Biomedical subjects

Shigeyasu Tanaka

Publications and source records attributed to Shigeyasu Tanaka.

25 records · Page 2Linked to original sources

PubMed2003

Molecular cloning of otoconin-22 complementary deoxyribonucleic acid in the bullfrog endolymphatic sac: effect of calcitonin on otoconin-22 messenger ribonucleic acid levels.

Anuran amphibians have a special organ called the endolymphatic sac (ELS), containing many calcium carbonate crystals, which is believed to have a calcium storage function. The major protein of aragonitic otoconia, otoconin-22, which is considered to be involved in the formation of calcium carbonate crystals, has been purified from the saccule of the Xenopus inner ear. In this study, we cloned a cDNA encoding otoconin-22 from the cDNA library constructed for the paravertebral lime sac (PVLS) of the bullfrog, Rana catesbeiana, and sequenced it. The bullfrog otoconin-22 encoded a protein consisting of 147 amino acids, including a signal peptide of 20 amino acids. The protein had cysteine residues identical in a number and position to those conserved among the secretory phospholipase A(2) family. The mRNA of bullfrog otoconin-22 was expressed in the ELS, including the PVLS and inner ear. This study also revealed the presence of calcitonin receptor-like protein in the ELS, with the putative seven-transmembrane domains of the G protein-coupled receptors. The ultimobranchialectomy induced a prominent decrease in the otoconin-22 mRNA levels of the bullfrog PVLS. Supplementation of the ultimobranchialectomized bullfrogs with synthetic salmon calcitonin elicited a significant increase in the mRNA levels of the sac. These findings suggest that calcitonin secreted from the ultimobranchial gland, regulates expression of bullfrog otoconin-22 mRNA via calcitonin receptor-like protein on the ELS, thereby stimulating the formation of calcium carbonate crystals in the lumen of the ELS.

Amino Acid Sequence↗

PubMed2003

Regulation of water absorption in the frog skins by two vasotocin-dependent water-channel aquaporins, AQP-h2 and AQP-h3.

A new frog aquaporin (AQP) cDNA was cloned from a cDNA library constructed from the ventral skin of the tree frog Hyla japonica. This AQP (Hyla AQP-h2) consisted of 268 amino acid residues with a high homology to mammalian AQP2. The predicted amino acid sequence contained the two conserved Asn-Pro-Ala motifs found in all the major intrinsic protein family members and the putative six transmembrane domains. The sequence also contained a mercurial compound: cysteine, one potential N-glycosylation site at Asn-124, and a putative phosphorylation site recognized by protein kinase A at Ser-262. In a swelling assay using Xenopus oocytes, AQP-h2 facilitated water permeability, especially in response to cAMP. Expression of AQP-h2 mRNA was restricted to several tissues including the ventral skin, kidney, and urinary bladder; but with immunofluorescence staining using an antipeptide antibody (ST-140) against the AQP-h2 protein, immunopositive cells were found only in the ventral skin and urinary bladder. In the ventral pelvic skin, the label for AQP-h2 was localized in the entire plasma membrane of the granular cells beneath the outmost layer of the skin and in the basolateral membrane of the granular cells in this layer. In response to vasotocin, however, the label for AQP-h2 became more intense in the apical membrane in the granular cells of the outermost layer, similar to the case for the earlier studied AQP-h3, which was specifically expressed in the ventral skin. Taken together, these findings suggest that not only AQP-h3, but also AQP-h2 acts as a regulator of the water balance in this frog.

Amino Acid Sequence↗

PubMed2003

Molecular cloning and expression of prohormone convertases PC1 and PC2 in the pituitary gland of the bullfrog, Rana catesbeiana.

We cloned cDNAs encoding PC1 and PC2 from a cDNA library constructed for the anterior pituitary gland of the bullfrog (Rana catesbeiana) and sequenced them. The bullfrog PC1 cDNA consisted of 2972 base pairs (bp) with an open reading frame of 2208 bp and encoded a protein of 736 amino acids, including a putative signal peptide of 26 amino acids. The protein showed a high homology to R. ridibunda PC1 (95.1%) and mammalian PC1 (72.6%). The bullfrog PC2 cDNA consisted of 2242 bp with an open reading frame of 1914 bp and encoded a protein of 638 amino acids, including a putative signal peptide of 23 amino acids. This protein showed a high homology to R. ridibunda PC2 (95.5%) and mammalian PC2 (84.8%). The catalytic triad of serine proteinases of the subtilisin family was found at Asp-168, His-209, and Ser-383 in the PC1 protein and at Asp-167, His-208, and Ser-384 in the PC2 protein. In situ hybridization staining revealed that PC2 mRNA was detected in corticotrope cells of the tadpoles, but not in those of the adults. In the adult, only PC1 mRNA was detected in the pars distalis but both PC1 and PC2 mRNAs were detected in the pars intermedia. The data also showed that PC1 mRNA was expressed in gonadotrope cells.

Amino Acid Sequence↗

PubMed2003

Comparative aspects of intracellular proteolytic processing of peptide hormone precursors: studies of proopiomelanocortin processing.

