Estrogen (17beta-estradiol) enhances glutamine synthetase activity in C6-glioma cells.

Glutamine synthetase (GS) is the major glutamine-forming enzyme of vertebrates and is accepted to be a marker of astroglial cells. Maturation of astroglial cells is characterized by an increase of GS activity, and the regulation of this enzyme is the topic of many publications. Because of the fundamental role of the GS in controlling brain glutamate and glutamine level, it is essential to understand the mechanism of expression of this enzyme. To our knowledge, the effect of estrogen (17beta-estradiol) on GS activity in glial cells has not been reported. We examined the effect of treatment with estrogen on glutamine synthetase enzyme activity in glial cells. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well-established model system. We adapted a colorimetric method to measure GS-catalyzed gamma-glutamyltransferase (GT) activity in C6-glioma cells. The assay monitors GT activity of glutamine synthetase by following the absorbance of the product gamma-glutamyl hydroxamate at 540 nm. We observed that, the absorbance of gamma-glutamyl hydroxamate significantly increased in estrogen treated cells (0.13 +/- 0.03), as compared to untreated cells (0.058 +/- 0.015). Estrogen also significantly increased concentration of glutamine in C6-glioma cells as measured by fluorometric assay. In addition, western blot analysis showed that estrogen significantly increased the amount of glutamine synthetase compared to control. This estrogen effect could have important physiological implications on cerebral glutamate and glutamine metabolism.

Cytoprotective effect of estrogen on ammonium chloride-treated C6-glioma cells.

The potential cytoprotective effects of estrogen in the brain are of special interest in aging, neurodegenerative diseases, exposure to toxins, and trauma. Estrogen effects on neurons have been widely explored, but less is known about estrogen effects on glia. Glial cells are primary targets of ammonia toxicity, which arises from liver disease or failure (such as from cirrhosis in alcoholics), urea cycle disorders, or inborn errors of metabolism. We examined the ability of estrogen to protect glial cells from ammonium chloride toxicity using an in vitro model system. C6-glioma cells in later passage have many astrocytic characteristics and provided a convenient and well established model system for this work. When C6-glioma cells were exposed to 15 mM ammonium chloride, we observed major cell death (only 32% cell survival relative to control) within 72 h. Pretreatment with 17beta-estradiol (10 microM) significantly protected C6-glioma cells from ammonia toxicity (99% cell survival relative to control). In addition to enhancing the viability of C6-glioma cells against ammonia challenge, estrogen pretreatment was also found to protect mitochondrial function as assayed using the MTT reduction assay. Mitochondrial function was reduced to 39% of control levels in ammonia-challenged cultures and was mostly protected by estrogen (72% of control levels). The findings are potentially relevant for the development of therapeutic strategies to protect glial cells against ammonia toxicity resulting from hepatic failure or other causes.

A comparison of the hormone levels in patients with Sjogren's syndrome and healthy controls.

OBJECTIVE: The purpose of this study was to compare the level and relative ratio of estrogen, progesterone, and prolactin in patients with Sjögren's syndrome and healthy controls. STUDY DESIGN: Serum samples were collected from 17 SS patients and 19 age-, sex- and race-matched controls. All subjects were postmenopausal females who were not currently on hormone replacement therapy. Prolactin levels were measured using ELISA and progesterone and estrogen were measured using EIA. RESULTS: Mann-Whitney U test revealed a significantly higher levels of prolactin among patients than controls (11.41 ng/ml vs. 6.74 ng/ml, p=0.003) with significantly higher prolactin/ progesterone (18.88 vs. 8.14, p=0.02) and estrogen/ progesterone (71.51 vs. 42.02, p=0.05) ratios. No significant differences were observed in the levels of estrogen and progesterone between patients and controls. CONCLUSION: Abnormal levels and relative ratios of hormones may play a role in the pathogenesis of Sjögren's syndrome.

The status of lactoferrin and total iron binding capacity of human parotid saliva in Sjögren's syndrome.

