Oxidative Damage to the Salivary Glands of Rats with Streptozotocin-Induced Diabetes-Temporal Study: Oxidative Stress and Diabetic Salivary Glands.

Objective. This study evaluated oxidative damage caused to the salivary glands in streptozotocin-induced diabetes (DM). Materials and Methods. Rats were divided into 4 groups: groups 1 and 2, control rats, and groups 3 and 4, DM rats. 8-Hydroxy-2'-deoxyguanosine (8-OHdG), protein carbonyl (PC), 4-hydroxynonenal protein adduct (4-HNE), oxidized and/or MDA-modified LDL-cholesterol (oxy-LDL/MDA), 8-isoprostanes (8-isoP), and oxidative stress index (OSI) were measured at 7 (groups 1 and 3) and 14 (groups 2 and 4) days of experiment. Results. The unstimulated salivary flow in DM rats was reduced in the 2nd week, while the stimulated flow was decreased throughout the duration of the experiment versus control. OSI was elevated in both diabetic glands in the 1st and 2nd week, whereas 8-isoP and 8-OHdG were higher only in the parotid gland in the second week. PC and 4-HNE were increased in the 1st and 2nd week, whereas oxy-LDL/MDA was increased in the 2nd week in the diabetic parotid glands. Conclusions. Diabetes induces oxidative damage of the salivary glands, which seems to be caused by processes taking place in the salivary glands, independently of general oxidative stress. The parotid glands are more vulnerable to oxidative damage in these conditions.

The skin remodeling in type 1 diabetes and insulin resistance animal models.

The skin matrix metalloproteinase 3, tissue inhibitors of matrix metalloproteinase 2 and collagen III content changes in type 1 diabetes and insulin resistance treated with insulin and metformin were studied. Healthy adult male Wistar rats were obtained from experimental animal house, Department of Experimental Pharmacology, Medical University in Bialystok. The rats were divided randomly into five groups of 8 rats each. Control rats were injected intraperitoneally by NaCl. Type IDDM was induced by a single injection of Streptozocin. Insulin resistance was induced by a high-fat diet. The chosen groups of rats were also treated with insulin or metformin. ELISA Kits (USCN Life Science, China) were used to measure content of matrix metallo-proteinase 3 (ELISA Kit for Matrix Metalloproteinase 3 - MMP3), tissue inhibitor of matrix metalloproteinase 2 (ELISA Kit for Tissue Inhibitors of Metalloproteinase 2 - TIMP2) and content of collagen type 3 (ELISA Kit for Collagen Type III - COL3). The results were reported as a median. The statistical significance was defined as p<0.05. Type 1 diabetes and insulin resistance have significantly reduced the quality of the skin, shown by the increase in content of matrix metalloproteinase 3 and the decrease in content of tissue inhibitors of matrix metalloproteinase 2. Type 1 diabetes and insulin resistance have reduced the quality of the skin expressed by type III collagen content decrease but for future studies it is recommend to determine rat interstitial collagenase, MMP-13, as well. Insulin and metformin treatment improved the quality of the diabetic skin, demonstrated by the type III collagen content increase.

Quantitative and qualitative evaluation of CART-containing cells in adrenal glands of male rats with hypertension.

Adrenal activity is stimulated and secretion of stress hormones is increased during advanced stages of renovascular hypertension. The literature suggests that the neuropeptide, cocaine and amphetamine regulated transcript (CART), might regulate adrenal secretory function and thus could influence its activity. We assessed potential quantitative and qualitative changes in the cells that contained CART in the adrenal glands of rats with renovascular hypertension. The renal arteries of ten rats were subjected to a clipping procedure, i.e., two-kidney one-clip (2K1C) model of arterial hypertension, and after 6 weeks each rat developed stable hypertension. CART was localized using immunohistochemistry. CART was detected in a large population of cells in the medulla, sparse nerve fibers in the cortex and the capsule of the adrenal gland. The population of CART-positive cells in adrenal glands of two kidney-one clip (2K1C) treated rats was greater and their immunoreactivity was increased compared to controls. Similarly, the length, width, area and diameter of CART-immunoreactive cells were significantly greater in the hypertensive rats than in controls. We demonstrated that renovascular hypertension alters the number and immunoreactivity of CART-containing cells in adrenal glands.

Antioxidant profile of salivary glands in high fat diet-induced insulin resistance rats.

