Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
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The MG1 population of mucins was isolated from human whole salivas by gel chromatography followed by isopycnic density gradient centrifugation. The reduced and alkylated MG1 mucins, separated by anion exchange chromatography, were of similar size (radius of gyration 55-64 nm) and molecular weight (2.5-2.9 x 10(6) Da). Two differently-charged populations of MG1 subunits were observed which showed different reactivity with monoclonal antibodies to glycan epitopes. Monosaccharide and amino acid compositional analyses indicated that the MG1 subunits had similar glycan structures on the same polypeptide. An antiserum recognizing the MUC5B mucin was reactive across the entire distribution, whereas antisera raised against the MUC2 and MUC5AC mucins showed no reactivity. Western blots of agarose gel electrophoresis of fractions across the anion exchange distribution indicated that the polypeptide underlying the mucins was the product of the MUC5B gene. Amino acid analysis and peptide mapping performed on the fragments produced by trypsin digestion of the two MG1 populations yielded data similar to that obtained for MUC5B mucin subunits prepared from respiratory mucus (Thornton et al., 1997) and confirmed that the MUC5B gene product was the predominant mucin polypeptide present. Isolation of the MG1 mucins from the secretions of the individual salivary glands (palatal, sublingual, and submandibular) indicate that the palatal gland is the source of the highly charged population of the MUC5B mucin. (+info)
Pancreatic lipase-related protein 1 mRNA in female mouse lacrimal gland.
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PURPOSE: Differential display analysis was used to look for gender differences in lacrimal gland gene expression. The expression of a female-specific mouse lacrimal gland mRNA that encoded pancreatic lipase-related protein 1 (PLRP1) was identified and characterized. METHODS: Differential display analysis of the exorbital lacrimal glands of male and female Swiss Webster mice detected a potential female-specific cDNA, designated Y2. Using the technique of rapid amplification of cDNA ends, a full-length cDNA of Y2 was obtained and the nucleotide sequence determined. To assess tissue-specific expression, a labeled Y2 cDNA probe was hybridized to RNA blots of male and female mouse lacrimal, harderian, parotid, mandibular, sublingual, and pancreas glands and liver. Y2 cDNA was also hybridized to RNA blots of male and female rat lacrimal gland and male rat pancreas. To determine subcellular localization, Y2 sense and antisense RNA probes were hybridized to female mouse lacrimal gland frozen sections. RESULTS: GenBank database sequence comparisons indicated that Y2 encoded mouse PLRP1. RNA blots documented that PLRP1 was expressed in female, but not in male, mouse lacrimal gland. PLRP1 mRNA was also expressed in male and female mouse sublingual gland and pancreas. Expression of PLRP1 was not detected in male or female rat lacrimal gland. In situ hybridization showed that PLRP1 was expressed in the acinar cells of the female mouse lacrimal gland. CONCLUSIONS: Lacrimal gland expression of PLRP1 mRNA was gender and species specific. Female, but not male, mouse lacrimal gland expressed PLRPI mRNA. Neither female nor male rat lacrimal gland expressed PLRP1 mRNA. PLRP1 protein may be secreted in mouse tears, where it may function as a lipolytic enzyme, modifying tear film lipids. (+info)
Evidence of a phenotypically determined ductal cell lineage in mouse salivary glands.
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The submandibular salivary gland of mice contains a parenchymal element, the granular duct, which matures peripubertally from the striated ducts. Granular duct cells also differentiate from intercalated ducts in the adult mouse submandibular gland. Using preproNGF-A as a signature protein of mature granular duct cells, this study inquired if phenotypic determination might have occurred earlier than the first signs of cellular differentiation. Results from RT-PCR indicate the presence of preproNGF-A transcripts at all postnatal stages of development of the submandibular glands, as well as in adult sublingual glands. The preproNGF-A transcript was also detected prenatally as early as embryonic day 17 in the submandibular/sublingual complex. Using an antibody directed specifically against the "pre" peptide, immunocytochemistry showed preproNGF-A localized in the granular ducts and striated ducts of the adult submandibular gland. In addition preproNGF-A was detected throughout the first order branches of the intercalated duct system. In the neonatal gland, preproNGF-A was found in the large tubules that differentiate to the striated ducts. The early appearance of preproNGF-A in the histological lineage that sequentially gives rise to striated ducts and then to granular ducts suggests that this lineage is phenotypically determined as early as birth. An undifferentiated stage of the phenotypically determined lineage also appears to be retained in the intercalated duct system to provide progenitors for subsequent differentiation in the adult gland. Throughout development of the sublingual gland, preproNGF-A was detectable in the striated ducts or in their predecessors, suggesting that they may also represent a phenotypically determined cell lineage similar to that of the submandibular gland. (+info)
Further characterization of human salivary anticandidal activities in a human immunodeficiency virus-positive cohort by use of microassays.