UNLABELLED: Sjögren's syndrome (SS) is chronic salivary gland disorder characterized by a reduction in salivary and lacrimal secretion. Elevation in salivary lactoferrin has been reported in SS patients. Fluctuation in the iron binding capacity of lactoferrin has been associated with cellular damage. OBJECTIVE: The purpose of this study was to compare the levels of salivary lactoferrin, total iron, and iron binding capacity in Sjögren's syndrome (SS) patients and healthy controls. METHODS: SDS-PAGE was used to examine the presence of lactoferrin in 102 patients and 20 healthy controls. A colorimetric assay was used to examine the level of total salivary iron and iron binding capacity in patients and controls. RESULTS: A higher number of SS patients exhibited elevated levels of lactoferrin as compared to controls (86% vs. 20%, respectively). No significant difference was observed in the mean level of total iron in the saliva between patients and controls (12.6 micrograms/100 ml vs. 11.1 micrograms/100 ml, respectively). However, the total iron binding capacity of lactoferrin was significantly lower among SS patients than healthy controls (38.2 micrograms/100 ml vs. 61.8 micrograms/100 ml, respectively), p = 0.019. CONCLUSION: The overall results of this study suggest a possible impairment of the iron binding capacity of saliva in SS patients. Such impairment may contribute to the cellular damage of the salivary glands observed in SS patients.

Frequency and predictive value of the clinical manifestations in Sjögren's syndrome.

The purpose of this study was to examine the frequency and predictive value of glandular and extraglandular manifestations in S ogren's syndrome (SS). The clinical profiles of 169 SS patients were compared to those of 44 non-SS controls. The specific symptoms examined were oral, ocular, vaginal, gastric, pulmonary, skin, joint and muscle pain. Statistical analyses were performed on both individual and grouped symptoms. Chi-squared analyses showed that the frequency of all symptoms was significantly higher among patients than controls. Stepwise discriminant analysis of individual symptoms suggests that the combined symptoms of dry mouth, sore mouth, and dry eyes correctly classified 93% of SS and 97.7% of the controls. While grouped gastric, muscle, psychological, vaginal, skin, nasal, and thyroid symptoms correctly classified 64.3% of SS and 86.1% of the controls. This is the first study to examine the diagnostic value of multi-system manifestation in SS. The overall results suggest that a comprehensive questionnaire of various symptoms may assist the diagnosis of SS. The high predictive value of the combined symptoms confirms their value in the evaluation of SS.

Evaluation of the effect of immediate grafting of mandibular postextraction sockets with synthetic bone.

Immediate mandibular complete dentures were placed in 18 patients. To prevent ridge resorption, bone grafts with hard tissue replacement synthetic bone were placed in sockets on one side of the mandible. The untreated sockets on the contralateral side served as controls. Bone density as shown in panoramic radiographs was reported at the time of placement, at 5 months to 6 months postsurgery, at 12 months to 14 months postsurgery, and at 20 months to 24 months postsurgery. The sites treated with hard tissue replacement synthetic bone not only prevented ridge resorption but also showed increased optical (socket) density of regenerating bone in the initial phase of the study, and maintained that elevated level at 24 months postextraction.

The effect of ammonium chloride on metabolism of primary neurons and neuroblastoma cells in vitro.

Hyperammonemia is a consistent finding in many metabolic disorders. The excess ammonia (NH4Cl) interferes with brain energy metabolism possibly in part by inhibiting the tricarboxylic acid (TCA) cycle. Inhibition of the TCA cycle may result in depletion of ATP in the brain cells. In this study, the acute and chronic effect of NH4Cl (7.5 mM and 15 mM) on the metabolism of isolated neurons and neuroblastoma cells was examined. These cells were treated with NH4Cl for 15 minutes and 24 hours. Morphologic and metabolic toxicity were greater in neuroblastoma cells than in primary neurons. Following 15 minutes treatment, concentration of lactate increased significantly in neuroblastoma cells but, the concentration of other metabolites did not change significantly in neuroblastoma cells and in primary neurons. Following 24 hours treatment, the glucose utilization increased in both cell types. This high utilization of glucose in neuroblastoma cells was in concert with an increase in lactate and decrease in glutamate and ATP. In primary neurons, following 24 hours treatment, the glucose utilization significantly increased, but the concentration of the other metabolites did not change significantly. Neuroblastoma cells consumed more glucose than primary neurons in absence of NH4Cl, but generated the same amount of lactate as neurons.

Responses in primary astrocytes and C6-glioma cells to ammonium chloride and dibutyryl cyclic-AMP.

