Streptozotocin-induced type 1 diabetes mellitus alters the morphology, secretory function and acyl lipid contents in the isolated rat parotid salivary gland

Diabetes mellitus (DM) is associated with numerous conditions including hypo-secretion of digestive enzymes. This study investigated the morphology, secretory function (alpha-amylase release) and acyl lipid contents in the isolated parotid gland of STZ-induced diabetic and age-matched control rats in order to provide insights into diabetes-induced salivary insufficiency. The techniques employed included light microscopy, colourimetric and gas chromatography (GC) analysis, respectively. Diabetes mellitus was induced in adult male Wistar rats by a single intraperitoneal (IP) injection of streptozotocin (STZ) (60 mg per kg body weight). Control animals were injected with a similar volume of citrate buffer. The animals were tested for DM 4 days after STZ injection and 2 months later when they were humanely killed for the experiment. The morphological results showed diabetic parotid glands to be extensively infiltrated with lipid droplets of various magnitudes, whereas glands from control animals display normal structure with the absence of lipid droplets. The analysis of parotid secretory function revealed a significant (p < 0.05) dose-dependent decrease in alpha-amylase release in response to noradrenaline (NA) in STZ-treated glands when compared to age-match control parotid glands. Furthermore, the levels of acyl lipids (16:0, 16:1, 18:0 and 18:1) in diabetic parotid glands was significantly (p < 0.01) reduced compared to control glands, along with a reduced ratio of 16:1/16:0. The results indicate DM can elicit changes in the morphology, secretory function and acyl fatty acid quantity in the isolated rat parotid gland.     

Low‐power laser irradiation decreases lipid droplet accumulation in the parotid glands of diabetic rats

Lipid droplet accumulation has been related to salivary gland hypofunction in diabetes. In this study, the effect of laser irradiation on the parotid glands (PGs) of diabetic rats was analyzed with regard to its effect on lipid droplet accumulation, intracellular calcium concentration and calmodulin expression. The animals were distributed into 6 groups: D0, D5, D20 and C0, C5, C20, for diabetic (D) and control animals (C), respectively. Twenty‐nine days following diabetes induction, PGs of groups D5 and C5; D20 and C20 were irradiated with 5 and 20 J/cm² of a red diode laser at 100 mW, respectively. After 24 hours, PGs were removed for histological, biochemical, and western blotting analysis. The diabetic animals showed lipid droplet accumulation, which was decreased after irradiation. Ultrastructurally, the droplets were nonmembrane bound and appeared irregularly located in the cytoplasm. Moreover, diabetic animals showed an increased intracellular calcium concentration. In contrast, after laser irradiation a progressive decrease in the concentration of this ion was observed, which would be in agreement with the results found in the increased expression of calmodulin in D20. These data are promising for using laser to decrease lipid droplet accumulation in PGs, however, more studies are necessary to better understand its mechanisms. Micrographs showing decreased lipid accumulation after laser irradiation in light micrographs (LM), and morphology of lipid droplet in transmission electron microscopic (TEM). LM: (A) PGs from nondiabetic rats that did not receive Laser irradiation (LI), (B) PGs from nondiabetic rats that received a dose of 20 J/cm², (C) lipid accumulation (arrows) in the secretory cells from diabetic rats that did not receive irradiation, (D) reduction of lipid accumulation in the secretory cells from diabetic rats that received a dose of 20 J/cm² and TEM: (E) scale bar = 5 μm, (F) scale bar = 1 μm, and (G) scale bar = 0.5 μm.      

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.     

