Von Ebner's Glands Research Articles

A novel lipocalin homologue from the venom gland of Deinagkistrodon acutus similar to mammalian lipocalins

Lipocalins are involved in a variety of functions including retinol transport, cryptic coloration, olfaction, pheromone transport, prostaglandin synthesis, regulation of the immune response and cell homeostatic mediation. A full-length cDNA clone (named d-lipo), isolated from the venom gland cDNA library of Deinagkistrodon acutus, contained an insert of 664 bp including an open reading frame that encodes a lipocalin homologue of 177 amino acids. Comparison of d-lipo and other related proteins revealed an overall amino acid identity of less than 21.5%. Primary structures of d-lipo carried three structurally conserved regions (SCR) showing homologies to those of lipocalins. The first conserved Cys residue - the essential amino acid residue for the catalytic activity and unique to lipocalin-type prostaglandin D synthase (L-PGDS) in the lipocalin protein family - was identified in d-lipo at amino acid position 58. Phylogenetic tree analysis showed that d-lipo was in-between the large L-PGDS cluster and the small von Ebner's-gland proteins (VEGP) cluster. Moreover, d-lipo gene presented a high-level expression in the venom gland and a low-level expression in the brain and its expression was significantly increased under pathological conditions, suggesting a possible relationship between d-lipo mRNA expression and the venom gland inflammatory disease. This is also the first report of a lipocalin homologous gene identified in the venom gland of a snake.

Morphologic diversity of the minor salivary glands of the rat: fertile ground for studies in gene function and proteomics

In this article the locations and histologic and ultrastructural features of all of the minor salivary glands of the rat are presented; similarities and differences among them are highlighted. These glands are almost as diverse morphologically as the major salivary glands of the rat. The acini of von Ebner's glands are serous; those of the anterior and posterior buccal glands and minor sublingual glands are mucous; and those of the glossopalatal, palatal, and Weber's glands are mucous with serous demilunes. The anterior buccal, minor sublingual and von Ebner's glands have striated and stratified columnar ducts, while only the minor sublingual and von Ebner's glands have intercalated ducts. The glossopalatal, palatal, posterior buccal and Weber's glands have none of these ducts; the tubulo-acini drain abruptly into short terminal ducts composed of stratified squamous epithelium. All of the mucous acini react with an antibody to a mucin (Muc19) of the rat major sublingual gland, but in some of the glands the reaction varies in intensity among the acinar cells. Ultrastructurally, the mucous secretory granules of the anterior buccal, glossopalatal, palatal and Weber's glands are biphasic, while those of the minor sublingual and posterior buccal glands are monophasic. Although there is a considerable body of literature concerning the development, innervation, physiology and proteomics of von Ebner's glands, investigation of the other minor salivary glands of the rat ranges from modest to nearly nonexistent.

Salivary PYY: A Putative Bypass to Satiety

Peptide YY3-36 is a satiation hormone released postprandially into the bloodstream from L-endocrine cells in the gut epithelia. In the current report, we demonstrate PYY3-36 is also present in murine as well as in human saliva. In mice, salivary PYY3-36 derives from plasma and is also synthesized in the taste cells in taste buds of the tongue. Moreover, the cognate receptor Y2R is abundantly expressed in the basal layer of the progenitor cells of the tongue epithelia and von Ebner's gland. The acute augmentation of salivary PYY3-36 induced stronger satiation as demonstrated in feeding behavioral studies. The effect is mediated through the activation of the specific Y2 receptor expressed in the lingual epithelial cells. In a long-term study involving diet-induced obese (DIO) mice, a sustained increase in PYY3-36 was achieved using viral vector-mediated gene delivery targeting salivary glands. The chronic increase in salivary PYY3-36 resulted in a significant long-term reduction in food intake (FI) and body weight (BW). Thus this study provides evidence for new functions of the previously characterized gut peptide PYY3-36 suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity.

