Biochemical quantitation and histochemical localization of glucose-6-phosphate dehydrogenase activity in the olfactory system of adult and aged rats

The activity of glucose-6-phosphate dehydrogenase, the rate-limiting enzyme of the hexose monophosphate shunt, was examined in olfactory epithelium, respiratory epithelium, olfactory bulb, and occipital cortex in Fisher 344 rats aged 4 and 24 months. Marked differences in this enzyme were found in olfactory compared to nonolfactory tissues. Olfactory epithelium and olfactory bulb have much greater glucose-6-phosphate dehydrogenase activity than respiratory epithelium and occipital cortex at both ages. Glucose-6-phosphate dehydrogenase remains fairly constant between adulthood and senescence in respiratory epithelium and occipital cortex. However, glucose-6-phosphate dehydrogenase activity decreases during the same time in both of the olfactory tissues examined. Previous studies of changes in this enzyme with aging have shown increases in enzyme activity in some brain regions, but never the decreases that we describe in olfactory tissues. Glucose-6-phosphate dehydrogenase histochemistry revealed intense staining of both the apical layer of olfactory epithelium and of Bowman's glands along with their ducts. Histochemistry of the olfactory bulb showed strongest staining in the nerve and glomerular layers of the bulb. The functional implications of these findings are discussed.     

Developmental expression of the calcium‐activated chloride channels TMEM16A and TMEM16B in the mouse olfactory epithelium

Calcium‐activated chloride channels are involved in several physiological processes including olfactory perception. TMEM16A and TMEM16B, members of the transmembrane protein 16 family (TMEM16), are responsible for calcium‐activated chloride currents in several cells. Both are present in the olfactory epithelium of adult mice, but little is known about their expression during embryonic development. Using immunohistochemistry we studied their expression in the mouse olfactory epithelium at various stages of prenatal development from embryonic day (E) 12.5 to E18.5 as well as in postnatal mice. At E12.5, TMEM16A immunoreactivity was present at the apical surface of the entire olfactory epithelium, but from E16.5 became restricted to a region near the transition zone with the respiratory epithelium, where localized at the apical part of supporting cells and in their microvilli. In contrast, TMEM16B immunoreactivity was present at E14.5 at the apical surface of the entire olfactory epithelium, increased in subsequent days, and localized to the cilia of mature olfactory sensory neurons. These data suggest different functional roles for TMEM16A and TMEM16B in the developing as well as in the postnatal olfactory epithelium. The presence of TMEM16A at the apical part and in microvilli of supporting cells is consistent with a role in the regulation of the chloride ionic composition of the mucus covering the apical surface of the olfactory epithelium, whereas the localization of TMEM16B to the cilia of mature olfactory sensory neurons is consistent with a role in olfactory signal transduction. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 657–675, 2014     

Protective activity of 28-kDa 1-Cys peroxiredoxin determines its peroxidase activity]

The 28 kDa peroxiredoxin from rat exhibited peroxidase activity only in the presence of dithiothreitol. Both organic and nonorganic peroxidases were found to be substrates for the 28-kDa peroxiredoxin activity. Analysis of the protective antioxidant activity of the 28-kDa peroxiredoxin revealed that it is accounted for by its peroxidase activity.     

A novel 45 kDa secretory protein from rat olfactory epithelium: primary structure and localisation.

cDNA clones encoding the 45 kDa protein were isolated from a rat olfactory epithelium cDNA library and their inserts were sequenced. The reconstructed protein sequence comprises 400 amino acids with a calculated molecular mass of 46,026 Da. A homology was revealed between the amino acid sequence of the 45 kDa protein and the proteins involved in the transfer of hydrophobic ligands. Using in situ hybridisation, the 45 kDa protein mRNA expression was detected in the layer of supportive cells of olfactory epithelium, apical region of trachea, surface layer of the ciliated bronchial epithelium in lung and in skin epidermis.     

Localization of prolactin receptor in the dorsal and ventral skin of the frog (Rana ridibunda)

The dorsal and ventral skin in amphibians plays an important role in osmoregulation. Prolactin hormone is involved in regulation of amphibian skin functions, such as water and electrolyte balance. Therefore, amphibians may be useful as a model for determining the sites of the prolactin receptor. In this study, prolactin receptor was detected in frog dorsal and ventral skin using immunohistochemical staining method. Prolactin receptor immunoreactivity was localized in all epidermal layers except stratum corneum of dorsal skin epidermis, stratum germinativum layer of ventral skin epidermis, myoepithelial cells, secretory epithelium and secretory channel cells of granular glands in both skin regions. The mucous glands and secretory granules of granular glands did not show immunoreactivity for the prolactin receptor. According to our immunohistochemical results, the more widespread detection of prolactin receptor in dorsal skin epidermis indicates that prolactin is more effective in dorsal skin. Presence of prolactin receptors in epidermis points out its possible osmoregulatory effect. Moreover, detection of receptor immunoreactivity in various elements of poison glands in the dermis of both dorsal and ventral skin regions suggests that prolactin has a regulatory effect in gland functions.     

