Development of xeno-free epithelial differentiation media for adherent, non-expanded adipose stromal vascular cell cultures.

INTRODUCTION: Reconstruction of respiratory epithelium is critical for the fabrication of bioengineered airway implants. Epithelial differentiation is typically achieved using bovine pituitary extract (BPE). Due to the xenogenic nature and undefined composition of BPE, an alternative for human clinical applications, devoid of BPE, must be developed. The goal of this study was to develop two different BPE-free media, with and without select pituitary hormone (PH), which could initiate epithelial differentiation for use in human implantation. METHODS: The ability of the two BPE-free media to initiate epithelial differentiation of adherent, non-expanded stromal-vascular cells grown on porcine small intestinal submucosa was compared to traditional BPE-containing media (M1). Nanostring® was used to measure differences in gene expression of stemness (MSC), basal cell (basal), and ciliated markers (muco-cil), and staining was performed support the gene data. RESULTS: Compared to baseline, both BPE-free media upregulated epithelial and stemness genes, however this was to a lower degree than BPE-containing media. In general, the expression of basal cell markers (COL17A1, DSG3, ITGA6, KRT6A, LOXL2) and secreted mucous proteins (PLUNC, MUC5B, SCGB2A1) was upregulated. The gene expression of ciliated markers C9orf24, TUBA3 and DNCL2B but not of the key transcription factor for cilagenesis FOXJ1 were upregulated, indicating that mucus-secreting cell differentiation occurs more rapidly than ciliogenesis. The ability of the adherent stromal vascular cells to upregulate gene expression of both epithelial and stemness markers suggests maintenance of the self-renewal capacity of undifferentiated and/or basal cell-like cells contributing to proliferation and ensuring a persisting source of cells for regenerative medicine applications. CONCLUSION: This study provides the initial step to defining a BPE-free epithelial differentiation medium for clinical translation. Thus, either of the proposed BPE-free medium are viable alternatives to BPE-containing medium for partial epithelial differentiation for human translational applications.

Neuropeptide glutamic acid-isoleucine (NEI)-induced paradoxical sleep in rats.

Neuropeptideglutamic acid-isoleucine (NEI) as well as melanin concentrating hormone (MCH) is cleaved from the 165 amino acid protein, prepro-melanin concentrating hormone (prepro-MCH). Among many physiological roles of MCH, we demonstrated that intracerebroventricular (icv) injection of MCH induced increases in REM sleep episodes as well as in non REM sleep episodes. However, there are no studies on the effect of NEI on the sleep-wake cycle. As for the sites of action of MCH for induction of REM sleep, the ventrolateral periaqueductal gray (vlPAG) has been reported to be one of its site of action. Although MCH neurons contain NEI, GABA, MCH, and other neuropeptides, we do not know which transmitter(s) might induce REM sleep by acting on the vlPAG. Thus, we first examined the effect of icv injection of NEI on the sleep-wake cycle, and investigated how microinjection of either NEI, MCH, or GABA into the vlPAG affected REM sleep in rats. Icv injection of NEI (0.61µg/5µl: n=7) significantly increased the time spent in REM episodes compared to control (saline: 5µl; n=6). Microinjection of either NEI (61ng/0.2µl: n=7), MCH (100ng/0.2µl: n=6) or GABA (250mM/0.2µl: n=7) into the vlPAG significantly increased the time spent in REM episodes and the AUC. Precise hourly analysis of REM sleep also revealed that after those microinjections, NEI and MCH increased REM episodes at the latter phase, compared to GABA which increased REM episodes at the earlier phase. This result suggests that NEI and MCH may induce sustained REM sleep, while GABA may initiate REM sleep. In conclusion, our findings demonstrate that NEI, a cleaved peptide from the same precursor, prepro-MCH, as MCH, induce REM sleep at least in part through acting on the vlPAG.

Identification of mRNAs coding for mammalian-type melanin-concentrating hormone and its receptors in the scalloped hammerhead shark Sphyrna lewini.