Two Newly Identified Cat Allergens: The von Ebner Gland Protein Fel d 7 and the Latherin-Like Protein Fel d 8

Introduction: Characterization of the complete IgE binding spectrum of cat allergens is important for the development of improved diagnosis and effective immunotherapeutics. While Fel d 1 remains unchallenged as the major cat allergen, we now report the isolation of two new allergens capable of binding similar concentrations of IgE in the allergic sera of some individuals. Materials and Methods: Cat tongue and submandibular salivary gland cDNA libraries were screened by DNA hybridisation and IgE immunoassay. The isolated DNA fragments were sub-cloned into an E. coli expression system and the IgE reactivity was examined with human cat-allergic sera using a DELFIA IgE quantitation assay. Results: Fel d 7, an 18 kDa von Ebner gland protein Can f 1 homologue, was isolated from the tongue library. Fel d 8, a 24-kDa latherin-like protein with homology to Equ c 5, was isolated from the submandibular library. The frequency of IgE binding of cat-allergic sera to recombinant Fel d 1, 7 and 8 was 60.5, 37.6 and 19.3%, respectively. Inhibition studies indicated some IgE binding cross-reactivity between Fel d 7 and dog dander extracts. Discussion: The study reports the isolation and characterization of two new cat allergens. The isolation of these allergens provides the opportunity to determine the role that IgE binding proteins other than Fel d 1 play in cat-allergic disease. For cat-allergic individuals with moderate to mild rhinoconjunctivitis these allergens may play a more important role in the manifestation of their allergic disease.

Immunohistochemical and Immunocytochemical Localization of Amylase in Rat Parotid Glands and von Ebner's Glands by Ion Etching-Immunoscanning Electron Microscopy

The distribution of amylase in rat parotid glands and von Ebner’s glands was examined using ion etching-immunoscanning electron microscopy, which enables both light and electron microscopic observations of identical semi-thin resin sections immunolabeled with anti-α-amylase and immunogold in association with silver enhancement. At the light microscopic level, most acinar secretory granules (SG) and striated duct secretions of parotid glands were strongly stained dark brown. In von Ebner’s glands, acinar SG and duct secretions were weakly to strongly stained light to dark brown. At the electron microscopic level, labeling was observed as bright gold-silver particles. The labeling intensity of acinar SG of parotid glands was higher than that of von Ebner’s glands. In parotid glands, weak labeling of SG in transitional cells between acini and intercalated ducts, very weak labeling of SG in intercalated ducts, and strong labeling of striated duct secretions were observed. In von Ebner’s glands, the secretions and some SG of interlobular ducts were strongly labeled compared to those of intralobular ducts and SG of acini. Less amylase was synthesized in von Ebner’s acini compared to parotid acini, whereas von Ebner’s ducts may secrete significantly more amylase to modify saliva than parotid ducts.

Necessity of the glossopharyngeal nerve in the maintenance of normal intake and ingestive bout size of corn oil by rats

Recent evidence in the literature suggests that signals carried by the glossopharyngeal nerve (GL), which supplies sensory and parasympathetic innervation of the posterior tongue, might be essential in the maintenance of normal gustatory responses to fat stimuli. Here, we report that GL transection (GLX) significantly decreased corn oil intake and preference in 23-h two-bottle tests relative to sham-operated controls (Sham). Drinking-pattern analysis of corn oil licking revealed that bout size, rather than the number of bouts initiated, was smaller in GLX than Sham rats. We also tested a range of glucose concentrations and found that total licks over daily 23-h sessions significantly decreased in GLX compared with Sham rats, but this difference failed to reach significance when intake or any bout parameter was measured. These results show that the signals in the GL normally contribute to processes involved with corn oil bout termination as opposed to bout initiation. GL-derived signals could potentially provide input to "reward" circuits in the ventral forebrain that could serve to maintain ingestion during a meal or, alternatively, could act at the level of the brain stem to attenuate the inhibitory potency of vagal signals, thus delaying the onset of satiation, or perhaps contribute to a cephalic phase reflex modulation of the gut. Parasympathetic efferents in the GL innervating the von Ebner's glands, which secrete lingual lipase, which is thought to break down corn oil into detectable ligands, could also be playing a role in driving corn oil intake. Whatever the mechanism, an intact GL is clearly necessary in maintaining normal intake of corn oil.