Distribution of prolactin receptor in frog (Rana ridibunda) dorsal skin during hibernation

The role of prolactin in the regulation of frog skin functions is still unclear particularly during environmental changes. In this study, prolactin receptor (PRLR) was detected in active and hibernating frog dorsal skin using immunohistochemical method. PRLR immunoreactivity in active frogs was observed in the epidermis, in the secretory epithelium of granular glands and the secretory channel cells of the glands. Myoepithelial cells of granular glands that started accumulating secretory material or those with a full lumen were PRLR immunoreactive, while some myoepithelial cells of empty granular glands were negative for PRLR. In hibernating frogs, this immunoreactivity was observed in the same regions; however, immunoreactivity was more intense than that in active frogs. PCNA was employed for detection of proliferative activity of PRL in the dorsal skin, and immunoreactivity was detected in the nuclei of a few epidermis cells and in the duct of glands of active frogs. The number of immunoreactive nuclei in these regions increased in hibernating and in prolactin injected groups. We conclude that prolactin provides morphological and functional integrity of skin stimulating the proliferation and regulating the function of granular glands and plays an important role in the adaptation of amphibians to the long winter period.     

Distribution of cytochrome P-450 monoxygenase enzymes in the nasal mucosa of hamster and rat

Deposition of inhaled particulates onto the respiratory mucosa is relatively great in that portion of the nasal cavity unprotected by ciliated, goblet, or keratinized superficial cells. The cytochrome P-450 system is an important enzyme system involved in the biotransformation of xenobiotics into metabolites that are more readily absorbed. To examine the transitional region caudal to the nasal vestibule, nasal tissues of hamster and rat were prepared for immunocytochemistry. Blocks of tissue representing four levels along the long axis of the nasal cavity were examined. Paraffin sections were processed through the avidin-biotin peroxidase procedure, with diaminobenzidine tetrahydrochloride as the chromagen. Enzyme localization was accomplished through the use of antibodies for three rabbit cytochrome P-450 isozymes; 2, 5, and 6 (subfamilies IIB, IVB, and IA, respectively); and for rabbit NADPH-cytochrome P-450 reductase. Enzyme distribution was similar in both hamster and rat nasal tissues except in cells of striated and intercalated ducts of nasal glands and in cells of the nasolacrimal duct where immunoreactivity was greater in the hamster. Immunoreactivity for reductase and isozyme 2 was intense in nonciliated cells lining the nonolfactory epithelium, in sustentacular cells of the olfactory epithelium, and in acinar cells of olfactory glands. Distribution of reaction products to isozyme 5 and 6 were similar to but not so intense as those of reductase and isozyme 2. Reaction products for reductase and isozyme 2 occurred generally in the same cellular and intracellular regions with the following exceptions: isozyme 2 was more concentrated in cells of striated ducts and of the nasolacrimal duct, and reductase was more abundant in intercalated ducts of nasal glands.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical detection of olfactory marker protein in tissues with ectopic expression of olfactory receptor genes

Olfactory marker protein (OMP) is a unique marker of mature olfactory neurons, which specifically express olfactory receptor genes. Widespread ectopic expression of olfactory receptor genes in numerous tissues outside olfactory system has also been reported, although the functional implication of this phenomenon remains unknown. We analyzed the presence of OMP in two rat tissues with ectopic expression of olfactory receptor genes (testis and circumvallate papillae of tongue) using immunohistochemistry. In testis, immunoreactivity against OMP was not detected. In circumvallate papillae of tongue, immunoreactivity was specifically localized to taste bud cells.     

Study of structure of secretory 28 kDa protein from the rat olfactory epithelium

Clone lambda a26.1 isolated from rat olfactory epithelium contains a full-length 28-kDa protein cDNA (1414 b.p.). The reconstructed protein sequence comprises 223 aa with a calculated molecular mass of 24,630 Da. A substantial homology was revealed between the amino acid sequence of the 28-kDa protein and those of thiol-specific antioxidants (peroxiredoxines). The 28-kDa protein belongs to the 1 Cys-subfamily of peroxiredoxines and is the first member of peroxiredoxines identified in the olfactory epithelium.     