Melanin-concentrating hormone (MCH) is a neuromodulator, synthesized in the hypothalamus, that regulates both appetite and energy homeostasis in mammals. MCH was initially identified in teleost fishes as a pituitary gland hormone that induced melanin aggregation in chromatophores in the skin; however, this function of MCH has not been observed in other vertebrates. Recent studies suggest that MCH is involved in teleost feeding behavior, spurring the hypothesis that the original function of MCH in early vertebrates was appetite regulation. The present study reports the results of cDNAs cloning encoding preproMCH and two MCH receptors from an elasmobranch fish, Sphyrna lewini, a member of Chondrichthyes, the earliest diverged class in gnathostomes. The putative MCH peptide is composed of 19 amino acids, similar in length to the mammalian MCH. Reverse-transcription polymerase chain reaction revealed that MCH is expressed in the hypothalamus in S. lewini MCH cell bodies and fibers were identified by immunochemistry in the hypothalamus, but not in the pituitary gland, suggesting that MCH is not released via the pituitary gland into general circulation. MCH receptor genes mch-r1 and mch-r2 were expressed in the S. lewini hypothalamus, but were not found in the skin. These results indicate that MCH does not have a peripheral function, such as a melanin-concentrating effect, in the skin of S. lewini hypothalamic MCH mRNA levels were not affected by fasting, suggesting that feeding conditions might not affect the expression of MCH in the hypothalamus.

Polymorphism of somatolactin-producing cells in the goldfish pituitary: immunohistochemical investigation for somatolactin-α and -ß.

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin family of adenohypophyseal hormones. In teleost fish, SL is encoded by one or two paralogous genes, namely SL-α and -ß. Our previous studies have revealed that pituitary adenylate-cyclase-activating polypeptide stimulates SL release from cultured goldfish pituitary cells, whereas melanin-concentrating hormone suppresses this release. As in other fish, the goldfish possesses SL-α and -ß. So far, however, no useful means of detecting the respective SLs immunologically in this species has been possible. In order to achieve this aim, we raised rabbit antisera against synthetic peptide fragments deduced from the goldfish SL-α and -ß cDNA sequences. Using these antisera, we observed adenohypophyseal cells showing SL-α- and -ß-like immunoreactivities in the goldfish pituitary, especially the pars intermedia (PI). Several cells in the PI showed the colocalization of SL-α- and -ß-like immunoreactivities. Then, using single-cell polymerase chain reaction with laser microdissection, we examined SL-α and -ß gene expression in adenohypophyseal cells showing SL-α- or -ß-like immunoreactivity. Among cultured pituitary cells, we observed three types of cell: those that possess transcripts of SL-α, -ß, or both. These results suggest a polymorphism of SL-producing cells in the goldfish pituitary.

EST dataset of pituitary and identification of somatolactin and novel genes in Chinese sturgeon, Acipenser sinensis.

Chinese sturgeon (Acipenser sinensis) is a rare and endangered species and also an important resource for the sturgeon aquaculture industry, however, a few genes have been identified in this species. We report here construction of a pituitary cDNA library from a 24 years old female Chinese sturgeon just after its spawning, and obtained 2,025 ESTs from the library. 885 unique sequences were identified, which were categorized into 12 functional groups. More than half of the unique sequences (57%) do not match with annotated sequences in the public databases. Three of these novel genes were further identified. Notably, a full-length of cDNA (1,143 bp) encoding somatolactin of 232 amino acids was identified. Phylogenetic analysis showed 97% amino acid identity with White sturgeon somatolactin. RT-PCR analysis indicated that the somatolactin mRNA was only detected in pituitary. Pituitary-specific expression of the somatolactin suggested that the protein may play important physiological functions in pituitary-endocrine system of the Chinese sturgeon.

Disulfide bond cleavage in TEMPO-free radical initiated peptide sequencing mass spectrometry.