Elevated brain ammonia levels are a major factor in the genesis of hepatic encephalopathy (HE). The mechanism of ammonium chloride (NH4Cl) neurotoxicity involves interruption of oxidative metabolism. This leads to decreased levels of ATP concentration and subsequent glial fibrillary acidic protein (GFAP) degradation of astrocytes and fibrous C6-glioma cells. Our study investigates NH4Cl toxicity by evaluating changes in ATP concentration and mitochondrial function as well as by evaluating alterations in GFAP expression. NH4Cl induced decreases in ATP were detected after 15 minutes in C6-glioma cells and 24 hours in both cell types. Mitochondrial function, assessed by MTT (2-4,5-dimethylthiazol A-yl)-2, 5-diphenyltetrazolium bromide) assay, was impaired in both cell types at 24 hours following NH4Cl treatment. GFAP was also significantly decreased in both cell types. Morphologic and metabolic toxicities were greater in C6-glioma cells. The data clearly indicate that NH4Cl interrupts oxidative metabolism. The greater toxicity seen in C6-glioma cells may be due to their greater dependence on oxidative metabolism. Lastly, the decrease in GFAP is probably a consequence of diminished ATP.

Effect of xerostomic medications on stimulated salivary flow rate in patients with Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to examine the effect of xerostomic medications on the salivary output of patients with Sjögren's syndrome. METHOD AND MATERIALS: Of 62 patients evaluated in this study, 23 were not using medication, and 39 were using between 1 and 6 medications with xerostomic side effect. RESULTS: The mean +/- SEM stimulated parotid output was 0.33 +/- 0.07 mL/min per gland for patients who were not using medication and 0.33 +/- 0.04 mL/min per gland for patients using (1 to 6) medications. Analyses did not reveal a significant difference in salivary output between these groups. The salivary output of patients using various numbers of medications (1 or 2; 3 or 4; 5 or 6) was also compared. Analysis revealed no significant difference in salivary output related to the number of xerostomic medications used. CONCLUSION: The use of xerostomic medications may not necessarily affect stimulated parotid flow rate in patients with Sjögren's syndrome. These results suggested that gustatory stimulation may be adequate to overcome the inhibitory effect induced by xerostomic medications.

A pilot study on the effect of radiation on calmodulin in rat submandibular salivary glands.

Xerostomia and loss of salivary gland secretion is one of the most common complications of the radiation treatment of head-and-neck malignancies. The secretory mechanism in the salivary glands can be modulated by the concentration of intracellular Ca2+. Calmodulin is a calcium-binding protein that is widely distributed in nature and is involved in regulating intracellular calcium. In this study the effect of radiation on the concentration of calmodulin in rat salivary glands was investigated. Fourteen rats were divided into three groups: R1 (n = 4) and R2 (n = 5) received a single dose of 15 Gy and group C (n = 5) received no radiation. R1 and R2 animals were killed at weeks 2 and 10 post-irradiation, respectively. The submandibular glands were removed, homogenized and their total calmodulin was determined. The mean calmodulin concentrations were 6.4+/-1.1 microg/gland for controls, 14.1+/-3.7 microg/gland for R1 and 68.2+/-14.4 microg/gland for R2. Kruskal-Wallis ANOVA revealed a significant increase in the concentration of calmodulin following irradiation (p = 0.003). The relationship between this increase and the loss of salivary gland function is not yet clear.

Salivary histatins in patients with recurrent oral candidiasis.

The level of salivary histatins was evaluated in 20 patients with a history of recurrent oral candidiasis and in age-, sex-matched healthy controls. Salivary pH, flow rate, total protein and histatins were compared. Results of this study indicated that salivary pH was significantly lower among patients than controls (5.9 vs. 6.3, respectively, P = 0.002). No significant difference was observed in the mean flow rate and total protein between patients and controls (0.39 ml/min/gland vs. 0.46 ml/min/gland and 112.4 mg% vs. 113.0 mg%, respectively). However, the mean salivary histatin levels were significantly higher among patients than controls, both in terms of concentration (16.8 mg% vs. 11.1 mg%, P = 0.016) as well as a ratio of total protein (15 mg/g vs. 10 mg/g, P = 0.018). These findings indicate that oral candidiasis is associated with increased levels of salivary histatin. Such a finding suggests that oral candidiasis may modulate the levels of salivary histatin.

Effect of alcohol on energy storage of primary astrocytes and C6-glioma cells in vitro.

