Influence of beta-carotene on lysosomal hydrolases and their natural substrates in major salivary glands of hamsters treated with 7,12-dimethylbenzanthracene (DMBA)

We evaluated the effects of beta-carotene, a precursor of vitamin A, on the activity of some lysosomal hydrolases and on the levels of their natural substrates in hamster major salivary glands during experimental oral 7,12-dimethylbenzanthracene (DMBA) carcinogenesis. Sixty-four hamsters (Cricetus auratus) were divided into four groups--group 1: untreated control; group 2: DMBA was painted three times a week in the left buccal pouch; group 3: beta-carotene was painted three times a week in the left buccal pouch; group 4: DMBA and beta-carotene were painted alternatively in the left buccal pouch. After 16 weeks, the animals were sacrificed and the activities of some lysosomal hydrolases and their natural substrates in the major salivary glands were measured. beta-Carotene when administered topically in DMBA treated animals (group 4) reduced the levels of the majority of enzymes and substrates closer to those of the untreated control group, thus outlining a mild protective effect of beta-carotene towards the DMBA carcinogenic stress. Nevertheless, the presence of some enzymes which responded negatively to the combined administration of DMBA and beta-carotene suggests the necessity for future studies on the effect of beta-carotene at different concentrations, the systemic administration and the possibility to combine the topical beta-carotene administration with other chemopreventive drugs.     

Comparative analysis of lysosomal hydrolases and natural glycoprotein substrates in the rat major salivary glands.

1. The activities of some lysosomal hydrolases and the concentrations of their natural substrates were studied in the submandibular and sublingual glands of male and female rats using biochemical procedures. 2. In sublingual gland enzyme activities and substrate concentrations show the highest values. 3. The enzyme activities appear, in general, lower and the natural substrate concentrations higher in the females with respect to males. 4. In both glands beta-galactosidase shows the highest activity and beta-glucosidase the lowest. 5. These findings suggest that metabolic turnover of glycoproteins is slower in females than in males, probably because the oestrogens control the activity of lysosomal hydrolases.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

Lysosomal enzymes in the rat harderian gland are altered by either bromocriptine treatment or hypophysectomy and hormone replacement therapy

Four groups of adult male hypophysectomized rats were injected subcutaneously twice daily between 0800-0900 hr and 1600-1700 hr with either saline diluent, 150 micrograms sheep prolactin and/or growth hormone (GH); intact rats received either saline or 150 micrograms bromocriptine twice daily. After 4 days of treatment, lysosomal enzyme assays revealed significant elevations in both acid phosphatase and alpha-mannosidase enzyme activities in the Harderian glands of saline-injected hypophysectomized rats compared to those in intact controls. beta-Glucuronidase levels were depressed and hexosaminidase activity unaffected by hypophysectomy treatment alone compared to intact controls. Lysosomal enzyme activities in hypophysectomized animals treated with prolactin were not different from the hypophysectomized control animals. However, treatment with GH alone or in combination with prolactin had a significant inhibitory effect on beta-glucuronidase, hexosaminidase, and alpha-mannosidase enzyme activities in the Harderian gland of hypophysectomized animals. Bromocriptine treatment in intact rats only elevated acid phosphatase activity. In summary, the patterns of responses did not reveal a role for prolactin in the control of Harderian gland lysosomal enzyme activities by the pituitary. However, some of the influence on this target system may be exerted by growth hormone.     

意大利蜜蜂工蜂唾腺和中肠消化酶活性比较研究

对意大利蜜蜂 Apis mellifera ligustica L工蜂唾腺和中肠中淀粉酶、蛋白酶、蔗糖酶、果胶酶和海藻糖酶5种消化酶进行活性测定,发现巾肠的蛋门酶和蔗糖酶活性较唾腺高,有显著性差异;唾腺的果胶酶活性较中肠高,有显著性差异;唾腺与中肠的淀粉酶和海藻糖酶没有显著性差异.表明意大利蜜蜂工蜂对蔗糖和蛋白的消化主要由中肠分泌的消化酶来完成,唾腺起辅助作用;对果胶的消化主要在唾腺,中肠起辅助作用;而在淀粉和海藻糖的消化过程中唾腺和中肠都有很重要的作用.     

