Hajime Amasaki

Distribution of Carbonic Anhydrase Isozyme VI in the Developing Bovine Parotid Gland

The distribution of bovine carbonic anhydrase isozyme VI (CA-VI), purified from bovine saliva, was studied immunohistochemically using antiserum against bovine CA-VI in bovine parotid glands during fetal and postnatal development. A weak expression of CA-VI in undifferentiated epithelial cells and ductal cells was observed in a 4- to 5-month-old fetus with a 26-cm crown-rump length. The reaction in both acinar and ductal cells subsequently persisted during late gestation and birth. Although anti-CA-VI reactivity was still seen in both regions immediately following birth, the reactivity had almost completely disappeared from most duct segments by 1 month following birth. Changes in the localization and time-dependent expression of the isozyme in parotid glands may reflect changes in the biological function of structurally closely related isozymes.

Centroacinar and intercalated duct cells as potential precursors of pancreatic endocrine cells in rats treated with streptozotocin.

The present study examined the possibility for regeneration of pancreatic endocrine cells from centroacinar (CA) and intercalated duct (ICD) cells in rat pancreas after 5 days of continuous streptozotocin (STZ) administration. Nine rats were divided into 3 experimental groups: 1) Control group, 2) Short term recovery group; three days after STZ administration (STZ 3), and 3) Long term recovery group; ten days post-STZ administration (STZ 10). The CA and ICD cells in the STZ 3 group had swollen cytoplasm, and sometimes contained a vesicle within the core. An insulin positive signal was detected in and around the CA and ICD cells. In the STZ 3 group, cytokeratin 20 signals were co-localized with insulin signals in both CA and ICD cells. Electron microscopically, endocrine cells and small pancreatic islets were in close contact with CA and ICD cells. Systemic biophysical serum data reflected these immunohistological results. The present results suggest that CA and ICD cells are involved in the regeneration of pancreatic B cells in rats following a lesion produced by five consecutive days of STZ administration.

Distributional changes of BrdU, PCNA, E2F1 and PAL31 molecules in developing murine palatal rugae

The distribution of cells incorporating bromodeoxyuridine (BrdU) and the expression of molecules involved in the control of cell proliferation (proliferating cell nuclear antigen [PCNA], a cellular factor in F9 teratocarcinoma cells that recognizes an adenovirus E1A inducible promoter 1 [E2F1] and proliferation-related acidic nuclear protein 31 [PAL31]) during morphogenesis of the murine palatine rugae (PR) was examined histochemically. Pattern formation of the PR rudiment was initiated with cell cycle related molecules in the epithelium of the primary palate. Cells which had incorporated BrdU were detected at the outer areas of the presumptive epithelial placode (EP) and the EP at 11.5-13.5 days post coitum (dpc) and the outer areas of the PR protrusion after 14.5 dpc. The number of PCNA-positive cells at the central area of the PR protrusion decreased after 16.5 dpc. E2F-positive cells were detected at the outer areas of the PR protrusion at 15.5 and 16.5 dpc. The number of PAL31-positive cells at the presumptive EP area and the already-formed EP area was decreased at 11.5-13.5 dpc. In two dimensional histological reconstructions, PAL31 expression approximately corresponded to the distribution of BrdU-positive cells at 11.5 and 13.5 dpc. EP placode formation might be regulated by spatiotemporal cell proliferation control involving the expression of the PAL31 molecule. Following EP formation, PR development and growth control involved the expression of E2F1 and PCNA molecules.

Comparative immunohistolocalization of carbonic anhydrase isozymes I, II and III in the equine and bovine digestive tract

SummaryImmunohistochemical localizations of carbonic anhydrase isozymes (CA-I, CA-II and CA-III) in equine and bovine digestive tracts were studied. In the horse, epithelial cells in both the oesophagus and non-glandular part of the stomach lacked all three isozymes. In contrast, surface epithelial and parietal cells in the glandular region of the stomach showed reactivity for CA-II. In the small intestine, absorptive columnar cells covering the villi in the duodenum were positive for CA-II. The epithelium of the jejunum and ileum lacked all three isozymes. In the large intestine, CA-II was detected in the columnar cells in the upper part of the crypt. In cattle, epithelial cells of the oesophagus showed reactions for CA-I and CA-III but not for CA-II. Although the absorptive epithelial cells of the small intestine lacked CA-I, CA-II and CA-III, those of the upper part of large intestine crypts were heavily stained for all three isozymes.

Morphological Changes in the Rat Endocrine Pancreas within 12 h of Intravenous Streptozotocin Administration

We examined early morphological changes in pancreatic endocrine cells within 12 h of intravenous streptozotocin (STZ) administration (60 mg/kg). Thirty rats were allocated either to a control group (vehicle alone) or to one of four experimental groups tested after 3, 6, 9 and 12 h. Karyopyknosis and cytoplasmic vacuoles were first observed in β‐cell cytoplasm 3 h after STZ administration (STZ‐3 h), and the most severe damage was found in β cells at STZ‐12 h. Insulin‐positive non‐islet cells were observed near the intercalated duct (ICD) and/or centroacinar (CA) cells at STZ‐6 h and their numbers peaked at STZ‐6 h. The distribution patterns of the insulin‐positive cells and those of nestin and insulin‐like growth factor‐1 were similar and their nuclei were positive for proliferating cell nuclear antigen. Thus, ICD cells and/or CA cells reacted immediately to transform into insulin‐secreting cells to replace injured β cells (or to compensate for the lack of β cells) within 12 h of STZ administration.

