Ultimobranchial glands in the teleost (Plecoglossus altivelis ryukyuensis): special references to changes of gland volume with maturation.

In lower vertebrates, the bone-mineralizing hormone "calcitonin" is secreted from ultimobranchial glands, which assume various shapes, e.g., follicles, cellular masses, or cell strands. Histological observations support the view that in some teleosts, the glands increase in size when females maturate. We determined the exact volume of the gland in juveniles and adult males and females of a teleost, Ryukyuayu (Plecoglossus altivelis ryukyuensis). Furthermore, we examined plasma Ca, Na and K levels. In this species, the gland was fundamentally composed of a single follicle. The gland volume was converted to numerical data under a certain condition. It thus became clear that the value of the follicle wall was significantly increased only in maturing and mature females with high plasma Ca levels and that the value of the lumen did not show any statistically significant changes during growth and maturation.

[Physiology of vertebrates under micro-gravity with special reference to the Ca metabolism].

On April 12, 1961, Major Yurii A. Gagarin of the former-U.S.S.R. Air Force circled the Earth in a spacecraft named "Vostok", a word which means "east". He spent 1 hour and 48 minutes in space. Since then, the U.S.S.R. and the U.S.A. have sent many astronauts into space. In one case, the stay in space exceeded a year in length, reaching 438 days. Through these experiences, it became clear that micro-gravity caused various problems in human physiology. One of the most serious problems was the loss of Ca from bones, as a result of the negative expenditure of Ca. Under 1G on the ground, bone absorption and bone formation proceed in accordance. Under micro-gravity, however, this balance is broken. Although this phenomenon has been widely analyzed from the viewpoint of molecular biology as well, studies to clarify the mechanism that causes the disorder of Ca metabolism in bones have just started. At present, no perfect treatment to prevent the loss of Ca from bones is available.

Molecular cloning of proopiomelanocortin cDNA from an elasmobranch, the stingray, Dasyatis akajei.

Recently, we have characterized a new MSH (named delta-MSH) which joins the group of MSHs (alpha, beta, gamma) in dogfish proopiomelanocortin (POMC). The present study has confirmed the presence of delta-MSH in POMC of another member of the elasmobranchian order, the stingray, Dasyatis akajei, by cDNA cloning from pituitary mRNAs. Overlapping partial cDNA clones corresponding to stingray POMC were amplified by PCR from single-strand cDNA prepared from pituitary poly (A)(+) RNA. Excluding the poly A tail, stingray POMC cDNA consists of 1077 base pairs (bp). It contains a 912-bp open reading frame encoding a signal peptide of 24 amino acids (aa) and a POMC of 280 aa. gamma-MSH, alpha-MSH, ACTH, delta-MSH, beta-MSH, and beta-endorphin are located at POMC (50-61), (115-127), (115-153), (182-193), (226-242), and (245-280), respectively. The stingray POMC is smaller than that of the dogfish POMC (294 aa) mainly due to the absence of a sequence of 11 consecutive aa between delta-MSH and beta-MSH. delta-MSH has been found only in the elasmobranchs and, therefore, delta-MSH might have evolved after the divergence of chondrichthians from the ancestral vertebrate lineage and before divergence of sharks and rays.

Anuran Calcitonins are Diverse in Lower Vertebrates.

We investigated the nucleotide sequences of cDNA fragments coding calcitonin from ultimobranchial glands in 2 species of urodelans (1 salamander and 1 newt) and 4 species of anurans (1 toad and 3 frogs) by the reverse transcription polymerase chain reaction (RT-PCR) method and rapid amplification of cDNA ends (RACE)-PCR method. The salamander and newt calcitonins were each 97% and 94% similar to the lungfish and caiman calcitonins that we have already reported, in the amino acid sequences. However, anuran calcitonins were not only dissimilar (63-81%) to the lungfish and caiman calcitonins but also diversified (59-91%) even among anurans. The sequence identity of toad calcitonin was always low (59-66%) among anurans.

Plasma calcium and calcitonin levels in eels fed a high calcium solution or transferred to seawater.

To examine the physiological role of calcitonin (CT) in calcium homeostasis of teleosts, we compared calcium and CT levels in freshwater eels fed a high calcium-consomme solution (Ca2+: 1.25 M; 1 ml/100 g body wt) into the stomach (Experiment I), and in freshwater eels transferred from freshwater to seawater (Experiment II). In experiment I, plasma calcium and CT levels in the high calcium-treated eels rapidly increased (calcium: 2.63 mM at 0 h to 8. 50 mM at 3 h; CT: below detection level at 0 h to 1118.2 pg/ml at 3 h). Plasma calcium and CT levels in the control eels remained below detection level during the 3 h of the experiment. In experiment II, the plasma CT levels did not increase, although the plasma calcium levels increased from 3.23 mM at 0 h to 4.10 mM at 8 h. Therefore, in eels, we demonstrate a correlation between plasma CT and plasma calcium raised by dietary calcium in the consomme form, but it does not participate in the initial processes of seawater adaptation.

