Purification and characterization of human salivary carbonic anhydrase

A novel carbonic anhydrase was purified from human saliva with inhibitor affinity chromatography followed by ion-exchange chromatography. The molecular weight was determined to be 42,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the human salivary enzyme is larger than the cytosolic isoenzymes CA I, CA II, and CA III (Mr 29,000) from human tissue sources. Each molecule of the salivary enzyme had two N-linked oligosaccharide chains which were cleaved by endo-beta-N-acetylglucosaminidase F but not by endo-beta-N-acetylglucosaminidase H, indicating that the oligosaccharides are complex type. The isoelectric point was determined to be 6.4, but significant charge heterogeneity was found in different preparations. The human salivary isozyme has lower specific activity than the rat salivary isozyme and the human red blood cell isozyme II in the CO2 hydratase reaction. The inhibitory properties of the salivary isozyme resemble those of CA II with iodide, sulfanilamide, and bromopyruvic acid, but the salivary enzyme is less sensitive to acetazolamide and methazolamide than CA II. Antiserum raised in a rabbit against the salivary enzyme cross-reacted with CA II from human erythrocytes, indicating that human salivary carbonic anhydrase and CA II must share at least one antigenic site. CA I and CA III did not crossreact with this antiserum. The amount of salivary carbonic anhydrase in the saliva of the CA II-deficient patients was greatly reduced, indicating that the CA II deficiency mutation directly or indirectly affects the expression of the salivary carbonic anhydrase isozyme. From these results we conclude that the salivary carbonic anhydrase is immunologically and genetically related to CA II, but that it is a novel and distinct isozyme which we tentatively designate CA VI.     

Adiponectin: Serum-saliva associations and relations with oral and systemic markers of inflammation

This study addresses gaps in our understanding about the validity and utility of using salivary adiponectin to index serum adiponectin levels. Matched blood and saliva samples were collected on a single occasion from healthy adults (n = 99; age 18–36 years, 53% male). Serum and saliva was assayed for adiponectin and inflammatory cytokines (IL-1β, IL-6, IL-8, TNFα), and saliva was also assayed for markers of blood contamination (transferrin), total protein (salivary flow rate) and matrix metalloproteinase-8 (MMP-8). We examined the extent to which salivary adiponectin was associated with serum adiponectin, and the influence of potential confounders on the serum-saliva correlation, including age, sex, body mass index, and markers of inflammation, oral health, salivary blood contamination, and flow rate. Findings revealed a modest serum-saliva association for adiponectin, and strong positive associations between salivary adiponectin and salivary levels of inflammatory cytokines, MMP-8, transferrin, and total protein. By contrast, salivary adiponectin was not related to serum levels of inflammatory activity. The magnitude of the serum-saliva association was strengthened when controlling for total protein in saliva, blood leakage into oral fluid, salivary inflammatory cytokines, and MMP-8. The pattern of findings extends our understanding of salivary adiponectin and its potential use as an index of circulating adiponectin levels.     

Characterization and identification of exflagellation-inducing factor in the salivary gland of Anopheles stephensi (Diptera: Culicidae)

Gamete activation factor (GAF) induces exflagellation of Plasmodium microgametes. We found GAF in the salivary glands of female mosquitoes, Anopheles stephensi. The exflagellation was induced in a concentration-dependent manner in the supernatant of salivary gland's crude homogenate. The exflagellation-inducing activity in the salivary gland was higher than that in the midgut and the head. GAF in the salivary glands was found to be heat stable and low molecular weight (<3000 molecular weight). Analysis of the supernatant by capillary electrophoresis and UV absorbance profile showed that the salivary glands contained xanthurenic acid, which was previously identified as GAF in the head of A. stephensi. The exflagellation-inducing activity in the salivary gland declined immediately after a blood meal, implying that GAF was in the saliva, and was delivered into the midgut together with the blood and induced exflagellation in the midgut.     

Immunohistochemical localization of estradiol, progesterone, and progesterone receptor in human salivary glands and salivary adenoid cystic carcinomas.

