Hydrazide and hydrazine reagents as reactive matrices for matrix-assisted laser desorption/ionization mass spectrometry to detect steroids with carbonyl groups.

Fifty-one reagents (37 hydrazides and 14 hydrazines) were examined to determine if they could function as reactive matrices for the detection of steroids with carbonyl groups in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Among these reagents, 3-hydroxy-2-naphthoic acid hydrazide reacted most efficiently with steroids with carbonyl groups, allowing detection of them at the 1pmol level. Results from accurate mass measurement with a JMS-S3000 Spiral-TOF suggested that the ion peaks corresponding to the protonated fragment of a hydrazone were observed, in addition to the protonated molecular peaks of the resulting hydrazones ([M(D)+H](+)) and their sodium adduct ([M(D)+Na](+)). 3-Hydroxy-2-naphthoic acid hydrazide also could detect cortisol (1.93ng) spiked in a human serum certified reference material, as well as cortisone and cortisol in a human saliva sample collected in the morning. Using a combination of 3-hydroxy-2-naphthoic acid hydrazide and α- cyano-4-hydroxycinammic acid (CHCA) (3-hydroxy-2-naphthoic acid hydrazide:CHCA=4:1, v/v), even cortisone and cortisol in a human saliva sample collected in the afternoon, when levels are typically low, could be detected effectively.

Identification of novel peptides from amphibian (Xenopus tropicalis) skin by direct tissue MALDI-MS analysis.

Twelve novel peptides (Pxt-1 to Pxt-12) were isolated from the skin of Xenopus tropicalis, diploid frogs, using topological MS analysis. Among them, Pxt-8, Pxt-9, and Pxt-10 were the N terminus of Pxt-1, N terminus of Pxt-3 and C terminus of Pxt-11, respectively. The Pxt-3 and Pxt-11 peptides shared significant sequence homologies with magainins 1, -2 and levitide, respectively, which all isolated from X. laevis. Pxt-12 was identical to the X. tropicalis XT-6-like precursor previously isolated by ESI-MS/MS. None of the Pxt peptides contained any Cys, Asp, Tyr or Trp, although Leu and Lys were frequently found as typical frog-skin peptides. RT-PCR analysis confirmed the gene expressions of Pxt-2, Pxt-3, Pxt-4, Pxt-5, Pxt-7 and Pxt-11 in X. tropicalis skin. Several ion peaks corresponding to all identified Pxt peptides were observed with MALDI-MS analysis of X. tropicalis secretory fluids, collected after in vivo stimulation, which suggested that Pxt peptides were definitely secretory molecules. CD studies and Schiffer-Edmundson helical wheel projections suggested that Pxt-5, as well as mastoparan, at least, could form a typical amphiphilic α helix without a phospholipid or a membrane-mimetic solvent (trifluoroethanol). Moreover, Pxt-2 and Pxt-5 showed growth inhibitory effects on both Escherichia coli (Gram-negative) and Staphylococcus aureus (Gram-positive). Measurements of dynamic light scattering and the surface tensions of Pxt peptides solutions suggested that both Pxt-2 and Pxt-5 could form associations as micelles and behave like a general surfactant. Moreover, the remarkable foaming properties of Pxt-2 and Pxt-5 were observed, as well as those of the secretory fluids of X. tropicalis.

Hydrazide and hydrazine reagents as reactive matrices for MALDI-MS to detect gaseous aldehydes.

The reagents 19 hydrazide and 14 hydrazine were examined to function as reactive matrices for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to detect gaseous aldehydes. Among them, two hydrazide (2-hydroxybenzohydrazide and 3-hydroxy-2-naphthoic acid hydrazide) and two hydrazine reagents [2-hydrazinoquinoline and 2,4-dinitrophenylhydrazine (DNPH)] were found to react efficiently with carbonyl groups of gaseous aldehydes (formaldehyde, acetaldehyde and propionaldehyde); these are the main factors for sick building syndrome and operate as reactive matrices for MALDI-MS. Results from accurate mass measurements by JMS-S3000 Spiral-TOF suggested that protonated ion peaks corresponding to [M + H](+) from the resulting derivatives were observed in all cases with the gaseous aldehydes in an incubation, time-dependent manner. The two hydrazide and two hydrazine reagents all possessed absorbances at 337 nm (wavelength of MALDI nitrogen laser), with, significant electrical conductivity of the matrix crystal and functional groups, such as hydroxy group and amino group, being important for desorption/ionization efficiency in MALDI-MS. To our knowledge, this is the first report that gaseous molecules could be derivatized and detected directly in a single step by MALDI-MS using novel reactive matrices that were derivatizing agents with the ability to enhance desorption/ionization efficiency.