A newly characterized melanotropin in proopiomelanocortin in pituitaries of an elasmobranch, Squalus acanthias.

Proopiomelanocortin (POMC) is a precursor for corticotropin (ACTH), three or fewer molecular types of melanotropin (MSH), and beta-endorphin. This protein is thought to have evolved by duplication of MSH genomic segments. Here we report that the POMC in the dogfish, an elasmobranch, contains a fourth type of MSH in addition to classical alpha-, beta-, and gamma-MSH. POMC cDNA was amplified by PCR from double-strand cDNA prepared from dogfish pituitary and ligated into lambdaZAP II. The POMC cDNA is composed of 1315 bp without a poly(A) tail. Northern blot analysis detected a 1.4-kb signal of dogfish POMC mRNA. An open reading frame of the POMC cDNA encodes 320 amino acids, including a signal peptide of 26 amino acids. The dogfish POMC includes gamma-MSH, ACTH, alpha-MSH, beta-MSH, and beta-endorphin at positions 50-61, 115-153, 115-127, 239-256, and 259-294, respectively. In addition to these classical peptides, a newly discovered MSH, which we have termed delta-MSH, is present in dogfish POMC at position (184-195). The four dogfish MSHs can be separated into two groups based on their sequence identities: one pair consists of alpha-MSH and gamma-MSH, and the other consists of beta-MSH and delta-MSH, suggesting that gamma-MSH and delta-MSH may have been duplicated evolutionarily from alpha-MSH and beta-MSH, respectively. gamma-MSH might first have appeared in early gnathostomes because it is absent in the most primitive vertebrate group, the agnathans. delta-MSH, which at this time is found only in chondrichthians, might have appeared after the divergence of chondrichthians from a lineage leading to osteichthyans and tetrapods.

Calcitonin cells in the intestine of goldfish and a comparison of the number of cells among saline-fed, soup-fed, or high Ca soup-fed fishes.

Calcitonin-immunoreactive cells were found in the intestine of goldfish. These cells were distributed mainly in the anterior part of the intestine, dispersed in the intestinal epithelium. The nucleus was located in the basal portion of the serosal side, and the cytoplasm was elongated to the luminal side. From the anterior part of the intestine, cDNA fragments with the same nucleotide sequence as that of the goldfish calcitonin gene were amplified by RT-PCR method. After administration of one of three kinds of solutions (saline, consommé soup, or high Ca consommé soup) into the digestive tract of the goldfish, the number of those cells was the largest in the consommé group at 6 h after ingestion, although blood Ca levels were the highest in the high Ca consommé group. The function of calcitonin cells in the intestine may be to restrain the acute absorption of nutrients and not to control blood Ca levels.

Fish calcitonin genes: primitive bony fish genes have been conserved in some lower vertebrates.

Using the polymerase chain reaction method, we amplified calcitonin genes from the cDNA of the ultimobranchial glands or from the genomic DNA of the blood cells and liver of various fishes. The fishes examined were primitive bony fishes (lungfish, polypterus, sturgeon, and gar), 16 species of teleosts, and cartilaginous fishes (stingray). Sequenced calcitonin genes were compared among fishes and with those of reptiles and chickens. The similarity of the calcitonin genes was the highest between the reptile and chicken groups and the primitive bony fishes (sequence similarity of the nucleotides 81-90%). The values between teleosts and the primitive bony fishes were 70-81%, with the exception of eels. Eel calcitonin genes were very similar to those of primitive bony fishes (83-88%). Stingray calcitonin genes were relatively more similar to those of the primitive bony fishes (74-78%) than to teleosts (63-73%). In goldfish and sardine, two types of calcitonin genes were found. We concluded that the genes of primitive bony fish are placed at a fundamental position in this hormone, at least among these vertebrates.

Salmon calcitonin induced hypocalcemia and hyperphosphatemia in an elasmobranch, Dasyatis akajei.

Salmon calcitonin (5 micrograms/kg body wt) was administered in an elasmobranch, Dasyatis akajei, to investigate the effects upon plasma calcium and inorganic phosphate. The hormone produced hypocalcemia and hyperphosphatemia in the stingray. It is concluded that calcitonin may have a role in calcium homeostasis by a mechanism different from that on bones.

Morphological and functional aspects of reptilian ultimobranchial gland.

