Salivary mucin MG1 is comprised almost entirely of different glycosylated forms of the MUC5B gene product.
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)
Biochemical and cytochemical studies on adenylate cyclase activity in the developing rat submandibular gland: differentiation of of the acinar secretory compartment.
To investigate membrane changes in development of the exocrine cells of the rat submandibular gland (SMG), biochemical and cytochemical studies of adenylate cyclase activity were performed on prenatal and postnatal glands. SMG rudiments and glands were studied from 15 days of gestation op to birth and 1, 2, 3, 4 and 24 weeks after birth. Glands were chemically assayed for adenylate cyclase activity using the procedures of Salomon and coworkers and cytochemically studied using a procedure which was verified biochemically. At 15-16 days of gestation basal adenylate cyclase activity was low and no staining could be observed. Adenylate cyclase activity rose six-fold from the 16th to the 18th day of gestation. Adenylate cyclase staining became evident along the surface of most of the cells of the rudiment at this time. Basal adenylate cyclase activity remained relatively constant from the 18th day of gestation up to 24 weeks of age. However, sequential changes were seen in the cytochemical localization, especially in relation to the apical plasma membrane of the developing secretory cells. (+info)
EGF precursor mRNA and membrane-associated EGF precursor protein in rat exorbital lacrimal gland.
This study was designed to demonstrate the presence of epidermal growth factor (EGF) in the rat exorbital lacrimal gland. EGF precursor gene transcription was demonstrated first by RT-PCR analysis of lacrimal gland RNA using a set of specific primers and second by Northern blot analysis of rat lacrimal gland mRNA. A rabbit polyclonal antibody (rEGF2) directed against rat submaxillary gland EGF was used to detect EGF-containing proteins by RIA. Results indicate that the rat lacrimal gland does not contain detectable soluble and mature EGF but that the EGF immunoreactivity is associated with the membrane-enriched fraction. Analysis of the detergent-solubilized membrane proteins by gel filtration shows that membrane-associated EGF immunoreactivity was present as a high-molecular-mass protein. Moreover, as shown by Western blot analysis, a specific anti-rat EGF precursor antibody (ppEGF1) can immunoprecipitate a 152-kDa EGF-containing protein. Taken together, these results demonstrate for the first time both EGF precursor gene transcription and EGF precursor protein expression in a lacrimal tissue, i.e., the rat exorbital lacrimal gland. The demonstration that EGF appears to be stored only as its full-length membrane precursor may provide important information to study the regulation of its secretory process. (+info)
Nasopharyngeal-associated lymphoreticular tissue (NALT) immunity: fimbriae-specific Th1 and Th2 cell-regulated IgA responses for the inhibition of bacterial attachment to epithelial cells and subsequent inflammatory cytokine production.
To investigate the antibacterial activity of mucosal Th1 and Th2 immune responses induced nasally and orally, mice were immunized with mucosal vaccine containing fimbrial protein of Porphyromonas gingivalis, a causative agent for a destructive chronic inflammation in the periodontium, and cholera toxin (CT) as mucosal adjuvant. Nasal vaccine containing low doses of fimbriae (10 micrograms) and CT (1 microgram) induced Ag-specific Th1/Th2-type response in CD4+ T cells in mucosal effector tissues, including nasal passage and submandibular glands, which accounted for the generation of Ag-specific IgA-producing cells. In contrast, oral immunization required higher amounts of fimbriae and CT for the induction of Ag-specific IgA responses. Fimbriae-specific IgA mAbs generated from submandibular glands of nasally immunized mice inhibited P. gingivalis attachment to and reduced subsequent inflammatory cytokine production from epithelial cells. These findings suggest that nasal vaccination is an effective immunization regimen for the induction of Ag-specific Th1 and Th2 cell-driven IgA immune responses that possess the ability to inhibit bacterial attachment to epithelial cells and subsequent inflammatory cytokine production. (+info)
Dynamics of parenchymal cell division, differentiation, and apoptosis in the young adult female mouse submandibular gland.
The submandibular salivary gland of the young adult female mouse has two secretory cell types, acinar and granular duct, which are separated by intercalated ducts. Based on the occurrence of autologous cell division in these cells, they have been traditionally classified as expanding populations. However, differentiation from stem or progenitor cells in the intercalated ducts, usually associated with renewing populations, has also been detected. The question of renewing or expanding populations is resolved by quantitating and integrating the rates of autologous cell division, differentiation, and apoptosis for each cell type. The integrated data shows that both acinar and granular duct cell populations exhibit a substantial positive growth index, whereas the growth index for the intercalated duct cells is moderately negative. On balance, it suggests that the submandibular gland of the young adult female mouse is still growing. Comparison of young female mice with older females suggests that, although overall parenchymal growth slows with age, there is no longer a net loss of intercalated duct cells. Comparison with young adult male submandibular glands indicates that gender differences exist in the rates and mechanisms used for maintaining the different cell populations. The acinar and granular duct cell populations in young adult female mouse submandibular glands are expanding at the expense of the intercalated duct cell population, which appears to be contracting. (+info)
Caffeine does not inhibit substance P-evoked intracellular Ca2+ mobilization in rat salivary acinar cells.