The multiplicity of post-translational modifications in pro-opiomelanocortin-derived peptides.

The precursor protein, pro-opiomelanocortin (POMC) undergoes extensive post-translational processing in a tissue-specific manner to yield various biologically active peptides involved in diverse cellular functions. The recently developed method of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for direct tissue analysis has proved to be a powerful tool for investigating the distribution of peptides and proteins. In particular, topological mass spectrometry analysis using MALDI-MS can selectively provide a mass profile of the hormones included in cell secretory granules. An advantage of this technology is that it is possible to analyze a frozen thin slice section, avoiding an extraction procedure. Subsequently, tandem mass spectrometry (MS/MS) has a profound impact on addressing the modified residues in the hormone molecules. Based on these strategies with mass spectrometry, several interesting molecular forms of POMC-derived peptides have been found in the fish pituitary, such as novel sites of acetylation in α-melanocyte-stimulating hormone (MSH), hydroxylation of a proline residue in ß-MSH, and the phosphorylated form of corticotropin-like intermediate lobe peptide.

Photouncaging nanoparticles for MRI and fluorescence imaging in vitro and in vivo.

Multimodal and multifunctional nanomaterials are promising candidates for bioimaging and therapeutic applications in the nanomedicine settings. Here we report the preparation of photouncaging nanoparticles with fluorescence and magnetic modalities and evaluation of their potentials for in vitro and in vivo bioimaging. Photoactivation of such bimodal nanoparticles prepared using photouncaging ligands, CdSe/ZnS quantum dots, and super paramagnetic iron oxide nanoparticles results in the systematic uncaging of the particles, which is correlated with continuous changes in the absorption, mass and NMR spectra of the ligands. Fluorescence and magnetic components of the bimodal nanoparticles are characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and elemental analyses using energy dispersive X-ray (EDX) spectroscopy and X-ray photoelectron spectroscopy (XPS). Bioconjugation of the nanoparticles with peptide hormones renders them with biocompatibility and efficient intracellular transport as seen in the fluorescence and MRI images of mouse melanoma cells (B16) or human lung epithelial adenocarcinoma cells (H1650). Biocompatibility of the nanoparticles is evaluated using MTT cytotoxicity assays, which show cell viability over 90%. Further, we combine MRI and NIR fluorescence imaging in C57BL/6 (B6) mice subcutaneously or intravenously injected with the photouncaging nanoparticles and follow the in vivo fate of the nanoparticles. Interestingly, the intravenously injected nanoparticles initially accumulate in the liver within 30 min post injection and subsequently clear by the renal excretion within 48 h as seen in the time-dependent MRI and fluorescence images of the liver, urinary bladder, and urine samples. Photouncaging ligands such as the ones reported in this article are promising candidates for not only the site-specific delivery of nanomaterials-based contrast agents and drugs but also the systematic uncaging and renal clearance of nanomaterials after the desired in vivo application.

A thiophene-containing compound as a matrix for matrix-assisted laser desorption/ ionization mass spectrometry and the electrical conductivity of matrix crystals.

The electrical conductivity of the matrix crystal might be a new factor to enhance matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) sensitivity. In MALDI-MS, several compounds are used as a standard matrix. Utilization of such compounds is based on an a posteriori approach, but there is no theoretical guidance for selecting a matrix. In an attempt to further understand performance in MALDI-MS, we utilized peptide detection for random screening of a chemical library (12,383 compounds) for compounds with matrix functions in MALDI-MS. A lot of thiophene compounds were found to be a matrix, in which 2-[5-(2,4-dichlorobenzoyl)-2-thienyl] acetic acid (DCBTA) provided an important clue to measure the electrical conductivity of the matrix crystal, because the structure of DCBTA is analogous to conductive polymers and organic solar cells. Most of the crystals of standard matrices, such as alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid [sinapinic acid, (SA)], and DCBTA showed electrical conductivity, whereas the conductivity of crystal was not observed in 2,5-dihydroxybenzoic acid (2,5-DHB). On the other hand, super-DHB using 2-hydroxy-5-methoxybenzoic acid [5-methoxysalicylic acid, (MSA)] as an additive to 2,5-DHB, improved the electrical conductivity of the crystal, that followed the enhancement of peak intensity in MS spectrum. These observations might indicate that the electrical conductivity of matrix crystals is a key consideration in obtaining efficient MALDI performance.