In this review, the mechanisms underlying the intracellular processing of peptide hormone precursors, with a focus on proopiomelanocortin (POMC), were discussed on the basis of recent information. POMC as well as other prohormones is processed to active peptides through proteolytic cleavage by prohormone convertases PC1 and/or PC2. However, the cleavage-specificity of PC1 and PC2 in mammals is somewhat different from that in amphibians. From the comparative endocrinological point of view, expression and tissue distribution of PC1 and PC2 were discussed here. In mammals, proteolytic processing of POMC occurs coordinately with the maturation of secretory granules. Studies using immunoelectron microscopy with DAMP (3-[2,4-dinitroanilino]-3'-amino-N-methyldipropylamine) as a pH probe revealed that the acidic pH in the secretory granules, generated by vacular type-H+-ATPase, provides a favorable environment for activating PC1 in AtT-20 cells, a mouse corticotrope tumor cell line. Recent data indicate that the 7B2 protein serves as a chaperone in the regulation of PC2 activation and to control the timing for activating the convertase. Together, secretory granules in endocrine and neuroendocrine cells provide proper sites for biosynthesizing hormones in addition to serving as storage sites and vehicles for the transport of peptide hormones.

Amino Acid Sequence↗

PubMed2002

Immunohistochemical characterization of the intracellular pool of water channel aquaporin-2 in the rat kidney.

Aquaporin-2 (AQP2) is a member of water channel proteins expressed in the kidney collecting duct cells, where it is stored in the intracellular compartment. Upon stimulation of antidiuretic hormone (ADH), AQP2 is recruited to the plasma membrane, and plays a critical role in urine concentration. We immunohistochemically characterized the intracellular compartment harboring AQP2 in the rat kidney using antibodies to the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, lysosome, and endosome. Aquaporin-2 did not colocalize with calnexin, TGN38, Golgi 58K, cathepsin D or Igp-110. Small portions of AQP2-bearing vesicles were positive for early endosome antigen 1. These localization patterns were basically the same in water-loaded and ADH-treated animals. These results indicate that AQP2-bearing vesicles constitute a unique intracellular compartment distinct from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network and lysosome. Partial colocalization of AQP2 with early endosomes suggests that the endosomal system might be involved in the trafficking of AQP2.

Animals↗

PubMed2002

Sequence analysis and expressional regulation of messenger RNAs encoding beta subunits of follicle-stimulating hormone and luteinizing hormone in the red-bellied newt, Cynops pyrrhogaster.

Two distinct cDNAs encoding beta subunits of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were cloned from the cDNA library constructed for the pituitary of the red-bellied newt, Cynops pyrrhogaster, and sequenced. The newt FSHbeta and LHbeta cDNAs encode polypeptides of 129 and 131 amino acids, including signal peptides of 20 and 19 amino acids, respectively. The number and position of cysteine and N-glycosylation in each of the beta subunits of FSH and LH, which are considered essential for assembly of the alpha subunit, are well conserved between the newt and other tetrapods. The high homology (41.6%) between the beta subunits of newt FSH and LH imply less specificity of FSH and LH in gonadal function. One cDNA encoding the common polypeptide chain alpha subunit of FSH and LH was also isolated from the newt pituitary gland. The mRNAs of FSHbeta, LHbeta, and the alpha subunit were expressed only in the pituitary gland among various newt tissues. Double-staining with in situ hybridization and immunohistochemistry revealed coexpression of FSHbeta and LHbeta in the same newt pituitary cells. Ovariectomy induced a significant increase in FSHbeta mRNA levels, but there was no significant change in LHbeta or alpha subunit mRNA levels compared with those in control animals. Taken together, these data suggest that two kinds of gonadotropins, namely FSH and LH, are expressed in the same gonadotropin-producing cells in the pars distalis of the newt as well as in other tetrapods and that the expression of FSHbeta is negatively regulated by the ovaries.

Amino Acid Sequence↗

PubMed2002

Immunocytochemical localization of prohormone convertases PC1 and PC2 in the mouse thyroid gland and respiratory tract.

We examined immunocytochemical localization of the prohormone convertases, PC1 and PC2, in the thyroid gland and respiratory tract of the adult mouse using the indirect enzyme- and immunogold-labeled antibody methods for light and electron microscopy, respectively. In the thyroid gland, PC1- and/or PC2-immunoreactive cells were cuboidal, scattered in the follicular epithelium and in the interfollicular spaces. When serial sections were immunostained with anti-calcitonin, anti-PC1, anti-calcitonin-gene-related-peptide (CGRP), and anti-PC2 sera, respectively, localization of both PC1 and PC2 was restricted to the calcitonin/CGRP-producing parafollicular cells. In the respiratory tract, only PC1 immunoreactivity was observed in the basal granulated neuroendocrine cells, which were scattered in the tracheal epithelium. Consecutive sections immunostained with anti-PC1 and anti-CGRP sera showed that a subpopulation of these PC1-immunoreactive cells contained CGRP. Double immunogold electron microscopy of the thyroid parafollicular cells revealed that calcitonin- and/or CGRP-immunopositive secretory granules were also labeled with both PC1 and PC2. These findings suggest that procalcitonin is proteolytically cleaved by PC2 alone or by PC2 together with PC1, and that the proCGRP is cleaved by PC1.

Animals↗