OBJECTIVE: There is no study analyzing the salivary antioxidant profile in the course of the insulin resistance. MATERIALS AND METHODS: Rats were divided into two groups. One group was fed with a normal diet, another one with a high fat diet for 5 weeks. The analysis included: catalase (CAT), superoxide dismutase, peroxidase activities, uric acid, and total antioxidant status concentrations. RESULTS: The activity of peroxidase in both kind of glands of insulin resistance rats was significantly reduced than in the control rats. The protein concentration, total amount of total antioxidant status in the parotid glands of insulin resistance rats were significantly lower than in the control glands The total amount of superoxide dismutase, CAT, and uric acid in the parotid glands of insulin resistance rats were significantly elevated in comparison with the control rats. The median values of the total amount of superoxide dismutase, CAT, peroxidase, total antioxidant status were significantly higher in the parotid than in the submandibular glands of the insulin resistance and control rats. CONCLUSION: Parotid and submandibular glands of rats react differently when exposed to insulin resistance condition; however, the parotid glands seem to be more affected. The main source of antioxidants is parotid glands of rats.

The unwounded skin remodeling in animal models of diabetes types 1 and 2.

Diabetes mellitus types 1 and 2 are chronic diseases that cause serious health complications, including dermatologic problems. The diabetic skin is characterized by disturbances in collagen metabolism. A tissue remodeling depends on the degradation of extracellular matrix through the matrix metalloproteinases, which are regulated by e.g. the tissue inhibitors of metalloproteinases. The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is essential to maintain homeostasis in the skin. The aim of this study was to determine the concentration of metalloproteinase 2, tissue inhibitor of metalloproteinase 3 and the concentration of collagen type 1 in unwounded skin of diabetes type 1 and 2 and healthy controls. The treatment of diabetes resulted in a significant decrease of MMP2, increase of TIMP3 and COL1 concentrations in the skin as compared to the untreated diabetic skin. The concentrations of MMP2 in the skin of treated rats did not show significant differences from the healthy control group. TIMP3 concentrations in the skin of treated rats are not returned to the level observed in the control group. Disturbances of the extracellular matrix of the skin are similar in diabetes type 1 and 2. Application of insulin in diabetes therapy more preferably affects the extracellular matrix homeostasis of the skin.

Activity of N-acetyl-beta-D-hexosaminidase (HEX) and its isoenzymes A and B in human milk during the first 3 months of breastfeeding.

PURPOSE: Milk contains free and bound oligo- and heteropolisaccharides, which protect newborns against pathogens and have nutritional value. N-acetyl-beta-D-hexosaminidase (HEX), the most active lysosomal exoglycosidase, modify and degrade oligo- and heteropolysaccharides. The objective of our study was to determine HEX activity and isoenzymes A and B in the progression of lactation. MATERIAL AND METHODS: Human milk samples were collected from 51 women on the 3rd, 21st and 100th day postpartum. Enzymatic activity was determined the Zwierz et al method modified by Marciniak et al. Protein and lactose concentrations were determined by a MilkoScan 4000 apparatus. RESULTS: The total HEX activity decreased by the 21st day in comparison to the 3rd day, and increased by the 100th day as compared to the 21st day. HEX A activity decreased by the 21st and the 100th day as compared to the 3rd day. HEX B activity decreased by 21st day and has the tendency to decrease by the 100th day as compared to the 3rd day. Protein concentration decreased and the lactose concentration increased in milk taken on the 21st day in comparison to concentration of protein and lactose on the 3rd day. HEX and its isoenzymes' activity significantly correlate with the progression of lactation. At the beginning of lactation, HEX A activity, which releases hexosamines from acidic oligosaccharides, dominates; later, HEX B releases hexosamines from neutral oligosaccharides. CONCLUSIONS: To better understand the degradation of human milk oligosaccharides, it would be useful to investigate and document their detailed structures and evaluate the activity of other exoglycosidases' activity in human breast milk over the course of lactation.

Fucosylation in synovial fluid as a novel clinical marker for differentiating joint diseases--a preliminary study.

OBJECTIVE: To investigate fucosylation of synovial fluid glycoproteins in patients with rheumatoid arthritis (RA), juvenile arthritis (JIA), gonarthrosis (GA) and reactive arthritis (ReA), referred to traumatized knee (TK). METHODS: Synovial fluid glycoproteins were separated by SDS-PAGE and either silver stained or blotted onto nitrocellulose and probed with the fucose-specific Aleuria aurantia lectin. Five bands were chosen for densitometric analysis. Total fucose content and density of fucosylated epitopes were analyzed. RESULTS: Fucose content was elevated in all patient groups and almost all bands, comparing to TK. The density of fucosylated epitopes was increased in the 42-kDa band of RA and JIA cases, and lowered in the 26-kDa band of RA and JIA, but not in GA. In all RA cases FR 42-kDa > FR 26-kDa. The relation was opposite in 8 out of 9 GA cases. CONCLUSION: The density of fucosylated epitopes differs significantly in particular glycoproteins of synovial fluid in joint diseases and may be of potential diagnostic value in differentiating diseases of inflammatory and degenerative origin.

