These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose-induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Thus, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives. The present review will discuss the trends in fructose consumption, the metabolic consequences of increased fructose intake, and the molecular mechanisms leading to fructose-induced lipogenesis, insulin resistance and metabolic dyslipidemia.Obesity and type 2 diabetes are occurring at epidemic rates in the United States and many parts of the world. The "obesity epidemic" appears to have emerged largely from changes in our diet and reduced physical activity. An important but not well-appreciated dietary change has been the substantial increase in the amount of dietary fructose consumption from high intake of sucrose and high fructose corn syrup, a common sweetener used in the food industry. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, perturbs glucose metabolism and glucose uptake pathways, and leads to a significantly enhanced rate of  The new millennium has witnessed the emergence of a modern epidemic, the metabolic syndrome, with frightful consequences to the health of humans worldwide. The metabolic syndrome, also referred to as "Diabesity"  describeThe main driving forces for the increased prevalence of insulin resistance are modern Westernized diets and patterns of eating associated with the dramatic rises in obesity. Insulin resistance is often linked to the macronutrient content in the diet. In the past, diets high in saturated fats have been shown to induce weight gain, insulin resistance, and hyperlipidemia in humans and animals -22. Receparticularly refined sugars high in fructose, appears to be at least one very important contributing factor.Nutrition represents a lifestyle element that can be controlled, and that can directly influence health; therefore preventative nutrition and weight control should become a main focus of consumers and prepared-food providers . The Weset al examined nutrient consumption in the United States between 1909 and 1997, and discovered there was a significant correlation in the prevalence of diabetes with fat, carbohydrate, corn syrup, and total energy intakes. Most striking was the fact that when total energy intake was accounted for, corn syrup was positively associated with type 2 diabetes, while protein and fat were not [type of common, general use sweeteners represent as large an impact as the dramatic increase in the use of these caloric sweeteners. Between 1909 and 1997, sweetener use increased by 86%; and specifically, corn syrup sweeteners now represent over 20% of total daily carbohydrate intake, at an increase of 2100% [The general increases in consumption of calories, and specifically of refined carbohydrates and fructose, is clear and correlates positively with an alarming increases in metabolic syndrome. Can these seemingly harmless nutrients actually be directly associated with metabolic syndrome? Recent studies appear to support this link. In a 2004 study, Gross were not . High frwere not . In 1970were not . The typof 2100% .These documented trends have inspired a number of consumption studies and recommendations towards HFCS intake. In 1992, the USDA recommended that only 40 g of extra sugars should be added to a standard 2000 calorie a day diet . The amoFructose is readily absorbed and rapidly metabolized by human liver. For thousands of years humans consumed fructose amounting to 16–20 grams per day, largely from fresh fruits. Westernization of diets has resulted in significant increases in added fructose, leading to typical daily consumptions amounting to 85–100 grams of fructose per day. The exposure of the liver to such large quantities of fructose leads to rapid stimulation of lipogenesis and TG accumulation, which in turn contributes to reduced insulin sensitivity and hepatic insulin resistance/glucose intolerance. These negative effects of fructose are the reason that fructose metabolism has gained recent research attention. Interestingly, small catalytic quantities of fructose can have positive effects, and actually decrease the glycemic response to glucose loads, and improve glucose tolerance. These effects are also observed without any changes in insulin responses and non-esterified fatty acid (NEFA) and TG levels ,41. In 1in vivo [et al. examined overweight men and women who consumed fructose-containing sucrose, as opposed to artificial sweeteners as supplements to their diet. Weight, fat mass, and blood pressure were found to be lower in the artificial sweetener-consuming group compared to the sucrose-consuming group, and the sucrose group did not decrease intake of other nutrients to compensate for their increased calorie consumption from the sucrose. Subjects consuming the sweetener did not exhibit increases in energy intake, weight, and blood pressure that seen in the sucrose-consuming subjects [Unfortunately, one out of every four children in the United States consumes above the recommended 25% of total energy intake from sweeteners  and the in vivo . A compasubjects . Researcsubjects . The lonvia the portal vein to the liver. It is believed that the ability of the liver to metabolize high doses of fructose is responsible for the disruption in energy stores and fuel metabolism observed [negatively regulated by phosphofructokinase, fructose can continuously enter the glycolytic pathway. Therefore, fructose can uncontrollably produce glucose, glycogen, lactate, and pyruvate, providing both the glycerol and acyl portions of acyl-glycerol molecules. These particular substrates, and the resultant excess energy flux due to unregulated fructose metabolism, will promote the over-production of TG .et al. determined the association between food intake and blood glucose, comparing glucose and a fructose mixture. Glucose was administered as a high GI preload, which resulted in lower mealtime energy intakes compared to the low GI preload of the glucose-fructose mixture. An inverse relationship was seen between GI (and blood glucose concentrations), and appetite with consequent increased food intakes seen with fructose [et al. studied the comparative effects of glucose and fructose on blood glucose, insulin, and acute food intake. When subjects drank equienergetic preloads of glucose or fructose before an ad libidum buffet lunch, glucose concentrations were lower in the fructose group compared to glucose, and insulin concentrations were 50% higher in the fructose group in type 2 diabetics than in non-diabetics. The authors concluded that fructose may be a suitable replacement for glucose in diabetic patients – although it was found that satiating efficiencies of fructose certainly offered no advantages [et al. showed that subjects served meals with either 30% glucose beverages, or 30% fructose beverages, had differing hormonal and metabolic responses. Glycemic excursions and insulin responses were reduced by 66% and 65%, respectively, in the fructose-consuming subjects. There was a concomitant reduction in circulating leptin both in the short and long-term as well as a 30% reduction in ghrelin (an orexigenic gastroenteric hormone) in the fructose group compared to the glucose group. A prolonged elevation of TG was also seen in the high fructose subjects [The glycemic index (GI) has been commonly used to differentiate and compare various nutrients, as well as to describe how different foods produce different plasma glucose levels after ingestion. The GI can range from 100 for glucose and baked potato compared to approximately 20 for fructose and whole barley . Foods wfructose . In 2002vantages . This stvantages . The varvantages . Glucosesubjects . Both fasubjects . The inssubjects . Chronicsubjects .et al. injected fructose into the cerebroventricles of rats, and observed enhanced food intake, whereas similar concentrations of injected glucose suppressed appetite-agonist stimulated food intake [de novo lipogenesis. In combination with alterations in insulin signaling and leptin regulation, weight gain and unregulated energy intake can occur [et al. found that fructose, administered in the form of the disaccharide sucrose, promotes obesity more than glucose because fructose does not stimulate thermogenesis [Animal studies have illustrated various differences between glucose and fructose metabolism. In 2002, Miller d intake . Feedingd intake . This isan occur . In 1986ogenesis . These hIncreasingly, questions have been raised as to whether dietary carbohydrate and fructose intake are directly related to the development of type 2 diabetes. As insulin resistance is often associated with circulating C-peptide concentrations, a cross-sectional study was performed to assess dietary fructose and carbohydrate, and glycemic loads related to C-peptide concentrations. It was found that the