Marked depletion of the water-channel protein, AQP5, in the canine nictitating membrane glands might contribute to the development of KCS.

The objectives of this study were to investigate the normal histological localization of aquaporin (AQP) 5 protein in the lacrimal and nictitating membrane glands and to compare this localization in healthy and keratoconjunctivitis sicca (KCS) dogs. Lacrimal and nictitating membrane glands of 5 healthy Beagles and nictitating membrane glands of 5 KCS dogs (3 Beagles and 2 mongrel dogs: 0-13 years) were used for the present study. The owners of the KCS dogs did not consent to perform biopsies of the lacrimal glands. The localization and distribution of AQP5 protein were investigated by an immunohistochemical technique. In immunohistochemical staining, AQP5 was localized in the apical site of acinar epithelial and ductal epithelial cells from both the lacrimal and nictitating membrane glands in healthy dogs. However, AQP5 was not detected in the 5 KCS dogs. These results for immunohistochemical AQP5 localization might correlate with the deficiency in tear secretion found in KCS dogs.

Immunohistolocalization and gene expression of the carbonic anhydrase isoenzymes (CA-II and CA-VI) in glands associated with the canine lacrimal apparatus.

Cytosolic and secretory carbonic anhydrase isoenzymes (CA-II and CA-VI, respectively) were detected by immunohistolocalization using specific canine CA-II and CA-VI antisera. CA-II and CA-VI were identified in glands associated with the canine lacrimal apparatus, such as lacrimal gland, superficial gland of the third eyelid (third eyelid gland) and tarsal gland. CA-II and CA-VI mRNA signals were also detected by reverse-transcriptase polymerase chain reaction in the same tissues. Some serous acinar cells and duct segments in the lacrimal gland and serous acinar cells in the third eyelid gland were immunopositive for anti-CA-II and CA-VI antisera. In particular, some immunopositive acini to CA-II and CA-VI on the edge of the third eyelid gland are histologically similar to sebaceous gland cells. Sebaceous gland cells in the tarsal and ciliary glands also showed immunopositivity to both CA antisera. CA-II and CA-VI gene transcripts were detected in the same regions. These results suggest that secreted CA-VI may form together with cytosolic CA-II, a high-activity isozyme mostly considered as a bicarbonate producer, in a mutually complementary system for the maintenance of bicarbonate levels to regulate pH in tear fluid and protect the corneal epithelia against injuries. In sebaceous gland cells in the lacrimal apparatus, CA-VI may be related to lipogenesis in an unknown function.

Histological structure and distribution of carbonic anhydrase isozymes (CA-I, II, III and VI) in major salivary glands in koalas.

While the mandibular glands usually consist of only mucous acinar cells or a combination of mucous and serous cells in other species of mammals, those of koalas were serous glands. Rabbit mono-specific polyclonal anti-canine CA-I, II, III or VI antiserum showed cross-reactivity against corresponding koala carbonic anhydrase (CA) isozymes. Although immunohistochemical reactions to CA-I, II and VI in ductal cells were moderate to strong in the tested salivary glands, no reaction or only slight reactions were observed against CA-III. In the sublingual glands, moderate immunohistochemical reactions to CA-I, II and VI were also evident in serous acinar cells and serous demilunes. However, no reactions to the tested isozymes were observed in mucous acinar cells in these glands. With the exception of the histological structure of the mandibular glands, histological features and the distributional profile of CA isozymes of the salivary glands in koalas are relatively close to results obtained from horses.

Gene expression of secretory carbonic anhydrase isozymes in striated ducts of canine salivary glands using laser microdissection system.

To clarify whether striated duct cells in canine salivary glands synthesize secretory carbonic anhydrase (CA-VI), as is the case with serous acinar cells, the present study utilized laser microdissection to harvest striated duct cells from canine parotid and submandibular glands, and total RNA extracted from these cells was then amplified by reverse transcription-polymerase chain reaction to assess CA-VI gene expression. The results confirmed the local expression of CA-VI mRNA in striated duct cells. This clarified that, in canine salivary glands, CA-VI is synthesized in not only serous acinar cells, but also striated duct cells.

Immunohistochemical examination of epiphyseal growth plates of Japanese Brown cattle with chondrodysplasia.