Gene expression profiles of the three major salivary glands in rats

The protein components of saliva reflect the condition of the whole body as well as the salivary glands. The aim of this study is to characterize the gene expression profiles in each of the rat major salivary glands-the parotid, submandibular, and sublingual glands. Gene expression was analyzed using DNA microarrays, and observed differences in expression of representative genes were confirmed by quantitative, real-time polymerase chain reaction. Among the glands, the contribution to the high expression of genes encoding various proteins, specifically mucin 10, proline-rich glycoproteins, proline-rich protein 2, proline-rich proteoglycans, cystatin 10, amylase, deoxyribonuclease I, and von Ebner's gland protein, was significantly greater in the parotid gland than the other glands. The submandibular and sublingual glands had similar gene expression profiles that differed from profile of the parotid gland. For example, the genes encoding mucin 19 and ovomacroglobulin were highly expressed only in the submandibular and sublingual glands. In summary, we characterized gene expression in the rat major salivary glands and provided basic information on salivary gland marker proteins.

흰쥐 혀침샘의 점액질에 미치는 N-Nitrosodimethylamine 독성에 대한 녹차의 완화 효과

Biology Education Major, Division of Science Education, College of Education, Pusan National University, Busan 609-735, KoreaAbstractThis study was performed to determine alleviating effects of green tea (GT) on the N-nitrosodimethylamine (NDMA) toxicity to mucins in the rat lingual salivary glands. Sprague-Dawley rats were divided into five groups: untreated (Control), one week-NDMA administrated (NDMA), one week-GT administrated after NDMA for one week (NDMAGT), one week-NDMA administrated with GT (GTNDMA), and one-week NDMA administrated with GT after GT for one week (GTGTNDMA). The mucin properties were elucidated by periodic acid Schiff (PAS) reaction, alcian blue (AB) pH 2.5, AB pH 2.5-PAS, aldehyde fuchsin (AF) pH 1.7-AB pH 2.5, and high iron diamine (HID)-AB pH 2.5 staining. The lingual von Ebner's glands of NDMA group showed some changes such as contraction and destruction of the serous acini, decrease and disappearance of the cytoplasmic granules, vacuolation of cytoplasm, and the mucigenous duct cells. Also, in the lingual mucous glands, enlarged lumens, fused acini, disappearance of granules, vacuolation of cytoplasm, and mucigenous duct cells were observed. All the GT administrated groups had a tendency to recover. GTGTNDMA group recovered almost up to the state of the control group. The lingual von Ebner's glands of NDMA group showed a decrease of neutral mucin and the lingual mucous glands showed a decrease of both neutral and acidic mucins decreased in comparison with control group, although mucous acini secreting strong sulfomucin decreased while those secreting non-sulfomucin (sialomucin) increased. All the GT administrated groups had a tendency to recover. The degree of recovery in the GTNDMA group was stronger than in NDMAGT group and that of the GTGTNDMA group was almost up to the state of the control group. Plus, lingual von Ebner's glands of GTGTNDMA group contained much more neutral mucin than in the control group. In conclusion, it is suggested that NDMA exhibit the toxicity which affects on the mucin properties and which green tea alleviates.Key words: green tea, epigallocatechin gallate, N-nitrosodimethylamine (NDMA), salivary glands, mucin

Shh and ROCK1 modulate the dynamic epithelial morphogenesis in circumvallate papilla development

In rodents, a circumvallate papilla (CVP) develops with dynamic changes in epithelial morphogenesis during early tongue development. Molecular and cellular studies of CVP development revealed that there would be two different mechanisms in the apex and the trench wall forming regions with specific expression patterns of Wnt11 and Shh. Molecular interactions were examined using in vitro organ culture with over-expression of Shh, important signalling molecules and various inhibitors revealed that there are two significant different mechanisms in CVP formation by Wnt11 and Shh expressions. Wnt, a well known key molecule to initiate taste papillae, would govern Rho activation and cytoskeleton formation in the apex epithelium of CVP. In contrast, Shh regulates the cell proliferation to differentiate taste buds and to invaginate the epithelium for development of von Ebner's gland (VEG). Based on these results, we suggest that these different molecular signalling cascades of Wnt11 and Shh would play crucial roles in specific morphogenesis and pattern formation of CVP during early mouse embryo development.