Immunohistochemical analyses of estradiol, progesterone and progesterone receptor were carried out in human salivary gland and salivary adenoid cystic carcinoma. Immunoreactivity to estradiol and progesterone was found in cytoplasm of the cells of the excretory duct system within normal salivary glands, whereas the progesterone receptor was restricted to nuclei of the cells where both sex steroids were positive. In addition, we demonstrated the presence of both sex steroids and the receptor for progesterone in salivary adenoid cystic carcinomas. These data indicate that the human salivary gland is one of the target tissues of estrogen. This also suggests the good possibility that tumors which express progesterone receptors will respond to endocrine therapy.     

Delayed inhibition of gap-junctional intercellular communication in the acinar cells of rat submandibular glands induced by parasympathectomy and cholinergic agonists

In this study, the effects of parasympathectomy and cholinergic agonists on gap-junctional intercellular communication and salivary secretion were investigated to clarify the involvement of salivary secretion in delayed uncoupling between acinar cells of rat submandibular glands. Gap-junctional intercellular communication was monitored as dye-coupling in the acinar cells of isolated acini by the transfer of Lucifer Yellow CH. Parasympathectomy induced dye-uncoupling in the acinar cells isolated from denervated salivary glands 12 hr after parasympathectomy-induced salivary secretion. Intraperitoneal application of carbachol (CCh), acetylcholine, pilocarpine, but not isoproterenol, stimulated salivary secretion, and then induced dye-uncoupling in the acinar cells 12 hr later. Atropine suppressed both the salivary secretion and delayed dye-uncoupling induced by parasympathectomy and CCh, when atropine was applied intraperitoneally before the induction of salivary secretion. However, atropine did not suppress the delayed dye-uncoupling by intraperitoneal application of CCh, when atropine was injected after the cessation of CCh-induced secretion. These results suggest that delayed inhibition of gap-junctional intercellular communication by parasympathectomy and cholinergic agonists in rat submandibular glands might be related to the change of secretory function after salivary secretion.     

Adsorption from saliva to silica and hydroxyapatite surfaces and elution of salivary films by SDS and delmopinol

The adsorption of proteins from human whole saliva (HWS) onto silica and hydroxyapatite surfaces (HA) was followed by quartz crystal microbalance with dissipation (QCM-D) and ellipsometry. The influence of different surface properties and adsorption media (water and PBS) on the adsorption from saliva was studied. The viscoelastic properties of the salivary films formed on the solid surfaces were estimated by the use of the Voigt-based viscoelastic film model. Furthermore, the efficiency of SDS and delmopinol to elute the adsorbed salivary film from the surfaces was investigated at different surfactant concentrations. A biphasic kinetic regime for the adsorption from saliva on the silica and HA surfaces was observed, indicating the formation of a rigidly coupled first layer corresponding to an initial adsorption of small proteins and a more loosely bound second layer. The results further showed a higher adsorption from HWS onto the HA surfaces compared to the silica surfaces in both adsorption media (PBS and water). The adsorption in PBS led to higher adsorbed amounts on both surfaces as compared to water. SDS was found to be more efficient in removing the salivary film from both surfaces than delmopinol. The salivary film was found to be less tightly bound onto the silica surfaces since more of the salivary film could be removed with both SDS and delmopinol compared to that from the HA surface. When adsorption took place from PBS the salivary layer formed at both surfaces seemed to have a similar structure, with a high energy dissipation implying that a softer salivary layer is built up in PBS as opposed to that in water. Furthermore, the salivary layers adsorbed from water solutions onto the HA were found to be softer than those on silica.     

Secretory and immunocompetent functions of the parotid and submaxillary salivary glands after thymectomy]

The dynamics of morphological, enzymatic and immunologic salivary glands indices in pubertal rates was investigated after thymectomy. The data obtained analysis demonstrated the functional salivary glands reconstruction, having immunologic and secretory characteristics: stimulation of the humoral and cellular immunity factors; reduction of the albuminous and rise of the mucous salivary glands component represented the remote effect of thymectomy /3 months]. Activation of the salivary glands immune characteristics may be explained by their belonging to the thymic-peripharyngeal complex.     

Morphological and Histochemical Study of Cephalic Glands of Bothrops jararaca (Ophidia,Viperidae)

Morphological and histochemical studies of the cell types in the cephalic glands of Bothrops jararaca have been performed. It is concluded: 1) mucous cells are found in the salivary labial, accessory glands; mucous-serous cells are found in the salivary labial, accessory and Harderian glands; serous-mucous cells are found only in the venom gland; 2) neutral mucosubstances and protein were found in the salivary labial, venom, accessory and Harderian glands; 3) hyaluronic acid was detected in the Harderian gland; 4) of the to sulfated acid mucosubstances, only chondroitin sulfate B was detected in the salivary labial and accessory glands; 5) sialic acid was detected in the salivary labial, accessory and Harderian glands.     