The gas-phase free radical initiated peptide sequencing (FRIPS) fragmentation behavior of o-TEMPO-Bz-conjugated peptides with an intra- and intermolecular disulfide bond was investigated using MS(n) tandem mass spectrometry experiments. Investigated peptides included four peptides with an intramolecular cyclic disulfide bond, Bactenecin (RLCRIVVIRVCR), TGF-α (CHSGYVGVRC), MCH (DFDMLRCMLGRVFRPCWQY) and Adrenomedullin (16-31) (CRFGTCTVQKLAHQIY), and two peptides with an intermolecular disulfide bond. Collisional activation of the benzyl radical conjugated peptide cation, which was generated through the release of a TEMPO radical from o-TEMPO-Bz-conjugated peptides upon initial collisional activation, produced a large number of peptide backbone fragments in which the S-S or C-S bond was readily cleaved. The observed peptide backbone fragments included a-, c-, x- or z-types, which indicates that the radical-driven peptide fragmentation mechanism plays an important role in TEMPO-FRIPS mass spectrometry. FRIPS application of the linearly linked disulfide peptides further showed that the S-S or C-S bond was selectively and preferentially cleaved, followed by peptide backbone dissociations. In the FRIPS mass spectra, the loss of •SH or •SSH was also abundantly found. On the basis of these findings, FRIPS fragmentation pathways for peptides with a disulfide bond are proposed. For the cleavage of the S-S bond, the abstraction of a hydrogen atom at C(ß) by the benzyl radical is proposed to be the initial radical abstraction/transfer reaction. On the other hand, H-abstraction at C(α) is suggested to lead to C-S bond cleavage, which yields [ion ± S] fragments or the loss of •SH or •SSH.

Molecular and expression characterization of growth hormone/prolactin family genes in the Prenant's schizothoracin.

The growth hormone/prolactin family genes are major participants in control of several complex physiologic processes, including growth, reproduction, and metabolism. In this study, total RNA was isolated from the pituitary of the Prenant's schizothoracin (Schizothorax prenanti), and SMART cDNA was synthesized from 100 ng total RNA. The full-length cDNAs of three GH/PRL family genes have been cloned and sequenced from the SMART cDNA library. The growth hormone of S. prenanti (SpGH) cDNA contains 1238 base pairs (bp) and encodes 215 amino acid (aa) residues; the prolactin (SpPRL) cDNA contains 1124 bp and encodes 209 aa residues; the cDNA sequence of somatolactin (SpSL) is 1290 bp in length, and encodes for a peptide of 234 aa residues. Although there is only about 22% aa sequence identity between the three deduced proteins, SpGH, SpPRL and SpSL, overall, the C-terminal region shows a higher identity. Multiple aa sequence alignments indicated the deduced SpGH, SpPRL and SpSL show high identities to that of goldfish and zebrafish, respectively. RT-PCR analysis revealed that the three hormones displayed different tissue distribution patterns. SpPRL transcripts were detected only in the pituitary of the Prenant's schizothoracin. SpSL was more widely distributed than SpPRL, high mRNA levels of SpSL were detected in the pituitary and low mRNA levels of SpSL were found in liver, kidney and heart. SpGH was expressed in nearly all tissues detected with the highest expression level in the pituitary.

New insights into the binding mode of melanin concentrating hormone receptor-1 antagonists: homology modeling and explicit membrane molecular dynamics simulation study.

Melanin concentrating hormone (MCH) is a cyclic 19-amino-acid peptide expressed mainly in the hypothalamus. It is involved in the control of feeding behavior, energy homeostasis, and body weight. Administration of MCH-R1 antagonists has been proved to reduce food intake and cause weight loss in animal models. In the present study, a homology model of the human MCH-R1 was constructed using the crystal structure of bovine rhodopsin (PDB: 1u19) as a template. Based on the observation that MCH-R1 can bind ligands of high chemical diversity, the initial model was subjected to an extensive ligand-supported refinement using antagonists of different chemotypes. The refinement process involved stepwise energy minimizations and molecular dynamics simulations. The refined model was inserted into a pre-equilibrated DPPC/TIP3P membrane system and then simulated for 20 ns in complex with structurally diverse antagonists. This protocol was able to explain the SAR of MCH-R1 antagonists with diverse chemical structures. Moreover, it reveals new insights into the critical recognition sites within the receptor. This work represents the first detailed study of molecular dynamics of MCH-R1 inserted into a membrane-aqueous environment.

