide of endocrine origin reported to be modified in such a manner. A non-phosphorylated form of CLIP was also present at lower concentrations. The main form of alpha-MSH was found to be N,-O-diacetyl-alpha-MSH, with the more familiar mono-N-acetyl-alpha-MSH present to a much smaller extent. Thus, in the rat neurointermediary lobe, the two main corticotropin-related peptides present are mostly in modified forms which are the result of posttranslational modifications. It is only by the use of methodology such as that described in this paper that small alterations in peptide structure may be identified.     

Kinetic models for peptide-induced leakage from vesicles and cells

In this article analytical expressions for peptide-induced membrane leakage are presented. Two different models for time-dependent leakage have been developed. In the first, the leakage is assumed to be coupled by pores formed by the peptides. In the second model the peptide is assumed to induce a stress/perturbation in the membrane, and in order to reduce the stress, rearrangements in the membrane are induced. The leakage is coupled to these rearrangements, and when equilibrium is achieved no more leakage occurs. From the kinetic models simple fitting routines have been developed involving only two fitting parameters, and these have been used to fit experimental data for two prion protein-derived peptides as well as the honey bee toxin melittin in both vesicles and erythrocytes with good results. The fitted parameters provide both a quantitative and a qualitative basis for interpreting the experimental results. In addition a model for the peptide concentration-dependent leakage is presented, which was used to fit experimental data for leakage induced by the prion protein-derived peptides. The models presented in this article are compared with other models for peptide-induced membrane leakage.