Sulfatides and arylsulfatase A activity in major salivary glands of hamster (Mesocricetus auratus) after adenocarcinoma induction in oral cavity

A biochemical study of sulfatides and arylsulfatase A (ASA) was carried out in the submandibular and sublingual glands of the male and female hamster Mesocricetus auratus after experimental induction of oral adenocarcinoma by 7,12-dimethylbenzanthracene (DMBA). Hamster experimental groups included control animals, animals treated with β-carotene, animals treated with DMBA, and animals treated with DMBA plus β-carotene. Oral cavity treatment with DMBA induced carcinogenesis in the buccal mucosa, but not in the major salivary glands, where nevertheless, the morphology and expression of both parameters examined changed. In fact, sulfatide concentrations and enzyme activity increased significantly, while in control and β-carotene-treated hamsters they were similar in both glands and sexes. After administration of DMBA plus β-carotene, sulfatide concentration decreased, as did ASA activity, slightly in the submandibular gland and remarkably so in the sublingual one of female hamsters. Thin-layer chromatography (TLC) analysis of lipid patterns, after DMBA treatment, revealed considerable differences, not only in sulfatides, but also in other lipid fractions, as well as between the two glands and two sexes. These findings show that oral cavity treatment with DMBA is not able to induce carcinogenesis in the major salivary glands examined; however, it does cause considerable metabolic changes.     

Alteration of Ca2+‐ATPase activity in the homogenate,plasma membrane and microsomes of the salivary glands of streptozotocin‐induced diabetic rats

Diabetes has been implicated in the dryness of the mouth, loss of taste sensation, sialosis, and other disorders of the oral cavity, by impairment of the salivary glands. The aim of the present study was to examine the plasma membrane, microsomal, and homogenate Ca²⁺‐ATPase activity in the rat submandibular and parotid salivary glands of streptozotocin‐induced diabetes. We have also examined the influence of the acidosis state on this parameter. Diabetes was induced by an intraperitoneal injection of streptozotocin and acidosis was induced by daily injection of NH₄Cl. At 15 and 30 days after diabetes induction, the animals were euthanized and the submandibular and parotid salivary glands were removed and analyzed. Ca²⁺‐ATPase (total, independent, and dependent) was determined in the homogenate, microsomal, and plasma membranes of the salivary glands of diabetic and control rats. Calcium concentration was also determined in the glands and showed to be higher in the diabetic animals. Ca²⁺‐ATPase activity was found to be reduced in all cell fractions studied in the diabetic animals compared with control. Similar results were obtained for the submandibular salivary glands of acidotic animals; however in the parotid salivary glands it was found an increase in the enzyme activity. Copyright © 2009 John Wiley & Sons, Ltd.     

Influence of streptozotocin-induced diabetes on hexokinase activity of rat salivary glands

The influence of diabetes on the enzyme hexokinase (HK) was examined in the salivary glands of rats. Diabetes was induced by an intraperitoneal injection of streptozotocin (60 mg/Kg body weight) in overnight fasted rats (180-200 g). The animals were killed 48 hours and 30 days after the induction of diabetes and the submandibular and parotid salivary glands extracted for use. Hyperglycemia was evaluated by determining the blood sugar. The area occupied by each intralobular component, acini, ducts, total parenchyma and stroma was measured, and no differences were observed compared with control. In the soluble fraction of the submandibular gland, no difference in the specific activity of HK was observed, between the diabetic and control animals, however, the activity per gland and per g of tissue showed lower values than control. The specific activity of the bound form was reduced in the diabetic gland. The results obtained for the parotid gland were different from the submandibular. The specific activity of both the soluble and bound forms were increased in the diabetic animals. The DEAE-cellulose column chromatography of the soluble and bound forms of the enzyme from both glands showed a first peak appearing during the washing of the column and two other peaks were eluted by the gradient. Thus, three isoenzymes in the submandibular and parotid salivary glands for the control and diabetic rats have been found.     