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Salivary anticandidal activities play an important role in oral candidal infection. R. P. Santarpia et al. (Oral Microbiol. Immunol. 7:38-43, 1992) developed in vitro anticandidal assays to measure the ability of saliva to inhibit the viability of Candida albicans blastoconidia and the formation of germ tubes by C. albicans. In this report, we describe modifications of these assays for use with small volumes of saliva (50 to 100 microl). For healthy subjects, there is strong inhibition of blastoconidial viability in stimulated parotid (75%), submandibular-sublingual (74%), and whole (97%) saliva, as well as strong inhibition of germ tube formation (>80%) for all three saliva types. The susceptibility of several Candida isolates to inhibition of viability by saliva collected from healthy subjects is independent of body source of Candida isolation (blood, oral cavity, or vagina) or the susceptibility of the isolate to the antifungal drug fluconazole. Salivary anticandidal activities in human immunodeficiency virus (HIV)-infected patients were significantly lower than those in healthy controls for inhibition of blastoconidial viability (P < 0.05) and germ tube formation (P < 0. 001). Stimulated whole-saliva flow rates were also significantly lower (P < 0.05) for HIV-infected patients. These results show that saliva of healthy individuals has anticandidal activity and that this activity is reduced in the saliva of HIV-infected patients. These findings may help explain the greater incidence of oral candidal infections for individuals with AIDS. (+info)
The serous demilune of rat sublingual gland is an artificial structure produced by conventional fixation.
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The ultrastructure of the secretory end-piece of the rat sublingual gland was examined in samples prepared by rapid freezing and freeze-substitution method, and results were analyzed in combination with 3-D images reconstructed by computer graphics from light micrographs of serial sections. Fixation by rapid freezing followed by freeze-substitution preserved cellular ultrastructures, especially the membrane structure, in perfect condition, and demonstrated the terminal portion of the sublingual gland to be a compound branched tubulo-alveolar gland with serous cells distributed throughout the end-pieces. All the serous cells aligned with mucous cells to surround a common lumen, leaving no demilune structure. In contrast, samples fixed by the conventional immersion method showed distended mucous cells displacing the serous cells toward the basal portion of the acinus to form the demilune structure. The luminal space was also compressed and appeared disconnected from the serous cells. From these observations, the serous demilune that for more than 130 years has been believed to be an actual histological entity was proved to be an artificial structure produced through compression by the hydrated and expanded mucous cells during immersion fixation. (+info)
The effects of sialoadenectomy and exogenous EGF on taste bud morphology and maintenance.
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Taste buds on the dorsal tongue surface are continually bathed in saliva rich in epidermal growth factor (EGF). In the following experiment, taste bud number and morphology were monitored following submandibular and sublingual salivary gland removal (sialoadenectomy), to determine if EGF plays a role in the maintenance and formation of taste buds. Adult male rats were divided into four groups: sialoadenectomized (SX, n = 4); sialoadenectomized with EGF replacement (SX + EGF, n = 5); sham-operated (SH, n = 4); and sham-operated with exogenous EGF (SH + EGF, n = 5). After a 3 week recovery, SX + EGF and SH + EGF animals were given 50 microg/day EGF in their drinking water for 14 days. At day 14, saliva was collected, the animals were killed and the presence of EGF determined by radioligand-binding assay. Tongues were removed and histologically examined for the presence and morphology of taste buds on fungiform and circumvallate papillae, or immunostained for the presence of EGF, TGFalpha (transforming growth factor alpha) and EGFR (EGF receptor). The removal of submandibular and sublingual salivary glands resulted in the loss of fungiform taste buds and normal fungiform papillae morphology. These effects were reversed by EGF supplementation, indicating a role for EGF in fungiform taste bud maintenance. In addition, supplementation of EGF to sham-operated animals increased the size of fungiform taste buds. In contrast, removal of salivary glands had no effect on the size, numbers, or morphology of circumvallate taste buds, suggesting that the formation and maintenance of taste buds in fungiform and circumvallate papillae may involve different and distinct processes. EGF, TGFalpha and EGFR were localized to distinct layers of the dorsal epithelium and to within both fungiform and circumvallate taste buds. Their expression within the epithelium or taste buds was not altered with sialoadenectomy, indicating that the actions of endogenous EGF and TGFalpha are distinct and not regulated by exogenous EGF and TGFalpha supplied in saliva. (+info)
Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice.
