Akikazu Yasuda

Primary structure of chum salmon prolactins: Occurrence of highly conserved regions

The complete amino acid sequence of prolactin from the pituitaries of salmon (Oncorhynchus keta) has been determined. Salmon prolactin comprised two variants, I and II, which were separated by reverse-phase high-performance liquid chromatography. Each variant was reduced, S-carboxymethylated, and then cleaved with cyanogen bromide and enzymes. The resulting fragments were separated by reverse-phase high-performance liquid chromatography, as well as gel filtration, and subjected to sequence analysis by the dansyl-Edman method. Both variants contain 187 amino acid residues with two disulfide linkages at residues 46-160 and 177-187, lack a linkage in the N-terminal portion of mammalian prolactins, and differ from each other by the replacement of only four amino acid residues. Salmon prolactin (sPRL) shows 31% sequence identify with ovine prolactin. Moreover, four restricted regions, i.e., sPRL (3-21), (46-60), (68-83), and (160-178), encompass this significant conservatism between the teleost and the mammalian hormone, with identities of 47, 87, 62, and 68%, respectively. Such considerable identity between these distant phylogenic species strongly suggests that these regions may be responsible for the biological activity of prolactin.

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.

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. J. Comp. Neurol. 496:135–147, 2006.

Primary structure of common carp prolactins

The complete amino acid sequence of common carp (Cyprinus carpio) prolactin (PRL) has been determined. One milligram of carp PRL was reduced, carboxymethylated, and then cleaved with cyanogen bromide and several enzymes. Another milligram of carp PRL was digested with lysyl endopeptidase. The resulting fragments were separated by reverse-phase high-performance liquid chromatography and subjected to sequence analysis by the automatic Edman method with a gas-liquid sequencer and the manual DNS-Edman method. The carp PRL consists of 186 amino acid residues with two disulfide linkages formed between residues 46-160 and 177-186. In addition, it lacks the linkage in the amino-terminal portion of mammalian prolactins, as do salmon and tilapia PRLs, and shows 77% sequence identity with salmon PRL and 36% identity with mammalian PRLs. The sequence comparison has been performed with 190 alignment positions. Four conserved segments of the alignment positions 1-32, 46-66, 71-94, and 155-182 that were found between salmon and mammalian PRLs are further confirmed in carp PRL. Variant residues are clustered in the region of residues 98-143 which has 48% sequence identity with salmon PRL and only 2% with mammalian PRL.

Primary structure of Eel (Anguilla japonica) growth hormone

Two molecular forms of growth hormone, GHs I and II, were isolated from a culture medium of the eel pituitary (Anguilla japonica). The complete amino acid sequence of GH I was determined in the present study. The hormone was reduced, carboxymethylated, and subsequently cleaved with cyanogen bromide and enzymes. Intact eel GH I was also digested with enzyme. The resulting fragments were separated by reverse-phase high-performance liquid chromatography (HPLC) and subjected to sequence analysis by automated gas-liquid sequencer employing an Edman method. Eel GH I consists of 190 amino acid residues with two disulfide linkages formed between residues 52-163 and 180-188. Sequence comparison with other vertebrate GHs revealed that eel GH I is closer to avian and mammalian GHs with 55% identity than to salmon GH with 48% identity.

Prepro-tachykinin gene expression in the brain of the honeybee Apis mellifera

We have recently identified a tachykinin-related peptide (AmTRP) from the mushroom bodies (MBs) of the brain of the honeybee Apis mellifera L. by using direct matrix-assisted laser desorption/ionization with time-of-flight mass spectometry and have isolated its cDNA. Here, we have examined prepro-AmTRP gene expression in the honeybee brain by using in situ hybridization. The prepro-AmTRP gene is expressed predominantly in the MBs and in some neurons located in the optic and antennal lobes. cDNA microarray studies have revealed that AmTRP expression is enriched in the MBs compared with other brain regions. There is no difference in AmTRP-expressing cells among worker, queen, and drone brains, suggesting that the cell types that express the prepro-AmTRP gene do not change according to division of labor, sex, or caste. The unique expression pattern of the prepro-AmTRP gene suggests that AmTRPs function as neuromodulators in the MBs of the honeybee brain.

The complete amino acid sequence of growth hormone of an elasmobranch, the blue shark (Prionace glauca).