The intention of this review is to compare studies on the morphology and histology (light and electron microscopic) of ultimobranchial glands of various groups of reptiles. Moreover, experiments (including our investigations) on suppression or stimulation of the ultimobranchial gland are included. Adult reptiles possess one (on the left side) or two ultimobranchial glands (UBG). The UBG lie just anterior to the heart. Light as well as electron microscopically, the gland has been shown to contain follicles and cell cords (cell aggregates). The follicular epithelium is lined by simple cuboidal or pseudostratified columnar cells. Ciliated and goblet cells may be present in the follicular epithelia in some groups. The lumen may contain a colloid-like substance with desquamated cells or debris. The UBG of reptiles seem to be an active secretory organ with influence on calcium regulation. Other functions of calcitonin have also been suggested in reptiles for example in neurotransmission, in volume regulation, phosphate balance and promotion of bone calcification (at least in juveniles).

Unpaired Ultimobranchial Glands of the African Lungfish, Protopterus dolloi.

The ultimobranchial glands of juvenile African lungfish (Protopterus dolloi) (14 individuals; total body length 25-205 mm) were immunohistochemically examined. In individuals larger than 36 mm, one ultimobranchial gland was close to the left afferent branchial arteries. The topography of the ultimobranchial gland was similar to that of salamanders and sharks, but not to teleosts. With body growth, the ultimobranchial gland was vascularized and the parenchymal cells were gradually immunostained with anti-calcitonin antibody. In all individuals examined, the ultimobranchial gland existed only on the left side of the pharynx. These observations are discussed from a phylogenetic viewpoint.

Primary structure and bioactivity of bullfrog calcitonin.

Calcitonin was isolated from the bullfrog, Rana catesbeiana, and the first amino acid sequence of an amphibian calcitonin was determined to be Cys-Ser-Gly-Leu-Ser-Thr-Cys-Ala-Leu-Met-Lys-Leu-Ser-Gln-Asp-Leu-His- Arg-Phe-Asn-Ser-Tyr-Pro-Arg-Thr-Asn-Val-Gly-Ala-Gly-Thr-Pro-NH2. Some portions of this sequence are specific to bullfrog calcitonin, and other portions are similar both to teleost calcitonins and to mammalian calcitonins. Administration of 5 pmol of bullfrog calcitonin to rats revealed a hypocalcemic potency similar to that of salmon calcitonin, at least for the first 3 hr.

Relationship between interleukin-8 levels and myeloperoxidase activity in human gastric mucosa.

Although interleukin (IL)-8 is well known as a chemotactic agent for neutrophil migration in vitro, the relationship between IL-8 activity and the degree of neutrophil infiltration in gastric mucosa is still unclear. In the present study, we investigated IL-8 and myeloperoxidase activity, a marker of neutrophil infiltration, in gastric antral mucosa using biopsy samples in 23 patients with no gastric lesions. The results indicate that there is a good correlation between IL-8 and myeloperoxidase activity (y = 0.173x + 13.9; r = 0.49, P < 0.01). Furthermore, IL-8 and myeloperoxidase activity are significantly higher in Helicobacter pylori-positive patients than in H. pylori-negative patients. In conclusion, an increase of IL-8 activity in the gastric mucosa causes increased neutrophil infiltration in human gastric mucosa and H. pylori infection accelerates these reactions in the mucosa.

Nucleotide sequences of reptile calcitonins: their high homology to chicken calcitonin.

The calcitonin genes of four species of reptiles (Reeve's turtle, rat snake, grass lizard, and spectacled caiman) were amplified from the genomic DNA, as well as from the mRNA of the ultimobranchial glands of the former three species, by the polymerase chain reaction (PCR) method, and were sequenced. Among several primer sets, only one primer set synthesized from the chicken calcitonin gene was compatible with those of the reptiles. The nucleotide sequences of the reptile calcitonin genes were highly homologous with that of chicken calcitonin (100% for turtle, 99% for caiman, 96% for lizard and 93% for snake). The products amplified from mRNA by the RT-PCR method matched completely those from genomic DNA in the turtle, snake and lizard.

Vitamin D3-induced calcemic and phosphatemic responses in the freshwater mud eel Amphipnous cuchia maintained in different calcium environments.