Antioxidant enzyme immunoreactivity in rat von Ebner gland after nickel treatment

Our study was designed to clarify the role of antioxidant enzymes in the rat von Ebner gland during acute nickel toxicity. After treatment with nickel acetate, we monitored ultrastructural alterations in acinar and ductal cells, immunohistochemical staining for glutathione peroxidase (GPx) and glutathione S-transferases (GST mu and GST pi), and immunoreactivity for malondialdehyde (MDA). Immunoreactivity for MDA was present only in the acinar cells, and it was enhanced at 3 h after Ni treatment. In contrast, immunoreactivities for GPx and GSTs did not change in acinar cells but significantly increased in ductal cells after Ni treatment. Cytoplasmic vacuoles increased in acinar cells at 3 h after Ni treatment, but they almost completely disappeared at 24 h. No morphological changes were observed in taste bud cells from Ni-treated rats. Because lipid peroxidation, as monitored by immunoreactivity for MDA, was only transiently increased in the acinar cells, the enhanced antioxidant enzyme immunoreactivity in ductal cells of the von Ebner gland plays a crucial role in the self-defense system against nickel toxicity in the rat oral cavity.

Synaptic Responses of Neurons Controlling the Parotid and von Ebner Salivary Glands in Rats to Stimulation of the Solitary Nucleus and Tract

Salivary secretion results from reflex stimulation of autonomic neurons via afferent sensory information relayed to neurons in the rostral nucleus of the solitary tract (rNST), which synapse with autonomic neurons of the salivatory nuclei. We investigated the synaptic properties of the afferent sensory connection to neurons in the inferior salivatory nucleus (ISN) controlling the parotid and von Ebner salivary glands. Mean synaptic latency recorded from parotid gland neurons was significantly shorter than von Ebner gland neurons. Superfusion of GABA and glycine resulted in a concentration-dependent membrane hyperpolarization. Use of glutamate receptor antagonists indicated that both AMPA and N-methyl-D-aspartate (NMDA) receptors are involved in the evoked excitatory postsynaptic potentials (EPSPs). Inhibitory postsynaptic potential (IPSP) amplitude increased with higher intensity ST stimulation. Addition of the glycine antagonist strychnine did not affect the amplitude of the IPSPs significantly. The GABA(A) receptor antagonist, bicuculline (BMI) or mixture of strychnine and BMI abolished the IPSPs in all neurons. IPSP latency was longer than EPSP latency, suggesting that more than one synapse is involved in the inhibitory pathway. Results show that ISN neurons receive both excitatory and inhibitory afferent input mediated by glutamate and GABA respectively. The ISN neuron response to glycine probably derives from descending connections. Difference in the synaptic characteristics of ISN neurons controlling the parotid and von Ebner glands may relate to the different function of these two glands.

Proteomic profiling of amniotic fluid in preterm labor using two-dimensional liquid separation and mass spectrometry