The monitoring of the menstrual status of female athletes by salivary steroid determination and ultrasonography

This study was designed to evaluate whether traditional plasma hormone determinations can be adequately replaced by measurements of salivary hormones. Eleven young sportswomen with menstrual irregularities attributed to strenuous physical exercise participated in this study. Mean body weight expressed as a percentage of ideal body weight was 92%, SD 4%. Their mean weekly training distance was 35 km, SD 15. Basal plasma endocrinological measurements revealed a hypo-oestrogenic status (mean plasma oestradiol values: 22 pg.ml-1, SD 8.8), and a deficient luteal phase (mean plasma progesterone: 2.9 ng.ml-1, SD 2.1). Pre-exercise salivary sex steroids were low. Salivary progesterone levels were 39.3 pg.ml-1, SD 9.5 (normal ranges in saliva: 25-60 pg.ml-1), salivary oestrone (E1) was 12.2 pg.ml-1, SD 2.3 (normal ranges in saliva: 7.5-25 pg.ml-1), and salivary oestradiol (E2) less than 1.9 pg.ml-1, SD 1.1 (normally 1.0-10.0 pg.ml-1). After a 21-km run, all salivary steroids appeared to increase. Mean salivary testosterone levels increased by 15.2% and salivary progesterone by 14.8%. Mean salivary oestrogens also increased (E1: +13.9%; E2: +21.1%). These findings confirm the results of earlier studies which found higher post-exercise plasma sex steroid levels. Since salivary measurements are believed to reflect non-protein-bound, thus free steroid levels, the results obtained by these techniques may provide a more realistic picture of the hormonal effects of physical exercise. In future, more accurate, cost-effective and easier techniques for salivary measurements may offer additional advantages.     

Measurement of salivary resistin, visfatin and adiponectin levels

Hormonal determination in saliva offers several advantages. Peptides enter the salivary glands either by active transport mechanisms or are expressed and secreted by the salivary glands themselves. The collection of saliva is a noninvasive, easily repeatable and less stressful technique than blood withdrawal. The purpose of the present study was to introduce a method for measuring salivary resistin, visfatin and adiponectin levels and to evaluate their associations with serum levels. Resistin, visfatin and adiponectin levels were measured in serum and saliva of 50 healthy adult volunteers (17 male and 33 female) using commercial enzyme immunoassay kits for serum with minor modifications. The present study documented the determination of resistin and adiponectin levels in saliva and the significant correlation of salivary levels with serum levels (r=0.441, p<0.01 and r=0.347, p<0.05, respectively). Moreover, the identification of visfatin in saliva was achieved, but no significant correlation with serum visfatin levels was observed. To our knowledge, this is the first study to report the determination of resistin and visfatin in saliva and the significant correlation of salivary resistin with serum levels, while it confirmed the significant association between salivary and serum adiponectin. The introduction of salivary determinations of adipokines could contribute to the elucidation of the physiology and the role of the specific adipokines in various clinical conditions (obesity, insulin resistance, inflammation, reproduction, energy imbalance and stress response).     

Salivary aldehyde dehydrogenase - temporal and population variability, correlations with drinking and smoking habits and activity towards aldehydes contained in food