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1. Intracellular pH (pHi) plays an important role in regulating fluid and electrolyte secretion by salivary gland acinar cells. The pH-sensitive, fluorescent dye 2', 7'-bis(carboxyethyl)-5(6)-carboxylfluorescein (BCECF) was used to characterize the mechanisms involved in regulating pHi during muscarinic stimulation in mouse sublingual mucous acinar cells. 2. In the presence of HCO3-, muscarinic stimulation caused a rapid decrease in pHi (0.24 +/- 0.02 pH units) followed by a slow recovery rate (0.042 +/- 0.002 pH units min-1) to the initial resting pHi in sublingual acinar cells. The muscarinic receptor-induced acidification in parotid acinar cells was of a similar magnitude (0. 25 +/- 0.02 pH units), but in contrast, the recovery rate was approximately 4-fold faster (0.181 +/- 0.005 pH units min-1). 3. The agonist-induced intracellular acidification was inhibited by the anion channel blocker niflumate, and was prevented in the absence of HCO3- by treatment with the carbonic anhydrase inhibitor methazolamide. These results indicate that the muscarinic-induced acidification is due to HCO3- loss, probably mediated by an anion conductive pathway. 4. The Na+-H+ exchange inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) amplified the magnitude of the agonist-induced acidification and completely blocked the Na+-dependent pHi recovery. 5. To examine the molecular nature of the Na+-H+ exchange mechanism in sublingual acinar cells, pH regulation was investigated in mice lacking Na+-H+ exchanger isoforms 1 and 2 (NHE1 and NHE2, respectively). The magnitude and the rate of pHi recovery in response to an acid load in acinar cells isolated from mice lacking NHE2 were comparable to that observed in cells from wild-type animals. In contrast, targeted disruption of the Nhe1 gene completely abolished pHi recovery from an acid load. These results demonstrate that NHE1 is critical for regulating pHi during a muscarinic agonist-stimulated acid challenge and probably plays an important role in regulating fluid secretion in the sublingual exocrine gland. 6. In NHE1-deficient mice, sublingual acinar cells failed to recover from an acid load in the presence of bicarbonate. These results confirm that the major regulatory mechanism involved in pHi recovery from an acid load is not Na+-HCO3- cotransport, but amiloride-sensitive Na+-H+ exchange via isoform 1. (+info)
Accumulation of common T cell clonotypes in the salivary glands of patients with human T lymphotropic virus type I-associated and idiopathic Sjogren's syndrome.
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To clarify the pathogenesis of human T lymphotropic virus type I (HTLV-I)-associated Sjogren's syndrome (SS), the TCR Vbeta gene usage by the infiltrating lymphocytes in the target organ was examined. The Vbeta families predominantly used in the labial salivary gland (LSG) from the HTLV-I-seropositive (HTLV-I+) SS patients were more restricted than those from the HTLV-I-seronegative (idiopathic) SS patients, and were commonly Vbeta5.2, Vbeta6, and Vbeta7. The single-strand conformation polymorphism analysis revealed that T cell clonotypes with Vbeta5.2, Vbeta6, and Vbeta7 accumulate in the LSG from the HTLV-I+ and idiopathic SS patients. Among junctional sequences of the most dominant Vbeta7 transcripts, the conserved amino acid motif (QDXG: X is any amino acid) was found in six of the five HTLV-I+ SS patients and was also detected in two of the five idiopathic SS patients. Using the probes specific to the motif, the Vbeta7 transcripts with the motif were detected in the LSG from all of the seven HTLV-I+ and five of the six idiopathic SS patients, but not from eight healthy subjects. The Vbeta7 transcripts with this motif were also detected in the HTLV-I-infected T cell lines obtained from the LSG of an HTLV-I+ SS patient. The accumulation of HTLV-I-infected T cells expressing TCR with the conserved motif was thus indicated. These T cells were commonly present in patients with idiopathic SS and are strongly suggested to most likely be involved in the pathogenesis of both HTLV-I-associated and idiopathic SS. (+info)