highest quintile of fructose intake had 13.9% higher C-peptide concentrations than the lowest quintile. Of note, subjects with high intakes of cereal fiber had 15.6% lower C-peptide concentrations, indicating that these types of nutrients may have opposing roles in the development of insulin resistance . A definAlthough fructose does not appear to acutely increase insulin levels, chronic exposure seems to indirectly cause hyperinsulinemia and obesity through other mechanisms. One proposed mechanism involves GLUT5, a fructose transporter that is found to have significantly higher expression levels in young Zucker obese rats compared to lean controls. As the rats age and become diabetic, GLUT5 abundance and activity is compromised, causing an even more marked insulin resistance over lean rats, implying a possible role of GLUT5 receptors in the pathology of metabolic syndrome associated with fructose feeding and insulin resistance . In ratset al. reported an induction of the hepatic SREBP-1 isoform and lipogenic gene expression including FAS, acetyl-CoA carboxylase (ACC), and stearoyl-CoA desaturase (SCD) in mice following 7 days on a 60% fructose diet [There is considerable evidence supporting the ability of high fructose diets to upregulate the lipogenesis pathway, leading to increased TG production . Insulinose diet . It is kose diet -81. Simiose diet ,83. Therose diet , with ERose diet . Despiteose diet .et al. found that overexpression of protein tyrosine phosphatase 1B (PTP-1B), which is associated with dysfunctional insulin signaling, leads to increased mRNA and promoter activity of SREBP-1c, and subsequent increases in the expression of FAS. PTP-1B may therefore regulate the lipogenesis and hypertriglyceridemia associated with insulin resistance syndrome [via an increase in protein phosphatase 2A activity [Emerging evidence suggests that a protein phosphatase, known as PTP-1B, may link high carbohydrate feeding, insulin resistance, and lipogenesis. Recently, PTP-1B has been linked to lipogenesis and SREBP regulation. Shimizu syndrome . In insusyndrome  occurs isyndrome . The autactivity . FAS, anactivity . Other sactivity . The humactivity . As discactivity . Again, activity . It has activity . In thesactivity . The mecactivity . All of in vivo evidence of fructose-induced insulin resistance as assessed by euglycemic hyperinsulinemic clamp studies. Taghibiglou et al. further characterized the fructose fed hamster model demonstrating the development of a metabolic dyslipidemic state characterized by high plasma levels of VLDL-TG and apolipoprotein B (apoB) due to hepatic lipoprotein overproduction [via both an increased secretion, and decreased clearance of VLDL [via dietary fructose [There is growing evidence that the insulin resistant state developed upon fructose feeding is also associated with stimulated hepatic VLDL secretion. Several animal models have been employed to examine the mechanisms of this induction of VLDL, and the subsequent increases in plasma TG observed. Mechanistic studies based on carbohydrate versus lipid metabolism have recently become important because carbohydrate induced hypertriglyceridemia shares a metabolic basis with high fat diet induced endogenous hypertriglycerolemia. The similarly induced dyslipidemias would therefore have the same or similar atherogenic risks . Carbohyoduction . Serum T of VLDL . Also, h of VLDL . Evidenc of VLDL . Induced of VLDL ,105. It  of VLDL . Increas of VLDL . Increas of VLDL . The malfructose .et al. hypothesized that pro-oxidant stress response pathways may mediate hepatic increases in VLDL secretion and delayed clearance upon fructose feeding. Hypertriglyceridemic fructose fed rats were treated with lipoxygenase inhibitors, which reversed the inflammatory protein activity response, and the lipid dysregulation observed [Another contributing factor to VLDL overproduction includes fructose effects on lipid peroxidation. High fructose diets can have a hypertriglyceridemic and pro-oxidant effect, and fructose fed rats have shown less protection from lipid peroxidation. Replacing the fructose in these diets with a more natural source of high fructose, honey, reduces this susceptibility and lowers plasma nitrite and nitrate levels . In 2004observed . Recent observed . Oxidatiobserved . LA treaobserved . Taken tet al. found that following 8 weeks of a high fructose diet, rats showed decreased PPARα mRNA and protein levels [et al., who found evidence for enhanced lipoprotein assembly, reduced intracellular apoB degradation, and increased microsomal triglyceride transfer protein (MTP) mass, mRNA and activity in the fructose fed hamster [de novo lipogenesis and MTP expression. Amplified MTP activity and expression would be expected to stimulate the assembly and secretion of apoB-lipoproteins, as an association has been demonstrated between MTP levels and VLDL production [et al. showed that fructose diets altered the structure and function of VLDL particles causing and increase in the TG: protein ratio, and an increased total cholesterol and phospholipid content [de novo lipogenesis. It is likely a combination of some or all of these factors that contribute to the elevated TG seen in a fructose rich carbohydrate fed model of metabolic disorder. High fructose, which stimulates VLDL secretion, may initiate the cycle that results in metabolic syndrome long before type 2 diabetes and obesity develop [Observations made in our own laboratory have also shown aberrant lipogenesis activity. In primary hepatocytes isolated from fructose fed hamsters, there were significant increases in LXRα, SREBP-1, FAS and SCD, which indicate increased activity of the lipogenic pathways (unpublished observations). Fructose has also been implicated in reducing PPARα levels in rat hepatocytes. PPARα is a ligand activated nuclear hormone receptor that is responsible for inducing mitochondrial and peroxisomal β-oxidation. Nagai n levels . In addin levels . Other m hamster . These m hamster . It appeoduction . As insuoduction , the uproduction . Howeveroduction  as well oduction  where inoduction . Recent oduction . Early s content . LDL par content  and ther develop .de novo lipogenesis and increased MTP availability. Comparison of plasma lipoproteins from fructose-fed animals showed a significant shift toward secretion of larger, less dense, chylomicrons in the insulin resistant animals [More recently, our studies have identified an interesting link between the development of insulin resistance and deregulation of intestinal lipoprotein metabolism . Chronic animals .de novo lipogenesis and TG synthesis, driven by the high flux of glycerol and acyl portions of TG molecules coming from fructose catabolism. These metabolic disturbances appear to underlie the induction of insulin resistance commonly observed with high fructose feeding in both humans and animal models. Fructose induced insulin resistant states are commonly characterized by a profound metabolic dyslipidemia, which appears to result from hepatic and intestinal overproduction of atherogenic lipoprotein particles. Taking into consideration that a typical western diet not only contains high levels of fructose but is also rich in both fat and cholesterol, synergistic interactions among these nutrients can readily occur leading to a greater degree of insulin resistance and dyslipidemia. In conclusion, emerging evidence from recent epidemiological and biochemical studies clearly suggests that the high dietary intake of fructose has rapidly become an important causative factor in the development of the metabolic syndrome. There is an urgent need for increased public awareness of the risks associated with high fructose consumption and greater efforts should be made to curb the supplementation of packaged foods with high fructose additives.The alarming increase in fructose consumption may be an important contributor to the epidemic of obesity and insulin resistant diabetes in both pediatric and adult populations. For thousands of years, the human diet contained a relatively small amount of naturally occurring fructose from fruits and other complex foods. Adaptation of humans to a high glucose/low fructose diet has meant that hepatic carbohydrate metabolism is designed to actively metabolize glucose with a limited capacity for metabolizing a small daily intake of fructose. The increasing application of high fructose sweeteners over the past few decades has resulted in a considerable rise in the dietary intake of fructose. A high flux of fructose to the liver, the main organ capable of metabolizing this simple carbohydrate, disturbs normal hepatic carbohydrate metabolism leading to two major consequences Figure : perturbACC: acetyl-CoA carboxylaseapoB: apolipoprotein BERK: extracellular signal related kinaseFAS: fatty acid synthaseGSK-3: glycogen synthase kinase-3HDL: high density lipoproteinHFCS: high fructose corn syrupIR: insulin receptorIRS: insulin receptor substrateLA: alpha-lipoic acidLDL: low density lipoproteinLXR: liver X receptorMAPK: mitogen activated protein kinaseMTP: microsomal triglyceride transfer proteinNEFA: non-esterified fatty acidsPA-1: plasminogen activator inhibitor-1PI3-kinase: phosphatidylinositol 3 kinasePKB: protein kinase BPKC: protein kinase CPPAR: peroxisome proliferator activated receptorPTP-1B: protein tyrosine phosphatase-1BSCD: stearoyl-CoA desaturaseSREBP: sterol regulatory element binding proteinTG: triglycerideVLDL: very low density lipoprotein