The present experiments were conducted to investigate the direct effects of ethanol on the energy metabolism of astrocytes and C6-glioma cells. Primary astrocytes were prepared from cerebral cortices of neonatal rats, and C6-glioma cells were purchased from American Type Culture Collection (ATCC). These cells were exposed to different concentrations of alcohol (100 mM, 200 mM, and 300 mM) for 15 minutes and 24 hours. The amount of ATP and PCr was measured by the method of Lowry and Passonneau (1972). Following 15 minutes treatment with different doses of ethanol the amount of ATP and PCr increased, in both cell types. Only the increase of ATP concentration with varying doses of ethanol (100 mM, 200 mM, and 300 mM) was statistically significant. Following 24 hours treatment of astrocytes with different doses of ethanol the concentration of ATP and PCr decreased. The decrease in concentration of ATP was significant with all three doses of ethanol, but the decrease of PCr concentration was only statistically significant with 300 mM ethanol. Following 24 hours treatment of C6-glioma cells to varying doses of ethanol, the concentration of PCr and ATP decreased. The decrease of PCr was statistically significant with all three doses of ethanol and the decrease of ATP concentration was only significant with 300 mM ethanol.

Evaluation of the levels of oral Candida in patients with Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to investigate the levels of oral Candida in patients with Sjögren's syndrome METHODS: The Candida count and salivary flow rate of patients with Sjögren's syndrome were compared with those of healthy control subjects. Candida cultures were obtained from oral rinses. The numbers of colony-forming units were determined through use of the Spiral System. RESULTS: The mean Candida count of patients with Sjögren's syndrome was 1672 +/- 1455 colony-forming units per mL; the count of healthy control subjects was 0.00 colony-forming units per mL. The mean salivary flow rate of patients with Sjögren's syndrome was significantly lower than that of healthy control subjects (0.16 +/- 0.13 mL/min/gland vs 0.55 +/- 0.24 mL/min/gland, respectively; p = 0.0001). However, Spearman rank correlation analyses did not reveal a significant correlation between salivary flow rate and Candida count (in colony-forming units per mL) among patients with Sjögren's syndrome. CONCLUSIONS: Alteration in the oral microbial flora in patients with Sjögren's syndrome may be enhanced by the reduction in salivary output.

Salivary electrophoresis in the diagnosis of Sjögren's syndrome.

OBJECTIVE: The purpose of this study was to investigate the potential use of salivary electrophoresis for the diagnosis of Sjögren's syndrome. METHODS: Salivary protein profiles of 43 patients and 39 healthy control subjects were compared on three different gel electrophoresis systems: sodium dodecylsulfate-polyacrylamide gel electrophoresis, anionic polyacrylamide gel electrophoresis, and immobilized pH gradient gel electrophoresis (isoelectric point, 3.5-5.0). RESULTS: Most of the patients with Sjögren's syndrome exhibited an electrophoretic profile that was different from that of the healthy control subjects. Among the three gel electrophoresis systems examined, the immobilized pH gradient system appeared to be the most reliable for Sjögren's syndrome. Tests of accuracy revealed that the immobilized pH gradient system exhibits high specificity (97%), sensitivity (95%), positive predictive value (97%), and negative predictive value (95%) in the diagnosis of Sjögren's syndrome. CONCLUSIONS: Our results suggest that salivary electrophoresis is a potentially useful test for the diagnosis of Sjögren's syndrome.

Prevalence of periodontal disease in patients with Sjögren's syndrome.

The periodontal status of 25 patients with Sjögren's syndrome was compared with that of an age-, sex-, and race-matched healthy controls. Clinical evaluation included determination of number of teeth, decayed/missing/filled surfaces, probing pocket depth, clinical attachment level, plaque index, gingival index, bleeding on probing, and measurement of alveolar bone level (examined with radiography). Results of the study suggest that patients with Sjögren's syndrome have a significantly higher plaque index score (p < 0.005), higher decayed/missing/filled surfaces scores (p < 0.05), increased alveolar bone loss (p < 0.05), deeper clinical attachment level (p < 0.05), and increased cementoenamel junction-alveolar bone crest distance (p < 0.005). Although no significant difference was found in the number of cases of "established periodontitis" between the Sjögren's syndrome and controls, odds ratio analysis suggests that patients with Sjögren's syndrome are at 2.2 times higher risk of having adult periodontitis than healthy controls.

Effect of 6-aminonicotinamide on metabolism of astrocytes and C6-glioma cells.