A new type of inherited chondrodysplasia is described in Japanese Brown cattle, but the basic defects of the epiphyseal growth plate (EGP) in the limb long bones, and proliferation and differentiation of the chondrocytes in the EGP, are not yet understood. In the present study, the EGPs of the limb long bones in eight cases of chondrodysplasia and four normal (control) cattle were examined histologically and immunohistochemically. In the control cattle, proliferative chondrocytes (PCs) and hypertrophic chondrocytes (HCs) were arranged in columns parallel to the long axis of the bone, and HCs were situated on the metaphyseal side of the EGP. In all the affected cattle, many chondrocytes with a hypertrophic appearance were detected in the inner areas of the central portion of the EGP. The PC columns were short and arranged irregularly. Bone tissue and small blood vessels were found frequently in these areas. Six affected cattle showed complete EGP-closure. Backscattered electron (BSE) imaging showed that the calcified cartilage matrix was restricted to the lower region of the hypertrophic zone (HZ) of the EGP in the control cattle, while the calcified cartilage matrix and bone tissue were scattered in the inner areas of the EGP in all the chondrodysplastic cattle. Immunohistochemistry revealed type X collagen in the HCs and cartilage matrix of the HZ in the control cattle. In all the affected cattle, type X collagen was detected in apparently hypertrophic chondrocytes in the inner areas of the EGP. Type II collagen was detected in the entire EGP in all the affected cattle, as in the controls. BrdU (5-bromo-2'-deoxyuridine), injected intravenously 1h before euthanasia was detected in many PCs in the EGP in the control cattle; none, however, was detected in the central portion of the EGP in any affected animal. These observations indicate that differentiation into HCs and calcification of cartilage matrix occur in the inner areas of the central portion of the EGP in chondrodysplasia of Japanese Brown cattle. Differentiation into the HCs at this abnormal site may be caused by the inadequate proliferation and disorganization of the PCs. Premature EGP-closure, observed commonly in chondrodysplasia of Japanese Brown cattle, was thought to be caused by replacement of the calcified cartilage in the inner areas of the EGP by bone tissue.

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 beta-cell cytoplasm 3 h after STZ administration (STZ-3 h), and the most severe damage was found in beta 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 beta cells (or to compensate for the lack of beta 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.

Cleavage line patterns in beagle dogs: as a guideline for use in dermatoplasty.

We prepared a map of the cleavage lines for beagle dogs, as a guideline for use of cleavage lines in dermatoplasty. The cleavage lines at the head resembled the orientation of the underlying muscles. Although the cleavage lines in the trunk were perpendicular to the body axis, those in the thoraco-abdominal region were parallel to the body axis. The cleavage lines at the limbs were parallel to the long axis of the limb on the cranial surface, but were perpendicular to the long axis of the limb on the lateral and caudal surfaces. Also, we recorded in detail the cleavage lines in the distal regions of the limbs.

Morphological relationship between intercalated duct and pancreatic islet in streptozotocin and/or camostat mesilate administrations in the chicken.

Present electron microscopical and immunocytochemistrical studies elucidated some morphological relationship between intercalated duct (ICD) and pancreatic islet cells in the chicken in streptozotocin (STZ) and/or camostat mesilate (CM) administrations. Twenty-one chickens were set into four experimental groups: (1) control group, (2) STZ administration group, (3) CM administration group, and (4) STZ + CM administration group. Cytoplasms of ICD cells stained more strongly with eosin in STZ administration group than other groups, and electron-dense materials and intercalated processes between ICD and islet cells were also increasing in time dependence in STZ administration. Number of pancreatic islet in STZ + CM co-administration was about 3.1 times larger than other groups. Many small sized cells were detected at surrounding area of ICD and they incorporated 5-bromo-2'-deoxyuridine better than other experimental groups. Present morphological data suggested that ICD cells might support some tolerances of pancreatic endocrine cells against toxic substances and also involve in regeneration of new pancreatic islet cells in STZ + CM co-administration.

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.

Purification of carbonic anhydrase isozyme VI (CA-VI) from swine saliva.