Activity of lysosomal exoglycosidases in saliva of patients with HIV infection.

PURPOSE: The aim of this work was to evaluate the influence of HIV infection on the catabolism of glycoconjugates in the oral cavity, by determination of the activity of lysosomal exoglycosidases in mixed saliva. METHOD: The specific activities of the following exoglycosidases were tested: N-acetyl-beta-hexosaminidase (HEX), its isoenzymes A (HEX-A) and B (HEX-B), alpha-mannosidase (MAN), beta-galactosidase (GAL) and alpha-fucosidase (FUC). RESULT: A significant increase of activity of HEX-A, GAL and FUC, and a significant decrease of the activity of HEX-B was found, but no significant changes in the HEX and MAN activity we noted. CONCLUSION: Our results indicate that following HIV infection, there is probably an increased rate of catabolism of glycoconjugates in saliva resulting from changes in the proportions of the activity of isoenzymes A and B of N-acetyl-beta-hexosaminidase, beta-galactosidase and alpha-fucosidase. An increase of HEXA activity can implicate the beginning of neoplastic changes developing in the oral cavity.

Saliva of patients with Type 1 diabetes: effect of smoking on activity of lysosomal exoglycosidases.

OBJECTIVE: The aim of our study was to evaluate the influence of smoking on the activity of N-acetyl-beta-hexosaminidase (HEX), its isoenzymes A (HEX-A) and B (HEX-B) and beta-galactosidase (GAL), in the saliva of patients with Type 1 diabetes. METHODS: In the supernatant HEX and its isoenzymes A and B, and beta-galactosidase were determined by the method of Chatteriee et al in modification of Zwierz et al (mKat kg(-1) of protein). Protein was determined by the Lowry et al method (mg ml(-1)). RESULTS: The results presented here suggest that diabetes and smoking modify activity of HEX and its isoenzymes, but only combination of diabetes and smoking give a significant increase in the specific activity of HEX and its isoenzymes. CONCLUSIONS: Type 1 diabetes slightly changes the composition of saliva. Smoking cigarettes significantly modifies the composition and properties of saliva in healthy individuals and patients with Type 1 diabetes.

Assessment of salivary levels of the chosen exoglycosidases in patients with aggressive periodontitis after treatment with doxycycline.

PURPOSE: The aim of the study was the clinical assessment of the periodontium in patients with aggressive periodontitis (AP) after treatment with doxycycline hyclate. Moreover, an attempt was made to evaluate the effect of the treatment on the salivary concentrations of beta-glucuronidase, HEX, HEX A and HEX B in AP patients. MATERIAL AND METHODS: Sixteen patients with aggressive periodontitis, aged 28-45 years, were enrolled in the study. The patients were treated with a doxycycline hyclate preparation (Periostat) for 2 months at a dose of 20 mg twice a day. The clinical examination was performed twice, directly prior to pharmacological treatment and after its termination. The following clinical parameters were evaluated: the plaque index (PI), the sulcus bleeding index (SBI), the pocket probing depth (PPD) and the clinical attachment level (CAL). Biochemical determination of beta-glucuronidase, HEX, HEX A and HEX B concentrations in non-stimulated saliva was performed before and after treatment. RESULTS: In AP patients, the values of PI, SBI and CAL before and after treatment were comparable. The mean pocket probing depth before treatment was 3.5 mm, which decreased significantly after treatment (3.2 mm). The values expressed as pKat/kg protein for specific enzymatic activities of HEX, HEX A, HEX B and beta-glucuronidase in the saliva of AP patients before and after doxycycline treatment were similar. CONCLUSIONS: A 2-month treatment with doxycycline is too short to obtain clinical changes. Although the assessment of the activity of such enzymes as beta-glucuronidase, HEX, HEX A and HEX B in the saliva of AP patients allows detection of periodontal inflammation, it cannot be used to determine the risk of its development and therefore has no practical significance.

Activity of lysosomal exoglycosidases in saliva of patients with HIV infection.