Objective. Simultaneous analysis of the protein composition of biological fluids is now possible. Such an approach can be used to identify biological markers of disease and to understand the pathophysiology of disorders that have eluded classification, diagnosis, and treatment. The purpose of this study was to analyze the differences in protein composition of the amniotic fluid of patients in preterm labor.Study design. Amniotic fluid was obtained by amniocentesis from three groups of women with preterm labor and intact membranes: (1) women without intra-amniotic infection/inflammation (IAI) who delivered at term, (2) women without IAI who delivered a preterm neonate, and (3) women with IAI. Intra-amniotic infection was defined as a positive amniotic fluid culture for microorganisms. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin (IL)-6 (≥2.3 ng/mL). Two-dimensional (2D) chromatography was used for analysis. The first dimension separated proteins by isoelectric point, while the second, by the degree of hydrophobicity. 2D protein maps were generated using different experimental conditions (reducing agents as well as protein concentration). The maps were used to discern subsets of isoelectric point/hydrophobicity containing differentially expressed proteins. Protein identification of differentially expressed fractions was conducted with mass spectrometry. Enzyme-linked immunosorbent assays (ELISA) as well as surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS)-based on-chip antibody capture immunoassays were also used for confirmation of a specific protein that was differentially expressed.Results. (1) Amniotic fluid protein composition can be analyzed using a combination of 2D liquid chromatography and mass spectrometry for the identification of proteins differentially expressed in patients in preterm labor. (2) While total insulin-like growth factor-binding protein-1 (IGFBP-1) concentration did not change, IGFBP-1 fragments at about 13.5 kDa were present in patients with IAI. (3) Proteins that were over-expressed in group 1 included von Ebner gland protein precursor, IL-7 precursor, apolipoprotein A1, tropomyosin sk1 (TPMsk1) fragment, ribosomal protein S6 kinase alpha-3, and alpha-1-microglobulin/bikunin precursor (AMBP). (4) Proteins that were over-expressed in group 3 included fibrinopeptide B, transferrin, major histocompatibility complex (MHC) class 1 chain-related A antigen fragment, transcription elongation factor A, sex-determining region Y (SRY) box 5 protein, Down syndrome critical region 2 protein (DSCR2), and human peptide 8 (HP8). (5) One protein, retinol-binding protein, was over-expressed in women who delivered preterm, regardless of the presence of IAI.Conclusions. A combination of techniques involving 2D chromatography, mass spectrometry, and immunoassays allows identification of proteins that are differentially regulated in the amniotic fluid of patients with preterm labor. Specifically, the amount of the IGFBP-1 fragments at approximately 13.5 kDa was found to be increased in patients with IAI, while the amount of the intact form of IGFBP-1 was decreased.

Ionotropic glutamate receptor expression in preganglionic neurons of the rat inferior salivatory nucleus

Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual–tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.

Perireceptor events in taste.

The microenvironment at chemical receptor sites is important for ligand-receptor interaction as it can influence the entry, residence time or exit of odorant and sapid molecules. The perireceptor milieu at apical taste cell microvilli consists of taste pore mucus and secretions from salivary glands. The majority of taste buds are sheltered in epithelial folds of the foliate and circumvallate papillae where saliva is provided predominantly by the lingual von Ebner's glands (VEGs). To investigate possible saliva-tastant interactions, we have characterized a prominent 18 kDa secretory protein expressed in human, rat and pig VEGs. The human and rat VEG proteins share 60% sequence identity and, by virtue of their protein and gene structure, can be assigned to the lipocalin superfamily of lipophilic ligand carrier proteins. VEG proteins might function as transporters of hydrophobic molecules, for example bitter substances, like the nasal odorant-binding proteins that belong to the same protein family. Because binding experiments using various bitter substances have so far failed, and in light of the species-specific expression, other functions for VEG proteins must be considered. These include the protection of taste epithelia, pheromone transport and lipid binding.

Effects of 5-Hydroxytryptamine and Substance P on Neurons of the Inferior Salivatory Nucleus