Desorption/ionization efficiency of peptides containing disulfide bonds in matrix-assisted laser desorption/ionization mass spectrometry.

In order to elucidate the role of desorption/ionization efficiency of peptides in MALDI-MS, we focused on peptides with disulfide bonds, which form a rigid tertiary structure. We synthesized seven sets of peptides with one disulfide bond (oxytocin, somatostatin, [Arg(8)]-vasopressin, [Arg(8)]-vasotocin, cortistatin, melanin-concentrating hormone, urotensin II-related peptide) and five sets of peptides with two disulfide bonds (tertiapin, α-conotoxin GI, α-conotoxin ImI, α-conotoxin MI and α-conotoxin SI). Each peptide set consisted of three peptides: the oxidized form (S-S type), the reduced form (SH type), and an internal standard peptide in which all cysteine residues were substituted with alanine residues. In the case of urotensin II-related peptide, tertiapin, α-conotoxin ImI and α-conotoxin MI, the reduced form showed higher desorption/ionization efficiency than the oxidized form. In contrast, the other peptides revealed higher desorption/ionization efficiency in the oxidized form relative to the reduced form. These results imply that a rigid structure of peptides formed by disulfide bonds does not correlate with desorption/ionization efficiency in MALDI-MS.

Characterization of triacetyl-α-melanocyte-stimulating hormone in carp and goldfish.

A triacetyl form of α-melanocyte-stimulating hormone (MSH) was found in carp (Cyprinus carpio) and goldfish (Carassius auratus), by selective detection of mass profile for cell secretory granules using direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry (MALDI-TOF MS) analysis during the investigation of fish pituitaries. The structure of triacetyl-α-MSH in carp and goldfish was further analyzed using a collision-induced dissociation with electrospray ionization mass spectrometry, and determined to be N,O-diacetyl Ser as the N-terminal residue and O-acetyl Tyr at position 2. These modifications for α-MSH in carp and goldfish are structurally different from that of medaka hormone, in which [N,O-diacetyl Ser(1), O-acetyl Ser(3)]-α-MSH has been identified. The profiles of four α-MSH variants, des-, mono-, di- and tri-acetyl forms in goldfish and medaka pituitaries were also examined by direct tissue MALDI-TOF MS analysis, and the percentages as a total of α-MSH molecules were compared for fish reared in a white or black tank for 5 days. Among structural variants, diacetyl-α-MSH was the predominant form in goldfish and N-desacetyl-α-MSH in medaka, respectively. In both species, the relative level of the predominant form in the pituitary of white-adapted fish tended to be lower than that of black-adapted fish. In goldfish, no significant difference was observed in the relative content of triacetyl-α-MSH in both backgrounds, whereas the lowest content of triacetyl-α-MSH was found in black-adapted medaka. These preliminary data indicate that it is difficult to elucidate the relations between the physiological roles and acetylated pattern of α-MSH molecule, depending on species.

Post-translational modifications of pro-opiomelanocrtin related hormones in medaka pituitary based on mass spectrometric analyses.

Direct tissue matrix-assisted laser desorption ionization with time-of-flight mass spectrometry analysis provides a selective detection of mass profile for the peptides contained into cell secretory granules. By this mass spectrometry with slice of pituitary, two novel molecular forms of pro-opimelanocrtin related hormone were found in the orange-red strain medaka (Oryzias latipes var.). The structures of [N,O-diacetyl Serine(1), O-acetyl Serine(3)]-α-melanocyte-stimulating hormone (MSH) and [hydroxyproline(15)]-ß-MSH, together with [phosphoserine(15)]-corticotropin-like intermediate lobe peptide, were determined for the first time using a collision-induced dissociation with electrospray ionization mass spectrometry. A combination of mass spectrometry analyses is thus a powerful tool to lead to the elucidation of the post-translational processing from the pre-prohormone.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae.