INTRODUCTION: The aim of this work was to evaluate the influence of HIV infection on the catabolism of glycoconjugates in oral cavity, by determination the activity of lysosomal exoglycosidases in resting whole saliva HIV positive patients. MATERIAL AND METHODS: Sample of resting whole saliva from HIV infected patients (divided into two groups, depending on lymphocyte CD4+ number in peripheral blood) and the control-HIV negative group were analyzed for exoglycosidases activity. Determinations the activities (muKat/kg of protein) of lysosomal exoglycosidases were performed according to Chatteriee et al., modified Zwierz et al. The protein content (mg/ml) was determined by the Lowry method. Statistical analysis was performed using packet Statistica 6.0. Results were expressed as the mean and SD. P values less than 0.05 were considered significant. RESULTS: Exoglycosidases activities were not statistically dependent on immunological status of HIV patients. We obtained insignificant increase activities of HEX, HEX A and GALp and insignificant decrease activity of HEX B along with the reduction of the CD4+ number. In both HIV positive groups the activities of HEX B were statistically lower and GALp statistically higher in comparison to the control. In the case of HEX A significant differences could be observed between patients with low immunological status and the control group. CONCLUSIONS: HIV infection intensifies catabolism glycoconiugates in saliva and changes activities of HEX, its isoenzymes A and B and beta-galactosidase. It may change susceptibility the cells lining oral cavity to viral and bacterial infections.

Antioxidant mechanism of hepatoprotection by ursodeoxycholic acid in experimental alcoholic steatohepatitis.

PURPOSE: The aim of this study was to evaluate the role of an antioxidant factor in the hepatoprotective effect of ursodeoxycholic acid (UDCA) in rat alcoholic steatohepatitis. MATERIAL AND METHODS: The effects of UDCA (40 mg/kg, i.g., 30 days) were studied using rats fed on a high-fat diet (52% calories as fat) and administered with ethanol via intragastric intubation (4 g/kg daily, 30 days). RESULTS: The livers of ethanol-treated animals were characterized by fatty dystrophy. The relative liver weight and the square of the sudanophylic area as well as the liver triglyceride content and the activity of the serum marker enzymes, aspartate aminotransferase and gamma-glutamyltransferase, were significantly increased. Elevated superoxide dismutase activity as well as increased contents of lipid peroxidation products (hydroxyalkenals, malone dialdehyde, etc.) and lucigenin-enhanced microsomal chemiluminescence were observed in the liver of ethanol-treated rats and the liver reduced glutathione content was decreased. An increase in monoenoic fatty acids, a decrease of the n-6 acid family and an enhancement of microsomal membrane viscosity were found in the liver of these animals. An elevation of the total cytochrome P-450 content and the activity of amidopyrine-N-demethylase were shown in liver microsomes of the ethanol-treated group. The UDCA treatment improved the liver morphology, decreased serum marker enzyme activities, liver triglyceride content and normalized all the indices of oxidative stress. UDCA lowered the viscosity of the microsomal membrane, as assessed by both the fluorescence probe techniques and the saturated/unsaturated fatty acid ratio. The microsomal cytochrome P-450 content and amidopyrine-N-demethylase activity were normalized in UDCA-treated rats. CONCLUSIONS: We can conclude that the hepatoprotective effect of UDCA stipulated by its antioxidant properties is indeed the factor enabling UDCA to control metabolic processes by changing the properties of liver membranes and membranous proteins.

The cleavage of vitamin E galactoside in the rat tissue homogenates.

The stability of alpha-tocopheryl beta-galactoside in the presence of endogenous galactosidases in selected tissue homogenates (liver, kidney, ileum and brain) was estimated. High degree release of alpha-tocopherol from alpha-tocopheryl beta-galactoside in tissues of ileum, kidney and brain was observed (82%, 75% and 72%, increase above endogenous alpha-tocopherol, respectively). A possible enzymatic mechanism of the galactoside decomposition was proposed.

[Non mucin proteins of saliva with high homology of polypeptide chains]. / Niemucynowe bialka sliny o wysokim stopniu homologii lancucha polipeptydowego.

To non mucin proteins of human saliva belong: cystatins, statherin, histatins and acidic proline-rich protein. These saliva proteins influence hard and soft tissues by forming a pellicle layer on oral mucosa and enamel, by taking part in removing bacteria or initiating of bacterial colonization. Most of them are able to inhibit the formation of dental calculus and control the calcium phosphate homeostasis.