To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P.Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N.Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation. Systemic thromboembolism represents a major complication in patients with mitral stenosis (MS), especially in those who have atrial fibrillation ,2. A hypThis study assessed systemic coagulation and platelet activities by measuring fibrinogen levels and platelet aggregation in mitral stenotic patients' peripheral venous blood and compared these measures to hematologic parameters in normal controls.Eighteen newly diagnosed patients with rheumatic MS were enrolled in the study. All were coded as having NYHA class I functional capacities. Two patients were excluded from this study, one due to pregnancy, the second due to current use of oral contraceptives. The study group thus consisted of 16 patients with MS . Peripheral venous coagulation activity and platelet activation tests were also evaluated in 16 control patients , who were normal volunteers. All participants gave informed consent.Patients were excluded from participating in the study for the following reasons: aortic and/or pulmonary valve disease, severe mitral regurgitation and/or left ventricular systolic dysfunction, atrial fibrillation, hypertension, diabetes mellitus, dyslipidemia, a history of renal or liver disease, malignancy, venous thrombosis, systemic or pulmonary embolism, congenital hemorrhagic disease, thrombocytopenia, thrombocytosis, acute or chronic inflammatory disease, autoimmune disease or current use of oral contraceptives or anticoagulant or anti-platelet drugs.In patients with MS, transthoracic echocardiography was performed with a 2.5-MHz transducer and a Hewlett Packard Sonos 4500 system to assess left atrial (LA) diameter, mitral valve area (MVA) and the transmitral mean pressure gradient (TMmPG). The LA anteroposterior diameter was determined using standard M-mode criteria , and mitPeripheral venous blood (for measurement of fibrinogen levels) was transferred into tubes containing 1:9 trisodium citrate . Blood samples were centrifuged within 1 hour of collection at 2,500 g for 10 minutes to separate out the plasma. An STA fibrinogen kit measured fibrinogen levels in plasma, using the clotting method of Clauss. The median inter-assay and intra-assay coefficients of variation for the assays were 3.57% and 3.65% for fibrinogen, respectively.5/ml were excluded from the study. For in vitro platelet aggregation tests, platelet-rich plasma (PRP) samples were obtained by centrifuging samples at 1,100 rpm for 10 minutes in a refrigerated centrifuge . Centrifuging samples at 3,000 rpm for 10 minutes yielded platelet-poor plasma (PPP) samples. The platelet counts of both PRP and PPP samples were performed using the same hemocytometer. Platelet-poor plasma samples were added to the PRP samples to obtain a total platelet count of 2 × 105/ml. These platelet-equalized plasma (PEP) samples were analyzed to determine in vitro platelet function, using ADP, Collagen and Epinephrine  in an automated aggregometer . Results were evaluated and calculated using maximum aggregation ratios.Blood samples were obtained from the forearm veins of participants, using standard blood drawing procedures . Several samples were drawn for different components of the study. Nine milliliters of blood was collected into special tubes containing 1 ml of 3.8% sodium citrate. Platelet count and other parameters, including white blood cell count and hemoglobin, were measured at the Ege University Hospital Hematology Laboratory using an automated hemocytometer . Patients with platelet counts less than 2 × 10PRP from healthy controls was centrifuged for 10 minutes, after which the supernatant was decanted, and normal saline added to the platelet fraction and centrifuged for 10 minutes. This procedure was repeated three times. Plasma obtained from patients with MS was added to the PPP from the control group, and platelet function tests were performed, using the same procedures mentioned above.Chi Square tests were employed to compare categorical variables, while Mann Whitney U tests were used, where appropriate, in the univariate analysis. Platelet aggregation changes were evaluated by repeated-measures analysis of variance for intra-group comparisons. Values of p < 0.05 were considered significant. SPSS 10.0 Statistical software was used in the statistical analysis.There were no significant differences between Groups P and N for the following variables: age, gender, current smoker, platelet, white blood cell and hemoglobin count. Clinical characteristics of the study groups are shown in Table Adenosine diphosphate, collagen and epinephrine-induced platelet aggregation ratios were significantly higher in Group P than in Group N . When the PPP obtained from Group P was added to Group N subjects' platelets, and platelet function tests were repeated, ADP and collagen-induced , but not epinephrine-induced (61.9 ± 16.2), aggregation ratios were statistically increased compared to baseline  truly reflects platelet activation in vivo [The results from this study emphasize the complexity of the events related to platelet activation in MS. Also compounding the challenges faced in this area is uncertainty about the gold standard method for assessing platelet activation and whether platelet aggregation ( in vivo .


In various people of the Western world, gastro-oesophageal reflux (GOR) has been reported to be a common problem. Various studies have also assessed the relationship between GOR and dental erosion. The authors are not aware of such studies in Nigerians. It is therefore the aims of the present study to estimate the prevalence of GOR; to estimate the prevalence of dental erosion in patients with GORD; to document the oral findings in patients diagnosed with GORD and to compare these findings with previous studies elsewhere.A total of 225 subjects comprising of 100 volunteers and 125 patients diagnosed with GORD were involved in this study. History of gastric juice regurgitation and heartburn were recorded. Oral examination to quantify loss of tooth structure was done using the tooth wear index (TWI) designed by Smith and Knight (1984).Twenty patients with GORD presented with dental erosion in the maxillary anterior teeth with TWI scores ranging from 1–3. The prevalence of erosion was found to be statistically significant between GORD patients (16%) and control (5%) (p < 0.05), but not significant between endoscopic diagnostic groups (p > 0.05).The present study supports the consideration of dental erosion as the extra-oesophageal manifestation of GORD. However the association between GORD and burning mouth sensation needs more investigation. Gastro-oesophageal reflux (GOR) is the passage of gastric contents into the oesophagus. Once past the upper oesophageal sphincter, the gastric juice may pass into the oral cavity. The continual exposure of the teeth and other oral structures to gastric refluxate may result in dental erosion and other soft tissue symptoms . Any aciCurrent understanding of GOR and GORD is that acid reflux into the oesophagus may be caused by three possible mechanisms :(1) Transient spontaneous or inappropriate relaxations of the sphincter;(2) Transient increase in intra-abdominal or intragastric pressure;(3) Functional abnormality of the lower oesophageal sphincter (LOS).et al .To document the oral findings associated with GORD, patients referred to the Gastro-intestinal (GIT) endoscopic unit for investigation of gastro-oesophageal tract disease were also evaluated for dental erosion and other soft tissue symptoms such as non-specific burning sensation in the mouth and sensitivity in the tongue. The dental evaluation included history to determine potential eatiological factors responsible for dental erosion. Patients were also examined clinically to quantify loss of tooth structure using the tooth wear index. The oral examination was performed by one of the authors (AO), blind as to the endoscopic diagnosis of subjects. Patients with positive endoscopic findings plus the occurrence of heartburn two or more times per-week were assessed to meet the criteria for GORD. When a