Protein tyrosine phosphorylation in cardiovascular system

Treatment of rat parotid acinar cells with sodium orthovanadate (an inhibitor of protein tyrosine phosphatase) caused a dose-dependent inhibition of phosphatase activity as measured by the hydrolysis of para nitrophenylphosphate (pNPP). Inclusion of 50 M sodium orthovanadate inin vitro gland cultures prevented the amylase secretion from both untreated control and isoproterenol-stimulated parotid acinar cells. Four different tyrosine-phosphorylated proteins with M_r 40, 45, 70 and 95 kDa, respectively, were identified in secretory granule preparations from rat parotid glands by immunoblot using a monospecific antibody for phosphotyrosine. An increase in the phosphorylation levels of these phosphoproteins was noted in the presence of 50 M sodium orthovanadate, suggesting that a protein tyrosine phosphatase (PTPase) is involved in parotid gland protein dephosphorylation reactions. Using antibody to Syp (a PTPase belonging to class 1D), a major fraction of subcellular activity was found to be associated with secretory granule membranes. These results suggest the possible involvement of a PTPase (Syp) in parotid gland secretory mechanisms.     

Characterization of human and rat immortalized clones of parotid acinar cells with respect to specific proteins and their mRNAs,and receptor-linked adenylate cyclase

Summary This study reports the isolation and characterization of a rat nontumorigenic parotid acinar cell clone (2RSG), a human nontumorigenic parotid acinar cell clone (2HPC8), and a human tumorigenic acinar clone (2HP₁G). The levels ofα-amylase mRNAs detected when usingα-amylase cDNA of 1176 and 702 bp for hybridization were higher in 2RSG and 2HPC8 cells than their respective whole parotid glands. The level of these mRNAs decreased in 2HP₁G cells. In contrast toα-amylase mRNAs levels, theα-amylase activity in cultured acinar cells was extremely low in comparison to whole glands, irrespective of species or cell status. The levels of proline-rich protein (PRP) mRNA and parotid secretory protein (PSP) mRNA detected when using PRP cDNA of 600 bp and PSP cDNA of 805 bp for hybridization were higher in 2RSG cells than those in rat parotid glands; the reverse was observed in 2HPC8 cells and human parotid glands. The levels of PRP mRNA and PSP mRNA in 2HPC8 and 2HP₁G acinar cells were similar. The level of mRNA was not detectable in murine neuroblastoma cells (NBP2) using the sameα-amylase cDNA, PRP cDNA and PSP cDNA for hybridization. The PSP level in rat parotid gland was lower than that found in 2RSG cells; the reverse was observed in 2HPC8 cells and human parotid glands. The level of PSP in 2HP₁G cells was higher than that found in 2HPC8 cells. Isoproterenol increased the cAMP level in 2RSG, 2HPC8, and 2HP₁G clones, being most effective in 2RSG cells, and least effective in 2HPG cells. Prostaglandin E₁ (PGE₁) also increased cAMP level, being most effective in 2HPC8 cells and ineffective in 2HP₁G cells, suggesting that the PGE₁ receptor-linked adenylate cyclase becomes inactive upon transformation. These results suggest that the three clonal acinar cells from rat and human parotid glands reported here can be useful in comparative studies on regulation of growth, differentiation, and transformation.     

Effects of ageing on morphology,amylase release,cytosolic Ca2+ signals and acyl lipids in isolated rat parotid gland tissue

Xerostomia (oral dryness sensation) is due to dryness of the oral cavity and it is more prevalent in the elderly. This study investigated the effect of ageing on parotid gland structure and function of control (2-6 months) and aged (12, 16-18 and 22-24 months) rats employing light microscopic, colorimetric, gas chromatographic and microspectrofluorimetric methods to investigate the morphological changes of the parotid glands, amylase release, endogenous lipid distribution and cytosolic free calcium levels, respectively. When compared to controls, age-related changes were apparent in glands obtained from rats aged 16-18 and 22-24 months, which included reduced acinar cell distribution, enlarged parotid ducts with fatty and connective tissue and mast cell infiltrations. Parotid acini from 12, 16-18 and 22-24-month-old glands showed significant (p < 0.05) age-related decreases in amylase release, compared to controls when challenged with acetylcholine (ACh). No change in basal calcium signals was observed in parotid acini from 2-6 to 16-18-month-old-animals. However, stimulation of 16-18-month-old parotid acini with 10(-5)M ACh resulted in a significant (p < 0.001) decrease in both peak and plateau phases of the cytosolic Ca2+ signal when compared to control. Gas chromatography of de novo and essential acyl lipids revealed no changes in the amount of either acyl lipid group in glands obtained from 2-6 to 22-24-month-old animals. Lipid analysis of phospholipid associated acyl chains showed a higher relative proportion of linoleic acid in older glands. The results reveal that ageing is associated with marked and distinct morphological changes including infiltrations of lipids and mast cells of the parotid gland and decreases in amylase release and cytosolic Ca2+ signals.     

Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse

Summary Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal -N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate -galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20–50% of these cells in all glands contained terminalN-acetylglucosamine residues. In contrast, terminal -N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.     

Lipid order and composition of synaptic membranes in experimental diabetes mellitus

The effect of diabetes in rats on lipid composition and order of synaptosomal membranes (SM) was determined in streptozotocin-induced diabetic rats after 6 weeks of chronic hyperglycemia. The cholesterol content was slightly, but not significantly, higher in diabetic SM (0.287±0.042 vs. 0.209±0.061 mol/mg protein). The phospholipid concentration in diabetic SM was significantly increased (0.515±0.042 vs. 0.305±0.041 mol/mg protein;P<0.005). Neither the molar ratios of cholesterol to phospholipids in the SM nor the fatty acid composition of the SM was significantly altered with diabetes. Diabetes did not affect membrane order or the thermotropic transition temperature of the SM as determined fluorometrically. On the other hand, the SM of diabetic rats had significantly increased concentration of lipid peroxidation products, namely conjugated dienes (the calculated O.D./mol phospholipids was 11.56±1.83 in controls and 19.95 ±4.1 in diabetic ratsP<0.01). Despite the accumulation of lipid peroxidation byproducts in SM of diabetic rats the overall membrane order and the cholesterol to phospholipid molar ratio do not appear to be significantly altered.     

High-level salivary gland expression in transgenic mice

A 7.1 kb mini-gene construct containing cloned DNA from the murine parotid secretory protein (PSP) gene with 6.2 kb of the promoter, has previously been shown to direct specific mRNA expression to the salivary glands in transgenic mice. However, the level of transgene expression in the parotid gland was only a few percent of the endogenous level. This indicated that elements necessary for high-level expression are still to be found. In this study, we have searched for such regulatory elements in additional flanking regions by using a 25 kb clonedPsp ^b fragment containing the complete structural gene, 11.4 kb of 5-flanking sequence, and 2.5 kb 3-flanking sequence as a transgene. To distinguish the expression of the transgene from that of the endogenous gene, we took advantage of an allelic difference, using an oligonucleotide that recognized the mRNA fromPsp ^b and the transgene but not that from the other allele,Psp ^a . The expression of the transgene was examined in animals homozygous forPsp ^a . Three independent integrations all exhibited a level of parotid-gland-specific expression that corresponded to that of the endogenous gene. Thus, sequences responsible for this high-level PSP mRNA expression are situated within the genomic DNA of the transgene.     

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.     