Differential binding patterns of three antibodies (VOBM1, VOBM2, and VOM2) in the rat vomeronasal organ and accessory olfactory bulb

Immunohistochemical properties of monoclonal antibodies raised against the rat vomeronasal epithelium were examined in adult rats. Three monoclonal antibodies, VOBM1, VOBM2, and VOM2, reacted specifically to the luminal surface of the sensory epithelium of the vomeronasal organ. In addition, the reactivities of VOBM1 and VOBM2 were detected in the vomeronasal nerve layer and the glomerular layer of the accessory olfactory bulb. Electron-microscopic study revealed differential patterns of the immunoreactivity of the three antibodies to the microvilli of vomeronasal sensory epithelium. VOBM1 immunoreactivity was found on the microvilli of the supporting cells, whereas VOBM2 immunoreactivity was found on those of the sensory cells. VOM2 immunoreactivity was observed on the microvilli of both the sensory and supporting cells. These results suggest that the three antibodies recognize different antigens on the vomeronasal sensory epithelium. In particular, VOBM2 antibody appears to react to an antigen specific to the microvilli of the vomeronasal sensory cells.     

Immunolocalisation of P2X and P2Y nucleotide receptors in the rat nasal mucosa

Purinoceptor subtypes were localised to various tissue types present within the nasal cavity of the rat, using immunohistochemical methods. P2X₃ receptor immunoreactivity was localised in the primary olfactory neurones located both in the olfactory epithelium and vomeronasal organs (VNO) and also on subepithelial nerve fibres in the respiratory region. P2X₅ receptor immunoreactivity was found in the squamous, respiratory and olfactory epithelial cells of the rat nasal mucosa. P2X₇ receptor immunoreactivity was also expressed in epithelial cells and colocalised with caspase 9 (an apoptotic marker), suggesting an association with apoptosis and epithelial turnover. P2Y₁ receptor immunoreactivity was found within the respiratory epithelium and submucosal glandular tissue. P2Y₂ receptor immunoreactivity was localised to the mucus-secreting cells within the VNO. The possible functional roles of these receptors are discussed.     

BM-derived cells restore expression of peroxiredoxin V in the airways following acute naphthalene injury in mice

BACKGROUND: Naphthalene-induced respiratory tract toxicity in mice is characterized by specific and rapid loss of the Clara cell population, which is restored only after several days. The sources of restoration of this cell population remain unclear. We investigated whether BM-derived cells participated in the process of epithelial restoration following naphthalene toxicity compared with bacterial infection. We further investigated the role of BM-derived cells in restoration of expression of peroxiredoxin V (PRXV), one of the major proteins of antioxidant defense, specifically expressed in the bronchial epithelium. METHODS: We transplanted GFP-tagged BM cells into 5 Gy-irradiated C57BL/6 recipients. Following 1 month of recovery, experimental animals were subjected to 250 mg/kg naphthalene i.p. An additional group of animals received intratracheal instillation of Escherichia coli to induce acute bacterial inflammation. Animals were killed at 1-12 days after naphthalene and analyzed immunohistochemically. RESULTS: Recipients' cells of bronchial epithelium demonstrated significantly reduced levels of PRXV expression following naphthalene. In animals with acute bacterial inflammation, PRXV levels were not reduced in epithelium and participation of BM-derived cells in epithelial restoration was minimal. Following naphthalene, GFP(+) cells were present in large numbers in lung parenchyma and epithelium of conducting airways starting at 1 day following injury. GFP(+) progeny of BM cells was the major source of PRXV in the epithelium. DISCUSSION: These data suggest that BM-derived cells may provide a source of antioxidant protection of airways by expression of PRXV in a model of acute epithelial respiratory tract toxicity.     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Immunohistochemical localization of vascular endothelial growth factor in the globule leukocyte/mucosal mast cell of the rat respiratory and digestive tracts