Regulation of food intake by melanin-concentrating hormone in goldfish.

Melanin-concentrating hormone (MCH), originally discovered in the teleost pituitary, is a hypothalamic neuropeptide involved in the regulation of body color in fish. Although MCH is also present in the mammalian brain, it has no evident function in providing pigmentation. Instead, this peptide is now recognized to be one of the key neuropeptides that act as appetite enhancers in mammals such as rodents and primates. Although there has been little information about the central action of MCH on appetite in fish, recent studies have indicated that, in goldfish, MCH acts as an anorexigenic neuropeptide, modulating the alpha-melanocyte-stimulating hormone signaling pathway through neuronal interaction. These observations indicate that there may be major differences in the mode of action of MCH between fish and mammals. This paper reviews what is currently known about the regulation of food intake by MCH in fish, especially the goldfish.

S38151 [p-guanidinobenzoyl-[Des-Gly(10)]-MCH(7-17)] is a potent and selective antagonist at the MCH(1) receptor and has anti-feeding properties in vivo.

Structure-activity relationships studies have established the minimal sequence of melanin-concentrating hormone (MCH) that retains full agonist potency at the MCH(1), to be the dodecapeptide MCH(6-17). The alpha-amino function is not required for activity since arginine(6) can be replaced by p-guanidinobenzoyl, further improving activity. We report that the deletion of glycine in this short potent agonist (EC(50) 3.4nM) turns it into a potent and new MCH(1) antagonist (S38151, K(B) 4.3nM in the [(35)S]-GTPgammaS binding assay), which is selective versus MCH(2). A compared Ala-scan of the agonist and antagonist sequences reveals major differences in the residues that are mandatory for affinity, including arginine(11) and tyrosine(13) for the agonist and leucine(9) for the antagonist, whereas methionine(8) was necessary for both agonist and antagonist activities. A complete molecular study of the antagonist behavior is described in the present report, with a particular focus on the description of several analogues, attempting to find structure-activity relationships. Finally, S38151 antagonizes food intake when injected intra-cerebroventricularly in the rat. This is in agreement with the in vitro data and with our previous demonstration of a good correlation between in vitro and in vivo data on MCH(1) agonists.

Functional amyloids as natural storage of peptide hormones in pituitary secretory granules.

Amyloids are highly organized cross-beta-sheet-rich protein or peptide aggregates that are associated with pathological conditions including Alzheimer's disease and type II diabetes. However, amyloids may also have a normal biological function, as demonstrated by fungal prions, which are involved in prion replication, and the amyloid protein Pmel17, which is involved in mammalian skin pigmentation. We found that peptide and protein hormones in secretory granules of the endocrine system are stored in an amyloid-like cross-beta-sheet-rich conformation. Thus, functional amyloids in the pituitary and other organs can contribute to normal cell and tissue physiology.

MCH receptor peptide agonists and antagonists.

Melanin-concentrating hormone (MCH) is an important neuropeptide hormone involved in multiple physiological processes. Peptide derivatives of MCH have been developed as tools to aid research including potent radioligands, receptor selective agonists, and potent antagonists. These tools have been used to further understand the role of MCH in physiology, primarily in rodents. However, the tools could also help elucidate the role for MCHR1 and MCHR2 in mediating MCH signaling in higher species.

Novel approach for chemotype hopping based on annotated databases of chemically feasible fragments and a prospective case study: new melanin concentrating hormone antagonists.