Vitamin D3 (100 ng 100 g body weight-1 day-1) was administered intraperitoneally (i.p.) to the freshwater mud eel Amphipnous cuchia kept in artificial freshwater, calcium-free freshwater, low-calcium freshwater (0.2 mmol/l CaCl2) or calcium-rich freshwater (13.4 mmol/l CaCl2) for 15 days. Analyses of serum calcium and phosphate levels were performed on days 1, 3, 5, 10 and 15 after the beginning of the experiment (six eels from each group at each interval). Administration of vitamin D3 elevated the serum calcium [maximum elevation occurred at day 10 in artificial freshwater (vehicle: 10.55 +/- 0.298, vitamin D: 13.90 +/- 0.324), low-calcium freshwater (vehicle: 11.17 +/- 0.220, vitamin D: 12.98 +/- 0.297) and calcium-rich freshwater (vehicle: 11.24 +/- 0.373, vitamin D: 14.24 +/- 0.208) whereas it occurred at day 5 (vehicle: 8.42 +/- 0.253, vitamin D: 11.07 +/- 0.328) in calcium-free freshwater] and phosphate levels [maximum elevation at day 15 in artificial freshwater (vehicle: 4.39 +/- 0.105, vitamin D: 5.37 +/- 0.121), calcium-free freshwater (vehicle: 4.25 +/- 0.193, vitamin D: 5.12 +/- 0.181), low-calcium freshwater (vehicle: 3.93 +/- 0.199, vitamin D: 5.28 +/- 0.164) and calcium-rich freshwater (vehicle: 3.77 +/- 0.125, vitamin D: 5.46 +/- 0.151)] of the fish maintained in the above mentioned environmental media, but the responses were more pronounced in the fish kept in calcium-rich media.

Corpuscles of Stannius-extract-induced rapid but transient hypocalcemia and hyperphosphatemia in stingray, Dasyatis akajei.

Aqueous extracts of goldfish Corpuscles of Stannius were tested in an elasmobranch, Dasyatis akajei, to investigate the effects upon plasma calcium and inorganic phosphate. The extract produced hypocalcemia and hyperphosphatemia in the stingray. Receptors with affinity for stanniocalcin are therefore widespread among the vertebrates.

K-ras point mutations in cancerous and noncancerous biliary epithelium in patients with pancreaticobiliary maljunction.

BACKGROUND: Pancreaticobiliary maljunction (PBM), an anomalous union of the pancreatic duct with the common bile duct, has frequently been shown to be associated with biliary carcinoma. However, the mechanism of carcinogenesis is unknown. METHODS: Mutations of the K-ras oncogene were examined in cancerous and noncancerous biliary tract epithelium of 20 patients with PBM by an extraction of DNA from surgically resected histologic specimens. DNA was analyzed by a polymerase chain reaction single strand conformation polymorphism (PCR-SSCP) method and direct sequencing. RESULTS: An abnormally mobilized DNA band was detected not only in cancerous epithelium but also in hyperplastic, metaplastic, and inflammatory epithelium of the gallbladder and/or common bile duct in patients with PBM. Among the biliary epithelium of patients with PBM, point mutation of K-ras oncogenes were detected in 4 of 5 (80%) cancerous epithelium, 7 of 12 (58%) hyperplastic and metaplastic epithelium, and 8 of 18 (44%) inflammatory epithelium, whereas no point mutation of the K-ras oncogene was detected in the gallbladder epithelium in 3 control patients without PBM. Direct sequence analysis of the K-ras oncogene revealed the mutation at codon 12 substituting the wild-type glycine (GGT) for aspartic acid (GAT) in all cancerous lesions of patients with PBM. Simultaneous two-point mutations from the wild-type glycine (GGC) to arginine (CGC) at codon 13 associated with the mutation at codon 12 were also found in one case of gallbladder carcinoma and one case of bile duct carcinoma. CONCLUSIONS: K-ras gene mutation is involved in the carcinogenesis of biliary tract epithelium in patients with PBM, and appears to be a high risk factor for carcinogenesis of the biliary tract.

The non-effect of parathyroidectomy in the aquatic limbless newt (Apoda, Amphibia).

In the aquatic limbless newt, Typhlonectes compressicauda (Apoda, Amphibia), serum Ca levels of parathyroidectomized newts were no lower than for the control newts at 1 week after the operation. In this species, the parathyroid glands may not be functional in raising the serum Ca levels.

Estrogen receptors in the stingray (Dasyatis akajei) ultimobranchial gland.

On the basis of our previous observation that estrogen stimulates calcitonin secretion by the ultimobranchial gland in the stingray, Dasyatis akajei, experiments were conducted to examine the presence of estrogen receptor (ER) and its mRNA in the gland, employing exchange assay and Northern blot analysis, respectively. The optimal incubation conditions of the exchange assay for cytosolic ER were found to be 25 degrees for 2 hr. Scatchard analysis of cytosolic ER revealed two components with dissociation constants (Kd) of 0.090 +/- 0.015 and 17.780 +/- 1.910 nM and with a maximum number of binding sites (Bmax) of 62.2 +/- 7.9 and 2290 +/- 180 fmol/mg protein, respectively. Furthermore, Sephadex G-100 filtration of the cytosol and immunoblot analysis using antiserum against a recombinant rat ER confirmed the presence of two specific binding components for estrogen (41 and 57 kDa). Total RNA extracted from the ultimobranchial gland was subjected to Northern blot analysis using a rat ER cDNA as a probe. Two positive signals were detected at 2.5 and 2.0 kb. Thus, the presence of ER was confirmed for the first time in nonmammalian calcitonin-secreting cells.