 Vascular endothelial growth factor (VEGF) is a potent angiogenic mitogen that also increases vascular permeability. Immunohistochemical localization of VEGF in the respiratory and digestive tracts of healthy adult rats was investigated at light and electron microscopic levels using a specific antibody. The results revealed solitary cells with strong VEGF immunoreactivity scattered in the epithelium of the respiratory tract as well as in the lamina propria and epithelium of the intestine. From ultrastructural features of their large cytoplasmic granules, VEGF-positive cells in the respiratory tract were identified as globule leukocytes (GL). The immunoreactivity was localized exclusively in the cytoplasmic granules of GL. Most of the VEGF-positive cells in the small intestine were located in the lamina propria, whereas those in the large intestine were found more frequently in the epithelium than in the lamina propria. They showed the same morphological features as respiratory tract GL and were identified as mucosal mast cells (MMC). When examined in serial sections, GL/MMC in the respiratory and digestive tracts showed only weak reactivity to anti-histamine antibody. In contrast, connective tissue mast cells (CTMC), which were located in the submucosa of the digestive tract and in the connective tissues of the respiratory tract and other organs, were intensely immunopositive for histamine, whereas they showed no reactivity to anti-VEGF antibody. The specific occurrence of VEGF in GL/MMC suggests that this cell type is involved in paracrine regulation of the permeability of nearby microvessels, and that VEGF immunoreactivity can be used as a histochemical marker to distinguish GL/MMC from CTMC. Accepted: 28 July 1998     

Comparative cytokeratin distribution patterns in cholesteatoma epithelium

Cytokeratins (CKs) are known as the intermediate filament proteins of epithelial origin. Their distribution in human epithelia is different according to the type of epithelium, state of growth and differentiation. We used monoclonal mouse antibodies against cytokeratins to study CK expression in the following human tissues: cholesteatoma, middle ear mucosa, glandular epithelium, and meatal ear canal epithelium. Immunohistochemical processing was performed using the labeled steptavidin peroxidase method to demonstrate the presence of CKs in cells of human epidermis. Positive reaction was obtained for CK4, CK34betaE12, CK10, CK14 in skin and cholesteatoma epithelium. However, a more extensive positive reaction with those CKs was observed in cholesteatoma epithelium. Positive immunoreactivity was seen with anti- CK19 in the glandular epithelium. Middle ear mucosa specimens revealed positive immunoreactivity with the antibodies against CK4. The expression of CK4 was definitely positive within the basal layers of the epidermis. The glandular epithelium showed no positive reaction with anti- CK4, anti- CK34betaE12, anti- CK14 and anti-CK10. Immunohistochemistry for CK18 showed no reaction in all examined tissues. Cholesteatoma is known as a proliferative disease in the middle ear which pathogenesis is not completely understood. Keratinocytes express hyperproliferation- associated CKs and after reaching the suprabasal layers they finally undergo apoptosis creating keratinous debris. Cytokeratin expression observed in the epithelium explains proliferative behavior of cholesteatoma which is associated with increased keratinocyte migration. Cytokeratins can be used as potential proliferative markers. It can also allow for searching the usefulness of inhibiting regulators in the treatment of hyperproliferative diseases.     

Identification of murine type I keratin 9 (73 kDa) and its immunolocalization in neonatal and adult mouse foot sole epidermis

The foot sole epidermis of the fore and hind feet of the adult mouse contains an acidic (type I) mRNA-encoded 73-kDa keratin polypeptide which cannot be detected in any other skin site of the mouse integument. Western blot analysis using an antibody specific for the 64-kDa keratin 9 of human and bovine callus-forming epidermis [A. C. Knapp et al. (1986) J. Cell Biol. 103, 657-667] demonstrates that the 73-kDa keratin represents the murine analog of keratin 9 of man and cow. Concomitant investigations in two related rodent species indicate that the size of this keratin varies more among species than that of any other orthologous keratin. Histological examination of adult mouse foot sole skin reveals an extremely thick and undulated epidermis covering the apical portion of the six footpads, whereas the epidermal-dermal junction of the lateral walls of these nodular protuberances as well as that of the remainder of the foot sole skin is essentially flat. If sections of adult foot sole skin are investigated by indirect immunofluorescence with the keratin 9-specific antibody, intense cytoplasmic staining is restricted to the apical rete pegs of the footpad epidermis in which virtually all suprabasal cells express keratin 9. However, we also observed keratin 9-negative cell columns ascending straight above the tips of the dermal papillae and separating the keratin 9-positive rete pegs from each other. At the transition from the strongly undulated apical epidermis to the flat epidermis of the lateral walls of the footpads, keratin 9-positive cells loose their coherence and gradually disappear toward the inter-footpad epidermis. This intimate relationship between the morphogenesis of epidermal ridges and inter-ridges and the expression of keratin 9 is also visible in foot sole epidermis of neonatal mice. Here we observed the appearance of keratin 9-positive suprabasal cells concomitant with the onset of pronounced folding of the apical footpad epidermis by about Day 3 after birth. Our findings confirm the view that the expression of keratin 9 is characteristic of a highly specialized pathway of epidermal differentiation. We propose a hypothesis for keratin expression in skin sites which are subject to pronounced mechanical wear and tear.