Salivary or secreted carbonic anhydrase (CA), which constitutes a new class of CA, designated CA-VI, was isolated. Swine CA-VI purified from swine saliva by inhibitor-affinity chromatography and ion exchange chromatography had a specific activity of 5,468 units/mg. The molecular weight was 250,000, as determined by gel filtration under non-denaturing conditions, and the subunit molecular weight was found to be 37,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that swine CA-VI consists of 7 subunits. The treatment of the enzyme with endo-N-acetylglucosaminidase F reduced its subunit molecular weight from 37,000 to 35,000 and 32,000. We raised a rabbit antibody against purfied swine CA-VI. Double immunodiffusion showed that anti-swine CA-VI serum reacted with swine CA-VI and swine saliva, but not with hemolysate (containing CA-I and CA-Il) or muscle extracts (containing CA-III). The concentration of CA-VI in swine saliva, measured using single radial immunodiffusion, was 0.027 +/- 0.017 mg/mg total protein.

Immunohistochemical localization of carbonic anhydrase isoenzymes in salivary gland and intestine in adult and suckling pigs.

Localizations of carbonic anhydrase isoenzymes (CA I, CA II and CA III) were investigated immunohistochemically in the salivary glands and intestine of mature and suckling pigs. Carbonic anhydrase isoenzymes were not detected in the salivary glands of sucklings, but were present in the adult. Bicarbonate ion in saliva might be important for the digestion of solid foods in mature pigs, but unnecessary for the digestion of milk in sucklings. Expressions of CA I and CA II were detected strongly in the large intestine of the adult and sucklings, and faintly only at duodenum in the small intestine. CA I and CA II isoenzymes in the large intestine may be involved, at least in part, in ion absorption and water metabolism during digestion and absorption of milk in suckling pigs. In addition, CA I and CA II expression in the duodenal villus enterocyte may support the process of bicarbonate absorption in the duodenum.

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.

Physiological roles of gammadelta T-cell receptor intraepithelial lymphocytes in cytoproliferation and differentiation of mouse intestinal epithelial cells.

In this study we aimed to elucidate the physiological role of gammadelta intraepithelial lymphocytes (IEL) in the mouse intestine. For this purpose, we used T-cell receptor (TCR) Vgamma4/Vdelta5 transgenic mice (KN 6 Tg: BALB/c background, H-2d), and compared the immunological and physiological characteristics of the intestinal tracts of KN 6 Tg and non-transgenic (non-Tg) littermates. In KN 6 Tg littermates, 95% of small intestinal (SI) and large intestinal (LI) IEL expressed gammadelta TCR, and their TCR was replaced by Tg gammadelta TCR. In these mice, class II major histocompatibility complex (MHC) expression was up-regulated in the SI epithelium, compared with the non-Tg littermates, under specific pathogen-free (SPF) conditions. Competitive reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the mRNAs of the I-Ealpha chain on the SI epithelial cells was higher in KN 6 Tg than in non-Tg littermates. However, in the LI, class II MHC molecules were not expressed in either KN 6 Tg or non-Tg littermates. The epithelial cell mitotic index in the SI, but not in the LI, was higher in KN 6 Tg than in non-Tg littermates under SPF conditions. However, differentiation markers for SI epithelial cells, such as alkaline phosphatase and disaccharidase (lactase, maltase and sucrase) activities, were similar in KN 6 Tg and non-Tg littermates. MHC class II molecule expression on the SI epithelium was absent in germ-free (GF) Tg mice, but was induced under SPF conditions, coinciding with the increase of interferon-gamma (IFN-gamma) mRNA in gammadelta TCR SI-IEL. These findings suggest that gammadelta TCR IEL regulate epithelial cell regeneration and class II MHC expression, but not cell differentiation in the SI. However, these functions were not observed in the gammadelta TCR IEL in the LI. In addition, the activation step in the gammadelta TCR SI-IEL is dependent on the presence of gut microflora.

Immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin during tooth development and degeneration in fetuses of minke whale, Balaenoptera acutorostrata.

The immunohistological distributions of fibronectin, tenascin, type I, III and IV collagens, and laminin were observed in the tooth buds of fetuses of minke whale, Balaenoptera acutorostrata. Distributions of extracellular matrices (ECMs) examined in this study except for tenascin were generally similar to those of terrestrial mammalian species during development of the tooth bud. Tenascin in the fetuses of minke whale showed characteristic distributions in the dental lamina and the enamel organ in the early tooth developmental stage. In the physiological degeneration stage of tooth bud development, immunoreactivity of the ECMs were very weakly and limitedly detected in the dental papilla and the surrounding mesenchyme. Immunoreactivity of tenascin and type I and III collagens were positively detected in the developing baleen plate germ which was associated with the degenerating tooth bud. These findings suggested that expressions of the ECMs were related to the formation of the tooth bud and baleen plate germ, and that the lack of the ECMs was related to the degeneration of the tooth bud in the fetal minke whale.