clear-cut endoscopic evidence of oesophageal inflammation was seen , a diagnosis of oesophagitis was made. Gastroduodenal ulcer was diagnosed through endoscopic evidence of an ulcer with a necrotic base or a clear-cut scar. This was grouped into those with gastric ulcer  and those with duodenal ulcer (ulcer in the bulbus). Only one diagnosis was given for each patient.Data collected were entered into a computer and analysed using chi-square. P value <0.05 were considered significant.Thirty-five of the 100 subjects attending the medical outpatient department had acid regurgitation and acidic taste sensation in the mouth. This occurs most of the time following a full stomach meal and is associated with belching. Table One hundred and twenty five patients were diagnosed with GORD. Twenty presented with dental erosion in the maxillary anterior teeth with TWI scores ranging from 1–3. The prevalence of erosion was found to be statistically significant between GORD patients (16%) and controls (5%) , Table The details of the orodental findings in the twenty patients who had dental erosion are shown in Table Burning mouth sensation and peppery sensation in the tongue where reported by 9 patients who had had gastrointestinal symptoms for 10–20 years.Various methods have been employed in the investigation of GOR including endoscopy with biopsy and contrast radiography. Although twenty-four hour oesophageal pH monitoring is considered the gold standard investigation of GOR , due to et al [et al [The prevalence of GOR and GORD in the present study was 35% and 11% respectively. The prevalence of GOR was much lower than the 60% reported in the U.K. and other Western societies , whereaset al . The prel [et al , and Schl [et al  respectil [et al , whereasl [et al ,25. Alsol [et al .The consumption of acidic beverages among the study population was low, hence the silence on its contribution to the overall disease level. Although this is controversial, the authors are of the opinion that intrinsic (gastric) acid results in palatally eroded sites  declare that they have no competing interests.AOO conceived of the study, participated in its design, performed the dental examination including the application of the tooth wear index (TWI), and participated in the initial draft and final write-up of the manuscript. EAA and DAN performed the endoscopic examination of patients with gastro-oesophageal reflux disease, participated in the initial draft and final write-up of the manuscript.The pre-publication history for this paper can be accessed here:


INO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Scs2 antagonizes the action of the transcriptional repressor Opi1 under conditions that induce the unfolded protein response (UPR) and, in turn, activate INO1. Whereas repressed INO1 localizes throughout the nucleoplasm, the gene is recruited to the nuclear periphery upon transcriptional activation. Recruitment requires the transcriptional activator Hac1, which is produced upon induction of the UPR, and is constitutive in a strain lacking Opi1. Artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the intranuclear localization of a gene can profoundly influence its mechanism of activation. Gene recruitment to the nuclear periphery, therefore, is a dynamic process and appears to play an important regulatory role.The spatial arrangement of chromatin within the nucleus can affect reactions that occur on the DNA and is likely to be regulated. Here we show that activation of  INO1 indicates that the recruitment of the gene to the nuclear membrane appears to play an important part in its regulationA study of the yeast gene  For over a hundred years, it has been recognized that chromatin is distributed non-randomly within the interphase nucleus . More reSaccharomyces cerevisiae, genes localized in proximity to telomeres are similarly transcriptionally silenced , which encodes inositol 1-phosphate synthase. The UPR is an intracellular signaling pathway that is activated by the accumulation of unfolded proteins in the endoplasmic reticulum (ER), which can be stimulated by treatment with drugs that block protein folding or modification or, in yeast, by starvation for inositol  in the promoters of most target genes to promote transcriptional activation . Howevernditions . Opi1 renditions . Positivromoters . Our preromoters .INO-controlled genes by the UPR, we have examined the molecular events leading to the activation of INO1. We find that Scs2, an integral protein of the nuclear and ER membrane that was recently shown to play a role in telomeric silencing -tagged Scs2. We observed specific recovery of Scs2-HA in Opi1-myc immunoprecipitates   . Consistnditions A. Real-ttutively B. AlthouINO1 promoter directly. Early gel-shift experiments using yeast lysates suggested that Opi1 might interact with DNA (INO1 promoter in vivo. We observed specific enrichment of the INO1 promoter by immunoprecipitation of Opi1 from cells grown in the presence of inositol (repressing condition) but no significant enrichment of the INO1 promoter by immunoprecipitation of Opi1 from cells starved for inositol  lacking the transmembrane domain, which was localized throughout the cell and was not excluded from the nucleus , which serves as a nuclear membrane–targeting signal  as the baseline for subsequent comparisons —did not improve the growth of the scs2Δ mutant in the absence of inositol. Consistent with the previous report that FFAT does not require Scs2 to promote nuclear membrane targeting, we observed approximately 50% membrane association of INO1 in the strain expressing the FFAT-Lac repressor . Thus, the defect in transcription of INO1 in the scs2Δ mutant could be rescued, at least partially, through artificial targeting of INO1 to the nuclear membrane. This result demonstrates that nuclear membrane association is functionally important for achieving INO1 transcriptional activation.The experiments described above indicate that there is a correlation between membrane association of analysis A or on the array C—or if tINO1 occurs at the nuclear membrane and requires the integral membrane protein Scs2. Moreover, artificial recruitment of INO1 to the nuclear membrane permits activation in the absence of Scs2, indicating that the precise intranuclear localization of a gene can profoundly influence its activation. Most importantly, we have shown that the localization of INO1 depends on its activation state; gene recruitment therefore is a dynamic process and appears to play an important regulatory role.It is becoming increasingly clear that the spatial arrangement of chromosomes within the nucleus is important for controlling the reactions that occur on DNA and might be regulated : We propose that the environment of membrane-tethered INO1 promotes late steps in the transcription activation—such as chromatin remodeling, discussed above—permitting INO1 to be expressed upon transient Hac1-induced Opi1 dissociation. Therefore, we envision that dissociation of Opi1 from the INO1 promoter is coupled to the delivery of the gene to an environment near the nuclear membrane that is permissive for its activation.Both Hac1  and Scs2Ascaris suum sperm that forms a cytoskeletal structure and confers motility to sperm cells. It is thus tempting to speculate that Scs2 might similarly self-associate in the plane of the nuclear membrane, perhaps providing a two-dimensional matrix on which membrane-associated reactions could be organized. One suggestion from our data is that Scs2 may function as a local sink for Opi1. But it is also clear that other nuclear membrane components are likely to participate in the reaction. Opi1, for example, still localizes to nuclear membranes even in scs2Δ cells, indicating that another, yet-unidentified Opi1 binding partner must exist . Monoclonal anti-Pgk1, rabbit polyclonal anti-GFP, goat anti mouse IgG-Alexafluor 594, and goat anti-rabbit IgG Alexafluor 488 were from Molecular Probes .Monoclonal anti- HA antibody HA11 was obtained from Babco . Monoclonal anti-All restriction endonucleases and DNA modification enzymes were from New England Biolabs . Unless indicated otherwise, all other chemicals and reagents were from Sigma .. Tags and disruptions marked with either the rkan gene from E. coli or the His5 gene from S. pombe were introduced by recombination at the genomic loci as described (r),(OPI1–13myc::kan JBY350-r1 (scs2Δr),:: kan JBY359 r),(SCS2-HA:: kan JBY356–1A (opi1Δ::LEU2), JBY356–1B (opi1Δ::LEU2 scs2Δr ),:: kan JBY356–1C (scs2Δr),:: kan JBY356–1D (wild-type control), JBY361 (scs2Δr),TMD-HA:: kan JBY370(INO2-HA3::His5+), JBY371 (INO4-HA3::His5+), JBY393 (INO4-myc::His5+ MATa), JBY397 r INO1:LacO128:URA3 HIS3:LacI-GFP),(SEC63–13myc:: kan JBY399 (SEC63–13myc::Kan^r INO1:LacO128:URA3 HIS3:LacI-FFAT-GFP), JBY401 (ino4Δr INO1:LacO128:URA3 HIS3:LacI-GFP MATα),::LEU2 