We used the Ca2+-sensitive fluorescent dye fura 2, together with measurements of intracellular D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], to assess the inhibitory effects of caffeine on signal transduction via G protein-coupled receptor pathways in isolated rat mandibular salivary acinar cells. ACh, norepinephrine (NE), and substance P (SP) all evoked substantial increases in the intracellular free Ca2+ concentration ([Ca2+]i). Responses to ACh and NE were markedly inhibited by prior application of 20 mM caffeine. The inhibitory effect of caffeine was not reproduced by phosphodiesterase inhibition with IBMX or addition of cell-permeant dibutyryl cAMP. In contrast to the ACh and NE responses, the [Ca2+]i response to SP was unaffected by caffeine. Despite this, SP and ACh appeared to mobilize Ca2+ from a common intracellular pool. Measurements of agonist-induced changes in Ins(1,4,5)P3 levels confirmed that caffeine inhibited the stimulus-response coupling pathway at a point before Ins(1,4,5)P3 generation. Caffeine did not, however, inhibit [Ca2+]i responses evoked by direct activation of G proteins with 40 mM F-. These data show that caffeine inhibits G protein-coupled signal transduction in these cells at some element that is common to the muscarinic and alpha-adrenergic signaling pathways but is not shared by the SP signaling pathway. We suggest that this element might be a specific structural motif on the G protein-coupled muscarinic and alpha-adrenergic receptors. (+info)
Cloning of Trp1beta isoform from rat brain: immunodetection and localization of the endogenous Trp1 protein.
The Trp gene product has been proposed as a candidate protein for the store-operated Ca2+ channel, but the Trp protein(s) has not been identified in any nonexcitable cell. We report here the cloning of a rat brain Trp1beta cDNA and detection and immunolocalization of the endogenous and expressed Trp1 protein. A 400-bp product, with >95% homology to mouse Trp1, was amplified from rat submandibular gland RNA. Rat-specific primers were used for cloning of a full-length rat brain Trp1beta cDNA (rTrp1), encoding a protein of 759 amino acids. Northern blot analysis demonstrated the transcript in several rat and mouse tissues. The peptide (amino acids 523-536) was used to generate a polyclonal antiserum. The affinity-purified antibody 1) immunoprecipitated human Trp1 (hTrp1) from transfected HEK-293 cells, 2) reacted with a protein of approximately 92 kDa, but not with hTrp3, in membranes of hTrp3-expressing HEK-293 cells, and 3) reacted with proteins of 92 and 56 kDa in human and rat brain membranes. Confocal microscopy and cell fractionation demonstrated that endogenous and expressed hTrp1 and expressed hTrp3 proteins were localized in the plasma membrane of HEK-293 cells, consistent with their proposed role in Ca2+ influx. The data demonstrate for the first time the presence of Trp1 protein in a nonexcitable cell. (+info)
Occurrence of permanent changes in vaginal and uterine epithelia in mice treated neonatally with progestin, estrogen and aromatizable or non-aromatizable androgens.
Female mice of the C57 Black/Tw strain were injected daily with 100 microng testosterone, 50 microng testosterone propionate (TP), 100 microng 5 alpha-dihydrotestosterone (DHT) or 50 microng 5 alpha-dihydrotestosterone propionate (DHTP), for 10 days from the day of birth. Two other groups of female mice were given neonatal injections with 20 microng estradiol-17 beta and 100 microng progesterone for 10 days, respectively. All mice were ovariectomized at 60 days of age and killed at 90 days. In 100% of neonatally estrogenized or androgenized, ovariectomized mice, the cranial part of the vagina was lined with stratified epithelium with either cornification or parakeratosis or mucification. Stratification only or stratification with superficial squamous metaplasia or cornification took place in the uterine epithelia of 18% of the TP-treated, 75% of the DHT-treated and 50% of the DHTP-treated, ovariectomized mice. In contrast, neonatally estrogenized, ovariectomized mice did not show the estrogen-independent, persistent uterine changes. Neonatal progesterone treatment failed to induce the permanent changes in the vaginal and uterine epithelia. (+info)