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.

Mass spectrometric map of neuropeptide expression and analysis of the gamma-prepro-tachykinin gene expression in the medaka (Oryzias latipes) brain.

Neuropeptides have important roles in modulating behavioral patterns such as social interaction. With the aim to determine the presence of neuropeptides known to be involved in social interaction as well as novel peptides, we used MALDI-TOF/MS to analyze neuropeptide profiles in some medaka brain regions. In the telencephalon, hypothalamus, and pituitary gland, 3, 6, and 10 peaks, respectively, were identified as neuropeptides (Arg-vasotocin [AVT], growth hormone-releasing hormone [GHRH], neuropeptide FF, substance P [SP], somatostatin-1 and -2, melanin-concentrating hormone [MCH], MCH gene-related peptide [Mgrp], melanocyte-stimulating hormone [MSH], corticotropin-like intermediate lobe peptide [CLIP], and beta-endorphin). The neuropeptide profile of telencephalon similar to that of the hypothalamus, but completely different from that of pituitary gland. For the future genetic analysis, we identified cDNAs encoding precursor proteins for the identified peptides. We also detect its expression of gamma-prepro-tachykinin gene encoding a SP precursor protein in both the telencephalon and hypothalamus. Our results indicated that the medaka brain contains some neuropeptides (AVT, SP, and somatostatins) that may be involved in modulating medaka behaviors such as social interaction.

Novel mastoparan and protonectin analogs isolated from a solitary wasp, Orancistrocerus drewseni drewseni.

Two novel biologically active peptides, Eumenine mastoparan-OD and Orancis-Protonectin, were isolated from a solitary wasp, Orancistrocerus drewseni drewseni (Eumeninae, Vespidae). MALDI-TOF MS analysis of a small amount of the crude venom gave two intensive molecular-related ion peaks at m/z 1269.9 and 1552.9 that were expected to be novel based on a peptide database search. Purification of the crude venom by HPLC gave two peptide fractions, P-1 and P-2. The amino acid sequence of P-1 (GRILSFIKAGLAEHL-NH2) and P-2 (ILGIITSLLKSL-NH2) were determined by ESI-MS/MS, automated Edman degradation, and amino acid analysis. According to the high sequence homology with those of mastoparan and protonectin, P-1 and P-2 were labeled Eumenine mastoparan-OD and Orancis-Protonectin, respectively. Orancis-Protonectin is the first example of a protonectin analog isolated from the venom of a solitary wasp. The hemolytic activities of these new peptides were more potent than that of mastoparan.

Synthesis and phosphorylation of ecdysteroids during ovarian development in the silkworm, Bombyx mori.

In the silkworm, Bombyx mori, it has been demonstrated that most free ecdysteroids in the ovary are converted to physiologically inactive ecdysteroid 22-phosphates, which are then transformed back to free ecdysteroids during early embryonic development. Two specific enzymes involved in the reciprocal conversion of ecdysteroids, namely, ecdysteroid 22-kinase (EcKinase) and ecdysteroid-phosphate phosphatase, have been isolated and characterized. In this study, we first attempted a phylogenetic analysis of EcKinase. The resulting phylogenetic tree showed that many proteins homologous to B. mori EcKinase are found not only in ecdysozoa, including insects and nematodes, but also in teleosts, fungi, and bacteria. We then investigated the sites where free ecdysteroids are synthesized and phosphorylated in the ovary. We found that (1) the mRNAs of two P450 enzymes involved in ecdysteroidogenesis, CYP306a1 (25-hydroxylase) and CYP314a1 (20-hydroxylase), are expressed mainly in follicle cells, (2) EcKinase mRNA localizes in the oocyte and nurse cells, and (3) EcKinase immunoreactivity localizes mainly in the external region of the oocyte, not in nurse cells or follicle cells. From these results, we suggest that ecdysteroids in the B. mori ovary are synthesized in follicle cells and transferred into the oocyte, where they are phosphorylated by EcKinase, whose mRNA originates from nurse cells and the oocyte itself.

Novel biologically active peptides from the venom of Polistes rothneyi iwatai.