In vivo effects of tunicamycin on the secretory processes of rat parotid glands

Summary The morphological and functional effects of tunicamycin were studied in rat parotid glands at the stage of the reformation of secretory granules following secretory stimulation by isoproterenol. Tunicamycin inhibited the incorporation of (³H)-mannose into the acid-insoluble fraction but had no effect on total protein synthesis as determined by the incorporation of (¹⁴C)-leucine. Thus the administration of tunicamycin in vivo inhibits the synthesis of mannose-rich glycoproteins in a manner similar to that in an in vitro system. The ultrastructure of the acinar cell showed little change following treatment with this drug, except that the number of reaccumulated secretory granules was greater than in the control. Amylase secretion stimulated by isoproterenol was inhibited in tunicamycin-treated cells, but did not decrease following treatment with N⁶,2-O-dibutyryladenosine 3-5-cyclic monophosphate, a secretory stimulator bypassing the -receptor. A radio-receptor assay using (³H)-dihydroalprenolol and direct localization using the fluorescent -adrenergic blocker 9-amino-acridin propranolol showed a marked reduction in the binding activity of -receptor following treatment with tunicamycin. Thus the inhibition of N-linked glycosylation appears to produce profound effects on the -adrenergic receptor-adenylate cyclase complex of acinar cells, although the steps of the transport and the exocytotic discharge of secretory materials are not affected.     

The ACh-induced whole-cell currents in sheep parotid secretory cells. Do BK channels really carry the ACh-evoked whole-cell K+ current?

As in other salivary glands, the secretory cells of the sheep parotid have a resting K⁺ conductance that is dominated by BK channels, which are activated by acetylcholine (ACh) and are blocked by tetraethylammonium (TEA). Nevertheless, perfusion studies indicate that TEA does not inhibit ACh-evoked fluid secretion or K⁺ efflux from intact sheep parotid glands. In the present study, we have used whole-cell patch clamp techniques to show that ACh activates K⁺ and Cl^– conductances in sheep parotid secretory cells by increasing intracellular free Ca²⁺, and we have compared the blocker sensitivity of the ACh-evoked whole-cell K⁺ current to the previously reported blocker sensitivity of the BK channels seen in these cells.The ACh-induced whole-cell K⁺ current was not blocked by TEA (10 mmol/l) or verapamil (100 mol/l), both of which block the resting K⁺ conductance and inhibit BK channels in these cells. Quinine (1 mmol/l) and quinidine (1 mmol/l), although only weak blockers of the resting K⁺ conductance, inhibited the ACh-evoked current at 0 mV (K⁺ current), by 68% and 78%, respectively. 4-Aminopyridine (10 mmol/l) partially inhibited the ACh-induced K⁺ current and caused it to fluctuate. It also caused the resting membrane currents to fluctuate, possibly by altering cytosolic free Ca²⁺. Ba²⁺ (100 mol/l), a blocker of the inwardly rectifying K⁺ conductance in sheep parotid cells, had no effect on the ACh-induced K⁺ current.We conclude that the ACh-induced K⁺ conductance in sheep parotid cells is pharmacologically distinct from both the outwardly rectifying (BK) K⁺ conductance and the inwardly rectifying K⁺ conductance seen in unstimulated cells. Given that in vitro perfusion and K⁺ efflux studies on other salivary glands in which BK channels dominate the resting conductance (e.g., the rat mandibular, rat parotid and mouse mandibular glands) have revealed an insensitivity to TEA, suggesting that BK channels do not carry the ACh-evoked K⁺ current, we propose that BK channels do not contribute substantially to the K⁺ current evoked by ACh in the secretory cells of most salivary glands.This project was supported by the Australian Research Council. We thank Dr. N. Sangster, Dr. J. Rothwell and Mr. R. Murphy for giving us access to their sheep.     

Functional activity of thyroid gland in male rats with acute and mild streptozotocin diabetes