SEC63–13myc::Kan JBY404 (opi1Δr INO1:LacO128:URA3 HIS3:LacI-GFP),::LEU2 SEC63–13myc::Kan JBY406 (opi1Δr INO1:LacO128:URA3 HIS3:LacI-FFAT-GFP),::LEU2 SEC63–13myc::Kan JBY409 r URA3:LacO128:URA3 HIS3:LacI-GFP),(SEC63–13myc::Kan JBY412 (INO2-myc::His5+), JBY 416 (hac1Δr LacO128:INO1 HIS3:LacI-GFP)::URA3 SEC63–13myc::Kan.All yeast strains used in this study were derived from wild-type strain CRY1 escribed . Strainsmyc was created by first amplifying the OPI1-myc coding sequence and 686 bp upstream from the translational start site from strain JBY345 using the following primers: OPI1 promoter Up (5′-GGGAGATACAAACCATGAAG-3′) and OPI1 down (5′-ACTATACCTGAGAAAGCAACCTGACCTACAGG-3′). The resulting fragment was cloned into pCR2.1 using the Invitrogen  TOPO TA cloning kit. The OPI1-myc locus was then cloned into pRS315 as a HindIII-NotI fragment. Plasmid pASF144 expressing GFP-lacI has been described (OPI1: LacI_FFAT1 (5′-AATTGGACGATGAGGAGTTTTTTGATGCCTCAGAGG-3′) and LacI_FFAT2 (5′-AATTCCTCTGAGGCATCAAAAAACTCCTCATCGTCC-3′). The orientation of the insert was confirmed by DNA sequencing. Both pAFS144 and pGFP-FFAT-LacI were digested with NheI, which cuts within the HIS3 gene, and transformed into yeast.Plasmid pRS315-Opi1-escribed . PlasmidINO1 coding sequence, with 437 bp upstream and 758 bp downstream, was amplified from yeast genomic DNA using the following primers: INO1_promoter_Up (5′-GATGAGGCCGGTGCC-3′) and INO1_3′down (5′-AAGATTTCCTTCTTGGGCGC-3′), and cloned into pCR2.1 using the Invitrogen TOPO TA cloning kit, to produce pCR2.1-INO1. INO1 was moved from pCR2.1 into pRS306 as a KpnI fragment, to produce pRS306-INO1. The Lac operator array was then cloned from pAFS52 into pRS306-INO1 as a HindIII-XhoI fragment, to produce plasmid 10.2. Because the Lac operator fragment was smaller than had been reported (2.5 kb instead of 10 kb), presumably reflecting loss of Lac operator repeats by recombination, the Lac operator array was duplicated by digesting plasmid 10.2 with HindIII and SalI and introducing a second copy of the 2.5-kb HindIII-XhoI fragment, as described  or the microsomal fraction  and incubated for 30 min at 4 °C; detergent-insoluble material was then removed by centrifugation at 21,000 × g, 10 min. Anti-myc agarose was added to the supernatant and incubated 4 h at 4 °C, while rotating. For the experiment in Cells were lysed using glass beads in IP buffer . The whole cell extract was used for coimmunoprecipitation of Ino2-.For immunoblot analysis, 25 μg of crude protein, prepared using urea denaturing lysis buffer , was sepmyc . After sonication, lysates were centrifuged 10 min at 21,000 × g to remove insoluble material and incubated for 4 h with anti-HA agarose or anti-myc agarose. After elution of immunoprecipitated DNA and reversal of crosslinks by heating to 65 °C for 8 h, DNA was recovered using Qiaquick columns from Qiagen . Eluted samples were analyzed by PCR using the following primers against the INO1 promoter or the URA3 gene: INO1_proUp2 (5′-GGAATCGAAAGTGTTGAATG-3′), INO1_proDown (5′-CCCGACAACAGAACAAGCC-3′), URAup (5′- GGGAGACGCATTGGGTCAAC-3′), and URADown (5′-GTTCTTTGGAGTTCAATGCGTCC-3′).Chromatin immunoprecipitation was carried out on strains expressing endogenous levels of tagged Ino2, Ino4, and Opi1 as described , with thINO1 promoter and the URA3 gene were calculated using this standard curve. The ratio of INO1 promoter to URA3 was corrected for each sample to make the input ratio equal to 1.0.PCR reactions were carried out as described  using a INO1 (NP_012382), Trl1 (NP_012448), Opi1 (NP_011843), Hac1 (NP_011946), Ino2 (NP_010408), Ino4 (NP_014533), Scs2 (NP_009461), Rap1 (NP_014183), URA3 (NP_010893), SSS1 (NP_010371), Pgk1 (NP_009938) lacI (NP_414879), and Ikaros (Q03267).The GenBank accession numbers of the genes and proteins discussed in this paper are Ire1 (NP_116622),


Bone metastases are common in many types of cancer. As screening methods different imaging modalities are available. A new approach for the screening of osseous metastases represents the measurement of bone metabolic markers. Therefore aim of this study was to evaluate the usefulness of the determination of bone metabolic markers aminoterminal propeptide of type I procollagen  and the carboxyterminal pyridinoline cross-linked telopeptide of type I collagen  for the detection of bone metastases associated with other malignancies.88 patients aged 21 – 82 years with malignant tumors were prospectively studied. The serum concentrations of PINP and ICTP were measured and compared to the results of bone scintigraphy, radiological bone series, CT, MRI and clinical follow-up.Osseous metastases were found in 21 patients. 19 of them were correctly identified by bone scintigraphy (sensitivity: 90%). For bone metabolic markers results were as follows: ICTP sensitivity: 71%, specificity: 42%; PINP sensitivity: 24%, specificity: 96%.As markers of bone metabolism PINP and ICTP showed low sensitivity and/or specificity for the detection of osseous metastases. The presented markers did not seem to be sufficient enough to identify patients with bone metastases or to replace established screening methods. Bone metastases are common in advanced cancers of the lung, breast, kidney, prostate and others. In autopsy studies the prevalence ranges from 47–85% . In patiThe screening for bone metastases is usually based on bone scintigraphy, confirmed by supplemental radiographic bone surveys, computer tomography, or magnetic resonance imaging. Bone scans are simple to perform and examine the whole skeleton, but although the sensitivity of this method is high its specificity is poor. For this reason, in many cases a positive scan requires confirmation by other imaging modalities, which leads to higher costs and time-consuming investigations. Therefore, an alternative cost-effective screening method with a similar sensitivity and a higher specificity would be very welcome.For the formation of osseous metastases the extracellular matrix consisting of collagens combined with noncollagenous glycoproteins and proteoglycans, including the basement membrane and the interstitial stroma play an important role. Normally, the extracellular matrix serves as a barrier for the attachment and invasion of malignant cells, but the proteolytic activity of tumor cells leads to the destruction of its collagenic components, thus facilitating the local invasion of malignant cells and finally the development of bone metastases . The majRecently, bone metabolic markers have been reported to be useful in diagnosing bone metastases ,5. NewlyBoth parameters have been identified as potential candidates for the early detection of bone metastases. Therefore, the purpose of this study was to evaluate the usefulness of PINP and ICTP in patients with newly diagnosed cancer for the screening of osseous metastases compared to current standard protocols.For this study 88 consecutive patients  with malignant tumors  between the ages of 21 and 82 were included. None of these patients were under a tumor specific therapy or presented primary bone disease such as osteoporosis or Paget's disease, which could interfere with the results of this study. All patients gave written consent to participate in this prospective study, which was approved by the local ethics committee.Blood samples for measuring PINP and ICTP were collected on the same day as bone scintigraphy. Due to higher PINP values at night, all samples were taken early in the morning and stored at -20°C until assayed. Apparent hemolytic serum was excluded.Serum concentrations of ICTP and PINP were measured by using commercially available RIA kits . According to the kit description, the normal range of ICTP was 1.6–5.3 μg/l for females and 1.4–5.2 μg/l for males. For PINP the normal range was 19–102 μg/l for females and 21–78 μg/l for males.99m-DPD . SPECT acquisitions were performed from suspected regions . The total acquisition time ranged from 100 to 130 min for planar and SPECT scans. The bone-scanning procedure was performed in accordance with procedural guidelines published by the Society of Nuclear Medicine [Two double-head gamma cameras  and a triple-head gamma camera  were used for planar and tomographic (SPECT) bone scans, respectively. Low-energy, high-resolution collimators 1024 × 256 matrix) were used and data acquisition was started 2–4 hrs after intravenous injection of 550–700MBq Tc matrix wTwo experienced nuclear medicine physicians interpreted planar bone and SPECT images. Initially, neither of the readers knew of the findings of the other or the results of other imaging modalities. Increased tracer-uptake located at joints or on the edge of vertebral bodies adjacent to disk spaces was interpreted as arthritis or osteophytes, respectively. Lesions were classified as fractures when they showed a typical linear and curved pattern