The parasympathetic secretomotor innervation of the salivary glands originates from a longitudinal column of neurons in the medulla called the salivatory nucleus. The neurons innervating the parotid and von Ebner salivary glands are situated in the caudal extremity of the column designated as the inferior salivatory nucleus (ISN). Immunocytochemical investigations have demonstrated the presence of a number of neuropeptides surrounding the ISN neurons. We have examined the neurophysiological effect of two of these neuropeptides on neurons of the ISN identified by retrograde transport of a fluorescent label. Both serotonin (5-HT) and substance P (SP) excited virtually all neurons in the ISN. Application of these neuropeptides resulted in membrane depolarization that was concentration dependent. Although the majority of ISN neurons that were depolarized by SP application exhibited an increase in input resistance, application of 5-HT induced widely varied change in input resistance. Membrane depolarization elicited action potential discharges that increased in frequency with increasing concentration of 5-HT and SP. Blocking action potential conduction from surrounding neurons did not eliminate the depolarizing effects of 5-HT and SP, indicating that both neuropeptides acted directly on the ISN neurons. Finally, the use of 5-HT agonists and antagonists indicates that 5-HT acts via a 5-HT(2A) receptor, and the use of SP agonists suggests that SP acts via neuokinin-1 and -2 receptors. These data show that 5-HT and SP excite most of the ISN neurons innervating the lingual von Ebner glands possibly modulating the synaptic drive to these neurons derived from afferent gustatory input.

Salivary histatins in human deep posterior lingual glands (of von Ebner)

Human saliva contains a family of low molecular weight histidine-rich proteins, named histatins, characterised by bactericidal and fungicidal activities in vitro against several microbial pathogens, such as Streptococcus mutans and Candida albicans. They represent a major component of an innate host non-immune defense system. In an earlier study we described the distribution of histatins in the glandular parenchyma of human major salivary glands, confirming that all human major salivary glands are involved in the secretion of histatins into saliva. In the present study we determined the expression and localisation of histatins in human posterior deep lingual glands (von Ebner's glands) by means of immunoelectron microscopy. Thin sections of normal human salivary glands, embedded in Epon resin, were incubated with rabbit polyclonal antibodies specific for human histatins and successively with a gold conjugated goat anti-rabbit IgG used as secondary antibody. Sections incubated with medium devoid of primary antibody or containing non-immune serum were used as controls. The serous secreting cells represented the main source of histatins in the glandular parenchyma of von Ebner's glands. At the electron microscopic level, labeling was associated with rough endoplasmic reticulum, Golgi complex and secretory granules that represented the main cytoplasmic site of histatin localisation. However, variability in the intensity of labeling was observed among adjacent cells. The present results show for the first time that human von Ebner's glands produce and represent a significant source of histatins, supporting the hypothesis of their important role in preventing microbial assaults on the tissues in the posterior region of the tongue and in the circumvallate papillae.

Association of Shh and Ptc with keratin localization in the initiation of the formation of circumvallate papilla and von Ebner’s gland

The development of gustatory papillae in mammalian embryos requires the coordination of a series of morphological events, such as proliferation, differentiation and innervation. In mice, the circumvallate papilla (CVP) is a specialized structure that develops in a characteristic spatial and temporal pattern in the posterior region of the tongue dorsal surface. The distinct expression patterns of Shh and Ptc, which play important roles in the development of other epithelial appendages, have been localized in the trench wall that gives rise to von Ebner's gland (VEG). To define the cellular mechanisms responsible for morphogenesis and differentiation during early development of CVP and VEG, the localization patterns of keratins (cytokeratins) K7, K8, K18, K19, K14 and connexin-43, which are dependent on Shh expression in other developmental systems, have been examined in detail. The distinct localization of keratins K7, K8, K18, K19, K14 and connexin-43 in the epithelium giving rise to the CVP and VEG suggests that cytodifferentiation is established prior to morphological changes. Interestingly, the localization of proliferating cell nuclear antigen, a marker for cell proliferation, is similar to that of Shh. An understanding of the regulatory roles of cell-cell interactions and signalling molecules in orchestrating a mutual network will bring us nearer to defining the molecular and cellular mechanisms underlying morphogenesis in mammalian taste bud development.

