Maebashi Gunma, Japan
Maebashi Gunma, Japan

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Azuma M.,University of Toyama | Suzuki T.,Protein Purify Company | Mochida H.,Protein Purify Company | Tanaka S.,University of Shizuoka | And 3 more authors.
Cell and Tissue Research | Year: 2012

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 -β. 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 -β. 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 -β cDNA sequences. Using these antisera, we observed adenohypophyseal cells showing SL-α- and -β-like immunoreactivities in the goldfish pituitary, especially the pars intermedia (PI). Several cells in the PI showed the colocalization of SL-α- and -β-like immunoreactivities. Then, using single-cell polymerase chain reaction with laser microdissection, we examined SL-α and -β gene expression in adenohypophyseal cells showing SL-α- or -β-like immunoreactivity. Among cultured pituitary cells, we observed three types of cell: those that possess transcripts of SL-α, -β, or both. These results suggest a polymorphism of SL-producing cells in the goldfish pituitary. © 2012 Springer-Verlag.


Ogushi Y.,University of Shizuoka | Tsuzuki A.,University of Shizuoka | Sato M.,University of Shizuoka | Mochida H.,Protein Purify Company | And 4 more authors.
American Journal of Physiology - Regulatory Integrative and Comparative Physiology | Year: 2010

Regions of specialization for water absorption across the skin of Bufonid and Ranid anurans were identified by immunohistochemistry and Western blot analysis, using antibodies raised against arginine vasotocin (AVT)-stimulated aquaporins (AQPs) that are specific to absorbing regions of Hyla japonica. In Bufo marinus, labeling for Hyla urinary bladder-type AQP (AQP-h2), which is also localized in the urinary bladder, occurred in the ventral surface of the hindlimb, pelvic, and pectoral regions. AQP-h2 was not detected in any skin regions of Rana catesbeiana, Rana japonica, or Rana nigromaculata. Hyla ventral skin-type AQP (AQP-h3), which is found in the ventral skin but not the bladder of H. japonica, was localized in the hindlimb, pelvic, and pectoral skins of Bufo marinus, in addition to AQP-h2. AQP-h3 was also localized in ventral skin of the hindlimb of all three Rana species and also in the pelvic region of R. catesbiana. Messenger RNA for AQP-x3, a homolog of AQP-h3, could be identified by RT-PCR from the hindlimb, pectoral, and pelvic regions of the ventral skin of Xenopus laevis, although AVT had no effect on water permeability. In contrast, 10-8 M AVT-stimulated water permeability and translocation of AQP-h2 and AQP-h3 into the apical membrane of epithelial cells in regions of the skin of species where they had been localized by immunohistochemistry and Western blot analysis. Finally, water permeability of the hindlimb skin of B. marinus and all the Rana species was stimulated by hydrins 1 and 2 to a similar level as seen for AVT. The present data demonstrate species differences in the occurrence, distribution, and regulation of AQPs in regions of skin specialized for rapid water absorption that can be associated with habitat and also phylogeny. Copyright © 2010 the American Physiological Society.


Azuma M.,University of Toyama | Suzuki T.,Protein Purify Company | Mochida H.,Protein Purify Company | Tanaka S.,University of Shizuoka | Matsuda K.,University of Toyama
Peptides | Year: 2013

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that stimulates the release of adenohypophyseal hormone from the pituitary in fish. In the goldfish, PACAP induces the release of somatolactin (SL), in particular, from cultured pituitary cells. SL belongs to the growth hormone and prolactin family, and comprises two molecular variants termed SL-α and SL-β in goldfish. However, there is no information about the involvement of PACAP in the regulation of SL-α and SL-β release and the expression of their mRNAs. Therefore, we examined the effect of PACAP on SL-α and SL-β release from cultured goldfish pituitary cells. Treatment with PACAP (10-10-10-7 M) increased the release of both SL-α and SL-β. The stimulatory action of PACAP (10 -9 M) on SL-α and SL-β release was blocked by treatment with a PACAP-selective receptor (PAC1R) antagonist, PACAP (6-38) (10-6 M). We also examined whether PACAP affects the expression of SL-α and SL-β mRNAs in cultured pituitary cells. Treatment with PACAP (10-9 and 10-8 M) for 6 h decreased the expression level of SL-α mRNA but increased that of SL-β mRNA. The action of PACAP (10-8 M) on SL-β mRNA expression was blocked by treatment with PACAP(6-38) (10-6 M), whereas PACAP(6-38) elicited no change in the expression of SL-α mRNA. These results indicate that in cultured goldfish pituitary cells, PACAP stimulates the release of SL-α and SL-β, and expression of SL-β mRNA, via the PAC1R-signaling pathway. However, the mechanism whereby PACAP inhibits the expression of SL-α mRNA does not seem to be mediated by PAC1R signaling. © 2013 Elsevier Inc.


Tokita Y.,Gunma University | Nakajima K.,Gunma University | Mochida H.,ProteinPurify Ltd | Iha M.,South Product Co. | Nagamine T.,Gunma University
Bioscience, Biotechnology and Biochemistry | Year: 2010

Fucoidan exhibits various biological properties. We raised a novel antibody against fucoidan extracted from Cladosiphon okamuranus and developed a sandwich ELISA method to measure fucoidan. The fucoidan antibody was specific and did not cross-react with other polysulfated polysaccharides. Fucoidan recovery from serum and urine by ELISA was 86-113%. Intra- and inter-assay CVs were 1.5-13.4%. Assay linearity was maintained after 3-fold dilution of each sample with phosphate-buffer saline (PBS). In the serum and urine of healthy volunteers (n = 10), fucoidan was not detected before administration, and the levels markedly increased 6 and 9h after oral administration. The molecular weight of the serum fucoidan determined by HPLC gel filtration remained unchanged, whereas that of urine fucoidan was significantly reduced. This is the first ELISA method of measuring serum and urine fucoidan levels after oral administration. The method is simple, reliable, and practical for the analysis of samples, especially urine samples.

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