A novel strategy for chemotype hopping, based on annotated databases of chemically feasible fragments and their oriented functionalization, is presented. A three-dimensional (3D) similarity analysis of project-oriented functionalized scaffolds provides a prioritized proposal for synthesis with the most appropriate linkers and optimal regiochemistry on R-groups. This strategy maximizes the potential of proprietary and commercially available compounds. A retrospective and prospective case study, on melanin concentrating hormone (MCH) antagonists, showing the impact on the drug discovery process of this new strategy by maintaining primary activity and improving key ADME/Tox property while enhancing intellectual property (IP) position is demonstrated.

cDNA cloning and isolation of somatolactin in Mozambique tilapia and effects of seawater acclimation, confinement stress, and fasting on its pituitary expression.

Somatolactin (SL) is a member of the growth hormone (GH)/prolactin (PRL) family of pituitary hormones, and is found in a variety of teleost species. Somatolactin is thought to be involved in a wide range of physiological actions, including reproduction, stress response, the regulation of Ca(2+) and acid-base balance, growth, metabolism, and immune response. We report here on the cDNA structure of SL from the pituitary of Mozambique tilapia, Oreochromis mossambicus, and its gene expression in response to seawater acclimation, stress, and fasting. Tilapia SL cDNA (1573bp long) encoded a prehormone of 230 amino acids. Sequence analysis of purified SL revealed that the prehormone is composed of a signal peptide of 23 amino acids and a mature protein of 207 amino acids, which has a possible N-glycosylation site at position 121 and seven Cys residues. Tilapia SL shows over 80% amino acid identity with SLalpha of advanced teleosts such as medaka and flounder, and around 50% identity with SLbeta of carp and goldfish. Acclimation to seawater had no effect on pituitary expression of SL or on hepatic expression of the putative tilapia SL receptor (GHR1). By contrast, seawater acclimation resulted in significant increases in pituitary GH expression and in hepatic expression of tilapia GH receptor (GHR2). Confinement stress had no effect on pituitary expression of either SL or GH, or on hepatic expression of GHR1, whereas a significant increase was seen in GHR2 expression in the liver. Fasting for 4 weeks resulted in significant reductions in SL transcripts both in fresh water and seawater. It is highly likely that SL is involved in metabolic processes in tilapia along with the GH/IGF-I axis.

Surface behavior and peptide-lipid interactions of the cyclic neuropeptide melanin concentrating hormone.

The kinetics and the thermodynamics of melanin concentrating hormone (MCH) adsorption, penetration, and mixing with membrane components are reported. MCH behaved as a surface active peptide, forming stable monolayers at a lipid-free air-water interface, with an equilibrium spreading pressure, a collapse pressure, and a minimal molecular area of 11 mN/m, 13 mN/m, and 140 A (2), respectively. Additional peptide interfacial stabilization was achieved in the presence of lipids, as evidenced by the expansion observed at pi > pi sp in monolayers containing premixtures of MCH with zwitterionic or charged lipids. The MCH-monolayer association and dissociation rate constants were 9.52 x 10 (-4) microM (-1) min (-1) and 8.83 x 10 (-4) min (-1), respectively. The binding of MCH to the dpPC-water interface had a K d = 930 nM at 10 mN/m. MCH penetration in lipid monolayers occurred even up to pi cutoff = 29-32 mN/m. The interaction stability, binding orientation, and miscibility of MCH in monolayers depended on the lipid type, the MCH molar fraction in the mixture, and the molecular packing of the monolayer. This predicted its heterogeneous distribution between different self-separated membrane domains. Our results demonstrated the ability of MCH to incorporate itself into biomembranes and supports the possibility that MCH affects the activity of mechanosensitive membrane proteins through mechanisms unrelated with binding to specific receptors.

Evolution of receptors for growth hormone and somatolactin in fish and land vertebrates: lessons from the lungfish and sturgeon orthologues.