Molecular cloning of trypsin-like cDNAs and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug Lygus lineolaris (Heteroptera: Miridae)

Using specific proteinase inhibitors, we demonstrated that serine proteinases in the tarnished plant bug, Lygus lineolaris, are major proteinases in both salivary glands and gut tissues. Gut proteinases were less sensitive to inhibition than proteinases from the salivary glands. Up to 80% azocaseinase and 90% of BApNAse activities in the salivary glands were inhibited by aprotinin, benzamidine, and PMSF, whereas only 46% azocaseinase and 60% BApNAse activities in the gut were suppressed by benzamidine, leupeptin, and TLCK. The pH optima for azocaseinase activity in salivary glands ranged from 6.2 to 10.6, whereas the pH optima for gut proteinases was acidic for general and alkaline for tryptic proteinases. Zymogram analysis demonstrated that approximately 26-kDa proteinases from salivary glands were active against both gelatin and casein substrates. Three trypsin-like cDNAs, LlSgP2-4, and one trypsin-like cDNA, L1GtP1, were cloned from salivary glands and gut, respectively. Putative trypsin precursors from all cloned cDNAs contained a signal peptide, activation peptide, and conserved N-termini (IVGG). Other structural features included His, Asp, and Ser residues for the catalytic amino acid triad of serine proteinase active sites, residues for the binding pocket, and four pairs of cysteine residues for disulfide bridges. Deduced trypsin-like proteins from LlSgP2, LlSgP3, and LlGtP1 cDNAs shared 98-99% sequence identity with a previously reported trypsin-like precursor, whereas the trypsin-like protein of LlSgP4 shared only 44% sequence identity with all other trypsin-like proteins, indicating multi-trypsin forms are present in L. lineolaris.     

Characteristics of sialidase in the rat salivary glands

Using 4-methylumbelliferyl-N-acetylneuraminic acid (4MU-NeuAc) as substrate, we measured sialidase activity in the salivary glands and other organs of the rat. The pH optima of salivary gland sialidase were between 4.0 and 4.5, which were similar to those of the enzyme in the brain, liver and kidney. Among the salivary glands, the submandibular one showed the highest sialidase activity followed by the parotid and the sublingual glands. However, sialidase activity in these glands was lower when compared with the activity in the brain, liver and kidney. From the subcellular distribution study, salivary gland sialidase was found to be mainly localized in the lysosomes. The pH optima of the lysosomal sialidase of the salivary glands were between 4.0 and 4.5; and Km values for 4MU-NeuAc approximately 0.09 mmol/l. In the submandibular and parotid glands, a soluble sialidase with a different pH optimum (5.5) and Km value (0.25 mmol/l) was also detected.     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.     

Chilled Galleria mellonella: Salivary glands may mechanically inhibit development

The possible rôle of the stiffened salivary glands in inhibiting metamorphosis of last-instar chilled Galleria mellonella was explored. Inhibitory effects of chilling on the salivary glands and delayed development of the whole animal were clearly correlated. In animals less than 3 days post-ecdysis, the salivary glands accounted for less than 4% of the total body weight; chilling these animals resulted in an increased number of supernumerary apolyses and accelerated developmental time. From 4 to 10 days post-ecdysis, when the salivary glands accounted for 5% or more of total body weight, chilling significantly delayed larval-pupal ecdysis. Reduction of the dimensions of the salivary glands by prior space deprivation significantly reduced the effects of chilling. Extirpation of the salivary glands eliminated the chill effect, whereas insertion of a 7 mm piece of tubing into the body mimicked the effect. These results suggest that chill-stiffened salivary glands may mechanically inhibit development.     