The complete amino acid sequence of growth hormone (GH) from a phylogenetically ancient fish, the blue shark (Prionace glauca), was determined. The shark GH isolated from pituitary glands by U. J. Lewis, R. N. P. Singh, B. K. Seavey, R. Lasker, and G. E. Pickford (1972, Fish. Bull. 70, 933-939) was purified by reversed-phase high-performance liquid chromatography. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting peptide fragments were separated by rpHPLC and submitted to sequence analysis by automated and manual Edman methods. The shark GH consists of 183 amino acid residues with a calculated molecular weight of 21,081. Sequence comparisons revealed that the elasmobranch GH is considerably more similar to tetrapod GHs (e.g., 68% identity with sea turtle GH, 63% with chicken GH, and 58% with ovine GH) than teleostean GHs (e.g., 38% identities with salmon GH and 42% with bonito GH) except for eel GH (61% identity), and substantiates the earlier finding derived from the immunochemical and biological studies (Hayashida and Lewis, 1978) that the primitive fish are less diverged from the main line of vertebrate evolution leading to the tetrapod than are the modern bony fish.

The complete amino acid sequence of growth hormone from the sea turtle (Chelonia mydas).

The complete amino acid sequence of growth hormone (GH) from a reptilian species (the sea turtle, Chelonia mydas) has been determined for the first time. The hormone was reduced, carboxymethylated, and subsequently cleaved in turn with cyanogen bromide and Staphylococcus aureus protease. The intact protein was also cleaved with lysyl endopeptidase and o-iodosobenzoic acid. The resulting fragments were exclusively separated by reversed-phase high-performance liquid chromatography and subjected to sequence analysis by automated gas-phase sequencer employing the Edman method. The sea turtle GH consist of 190 amino acid residues with two disulfide linkages formed between residues 52-160 and 180-188, and possesses a microheterogeneity, indicated by the presence or absence of an additional alanine residue at the N-terminus. Sequence identities of sea turtle GH to other species of GH are 89% with chicken GH, 79% with rat GH, 68% with blue shark GH, 58% with eel GH, 59% with human GH, and 40% with a teleostean GH such as chum salmon. On the basis of amino acid sequence comparisons, a molecular phylogenetic tree is proposed.

Isolation and characterization of Japanese eel prolactins

A highly purified prolactin (PRL) was isolated from the pituitary of the Japanese eel (Anguilla japonica) by extraction with acid-acetone, gel filtration on Sephadex G-75, and reversed-phase HPLC on TSK-gel ODS 120T and on TSK-gel TMS 250. Eel PRL is comprised of two variants (ePRL I and II), which were separated by HPLC on an ODS column. The two PRLs were also secreted by organ-cultured pituitaries in a defined medium. After being dialyzed against distilled water and lyophilized, the medium was dissolved in 0.01 M ammonium acetate (pH 9.0), and then the insoluble fraction was subjected to HPLC on an ODS column to isolate the secreted PRLs. The ePRL I and II were equipotent in retaining plasma Na in the hypophysectomized killifish, Fundulus heteroclitus, transferred from sea-water to fresh water. The putative PRL-producing cells in the rostral pars distalis of the eel pituitary were specifically stained with the antiserum against the mixture of ePRL I and II. Both PRLs had a molecular weight of 22 kDa in SDS-PAGE, an isoelectric point of 10.1 by gel electrofocusing, and an N-terminal residue of valine. Amino acid compositions and the partial amino acid sequences of ePRL I and II show that they are highly homologous with a limited number of substitutions, and that they are more closely related to those of teleostean PRLs than to those of mammalian PRLs.

Purification and characterization of bullfrog growth hormone

A highly purified growth hormone (GH) was isolated from an unadsorbed fraction obtained by subjecting acid acetone extract of bullfrog pituitary glands to DEAE-cellulose column chromatography, a side fraction obtained during the purification of prolactin, by cation-exchange chromatography on CM-Toyopearl and high-performance liquid chromatography on ODS with a yield of 5.6 mg/g protein of the starting material. Intraperitoneal injections of GH to hypophysectomized Xenopus resulted in a considerable elevation of chondroitin sulfate synthesis in the xiphisternal cartilage as measured in vitro. The bullfrog GH had a molecular weight of 22,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The isoelectric point of bullfrog GH was estimated to be 7.8 by gel electrofocusing. The partial amino acid sequences of bullfrog GH at both terminal regions were determined.