Type 1 diabetes mellitus (DM1) and dysfunction of the thyroid gland (TG) are the most common endocrine diseases, which are interrelated. However, the molecular mechanisms of thyroid dysfunction in DM1 and the role of adenylyl cyclase signaling system (ACSS) in this process remain poorly understood. Typically for studying etiology and pathogenesis of thyroid diseases in DM1 the models of acute DM1 induced by high doses of streptozotocin (STZ) are used. At the same time, a suitable model for this purpose is the model of mild DM1 initiated by moderate doses of STZ, which more closely resembles human DM1. The aim of this study was a comparative study of the functional state of the thyroid gland in rats with 30-day acute DM1 induced by injection of STZ at a dose of 65 mg/kg, and in rats with 30- and 210-day mild DM1 induced by three consecutive injections of STZ at medium doses (30–40 mg/kg). For this purpose in diabetic animals the levels of thyroid hormones and TSH and the functional activity of hormone-sensitive ACSS in membranes isolated from thyroid gland were studied. It was shown that in blood of rats with acute DM1 the levels of fT₄, fT₃, and tT₃ were decreased by 45, 23 and 19%, respectively, while the level of TSH did not change significantly. In rats with the 30-day mild DM1 the concentration of fT₄ was decreased by 32%, while the levels of tT₄, tT₃, and TSH were similar to that in control. In rats with prolonged mild DM1 after 150 and 210 days following the first treatment with STZ the levels of tT₄, fT₄, and tT₃ were significantly reduced, but the concentration of TSH in rats with 210-day mild DM1 was increased by 119%. The results obtained in the study of thyroid status and TSH levels in rats with prolonged mild DM1 are in good agreement with the data obtained in the study of thyroid diseases in patients with DM1. It was found that the AC basal activity in the membranes isolated from the thyroid gland of diabetic rats did not change, except for the rats with the prolonged mild DM1 where this activity was increased by 21%. In all groups of diabetic rats the decrease of AC stimulating effects of GppNHp (10^?5 M) and TSH (10^?8 M) was found, and in the rats with prolonged mild DM1 the AC effect of PACAP-38 (10^?6 M) was also reduced. The decrease of AC effect of TSH varied among different groups of the diabetic animals: in the rats with acute DM1 this effect was reduced by 46% and in the rats with 30- and 210-day mild DM1-by 18 and 34%. Thus, it was concluded that the key cause of the thyroid resistance to TSH under conditions of DM1 is a weakening of the signal transduction generated by TSH via the ACSS.     

Circadian Variations in the Activities of 6‐Phosphogluconate Dehydrogenase and Glucose‐6‐Phosphate Dehydrogenase in the Liver of Conrol and Streptozotocin‐Induced Diabetic Rats

The aim of this study was to examine: the 24 h variation of 6‐phosphogluconate dehydrogenase and glucose‐6‐phosphate dehydrogenase activities, key enzymes for the maintenance of intracellular NADPH concentration, in rat liver in control and streptozotocin‐induced diabetic animals. Adult male rats were fed ad libitum and synchronized on a 12:12 h light‐dark cycle (lights on 08:00 h). One group of animals was treated with streptozotocin (STZ, 55 mg/kg, intraperitoneal) to induce experimental diabetes. Eight weeks after STZ injection, the animals were sacrificed at six different times of day—1, 5, 9, 13, 17 and 21 Hours After Lights On (HALO)—and livers were obtained. Enzyme activities were determined spectrophotometrically in triplicate in liver homogenates and expressed as units per mg protein. 6‐phosphogluconate dehydrogenase activity was measured by substituting 6‐phosphogluconate as substrate. Glucose‐6‐phosphate dehydrogenase activity was determined by monitoring NADPH production. Treatment, circadian time, and interaction between treatment and circadian time factors were tested by either one or two way analysis of variance (ANOVA). Two‐way ANOVA revealed that 6‐phosphogluconate dehydrogenase activity significantly depended on both the treatment and time of sacrifice. 6‐phosphogluconate dehydrogenase activity was higher in control than diabetic animals; whereas, glucose‐6‐phosphate dehydrogenase activity did not vary over the 24 h in animals made diabetic by STZ treatment. Circadian variation in the activity of 6‐phosphogluconate dehydrogenase was also detected in both the control and STZ treatment groups (one‐way ANOVA). Time‐dependent variation in glucose‐6‐phosphate dehydrogenase activity during the 24 h was detected in control but not in diabetic rats. No significant interaction was detected between STZ‐treatment and time of sacrifice for both hepatic enzyme activities. These results suggest that the activities of NADPH‐generating enzymes exhibit 24 h variation, which is not influenced by diabetes.     