Four novel peptides, polistes-mastoparan-R1, 2, 3, and polistes-protonectin, were isolated from the venom of a paper wasp, Polistes rothneyi iwatai. MALDI-TOF MS analysis of a small amount of the crude venom gave six molecular-related ion peaks. Among them, m/z 1565 was expected to be a novel peptide. Purification of the crude venom by HPLC gave two known kinins, Thr6-bradykinin and Ala-Arg-Thr6-bradykinin, and four novel peptides named polistes-mastoparan-R1, 2, and 3, and polistes-protonectin. Polistes-mastoparan-R1, 2, and 3 (Pm-R) were tetradecapeptides that possess high sequence homology with that of mastoparan. The sequence of polistes-protonectin was similar to that of protonectin isolated from a Brazilian paper wasp. Histamine-releasing activities of Pm-R1, 2, and 3 were more potent than that of mastoparan. Polistes-protonectin exhibited the most potent hemolytic activity in comparison with the four novel peptides and mastoparan.

Characteristic expression patterns of allatostatin-like peptide, FMRFamide-related peptide, orcokinin, tachykinin-related peptide, and SIFamide in the olfactory system of crayfish Procambarus clarkii.

The olfactory system plays important roles in various crustacean behaviors. Despite numerous studies on different aspects of the olfactory neural pathway, only the decapod-tachykinin-related peptide (decapod-TRP) has been identified as a neuromodulator in this processing to date. To establish the functions of other related neuropeptides, we initially performed cDNA cloning of FMRFamide-related peptide (FaRP) and allatostatin (AST)-like peptide from the crayfish Procambarus clarkii, followed by in situ hybridization (ISH) analysis of these peptides, along with decapod-TRP, orcokinin, and crustacean-SIFamide. Cloned FaRP cDNA encodes seven copies of C-terminal RN(F/Y)LRFamide-containing peptide, whereas AST-like peptide cDNA comprises 29 copies of AST-like peptide (-YXFGLamide) and three additional putative peptides. ISH analysis of the brain revealed specific expression of crustacean-SIFamide mRNA in most projection neurons (cell cluster 10), and predominant localization of other mRNAs to interneurons. The data suggest that the crustacean-SIFamide neuropeptide is involved in output of the deutocerebrum to the protocerebrum. Double-fluorescence ISH data further disclose that, in cluster 9, orcokinin is coexpressed in decapod-TRP-specific interneurons, whereas AST-like peptide-containing cells do not overlap with orcokinin-expressing cells. On the other hand, FaRP-expressing cells overlap with both orcokinin- and AST-like peptide-specific cells. In cluster 11, where signals for AST-like peptide are absent, a number of interneurons express both decapod-TRP and orcokinin, emphasizing a close relationship between these two factors with regard to olfactory processing, and possibly tactile and/or visual sensory systems. These characteristic expression patterns of neuropeptides support their distinct involvement in the modulation of olfactory processing.

Posttranslational processing of proopiomelanocortin family molecules in sea lamprey based on mass spectrometric and chemical analyses.

In gnathostomes, adrenocorticotropic hormone (ACTH), melanophore-stimulating hormones (MSHs), and beta-endorphin (beta-END) are derived from a common precursor, proopiomelanocortin. In sea lamprey, ACTH and two forms of MSHs are contained in independent precursors, proopiocortin (POC), and proopiomelanotropin (POM), respectively, together with a distinct beta-END. Here, we characterized products from POC and POM. An analysis of previously purified ACTH by mass spectrometry (MS) detected four peptides with a molecular weight of 6469.4, 6549.6, 6556.6, or 6636.1. The sequence analysis of an ACTH preparation following enzymatic and chemical cleavage revealed the presence of ACTH(1-59) and ACTH(1-60) corresponding to a molecular weight of 6469.4 and 6556.6, respectively, and of ACTH(1-59) and ACTH(1-60) modified at Ser(35) by a group having a mass of 80, giving the molecular weight 6549.6 and 6636.1, respectively. The modification could be due to phosphorylation based on the increase in molecular weight of 80. Analyses of frozen pituitary slices with MALDI-TOF MS detected several mass numbers corresponding to POC-derived peptides such as ACTH(1-60), modified ACTH(1-60), and (POC)beta-END, and those corresponding to POM-derived peptides such as MSH-A, MSH-B, and the C-terminal fragment of (POM)beta-END lacking a Met-enkephalin segment. The present results together with previous characterizations show that in sea lamprey pituitary the major products derived from POC in the PD by posttranslational processing are ACTH and beta-END as in gnathostomes. The posttranslational processing of POM in the PI is similar to that in gnathostomes in the sense of the occurrence of MSH, however, it differs in that beta-END is further cleaved, thus generating Met-enkephalin.