Brain tissue cells have been shown to use two predominant pathways for energy production. The first of these is the pentose phosphate shunt, and the second is glycolysis, followed by the TCA cycle. Inhibition of these pathways can result in a reduction of ATP, and changes in the concentration of various metabolites. In the present study, the acute and chronic effect of 6-aminonicotinamide (6-AN) (0.01, 0.02, and 0.03 mg/ml) was examined on astrocytes and C6-glioma cells. Following this treatment, glucose, lactate, glutamate, ATP, and PCr were assayed according to the procedures of Lowry and Passonneau. Our data indicated that following 15 minutes treatment of astrocytes and C6-glioma with 6AN there was no significant difference in the concentration of metabolites measured. However, following 24 hours treatment there was a significant increase in glucose concentration and significant reduction in the concentration of ATP, PCr, lactate and glutamate in both cell types. Morphological changes appeared later following 48 hours treatment with 6-AN in both cell types. Glucose accumulation can be explained by the fact that it is the precursor to both glycolysis and the pentose phosphate shunt. If these processes are inhibited, glucose will obviously accumulate and products like ATP, PCr, lactate and glutamate will decrease. Additionally, there was significant differences in concentration of glucose and lactate between astrocytes and C6-glioma cells. The significance of these differences has been discussed.

Effect of ammonium chloride on energy metabolism of astrocytes and C6-glioma cells in vitro.

Increased ammonia has been considered a key factor in the pathogenesis of hepatic encephalopathy. The high concentration of ammonia interferes with oxidative metabolism in the brain through an inhibitory effect on the tricarboxylic acid cycle (TCA). Inhibition of the TCA cycle may result in depletion of ATP. Due to the involvement of astrocytes in brain detoxification of ammonia, these cells are good candidates for studying ammonia's effect on energy stores in the brain. C6-glioma cells, which have altered glycolytic rates, may show greater sensitivity to the toxicity of ammonium chloride than astrocytes. To study the effect of ammonium chloride on energy storage of both astrocytes and C6-glioma, we observed the acute and chronic effects of NH4Cl (7.5 or 15 mM) on the metabolism of isolated astrocytes and C6-glioma cells. Primary astrocytes were isolated from the cerebral hemispheres of 1-2 day old Sprague-Dawley rats, and C6-glioma cells were purchased from the American Type Culture Collection (ATCC). Following treatment of the cells with ammonia, glucose, lactate, glutamate, ATP, and PCr were assayed. Our data showed that at 15 min following treatment with NH4Cl, there were no significant differences in the concentration of metabolites measured in astrocytes. However, following 15 min of treatment with NH4Cl, the concentration of some metabolites, for example, ATP and lactate, changed significantly in C6-glioma cells. We have shown that 24 h of treatment was sufficient time to see significant biochemical changes but not morphological changes in either cell type. Simultaneous biochemical and morphological changes were observed 48 h following treatment in C6-glioma cells and at 9-10 days following treatment in primary astrocytes. In primary astrocytes at 24 h following treatment, glucose utilization increased. This high utilization of glucose was in accordance with the increase in lactate and glutamate production and the decrease in ATP and PCr formation. In C6-glioma cells the utilization of glucose increased but this high utilization of glucose was consistent with a significant decrease in the concentration of lactate, glutamate and ATP.

Calcium influx in Trypanosoma brucei can be induced by amphiphilic peptides and amines.

The following study was undertaken to determine whether an inducible calcium influx pathway is present in intact bloodstream forms of Trypanosoma brucei. Fura-2 fluorescence was used to demonstrate that amphiphilic peptides and amines, including melittin, mastoparan and compound 48/80, each produced a dose dependent calcium influx across the plasma membrane. Calcium influx did not result from general disruption of membrane integrity, since a corresponding influx of ethidium bromide or other divalent cations was not observed. Instead, the calcium influx was selectively blocked by the calcium channel antagonists, La3+, Cd2+ or Ni2+, and was not affected by the Na+ channel antagonists, tetrodotoxin or amiloride. Activation of the trypanosome calcium influx pathway was dependent upon an intact membrane potential, and the rise in intracellular free calcium concentration ([Ca2+]i) was reversed upon membrane depolarization with gramicidin D. Changes in Ins(1,4,5)P3 did not accompany the calcium influx. Overall, these data provide the first evidence of an inducible calcium influx pathway in T. brucei, and describe methods to selectively manipulate this pathway.

Alcohol consumption by nursing rats and its effect on the cerebellum of the offspring.

To study the effect of alcohol on Purkinje cells and on myelination of the cerebellum of neonate rats, female Sprague-Dawley rats were fed alcohol (ethanol) during their nursing period. The alcohol was given in amounts of 5% or 10% per volume of drinking water. Food and liquids, equal in calories, were distributed among dams receiving alcohol. One group of control rats received similar diet but no alcohol. The results were then compared with those of a second group of controls, receiving no alcohol but having access to unlimited supplies of food and water. Histological examination of pups revealed degenerative changes in Purkinje cells and delayed myelination of the cerebellum.