Comparative Proteomics of Nasal Fluid in Seasonal Allergic Rhinitis

A comparative proteomic approach was applied to examine nasal lavage fluid (NLF) from patients with seasonal allergic rhinitis (SAR, n = 6) and healthy subjects (controls, n = 5). NLF samples were taken both before allergy (pollen) season and during season, and proteins were analyzed by two-dimensional gel electrophoresis (2-DE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) after tryptic cleavage. Twenty proteins were selected and quantified. During allergy season, the levels of six sialylated isoforms of PLUNC (palate lung nasal epithelial clone) were lower in SAR patients than controls, as were the levels of six isoforms of von Ebner's gland protein (VEGP), including a previously undescribed form with N-linked glycosylation, and of cystatin S. PLUNC is a new innate immunity protein and VEGP and cystatin S are two endogenous proteinase inhibitors. By contrast, the levels of an acidic form of alpha-1-antitrypsin were higher in SAR patients than controls. One previously unidentified NLF protein was found in all samples from the SAR patients during allergy season but not in any sample before allergy season: this protein was identified as eosinophil lysophospholipase (Charcot-Leyden crystal protein/galactin 10). MS/MS analysis of the N-terminus of the protein showed removal of Met and acetylation of Ser. Altogether, these findings illustrate the potential use of proteomics for identifying protein changes associated with allergic rhinitis and for revealing post-translational modifications of such new potential markers of allergic inflammation.

Histological and biochemical characterization of von Ebner's glands in the Syrian hamster; comparison with rat von Ebner's glands

We report here for the first time a morphological description and observations on some of the secretory proteins of the von Ebner's lingual salivary glands (VEG) of the Syrian hamster. Hamster VEG were macroscopically less distinct, but histologically similar to rat VEG. VEG extracts of hamster and rat were assayed for lipase, α-amylase and peroxidase activities. Unlike rat VEG, which is rich in lipase activity, hamster VEG extract had no detectable lipase activity and did not react with antibodies to either rat lingual lipase or human gastric lipase in Western blots. Immunohistochemical reactions with the anti-rat lingual lipase antibody were very weak in hamster VEG and strong in rat VEG. Moderate α-amylase enzyme activities and immunohistochemical reactions were demonstrated in both hamster and rat VEG. Peroxidase activity was negligible in the VEG, unlike the high activity in the submandibular glands of both species. An 18 kDa von Ebner's gland protein (VEGP), a member of the lipocalin superfamily of hydrophobic ligandbinding proteins, was abundant in rat VEG, but not detected in hamster VEG. Thus, hamster VEG differs from rat VEG in macroscopic appearance and the absence of lipase and VEGP. It is similar to rat VEG histologically and with regard to the presence of α-amylase and absence of peroxidase.

Expression of IGFBPs in the developing mouse submandibular and von Ebner’s glands

The expression of insulin-like growth factor binding proteins (IGFBPs) in the developing mouse submandibular and von Ebner's glands was determined by in situ hybridization and by an immunohistochemical method. In the submandibular glands, IGFBP-2 and IGFBP-4 mRNAs were expressed in the terminal end-buds (TEB) at E13-E17, concomitant with epithelial branching. IGFBP-3 mRNA was expressed in the mesenchyme surrounding the TEB; and IGFBP-5 mRNA, in the ducts. At E17, IGFBP-5 mRNA expression was observed not only in the ducts but also in the TEB. Similarly, IGFBP-4 mRNA expression was observed not only in the TEB but also in the mesenchyme. After birth, IGFBP-4 expression was observed only in the connective tissue and disappeared by P14. That of IGFBP-7 appeared at P1 and was observed in the connective tissue until P21. The IGFBP-5 mRNA expression pattern after birth was the same as that seen at E17, but at P21 IGFBP-5 was immunohistochemically expressed only in the duct. The mRNA level of IGFBP-2 expression at postnatal days was weak, but its protein was detected in the ducts and acini at P14-P21. In von Ebner's glands, which appeared at the base of the circumvallate papillae at E17, only IGFBP-2 and IGFBP-4 mRNAs were expressed in the ducts and acini. Postnatally, IGFBP-4 was substituted by IGFBP-5 in the same region. Immunohistochemically, IGFBP-5 and IGFBP-2 were expressed in the ducts and acini at P14-P21. Throughout the study, IGFBP-6 was not detected by in situ hybridization, the immunoreactivity for it was observed in the nerve fibers of submandibular and von Ebner's glands. These data support a role for these molecules as local mediators of salivary growth and differentiation.