Substrate specificity of kallikrein-related peptidase 13 activated by salts or glycosaminoglycans and a search for natural substrate candidates

KLK13 is a kallikrein-related peptidase preferentially expressed in tonsils, esophagus, testis, salivary glands and cervix. We report the activation of KLK13 by kosmotropic salts and glycosaminoglycans and its substrate specificity by employing a series of five substrates derived from the fluorescence resonance energy transfer (FRET) peptide Abz-KLRSSKQ-EDDnp. KLK13 hydrolyzed all these peptides only at basic residues with highest efficiency for R; furthermore, the S₃ to S₂′ subsites accepted most of the natural amino acids with preference also for basic residues. Using a support-bound FRET peptide library eight peptide substrates were identified containing sequences of proteins found in testis and one with myelin basic protein sequence, each of which was well hydrolyzed by KLK13. Histatins are salivary peptides present in higher primates with broad antifungal and mucosal healing activities that are generated from the hydrolysis from large precursor peptides. KLK13 efficiently hydrolyzed synthetic histatin 3 exclusively at R²⁵ (DSHAKRHHGYKRKFHEKHHSHRGYR²⁵↓SNYLYDN) that is the first cleavage observed inside the salivary gland.In conclusion, the observed hydrolytic activities of KLK13 and its co-localization with its activators, glycosaminoglycans in the salivary gland and high concentration of sodium citrate in male reproductive tissues, indicates that KLK13 may play a role in the defense of the upper digestive apparatus and in male reproductive organs.     

Cytochemical demonstration of enzymatic activities of the parotid, submandibular, and sublingual glands of Praomys (Mastomys) Natalensis

Histochemical techniques were applied to salivary glands removed from adult multimmate rodents (Praomys) of either sex to detect and localize the following enzymatic activities: acid and alkaline phosphatase, arylsulphatase, ali-esterases, beta-glucuronidase, N-acetyl-betaglucosaminidase, and L-leucyl-aminopeptidase. No reaction was observed for alkaline phosphatase and glucuronidase. The glands reacted differently to the other enzymatic activities. Alkaline phosphatase and glucosaminidase were present only in one glandular type whereas arysulphatase and esterases were present in all types although demonstrating a variable staining intensity in different glands. Sharp differences in some enzymatic activities of the submandibular and parotid glands were related to the sex of the animal.     

Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutus (Hemiptera: Miridae)

Protease activities in the secreted saliva, salivary glands and midgut of the green mirid, Creontiades dilutus, were investigated. The saliva and salivary glands had more protease activity than the midgut, but no differences in protease activity levels were detected between male and female mirids, adult mirids and third instar nymphs, or between fed and starved mirids.In the salivary glands, chymotrypsin-like serine proteases predominated, as characterised by inhibitor specificity, basic pH optima, and hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide and N-succinyl-ala-ala-pro-leu p-nitroanilide. The pH optimum of midgut extracts was acidic (pH 4), implying that acidic proteases predominate. However, protease activity was inhibited substantially by both aprotinin and E-64, suggesting the presence of both serine and cysteine proteases in the midgut of the green mirid.     

Phenomenon of hypertrophy of the testes after amputation of the lower incisors in rats]

Repeated amputation of the lower incisors in rats carried out at the early postnatal on togenesis causes an enlargement of the salivary glands and hypertrophy of the testis with the phenomena of acceleration of their differentiation. Hypertrophy of the testes withthe mentioned action was much greater (76%) than in the case of unilateral castration at the same age. In adult animals repeated amputation of the lower incisors caused hypertrophy of the salivary glands, but failed to influence the hypertrophy of the testes.     