Two cognate hormones, growth hormone (GH) and somatolactin (SL), control several important physiological processes in vertebrates. Knowledge about GH and its receptor (GHR) has accumulated over the last decades. However, much less is known about SL and its receptor (SLR). SL is found only in fish (including lungfish), suggesting that it was present in the common ancestor of vertebrates, but was lost secondarily in the lineage leading to land vertebrates after the lungfish branched off. SLR was suggested to be a duplicated copy of GHR acquired only in teleosts via the fish-specific genome duplication (FSGD). This scenario (i.e., the existence of SL but not SLR in the vertebrate ancestors) is intriguing but contested. In this study, we first evaluated the plausibility of this scenario through synteny analyses and found that the loci for GHR and SLR are located in syntenic genomic positions, whereas the loci for GH and SL are not. Next, we cloned GHRs of lungfish and sturgeon, which possess SL but did not undergo the FSGD (i.e., they should not possess SLR). Their phylogenetic positions in the GHR/SLR gene tree further support the fish-specific scenario for the GHR-SLR duplication. Interestingly, their sequences share greater similarity with teleost SLRs and reptilian/amphibian GHRs than with the GHRs of mammals, birds, and teleosts. On the basis of these results, we discuss the validity of the nomenclature of the teleost-specific copy of GHR as SLR and an ancestral receptor(s) for SL before the evolution of SLR during the FSGD.

Peptide ligands for the melanin-concentrating hormone (MCH) receptor 1.

The melanin-concentrating hormone receptor 1 (MCH-1R) has been recognized as a receptor which mediates effects of the endogenous melanin-concentrating hormone (MCH) on appetite and body weight gain in rodents. In the last several years, a number of hMCH analogs have been designed which were potent and selective ligands for hMCH-1R. These peptidic agonists and antagonists have served as research tools in animal studies that showed a key role of the MCH-1R in the development of obesity and proved that MCH-1R antagonism can produce anti-obesity effects in rodents.

Isolation of Atlantic halibut pituitary hormones by continuous-elution electrophoresis followed by fingerprint identification, and assessment of growth hormone content during larval development.

Continuous-elution electrophoresis (CEE) has been applied to separate putative hormones from adult Atlantic halibut pituitaries. Soluble proteins were separated by size and charge on Model 491 Prep Cell (Bio-Rad), where the homogenate runs through a cylindrical gel, and protein fractions are collected as they elute from the matrix. Protein fractions were assessed by SDS-PAGE and found to contain purified proteins of molecular size from 10 to 33 kDa. Fractions containing proteins with molecular weights of approximately 21, 24, 28 and 32 kDa, were identified as putative growth hormone (GH), prolactin, somatolactin and gonadotropins, respectively. These were analyzed further by mass spectrometry and identified with peptide mass protein fingerprinting. The CEE technique was used successfully for purification of halibut GH with a 5% yield, and appears generally well suited to purify species-specific proteins often needed for research in comparative endocrinology, including immunoassay work. Thus, the GH obtained was subsequently used as standards and iodination label in a homologous radioimmunoassay, applied to analyze GH content through larval development in normally and abnormally metamorphosing larvae. As GH is mainly found in the pituitary, GH contents were analyzed in tissue extracts from the heads only. The pituitary GH content increases proportionally to increased larval weight from first feeding to metamorphic climax. No difference in relative GH content was found between normal and abnormal larvae and it still remains to be established if GH has a direct role in metamorphosis.

Peptide-cleaving catalyst selective for melanin-concentrating hormone: Oxidative decarboxylation of N-terminal aspartate catalyzed by Co(III)cyclen.

To provide a firm basis for the new paradigm of drug discovery based on catalysts for oxidative cleavage of N-terminal aspartate (Asp) residues of oligopeptides, oligopeptide-cleaving catalysts were searched by using melanin-concentrating hormone (MCH) as the substrate. MCH is a target for designing drugs to reduce obesity. Catalyst candidates containing the Co(III) complex of cyclen as the catalytic center were prepared by multicomponent condensation reactions. From three kinds of chemical libraries containing about 19,000 catalyst candidates, one compound was identified as the MCH-cleaving catalyst. On incubation with the catalyst, the N-terminal Asp residue of MCH was converted to the pyruvate residue by oxidative decarboxylation. Detailed kinetics analysis revealed the catalytic nature of the action of the catalyst. In addition, the kinetics data indicated that MCH can be cleaved with half-lives of 3 h or less with submicromolar catalyst concentrations if the structure of the catalyst is further improved.