Fluorimetric method based on oxidation of the fluorogenic 6-methoxy-2-naphthaldehyde was applied to evaluate temporal and population variability of the specific activity of salivary aldehyde dehydrogenase (ALDH) and the degree of its inactivation in healthy human population. Analyzed was also its dependence on drinking and smoking habits, coffee consumption, and its sensitivity to N-acetylcysteine. Both the specific activity of salivary ALDH and the degree of its inactivation were highly variable during the day, with the highest activities recorded in the morning hours. The activities were also highly variable both intra- and interpersonally, and negatively correlated with age, and this correlation was stronger for the subgroup of volunteers declaring abstinence from alcohol and tobacco. Moderately positive correlations of salivary ALDH specific activity with alcohol consumption and tobacco smoking were also recorded (r(s) ~0.27; p=0.004 and r(s) =0.30; p=0.001, respectively). Moderate coffee consumption correlated positively with the inactivation of salivary ALDH, particularly in the subgroup of non-drinking and non-smoking volunteers. It was found that mechanical stimulation of the saliva flow increases the specific activity of salivary ALDH. The specific activity of the salivary ALDH was strongly and positively correlated with that of superoxide dismutase, and somewhat less with salivary peroxidase. The antioxidant-containing drug N-acetylcysteine increased activity of salivary ALDH presumably by preventing its inactivation in the oral cavity. Some food-related aldehydes, mainly cinnamic aldehyde and anisaldehyde, were excellent substrates of the salivary ALDH3A1 enzyme, while alkenals, particularly those with short chain, were characterized by lower affinity towards this enzyme but high catalytic constants. The protective role of salivary ALDH against aldehydes in food and those found in the cigarette smoke is discussed, as well as its participation in diminishing the effects of alcohol- and smoking-related oxidative stress.     

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe): possible digestion of symbiont bacteria transferred by trophallaxis

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood‐feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.     

性别及日龄对转人神经生长因子基因小鼠唾液中蛋白分泌的影响

神经生长因子(Nerve growth factor,NGF)是一种能促进神经元发育、分化、再生的蛋白。为高效生产药效更佳的人源NGF (hNGF)药物,最近,笔者实验室构建出唾液腺特异表达hNGF的转基因小鼠,并从该转基因小鼠唾液中纯化获得具有高生物学活性的h NGF蛋白。为了选择性别和日龄最适宜的转hNGF基因小鼠用于收集纯化hNGF蛋白,文中比较了28日龄(性成熟前)雄性、雌性,63日龄(性成熟后)雄性、雌性转hNGF基因小鼠,共4组转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液鼠源NGF (mNGF)蛋白量和唾液h NGF蛋白量等指标。结果显示,63日龄的转hNGF基因小鼠分泌的唾液量、唾液总蛋白量、唾液mNGF蛋白量和唾液hNGF蛋白量显著高于28日龄同一性别的转hNGF基因小鼠,且63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF蛋白量显著高于同一日龄的雌性转hNGF基因小鼠;在4组小鼠中,63日龄的雄性转hNGF基因小鼠分泌的唾液hNGF含量最高,比28日龄雌性转hNGF基因小鼠高出约46倍,最适宜用于收集唾液并从中纯化hNGF。     

斯氏按蚊生长发育中唾液腺配子体激活因子的变化

为探讨斯氏按蚊生长发育过程中唾液腺抽提物配子体激活因子的消长,应用体外雄配子体出丝观察方法比较羽化后未吸血及吸血组雌性斯氏按蚊唾液腺抽提物中配子体激活因子对柏氏疟原虫雄配子体出丝诱导活性的动态变化。羽化后未吸血组按蚊唾液腺配子体激活因子活性与按蚊生长、发育呈同步变化。羽化后当日至羽化后第6 d,吸血组按蚊唾液腺抽提物的GAF活性变化与未吸血组相似,吸血后该组GAF活性下降,羽化后14 d恢复到吸血前水平。吸血后斯氏按蚊唾液腺配子体激活因子活性的降低与蚊卵发育可能相关。     

Tec29 controls actin remodeling and endoreplication during invagination of the Drosophila embryonic salivary glands

Epithelial invagination is necessary for formation of many tubular organs, one of which is the Drosophila embryonic salivary gland. We show that actin reorganization and control of endocycle entry are crucial for normal invagination of the salivary placodes. Embryos mutant for Tec29, the Drosophila Tec family tyrosine kinase, showed delayed invagination of the salivary placodes. This invagination delay was partly the result of an accumulation of G-actin in the salivary placodes, indicating that Tec29 is necessary for maintaining the equilibrium between G- and F-actin during invagination of the salivary placodes. Furthermore, normal invagination of the salivary placodes appears to require the proper timing of the endocycle in these cells; Tec29 must delay DNA endoreplication in the salivary placode cells until they have invaginated into the embryo. Taken together, these results show that Tec29 regulates both the actin cytoskeleton and the cell cycle to facilitate the morphogenesis of the embryonic salivary glands. We suggest that apical constriction of the actin cytoskeleton may provide a temporal cue ensuring that endoreplication does not begin until the cells have finished invagination.