e.g. adjacent lesions in the ribs. Multiple lesions of varying size, shape and intensity, elongated rib lesions or photopaenic areas (cold spots) were classified as osseous metastases or suspicious lesions, where further analysis or imaging methods were necessary. In general, interpretation was performed following the criteria described by Krasnow et al. . FinallySensitivity and specificity were calculated. Values are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS-Software version 10.0 . The Mann-Whitney U test was applied to compare concentrations of PINP and ICTP, respectively. A value of p < 0.05 was considered to be statistically significant. Furthermore, we performed a receiver operating characteristic (ROC) analysis to assess the impact of bone metabolic markers in detecting bone metastases.Osseous metastases were found in 21 patients with the following tumors: 8 patients with breast cancer, 3 patients with head/neck cancer and local osseous tumor invasion, 3 prostatic cancer, 2 plasmocytoma, 1 each lung cancer, histiocytosis X, thyroid cancer and sarcoma and 1 patient had breast cancer and hypernephroma.Using planar and tomographic bone scintigraphy, 19 patients were correctly diagnosed as patients with osseous metastases by both investigators independently and confirmed by other imaging modalities or follow-up (sensitivity: 90%). Two patients were false negative, 1 patient with histiocytosis X  and 1 had breast cancer and hypernephroma . In 43 patients there was no evidence for osseous metastases by bone scintigraphy or other imaging modalities. In 24 patients, changes in bone scintigraphy were described not typical for osseous metastases, but additional imaging methods were recommended for verification. Most of these lesions were located in the ribs (singular focus) and the spine, typically for fractures, traumatic injuries or osteoporotic changes (compression fracture). Additional verification was recommended especially in those patients where no history of traumatic injuries, degenerative processes or osteoporosis was known. Verification was done in most cases by plain radiography, computed tomography or within the staging by FDG-PET.Serum ICTP was elevated above the upper reference limit  in 56 patients . In females the mean value was 7.64 ± 4.25 μg/l. In the group with metastases (n = 10) the mean value was 10.71 ± 5.90 μg/l compared to 6.41 ± 2.66 μg/l in the other group (n = 25) without metastases (p = 0.11). In males the mean value was 8.74 ± 9.49. The patients with metastases (n = 11) showed a value of 9.23 ± 7.62 compared to 8.61 ± 9.99 in patients (n = 42) without bone metastases (p = 0.24). Figures Serum PINP was elevated above the upper reference limit  in 8 patients 5 male, 3 female). In females the mean value was 57.42 ± 38.50 μg/l. In the group of patients with metastases (n = 10) the mean value was 73.29 ± 62.38 μg/l compared to 51.07 ± 22.23 μg/l in the group (n = 25) without metastases (p = 0.95). In males the mean value was 45.59 ± 43.20. The patients with metastases showed a value of 60.70 ± 83.86 compared to 41.63 ± 23.99 in patients without bone metastases (p = 0.70). Figures  female. Neither parameter achieved statistical significance by ROC analysis. According to the ROC analysis, the optimal cut-off level of ICTP that maximizes sensitivity and specificity was 7.5 μg/l in females (60% sensitivity and 88% specificity) and 5.9 μg/l in males (73% sensitivity and 57% specificity), for PINP 85.5 μg/l in females (40% sensitivity and 96% specificity) and 25.8 μg/l in males (46% sensitivity and 74% specificity).Figure The types of tumors in our study were very heterogeneous, which reflects the normal day-to-day situation seen in a department of nuclear medicine. To obtain additional information concerning special tumor types, we performed a separate examination of two different tumor types with a higher number of patients: breast cancer and head/neck cancer.The group with breast cancer consisted of 20 patients. Subsequently, bone metastases could be verified in eight of these patients. Six cases could be identified clearly by bone scan, in one patient additional plain radiographic analysis was recommended to confirm the diagnosis and one patient was false positive . For bone markers the following results were observed:ICTP: sensitivity: 63%, specificity: 25%. PINP: sensitivity: 25%, specificity: 100%. Figure 19 patients with head/neck cancer were examined . In these patients determination of whether local osseous structures are involved is essential for the preoperative planning of further treatment. In three patients osseous structures were affected by the tumor and all of these were correctly diagnosed by bone scintigraphy. ICTP and PINP were right positive in two separate patients. Sensitivity for PINP and ICTP was 33%, specificity for ICTP was 56% and for PINP 94%.Metastatic bone disease is a serious clinical problem. Complications associated with osseous metastases are pain, fractures, spinal cord compression, paralysis, etc., which lead to a significant reduction in the quality of life of tumor patients. Several diagnostic tools are available to detect bone metastases, including plain radiography, computer tomography, magnetic resonance imaging and bone scintigraphy. Of these, radionuclide bone scanning using Tc-99m labeled diphosphonates is the most widely used and accepted method for the detection of bone metastases. Bone scintigraphy can detect osseous metastases several months before changes in plain radiographs can be seen, thus making bone scintigraphy an excellent diagnostic tool. However, this method is expensive, is not always available in every hospital and has the disadvantage of showing a positive reaction even to bone inflammation, degenerative changes and fractures or flare reaction, which leads to a reduced specificity. This is the reason why many authors have described outcomes regarding diagnosis of bone metastases and observation of the clinical course using markers of bone turnover.In recent years, there have been important advances in the field of biochemical markers of bone turnover and new methods have emerged . MeasureYoshida et al. reported on the serum concentration of type I collagen metabolites as a quantitative marker of bone metastases in patients with prostate cancer . They coFor other tumor types we did not observe a similar high correlation. Horiguchi et al. reported on the usefulness of ICTP as a marker for bone metastases in patients with lung cancer . He suggAnother major group of patients in our study were females with breast cancer. The literature on these tumors and the value of bone metabolic markers for detection of osseous metastases is very controversial. Blomqvist et al. reported a positive and significant correlation between ICTP and PICP and the number of bone metastases, plus Wada et al. suggested that ICPT might be a useful marker for screening and monitoring bone metastases in breast cancer ,13. In cThe types of tumors in our study were very heterogeneous, which seems to reflect the circumstances seen on a daily basis. Due to the fact that tumors can metastasize to bone in different ways (osteoblastic and/or osteolytic) we established parameters for both possibilities. For PINP, the marker for osteoblastic activity, the specificity was high but with a poor sensitivity. For ICTP, the marker for osteolytic activity, both characteristics of sensitivity and specificity were low, such that a general recommendation for the use of this marker as a screening parameter cannot be made.Potential indications for bone metabolic markers might be the therapy control in patients with bone metastases, in which increased parameters had been proven before therapy. Particularly in these patients, the so-called "bone flare reaction", the repair of destroyed bone structures by tumor cells, complicates the assessment of bone scintigraphy. Increased activity in known osseous metastases after or during therapy can be caused by either repair or further tumor growth. Blomqvist and colleagues and Koizumi et al. reported on patients with breast cancer and osseous metastases, where only patients with progressive disease showed an increase in ICTP values during therapy compared to other patients with response to therapy ,15. AnotIn summary, when a new method is recommended for the diagnostic work up, it is necessary to demonstrate that this new modality is as sensitive and specific as existing routine imaging procedures. The determination of bone metabolic parameters like ICTP or PINP is less expensive than bone scanning, but this prospective study has shown that the results from bone metabolic markers are not yet sufficient enough to demonstrate a bone involvement in different type of malignancies with high sensitivity and specificity.The author(s) declare that they have no competing interests.JS and CE designed the study, performed the statistical analysis and drafted the manuscript.SR and EW recruited patients and carried out the nuclear medicine investigations.