Melatonin Influences the Imbalance in Neural Cell Adhesion Molecule (NCAM) Isoforms in Diabetes Mellitus

Diabetes mellitus is associated with significant cognitive deficiencies, which develop in a parallel manner with neurophysiological and structural changes in the brain. Intravenous or intraperitoneal injections of a cytotoxic agent influencing the cells, streptozotocin (STZ), is most often used to create animal models of diabetes. The pathogenesis of diabetic encephalopathy is not yet understood, but an impairment of spatial learning occurs in association with distinct changes in hippocampal synaptic plasticity. Cell adhesion molecules are good candidates to participate in synaptogenesis on neuronal plasticity. It has been proposed that neural cell adhesion molecule mediates synaptic plasticity during learning and memory formation.     

Further investigations on the action of ecdysteroids on the salivary glands of the female tickAmblyomma americanum

Two phytecdysteroids (abutasterone, makisterone A) and five synthetic ecdysteroid analogues, all at 1 g/ml, were tested on salivary glands from the female tick,Amblyomma americanum L. (Acari:Ixodiae), held in organ culture for four days. All of these substances caused a significant reduction in fluid secretory competence of salivary glands in vitro. This constitutes further evidence that the structural requirements for causing salivary-gland degeneration in ticks are similar to those generally required for ecdysteroid activity in other arthropods. Although vertebrate steroids are known to augment fluid secretory competence by salivary glands in organ culture, -estradiol was not able to attenuate the degenerating effect of 20-hydroxyecdysone, supporting the suggestion that ecdysteroids and vertebrate steroids have distinct sites/mechanisms of action on this tissue.     

Selenium–Vitamin E Combination and Melatonin Modulates Diabetes-Induced Blood Oxidative Damage and Fetal Outcomes in Pregnant Rats

Oxidative stress is considered to be the main cause of diabetic complications. In the current study, we investigated the effect of selenium–vitamin E combination and melatonin on lipid peroxidation (LPO) and scavenging enzyme activity in the blood of streptozocin (STZ)-induced diabetic pregnant rats. Forty female Wistar rats were randomly divided into five groups. The first and second groups were used as the non-pregnant control and pregnant control groups, respectively. The third group was the pregnant diabetic group. Vitamin E plus selenium and melatonin were administered to the diabetic pregnant rats consisting fourth and fifth groups, respectively. Diabetes was induced on day 0 of the study by STZ. Blood samples were taken from all animals on the 20th day of pregnancy. LPO level was higher in diabetic pregnant rats than in control, although superoxide dismutase, catalase, and glutathione peroxidase activities were lower in diabetic pregnant animals than in control. LPO levels were lower both in the two treatment groups than in the diabetic pregnant rats, whereas selenium–vitamin E combination and melatonin caused a significant increase in the activities of these antioxidant enzymes (p < 0.01). In conclusion, vitamin E plus selenium seems to be a more potent antioxidant compared to melatonin in diabetic pregnant rats. Melatonin did not significantly affect the elevated glucose concentration of diabetic pregnant treated with melatonin group. Vitamin E plus selenium may play a role in preventing diabetes-related diseases of pregnant subjects.     

