Cellular Distribution and Subcellular Localization of mCLCA1/2 in Murine Gastrointestinal Epithelia

mCLCA1/2 are members of the CLCA protein family that are widely expressed in secretory epithelia, but their putative physiological role still awaits elucidation. mCLCA1/2 have 95% amino acid identity, but currently no specific antibody is available. We have generated a rabbit polyclonal antibody (pAb849) against aa 424–443 of mCLCA1/2. In HEK293 cells transfected with mCLCA1; pAb849 detected two specific protein bands at ∼125 kDa and 90 kDa, representing full-length precursor and N-terminal cleavage product, respectively. pAb849 also immunoprecipitated mCLCA1 and labeled the protein by immunostaining. But pAb849 crossreacted with mCLCA3/4/6 despite ≤80% amino acid identity of the antigenic epitope. We therefore investigated the cellular localization of mCLCA1/2 in epithelial tissues, which do not express mCLCA3/4/6 (salivary glands, pancreas, kidney) or express mCLCA3/6 with known localization (mucus cells of stomach and small intestine; villi of small intestine). mCLCA1/2 mRNA and protein expression were found in both parotid and submandibular gland, and immunohistochemistry revealed labeling in parotid acinar cells, in the luminal membrane of parotid duct cells, and in the duct cells of submandibular gland. In exocrine pancreas, mCLCA1/2 expression was restricted to acinar zymogen granule membranes, as assessed by immunoblotting, immunohistochemistry, and preembedding immunoperoxidase and immunogold electron microscopy. Moreover, mCLCA1/2 immunolabeling was present in luminal membranes of gastric parietal cells and small intestinal crypt enterocytes, whereas in the kidney, mCLCA1/2 protein was localized to proximal and distal tubules. The apical membrane localization and overall distribution pattern of mCLCA1/2 favor a transmembrane protein implicated in transepithelial ion transport and protein secretion. (J Histochem Cytochem 58:653–668, 2010)     

Ultrastructure and histochemistry of the subepithelial glands of the nasal septal island in dromedaries with special reference to the possible functions

The nasal septal island (NSI) is a sensory patch of neuroepithelium located within the soft tissue of the nasal septum in dromedaries. The island has unique anatomical features, including the specialized subepithelial glands. The aim of the present study was to describe the microscopic features and ultrastructure of these subepithelial glands and to speculate the possible functions. A total of 10 camel heads were used for the study. Unlike the serous and mucous airway glands, the NSI glands’ ultrastructural features were typical for cells of the (Amine Precursor Uptake and Decarboxylation, APUD) system. These features were included, membrane bound secretory vesicles of varying electron density, smooth endoplasmic reticulum in the form of vesicles; electron dense mitochondria, abundant rough endoplasmic reticulum and free ribosomes. Alcian-PAS identifiable mucus granules were not observed, except for few clusters of cells, located at the luminal surface. The probable functions were discussed on basis of cellular morphology and context. In a conclusion, the NSI subepithelial glands in dromedaries had unique anatomical structures, and as many other APUD cells, they had the machinery required for synthesis of a variable number of biologically active peptides, amines and chemical mediators.     

Immune responses of locusts to challenge with the pathogenic fungus Metarhizium or high doses of laminarin

Two isolates of Metarhizium anisopliae var acridum were tested for their effects on the locust immune system and for comparison with the effects of challenge by injection with laminarin. Isolate IMI 330189 (referred to hereafter as Met 189) is highly pathogenic whether applied topically as conidia or injected as blastospores. However, isolate ARSEF 728 (referred to hereafter as Met 728) is pathogenic only when injected as blastospores, suggesting that the lack of pathogenicity of topically applied conidia from this isolate is due to a failure to penetrate the insect cuticle and gain access to the haemocoel. After topical application of conidia from Met 189, no activation of prophenoloxidase is detected, but injection of blastospores from Met 189 brings about a transient increase in phenoloxidase activity in the haemolymph in both adult locusts and 5th instar nymphs, although this does not prevent fungal-induced mortality. Co-injection of adipokinetic hormone-I (AKH-I) with blastospores prolongs the activation of prophenoloxidase in the haemolymph of adult locusts, and enhances it in nymphs. It is argued that the lack of activation of prophenoloxidase in nymphs shown previously (Mullen, L., Goldsworthy, G., 2003. Changes in lipophorins are related to the activation of phenoloxidase in the haemolymph of Locusta migratoria in response to injection of immunogens. Insect Biochemistry and Molecular Biology 33, 661-670), reflects differences in the sensitivity of the immune system between adults and nymphs rather than distinct qualitative differences, and this is confirmed in this study by the demonstration that doses of laminarin higher than those used previously (>or=100 microg) do activate the prophenoloxidase cascade in 5th instar nymphs. Nodules are formed in locusts of all ages in response to fungal infection or injection of laminarin, although there is wide variation in the number, size and distribution of nodules formed. During the examination of 5th instar nymphs for nodule formation, a previously unknown phenomenon was observed in which the salivary glands melanise in response to injections of blastospores or high doses of laminarin. In c. 85% of such nymphs, this reaction is so strong that the whole salivary gland is intensely black. Such a response is not observed in the salivary glands of mature adult locusts.     