Nucleotide sequence and expression of three subtypes of proopiomelanocortin mRNA in barfin flounder.

Melanophore-stimulating hormone (MSH) has been shown to be associated with food intake in addition to body color change in teleosts. MSH is encoded by a proopiomelanocortin (POMC) gene together with endorphin (END). To assess the significance of MSH to biological activities, we determined the structure and evaluated the expression of POMC mRNA in barfin flounder (bf), Verasper moseri, a member of a group of teleosts, Pleuronectiformes. Three subtypes of POMC cDNAs (A, B, and C) were amplified from bf pituitary glands. These bfPOMCs contained segments for N-POMC, alpha-MSH, beta-MSH, and beta-END as do other teleost POMCs, while POMC-C showed remarkable variations in the segments corresponding to N-POMC and beta-END. A phylogenetic tree of ray-finned fish POMCs constructed by the neighbor joining method revealed that the three POMC subtypes may have appeared as a result of duplication events occurring at least twice during the course of bf evolution. The first duplication may have generated the lineage leading to an ancestor of bfPOMC-A and -B and that leading to bfPOMC-C, and then the lineage of bfPOMC-A may have diverged from that of bfPOMC-B. All peptides flanked by processing signals excluding N-POMC-C (1-14) were identified in a single pituitary extract by mass spectrometry, and the cDNAs of three POMCs were amplified from a single pituitary by reverse transcription polymerase chain reaction. These results demonstrated that the three POMC genes are expressed in a single individual. While the bfPOMC-A gene was exclusively expressed in the pituitary, the bfPOMC-B and -C genes were expressed in non-pituitary tissues such as brain, gill, heart, spleen, liver, stomach, intestine, testis, muscle, blood, and skin in addition to the pituitary. The expression levels of the POMC-A, -B, and -C genes in pituitary neurointermediate lobe were greater in the fish reared with a black background than the fish reared with a white background, indicating that MSH derived from all of the three bfPOMC genes was associated with body color change. No difference was observed in the expression levels of bfPOMC-C in the brain in response to feeding status.

Possible involvement of melanin-concentrating hormone in food intake in a teleost fish, barfin flounder.

We investigated the involvement of MCH in food intake in barfin flounder. The structure of barfin flounder MCH was determined by cDNA cloning and mass spectrometry. In fasted fish, the MCH gene expression and the number of MCH neurons in the brain were greater than controls. In white-reared fish, the MCH gene expression and the number of MCH neurons in the brain were greater than black-reared fish. Furthermore, white-reared fish grew faster than black-reared fish. These results indicate that a white background stimulated production of MCH and MCH, in turn, enhanced body growth, probably by stimulating food intake.

APSGFLGMRamide is a unique tachykinin-related peptide in crustaceans.

We report here the identification of a tachykinin-related peptide (TRP) in crustaceans. Direct MALDI-TOF MS with brain slices of the crayfish Procambarus clarkii indicated the presence of a peptide having an MS number of around 934. Quadrupole orthogonal acceleration time-of-flight (Q-TOF) MS/MS analysis implied the sequence to be APSGFLGMRamide, identical to that of CabTRP Ia, isolated previously from the crab Cancer borealis, and Pev-tachykinin, from the shrimp Penaeus vannamei. The peptide has been shown to be myoactive in the cockroach hindgut, but the structure of its precursor protein had not been elucidated. A cDNA encoding crayfish preproTRP was cloned, revealing that the 225-residue protein contains seven identical copies of the peptide APSGFLGMRamide. This is unique because TRPs identified in other invertebrates were known to exist in several related forms in each species. The conserved structure of TRP in crustaceans was confirmed by cloning preproTRP from the spiny lobster Panulirus interruptus. RT-PCR and Northern blotting analyses suggested that the crayfish preproTRP mRNA is expressed throughout the nervous system, and in situ hybridization studies of the brain revealed that the transcript predominantly localizes to cell clusters 11 (dorsal lateral cells) and 9 (ventral lateral cells).