Expression of Nestin and IGF-1 in Rat Pancreas after Streptozotocin Administration

The present study examines whether centroacinar (CA) and intercalated duct (ICD) cells can serve as stem cells, after administration of the diabetogenic agent streptozotocin (STZ). Thirty rats were divided into five experimental groups: (1) control, (2) 1 day after STZ (STZ‐1), (3) 3 days after STZ (STZ‐3), (4) 7 days after STZ (STZ‐7) and (5) 14 days after STZ (STZ‐14). Many small pancreatic islets were observed in the STZ‐7 group than in the other experimental groups, and many of these small islets were in close contact with ICD and CA cells. A higher number of nestin, insulin‐like growth factor‐1 (IGF‐1) and IGF‐1‐receptor positive ICD and CA cells were observed at STZ‐3 and STZ‐7 than at the others. These expression patterns coincided well with the proliferating cell nuclear antigen pattern. The results suggest that rat pancreatic endocrine cells after damage by STZ administration might be recovered from newly generated cells derived from ICD and CA cells.

Conjunctival expression of the P2Y2 receptor and the effects of 3% diquafosol ophthalmic solution in dogs.

Conjunctival epithelial and goblet cell P2Y2 nucleotide receptors regulate ion transport and secretory function. Diquafosol is a P2Y2 purinergic receptor agonist that stimulates secretion of aqueous tear components from conjunctival epithelial cells and secretion of mucin from conjunctival goblet cells. In humans suffering from keratoconjunctivitis sicca (dry eye), topical administration of diquafosol improves corneal epithelial integrity and stabilises the tear film. The aim of the present study was to investigate P2Y2 receptor expression and to determine the effect of topical administration of diquafosol on mucin and aqueous tear production in dogs. Canine conjunctival P2Y2 receptor expression was evaluated by Western blotting and immunohistochemical analysis. The effect of diquafosol on mucin secretion was evaluated by examining mucin-5 subtype AC (MUC5AC) concentration in tears. The effect of diquafosol on aqueous secretions was evaluated by performing the Schirmer tear test (STT) and phenol red thread test. Expression of the P2Y2 receptor was confirmed in canine bulbar and palpebral conjunctivae and receptors were identified at the conjunctival epithelial and goblet cell surface. Tear MUC5AC concentration significantly increased after administration of 3% diquafosol ophthalmic solution, although neither STT nor phenol red thread test values showed any significant change after diquafosol instillation. Topical ocular administration of 3% diquafosol might improve corneal epithelial disorders in dogs through stabilisation of the tear film, by virtue of an increase in MUC5AC secretion.

Distributions of Fibronectin, Laminin, Type I and IV Collagens and Carbonic Anhydrase Isozyme III during Bovine Ruminal Epithelial Development

The present study describes the immunohistochemical distributions of extracellular matrices and the carbonic anhydrase isozyme III (CA-3) in the bovine ruminal epithelium during fetal development. Fibronectin (FN), laminin and type I and IV collagens were distributed in the ruminal subepithelial mesenchymal regions with nonspecific regionality during the gestational periods. At stages after about 59 cm crown-rump length (CRL), FN of the epithelial basement membrane disappeared, and CA-3 appeared in the basal epithelial cells of the root of the ruminal papillae, suggesting that the functional differentiation of the ruminal epithelium might start at around 59 cm CRL in bovine fetuses.

Epithelial cell proliferation and apoptosis in the developing murine palatal rugae

Epithelial cell proliferation and apoptosis during morphogenesis of the murine palatal rugae (PR) were examined histochemically by using anti‐bromodeoxyuridine (BrdU) and the terminal deoxynucleotidyl transferase‐mediated UTP nick‐end‐labelling (TUNEL) technique. Formation of the PR rudiment was observed as an epithelial placode in fetuses at 12.5 days post‐coitus (dpc). During the PR formation, BrdU‐positive cells were detected mainly in the epithelium of the interplacode and interprotruding areas in fetuses administered BrdU maternally at 2 h before killing. TUNEL‐positive cells were detected only at the epithelial placode area in 12.5–14.5 dpc. At 16.5–18.5 dpc, the BrdU‐positive cells were decreased in number in the epithelial cells at the interprotruding area of the PR. Only a few TUNEL‐positive cells were observed in the protruding area of the PR at 16.5 dpc. These results suggest that cell proliferation and apoptosis in the palatal epithelium are involved spatiotemporally in the murine PR morphogenesis.

Development of the External Genitalia in Fetuses of the Southern Minke Whale, Balaenoptera acutorostrata

The developmental changes of the anogenital distance and external genitalia were studied in 81 fetuses of the southern minke whale. It was difficult to sex the fetuses with less than 55.8 mm crown-rump length (CRL) even by histological means, but it was easy in the fetuses with more than 77.0 mm CRL and less than 110.0 mm CRL. The histologically determined male fetuses had a ratio of anogenital distance to CRL or body length of more than 5%, while females had a value of less than 4%. At later stages with a CRL of more than 113.0 mm, male fetuses had an umbilicus-directed genital tubercle, while in females a tail-directed tubercle was observed. The present study suggests that the stage with sexual dimorphism might be earlier in the southern minke whale than that previously reported.