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Sales data indicate a major increase in the prescription of antifungal drugs in the last two decades. Many new agents for systemic use that only recently have become available are likely to be prescribed intensively in acute care hospitals. Sales data do not adequately describe the developments of drug use density. Given the concerns about the potential emergence of antifungal drug resistance, data on drug use density, however, may be valuable and are needed for analyses of the relationship between drug use and antifungal resistance.Hospital pharmacy records for the years 2001 to 2003 were evaluated, and the number of prescribed daily doses  per 100 patient days were calculated to compare systemic antifungal drug use density in different medical and surgical service areas between five state university hospitals.The 3-year averages in recent antifungal drug use for the five hospitals ranged between 8.6 and 29.3 PDD/100 patient days in the medical services , and between 1.1 and 4.0 PDD/100 patient days in the surgical services, respectively. In all five hospitals, systemic antifungal drug use was higher in the hematology-oncology service areas  than in the medical intensive care units  or in the surgical intensive care units . Fluconazole was the most prescribed antifungal drug in all areas. In 2003, amphotericin B consumption had declined to 3 PDD/100 in the hematology-oncology areas while voriconazole use had increased to 10 PDD/100 in 2003.Hematology-oncology services are intense antifungal drug prescribing areas. Fluconazole and other azol antifungal drugs are the most prescribed drugs in all patient care areas while amphotericin B use has considerably decreased. The data may be useful as a benchmark for focused interventions to improve prescribing quality. There has been a major increase in the prescription of antifungal drugs after the introduction of fluconazole into the market in the late 1980s, and again in the late 1990s. The systemic antifungal market has continued to experience growth since 1999, increasing in value from $2.1 billion to $3.3 billion in 2003. The azoles dominate the systemic antifungal market, accounting for 52% of total sales in 2003 ,9-11. NeAlthough multiple current and projected market and sales data on systemic antifungal drugs are available, few studies have provided estimates of antifungal drug use density especially in hospitals. Alvarez-Lerma and colleagues reported a prescription rate of 14% in intensive care unit patients . In a suPharmacy data on systemic antifungal drug use in the medical and surgical services of five university hospitals located across Germany were obtained for the period 2001 to 2003. The five university hospitals included, here designated A through E, varied in size from ~1,000 to ~1,700 beds, and differed from each other in structure, special services offered, and in the availability of interdepartmental guidelines and an antiinfective therapeutics committee, drug formularies, formulary restrictions, and infectious disease consultation services. which defines lower doses for amphotericin B, fluconazole, and itraconazole (Table 2). Antifungal drug use density was calculated as yearly PDD/100 patient days (i.e. occupied bed days). Separate data were calculated for the medical ICU (MICU), the surgical ICU (SICU), and the hematology-oncology services, respectively. We also calculated yearly means of overall and specific antifungal use densities to assess time trends.We used a consensus definition of  prescribed daily doses (PDD) in adults Table  accordinThe yearly antifungal drug use densities differed between the five hospitals in particular for the medical services. Hospital A showed use density values of consistently >20 PDD/100 patient days while hospital E values were consistently <10 PDD/100 patient days , but only from 2.1 to 2.2 PDD/100 patient days in the surgical services (+5%). Applying the WHO/ATC definition of daily defined doses , corresponding values for the years 2001 and 2003 were calculated to be 22.8 to 26.3 DDD/100 patient days (+15%) in the medical services, and 4 to 4.1 DDD/100 patient days (+4%) in the surgical services, respectively (data not shown).As in other reports , fluconaAs expected, antifungal drug use was much more intense in the hematology-oncology services and intensive care areas Figure  than in Of note, hospital E had a moderately active infectious disease consultant service with an antimicrobial agents management program, and this was previously associated with low antibacterial drug use in the medical service ,19. AccoIn hospital C, there was a program in the MICU attempting to decrease the use of fluconazole based solely on positive cultures for yeasts in tracheal or bronchial secretions. This program, which was primarily a focused infectious diseases consultation program was started in 2002, and appeared to be effective in decreasing fluconazole use from 13.5 to 5 PDD/100 patient days without changing the use density of other systemic antifungal drugs  declare that they have no competing interests.KdW and WVK analysed and interpreted the data and wrote the article. MSB is data manager, analysed the data and presented them through a searchable database. HK, FD, ES, UR and LM checked the data for consistency and correctness, provided them in electronic format, and helped with interpretation of the data and revision of the manuscript.The pre-publication history for this paper can be accessed here:


The Specificity proteins (Sp) are a family of transcription factors that have three highly conserved zinc-fingers located towards the carboxy-terminal that bind GC-boxes and assist in the initiation of gene transcription. Human Sp1-7 genes have been characterized. Recently, the phenotype of Sp8 null mice has been described, being tailless and having severe truncation of both fore and hind limbs. They also have malformed brains with defective closure of the anterior and posterior neuropore during brain development.2H2 zinc-fingers. Sp8 mRNA expression was detected in a wide range of tissues at a low level, with the highest levels being found in brain. Treatment of the murine pluripotent cell line C3H10T1/2 with 100 ng/mL BMP-2 induced Sp8 mRNA after 24 hours.The human Sp8 gene is a three-exon gene that maps to 7p21.3, close to the related Sp4 gene. From an osteosarcoma cell line we cloned two transcript variants that use two different first exons and have a common second exon. One clone encodes a 508-residue protein, Sp8L (isoform 1) and the other a shorter 490-residue protein, Sp8S (isoform 2). These two isoforms are conserved being found also in mice and zebrafish. Analysis of the Sp8L protein sequence reveals an amino-terminal hydrophobic Sp-motif that is disrupted in Sp8S, a buttonhead box and three CThere is conservation of the two Sp8 protein isoforms between primates, rodents and fish, suggesting that the isoforms have differing roles in gene regulation. Sp8 may play a role in chondrogenic/osteoblastic differentiation in addition to its role in brain and limb development. Drosophila gene buttonhead (Btd) ) was used as a measure of abundance. A comparative Ct method was used to determine gene expression. Expression levels in each cDNA sample were normalised to the expression levels of the control gene (ΔCt). The ratios of Sp8 mRNA/control gene RNA from each cDNA were standardised to that of the untreated cells on day 0 that was taken as 100% (ΔΔCt). The formula E-ΔΔCt was used to calculate relative expression levels where E is the efficiency of the PCR per cycle. Statistically significant changes in gene expression were determined using the t-test on data from three replicate experiments. The amplification specificity was confirmed by melting curve analysis and agarose gel electrophoresis.Utilising a CAS-1200 robotic precision liquid handling system, PCR was carried out on a Rotor Gene 3000  using a SYBR Green I double-stranded DNA binding dye assay. Copy DNA derived from 200 ng of total RNA from each sample was amplified by PCR using Taq Gold polymerase using PCR primers to a final concentration of 50 nM. The primers were: mouse Sp8 3'UTR, CCATTCAGCTCTGGCTAGGTCTT and GATTCCCGTTCGCAGAACTC producing a 67 bp amplicon. Beta-actin mRNA was used as a control gene as previously described . The ampAJE conceived of the study, and participated in its design and coordination. MAM, JEG and AJE carried out the cell culture work, MAM and AJE carried out the expression studies and MAM and AJE carried out the gene cloning. All authors participated in writing the manuscript, read, and approved the final manuscript.