Permeability of Oral Tissues to Blood-borne Coxsackievirus B-1

The ability of coxsackievirus B-1 to pass the barriers of the circulatory system into whole saliva has been shown previously. In this investigation, the major salivary glands and the oral mucosa were studied, and their role as participants in the excretion of coxsackievirus B-1 during viremia was evaluated. The effect of the salivary-gland stimulant pilocarpine nitrate on both the salivary flow rate and the recovery of virus during viremia was determined. A comparison was made between the amount of virus recovered from whole saliva during viremia in animals deficient in one or both of the major salivary-gland pairs and animals with a complete complement of salivary glands. The salivary glands in other animals were cannulated, and pure glandular secretions were collected during viremia and assayed for the presence of virus The amount of virus passing from the capillaries of the oral mucosa to the surface was also determined to evaluate this route as a possible site for the excretion of virus into saliva during viremia. The major salivary glands did not excrete appreciable quantities of virus during viremia. The submaxillary-gland secretions did not contain virus, and the parotid-gland secretions showed virus only at extremely high blood virus levels. Either removal of the major salivary glands or decreased salivary flow rates increased the concentration of virus in whole saliva. This observation suggested that the production of saliva by the major salivary glands tends to dilute the virus in the oral cavity. A 0.88-cm² sample of the oral mucosa excreted significantly large amounts of virus during viremia and suggested that the passage of virus through the oral mucosa was the major route for the excretion of virus into saliva during viremia.     

Antioxidant enzymatic defense in salivary glands of streptozotocin‐induced diabetic rats: a temporal study

Hyperglycemia induces overproduction of superoxide and it is related to diabetic complications. In this study, we analyzed the antioxidant enzymatic defense and the lipid peroxidation of rat salivary glands in six different periods of diabetic condition. Ninety‐six rats were divided into 12 groups: C7/14/21/28/45/60 (non‐diabetic animals) and D7/14/21/28/45/60 (diabetic animals). Diabetes was induced by streptozotocin and the rats were euthanized after 7, 14, 21, 28, 45, or 60 days. Their parotid (PA) and submandibular (SM) glands were removed soon after the sacrifice and the total protein and malondialdehyde (MDA) concentrations, as well as, the superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) activities were determined. Twenty‐one days after the diabetes induction, the SM glands showed an increase in SOD, CAT, and GPx activities, as well as, MDA concentration. Concerning the PA glands, an increase in the CAT activity and MDA content was observed throughout the observation period. The results suggest that diabetes can cause alterations on the salivary glands and that PA and SM glands react differently when exposed to diabetes condition. However, no impairment of antioxidant system was observed in the group whose diabetic condition had been induced 60 days earlier, herein named 60‐day group. Copyright © 2010 John Wiley & Sons, Ltd.     

Morphology and electrophoretic protein profiles of female salivary glands in four Oriental black fly species (Diptera: Simuliidae).

Saliva of female flies is responsible for localized hypersensitivity reactions and life-threatening systemic hemorrhagic syndromes in humans and animals. In this study, morphology and electrophoretic protein profiles of female salivary glands of Oriental black flies in the subgenus Simulium Latreille s. str., Simulium (Simulium) nigrogilvum, S. (S.) rufibasis, S. (S.) nodosum, and subgenus Gomphostilbia Enderlein, S. (Gomphostilbia) asakoae were analyzed. The paired female salivary glands of the four simuliid species were morphologically similar and situated on either side of the esophagus. Each gland is composed of two main parts, a secretory arm and a reservoir. In each species, the size of the gland correlated with salivary gland protein contents. SDS-PAGE analysis revealed differences of electrophoretic protein profiles and specifically major protein bands of the female salivary glands in each species, suggesting that protein profiles might be useful for construction of an additional tool to distinguish these black fly species. The information obtained from this study is an initial step for further research on salivary proteins that are involved in vertebrate hemostatic response.     

The effect of the physiological age of Dermacentor marginatus (Ixodidae) ticks on their infection with and the infiltration of the tick-borne encephalitis virus into the saliva

The decisive effect of physiological age of ticks on the infection of their saliva was revealed by means of parenteral infection of D. marginatus females with tick-borne encephalitis virus. The virus was not found in the saliva of young individuals of instar II. Maximum number of cases of saliva infection was recorded in mature ticks of instar III (50%). It was established that the infection of saliva did not depend on the amount of virus in the tick's body. Less susceptibility to the virus of young individuals, associated probably with the way of infection, was noted. Under light microscopy there were observed no significant age differences in salivary glands of ticks of instars II and III. The infection of saliva of young individuals is, apparently, opposed by the barrier of fat body on the way of virus to salivary glands, depending on the age of ticks. The barrier is supposed to have an influence under natural transphase infection of ticks.