Myocardial and metabolic dysfunction in type 2 diabetic rats: impact of ghrelin

Diabetes mellitus (DM) is commonly associated with metabolic and cardiac dysfunctions. The aim of this study was to examine the effect of ghrelin on metabolic and cardiac dysfunctions in a type-2 diabetes mellitus (T2DM) rat model. For this, 48 male adult Sprague-Dawley rats were divided equally into 4 groups: Group I, fed normal chow, served as normal control group; Groups II-IV, were fed a high-fat diet for 2?weeks followed by injection of streptozotocin (STZ) (35?mg/kg body mass) to create a model of T2DM; Group II, were not treated; Group III, were treated with the vehicle (saline); Group IV, were treated with ghrelin (40?μg/kg body mass) twice daily for 10 days. The untreated diabetic rats showed a significant increase in serum fasting blood glucose, insulin homeostasis model assessment (HOMA) index, triglycerides (TGs), low-density lipoprotein cholesterol (LDL-C), total serum cholesterol (TC), and body mass, with a decrease in high-density lipoprotein cholesterol (HDL-C) (p?< 0.05). Hearts isolated from diabetic rats showed a significant increase in myocardial fat content, a significant decrease in GLUT4, and an increase in acyl-CoA oxidase enzyme mRNA (p?< 0.05). Ghrelin administration for 10?days caused a significant improvement in lipid profile, HOMA index, and body mass, and significantly corrected the myocardial mass, significantly reduced the fat content of the myocardium, significantly increased GLUT4, and decreased acyl CoA oxidase mRNA (p?< 0.05). Thus, ghrelin improves both the metabolic functions and the disturbed energy metabolism in the cardiac muscle of obese diabetic rats.     

Structural organization of ultrarapidly frozen barley aleurone cells actively involved in protein secretion

The ultrastructural organization of actively secreting barley (Hordeum vulgare L. cv. Himalaya) aleurone cells was examined using ultrarapid-freezing (<-10 000°C s^-1) followed by freeze-fracture and freeze-substitution. Our analysis indicates that much of the evidence supporting a direct pathway from the endoplasmic reticulum (ER) to the plasma membrane (i.e. bypassing the Golgi apparatus) for the secretion of -amylase (EC 3.2.1.1) may not be valid. Cryofixed ER cisternae show no sign of vesiculation during active -amylase secretion in gibberellic acid (GA₃)-treated cells. At the same time, Golgi complexes are abundant and numerous small vesicles are associated with the edges of the cisternae. Vesicles appear to be involved in the delivery of secretory products to the plasma membrane since depressions containing excess membrane material appear there. Treatment with GA₃ also induces changes in the composition of Golgi membranes; most notably, the density of intramembrane particles increases from 2700 m^-2 to 3800 m^-2 because of an increase of particles in the 3–8.5-nm size range. A slight decrease in 9–11-nm particles also occurs. These changes in membrane structure appear to occur as the Golgi complex becomes committed to the processing and packaging of secretory proteins. We suggest that secretory proteins in this tissue are synthesized in the abundant rough ER, packaged in the Golgi apparatus, and transported to the plasma membrane via Golgi-derived secretory vesicles. Mobilization of reserves is also accompanied by dynamic membrane events. Our micrographs show that the surface monolayer of the lipid bodies fuses with the outer leaflet of the bilayer of protein-body membranes during the mobilization of lipid reserves. Following the breakdown of the protein reserves, the protein bodies assume a variety of configurations.Abbreviations ER endoplasmic reticulum - GA₃ gibberellic acid - P protoplasmic - E exoplasmic     

Amplification of the amyE-tmrB region on the chromosome in tunicamycin-resistant cells of Bacillus subtilis

Summary In a class of tunicamycin-resistant mutants (tmrA7) of Bacillus subtilis, the production of extracellular -amylase is increase by about five fold. The tmrA7 characteristics (tunicamycin resistance and hyperproduction of extracellular -amylase) can be transferred to recipient cells by transformation. In the transformants and the original tmrA7 mutant, typical amplification of the region from 4 kb upstream of the amyE gene to the tmrB gene on the chromosome was detected. The repeating unit, 16 kb in size, repeats tandemly about five and ten times in the mutant and transformants, respectively, and the -amylase production is proportional to the copy number of the amyE gene. Simultaneous amplification of the tmrB gene, which is responsible for tunicamycin resistance in the multicopy state, and the -amylase structural gene (amyE) seems to be the cause of the pleiotropy of the tmrA7 mutation.