Distribution of cytokeratin polypeptides detected by monoclonal antibodies K8.13 and K8.12 in the fetal bovine ruminal epithelium.

Temporal and spatial distributions of cytokeratin (CK) polypeptides were detected by monoclonal antibodies (mAbs) K8.13 and K8.12 during the development of the bovine ruminal epithelium. By the Western blotting analysis after the sodium dodecyl sulfate-polyacrilamide gel electrophoresis, mAb K8.13 confirmed 60.8 and 63.0 kD CK polypeptides in the fetal ruminal epithelial extract, and mAb K8.12 also 48.0 and 54.0 kD CK polypeptides. Immunohistochemical reactivities against both mAbs were detected only in the epithelial cells throughout the fetal periods. Distributions of CK polypeptides detected only by mAb K8.13 were observed on the basal side of the epitherial layer, but not by mAb K8.12 in the 7 cm fetus in crown-rump length. MAb K8.13 reacted also intensely with columnar-shaped cells in the basal layer in the fetuses of the later developmental periods. These results suggest that CK polypeptides detected by mAb K8.13 might be involved in the differentiation and/or the maintenance of the basal layer in the ruminal epithelial development.

Identification of bovine decorin in the fetal bovine rumen.

Immunohistochemical localization of bovine decorin was examined with its biological analysis in the fetal bovine rumen. By immunohistochemical staining, monoclonal antibody (mAb) 2B6, which recognizes chondroitin 4-sulfate and/or dermatan sulfate (DS), reacted specifically to the lower mesenchymal region in the developing ruminal wall. Biochemical analysis of the extract from the developing rumen revealed that molecule detected immunohistochemically by mAb 2B6 was small DS proteoglycan, bovine decorin. These results support the view that bovine decorin is involved in organization of the fetal bovine ruminal mesenchyme as a collagenous tissue.

Expression and distribution of myoepithelial cells in developing bovine parotid gland.

The expression and distribution of myoepithelial cells in relation to parotid acinar cells were studied immunohistochemically in bovine fetuses and neonates. Definitive myoepithelial cells first appeared as slender, brown short lines around the perimeters of developing secretion acini at four months of fetal age (in a 26 cm long fetus). At this time, parotid acinar cells possessed no distinct secretory granules in the supranuclear region. The differentiation of myoepithelial cells subsequently progressed during late gestation and birth. In neonates, the myoepithelial cells surrounded the secretory acini and parts of the intercalated ducts.

Distribution of chondroitin sulphate proteoglycans and peanut agglutinin-binding molecules during bovine fetal palatine ridge formation.

The expression of proteoglycans detected by the monoclonal antibodies MO-225, 3B3 and 2B6 and of peanut agglutinin-binding molecules was examined histochemically during the development of bovine palatine ridge (PR) rudiments in fetuses with crown-rump lengths of 4-60 cm. The bovine PR rudiment has 2 characteristic developmental stages: the 1st is the positioning of the epithelial placode (EP) to the predetermined site of PR rudiment formation and the 2nd is the reorientation of the apical edge of PR rudiments to form wave-like patterns from mouth to pharynx. During the 1st stage, chondroitin 6-sulphate (C6S) was expressed strongly at the epithelial basement membrane just beneath the EP. During the 2nd stage, both C6S and disaccharide unit of Glc2sulphate-GalNAc6sulphate (GlcAsSO4-GalNAc6SO4) were distributed at the basement membrane and the mesenchyme just beneath the steeper wall of the PR rudiment. Peanut agglutinin-binding molecules were also detected in the mesenchyme, their distribution being similar to that for C6S and GlcA2SO4-GalNAc6SO4, and additionally in the epithelial cells after formation of the wave-like PR. The distribution of dermatan sulphate was not directly related to the developmental changes of PR rudiments, but it was detected in the lower mesenchymal layer which supported the protruding site of the PR rudiment after the 2nd stage. The results suggest that 2 different types of proteoglycan may be involved in the critical periods during the morphogenesis of the bovine PR rudiments.