Nitric oxide synthase in human salivary glands

The distribution of the three nitric oxide synthase (NOS) isoforms was determined immunohistochemically in the human minor and major salivary glands with comparison to that of rat salivary glands. In contrast to rat glands, which contained a dense plexus of neuronal NOS-immunoreactive nerve fibers, only a minority of the nerve fibers in human glands showed neuronal NOS immunoreactivity. Human labial and submandibular glands contained sparse NOS-immunoreactive fibers, while only occasional nerve fibers in the parotid or sublingual glands were stained. Furthermore, in contrast to the animal glands, most duct epithelial cells in all human salivary glands were immunoreactive for neuronal NOS. No specific immunoreactivity for inducible or endothelial NOS were observed in the nerve fibers or duct epithelium. We provide evidence to suggest that the role of nitric oxide in the regulation of salivary gland function is different in human as compared to experimental animals. Nitricergic innervation in human tissue is very sparse and thus nitric oxide is probably of minor importance as a neural regulator of salivary glands. Instead, NOS localized in duct epithelial cells suggests that nitric oxide might directly regulate saliva secretion and it is a putative source of nitrates previously reportedly secreted into the saliva.     

ANG II-induced attenuation of duck salt gland secretion does not depend upon the release of adrenal catecholamines

Pekin ducks (Anas platyrhynchos) were bilaterally adrenalectomized (ADX) using a two-stage procedure and given daily i.m. injections of 1 mg kg bw⁻¹ of dexamethasone (DEXA), a steroid lacking mineralocorticoid activity, and 0.9% saline drinking water ad libitum to counterbalance renal losses of salt and water. Mean arterial blood pressure (mmHg) fell from 161±3.7 (intact controls) to 116±6.9 (bilateral ADX+DEXA), a decrease of 27%, but heart rates (HR) were unchanged. The nasal salt glands were fully active after ADX+DEXA. Rates of fluid secretion and electrolyte and osmolal concentrations were unchanged. Secretion stopped, then rebounded several minutes later if ADX+DEXA ducks were injected i.v. with 1 μg of [Asn¹,Val⁵]-angiotensin II (ANG II) kg bw⁻¹ which showed that attenuation was not adrenal catecholamine-dependent.     

Morphogenetic and diurnal variation of hypericin in some Hypericum species from Turkey during the course of ontogenesis

The genus Hypericum has received considerable interest from scientists, as it is a source of a variety of biologically active compounds including the hypericins. The present study was conducted to determine ontogenetic, morphogenetic and diurnal variation of the total hypericins content in some species of Hypericum growing in Turkey namely, Hypericum aviculariifolium subsp. depilatum var. depilatum (endemic), Hypericum perforatum and Hypericum pruinatum. The Hypericum plants were harvested from wild populations at vegetative, floral budding, full flowering, fresh fruiting and mature fruiting stages four times a day. Plants were dissected into stem, leaf and reproductive tissues, which were dried separately, and subsequently assayed for total hypericin content. The density of dark glands on leaves at full flowering plants was determined for each species. Floral parts had the highest hypericin content in all species tested. But diurnal fluctuation in the hypericin content of whole plant during the course of ontogenesis varied among the species. It reached the highest level at floral budding and tended to increase at night in H. aviculariifolium subsp. depilatum var. depilatum and H. pruinatum, whereas in H. perforatum hypericin content was the highest at full flowering and no diurnal fluctuation was observed. In general, hypericin content of leaves and whole plant was higher in H. aviculariifolium subsp. depilatum var. depilatum whose leaves had more numerous dark glands than those of the two other species.     

Structural and ultrastructural features of boar bulbourethral glands

The morphological features of boar bulbourethral glands were examined by light and transmission microscopy. Bulbourethral glands are compound tubuloalveolar glands surrounded by a capsule of dense connective tissue and arranged in multiple lobules formed by endpieces and excretory ducts. Endpieces and excretory ducts are both lined by a single epithelium of mucous cells with a basal nucleus. Epithelial cells accumulate secretory granules containing neutral and carboxylated acid mucosubstances and a small amount of sulphated acid mucosubstances. The ultrastructure of epithelial cells varies according to the secretory cycle. In initial stages, the cells show a columnar shape and secretory granules unevenly distributed in the cytoplasm. As the synthesis of mucosubstances progresses, the amount of the secretory granules increases and the cellular shape becomes pyramidal. Secretory granules can contain inclusions and present differences among them according to their different phases of formation. In pyramidal cells, secretory products are released into the lumen by a merocrine mechanism.     

Differential resistance and the importance of antibiotic production in Acromyrmex echinatior leaf-cutting ant castes towards the entomopathogenic fungus Aspergillus nomius

Paired exocrine metapleural glands are present in almost all ants and produce compounds with antibiotic properties towards a variety of pathogenic fungi and bacteria. In Acromyrmex leaf-cutting ants, small workers have relatively large metapleural glands compared to large workers, and thus harbour approximately half the number of gland cells of large workers, despite being only one-fifteenth their body mass. Here we present results showing that when the two worker castes of A. echinatior are treated with spores of the pathogenic fungus Aspergillus nomius in doses that correspond to the difference in metapleural gland cell numbers they do not differ in survival. However, we also show, for the first time, that small workers survive significantly longer than large workers when both are challenged with a dose of spores that corresponds to their difference in body mass. Furthermore, the time until Aspergillus nomius hyphae and spores appear on the cadavers of workers dead from infection, is significantly increased in the small worker caste. In addition to supporting previous findings that the metapleural glands have an important defence function, the results of this study indicate that the relatively large glands in small workers makes this caste particularly well adapted to preventing pathogenic microorganisms from entering the colony. Received 23 January 2006; revised 7 April 2006; accepted 11 April 2006.