Recently reported prevalences of myopia in primary school children vary greatly in different regions of the world. This study aimed to estimate the prevalence of refractive errors in an unselected urban population of young primary school children in eastern Sydney, Australia, between 1998 and 2004, for comparison with our previously published data gathered using the same protocols and other Australian studies over the last 30 years.Right eye refractive data from non-cycloplegic retinoscopy was analysed for 1,936 children aged 4 to 12 years who underwent a full eye examination whilst on a vision science excursion to the Vision Education Centre Clinic at the University of New South Wales. Myopia was defined as spherical equivalents equal to or less than -0.50 D, and hyperopia as spherical equivalents greater than +0.50 D.The mean spherical equivalent decreased significantly (p < 0.0001) with age from +0.73 ± 0.1D (SE) at age 4 to +0.21 ± 0.11D at age 12 years. The proportion of children across all ages with myopia of -0.50D or more was 8.4%, ranging from 2.3% of 4 year olds to 14.7% of 12 year olds. Hyperopia greater than +0.50D was present in 38.4%. A 3-way ANOVA for cohort, age and gender of both the current and our previous data showed a significant main effect for age (p < 0.0001) but not for cohort (p = 0.134) or gender (p = 0.61).Comparison of our new data with our early 1990s data and that from studies of over 8,000 Australian non-clinical rural and urban children in the 1970's and 1980's provided no evidence for the rapidly increasing prevalence of myopia described elsewhere in the world. In fact, the prevalence of myopia in Australian children continues to be significantly lower than that reported in Asia and North America despite changing demographics. This raises the issue of whether these results are a reflection of Australia's stable educational system and lifestyle over the last 30 years. The prevalence of myopia is currently receiving worldwide attention as many recent studies report dramatic increases over the last 20 years ,2. MyopiDespite much research interest over the last half century, there have been surprisingly few well-designed epidemiological studies of refractive error with large numbers of randomly selected younger school children to form the basis of valid world wide comparisons of the earliest stages of development of myopia ,9,10. HoIn general, estimates of the prevalence of myopia have shown less increase in the Western world than in Asia, and less increase in rural than in urban populations ,10,12-16It has often been suggested that myopia is more prevalent in ethnic Chinese (reviewed ), but onBy comparison, the epidemiology of refractive error for young Australian school children is relatively well documented and presents a very different profile. A number of studies were carried out in the early 1970s and the 1980s on relatively large groups of unselected primary school children from the socio-economic extremes , and indicated a prevalence of myopia ranging from approximately 3% to 13%   school vPermission was obtained from the NSW Department of Education and the NSW Catholic Education Office to approach all schools in the eastern region of Sydney  to send entire classes to the VEC. A flyer was sent describing the VEC science excursion and age-appropriate eye examination, inviting Years 1, 3 and 5 particularly to participate.The group of 1,936 children examined came from the eastern suburbs along the southern beaches of Sydney, and may be thought of as randomly selected with little likelihood of bias to the data as individual classes were free to respond. Children were drawn from twelve government and non-government primary schools and one pre-school and attended the clinic only once. During the 1996 Australian Bureau of Statistics census 14,785 children aged 4 to 12 years were recorded in this region (Randwick and Waverley precincts of Eastern Sydney) who came from a very broad range of ethnic and socio-economic backgrounds present, where 37 different languages might be spoken in the home . This waThe comprehensive optometric examination by experienced paediatric practitioners included all age-appropriate tests meeting Australian Optometric Competency Standards, except that parents/guardians were not present to ratify history. Refractive error was determined by non-cycloplegic retinoscopy with optical fogging while the child maintained fixation on a distant non-accommodative (6 metre) target. In most cases refractive status was confirmed by subjective refraction. Other tests included letter visual acuity at 6 m and 33 cm, cover test for strabismus, motilities, saccades, pupil reactions, near point of convergence, heterophoria, stereopsis, accommodative facility, colour vision and ophthalmoscopy.Cycloplegic retinoscopy was not undertaken for many reasons including the fact that VEC studies started prior to the 2000 convention suggesting use of cycloplegic retinoscopy for studies of refractive error prevalence . SecondlAutorefractors were not employed as hand-held versions were unavailable when the first cohort was seen. Equally as important, there is no convincing evidence that the proportion of myopes identified in the sample would have changed .To compare the estimated prevalence of myopia in this urban population of 'Australian children' over the last decade, this more recent 2000s data set was analysed against data from an earlier cohort of 2,322 children with similar demographics seen in the early 1990's, using the same testing protocols and seen at the same venue . The optData was analysed by Analysis of Variance ANOVA (StatView software). Only refractive data from right eyes was used for the current refractive class analysis, as the correlation between right and left eye refractions was extremely high (p < 0.0005). The preferred criterion to define myopia in this study is that used clinically in Australia: a spherical equivalent equal to or more minus than -0.50 D. However, as myopia more minus than -0.50 D has occasionally been used to define myopia in epidemiological studies ,19,41, aThe records of 1,936 children aged 4 to 12 years from a non-clinical unselected population examined during the six years from March 1998 to May 2004 were analysed retrospectively to estimate the prevalence of different types of refractive error. Primary schools of their own choice sent more children from years 1, 3, and 5, which resulted in unequal numbers of children in each of the age groups. There were 925 boys and 951 girls, and the relative numbers for both males and females in each age group are shown in Table The mean spherical equivalent refraction of all 1,936 children was +0.45 ± 0.02 DS, however it should be noted that there is a preponderance of children aged 5–6, 9 and 11 years old corresponding with Years 1, 3, and 5 of primary school. Overall, there was no significant difference in spherical equivalent refractive error between girls and boys (p = 0.697). In general, mean refraction demonstrates a highly significant shift towards less hyperopia with increasing age (p < 0.0001) from 0.73 ± 0.1DS for 4 year olds to 0.21 ± 0.11 for 12 year olds, however this is more noticeable after the age of 9 years as seen in Fig. A summary of the relative proportions of myopia and hyperopia for this cohort of children of all ages seen during the six years ('2000s' data) is given in Table The number of children per age group for the two cohorts is shown in Fig. A three-way analysis of variance was carried out for cohort, age and gender. There was a main effect for age (p < 0.0001), but no main effect for either cohort (p = 0.134) or gender (p = 0.61). However, there were age/gender/cohort interactions that indicate a trend towards an increasing shift away from the hyperopic refraction in the later cohort.An analysis of the prevalence of refractive errors in young school children in eastern Sydney during the last thirteen years has been presented. The latest data gathered from 1,936 unselected primary school-aged children in the last 6 years, indicates that the prevalence of myopia remains quite low compared to that reported for the western world and Asia, especially as refractive error was established by non-cycloplegic retinoscopy (as will be discussed later). These findings are not significantly different (p = 0.13) to our previous report  indicatiTherefore, if we take the total 4,258 children seen since 1990, the relative frequency of refractive error across all is: 54.2% emmetropic by our criteria, 32.3% low to moderate hyperopes, 5.3% myopic greater than -0.50D spherical equivalence and 7.4% myopic by at least -0.50 DS. The number with myopia of at least -4.00 DS was an extremely small 0.6%.As alluded to in the introduction, the proportion of Sydney children with myopia is dramatically less than in Asia. Indeed, the proportion appears significantly lower than in the USA  and CanaIn the past, a lack of internationally accepted definitions for 'myopia' has hampered valid comparisons across the various studies . CommonlComparison across studies is also difficult when only an 'overall' mean refraction is presented covering all children in a study, due to the well known increasing prevalence of myopia with age. Indeed, the comparison of data from our own two data sets is confounded to some extent by the slightly different age profiles for each cohort. However, in neither cohort was the age range nor mean significantly different, so the similar proportion of myopes is not unexpected.The question of optimal ocular conditions for comparison of the prevalence of refractive errors remains controversial. A cycloplegic agent is typically proposed as the gold standard ,43,44 inThus there appears to be no scientific concurrence regarding the efficacy of cycloplegia for studies on the prevalence of myopia -37, withAs our refractive data was derived from non-cycloplegic retinoscopy we readily concede that mean refractive error may be less hyperopic than if a cycloplegic had been used. However, we suggest that as the influence of a cycloplegic is uncertain and is of least concern for myopes, the estimated prevalence of myopia will not be significantly altered by our decision to not use a cycloplegic. In support of this notion are new conference data from Rose et al ,48 reporWorldwide patterns of the prevalence of myopia suggest significant differences are likely to be due to the different demographics and lifestyles ,10,49. ZWhatever way it is argued, our results indicate little evidence for an epidemic of myopia although there is a developmental trend towards an earlier decrease in hyperopia to the point of myopia. Thus, the question of whether it is a matter of lifestyle, or perhaps familial environmental stress, or more, remains. Certainly, the education system and housing has changed little in Australia the last 30 years. By comparison, most Asian children participating in myopia epidemiological studies reportedly are more likely to live in high-rise residential blocks  and haveIt is concluded that despite some differences in methodology across earlier studies, the prevalence of myopia in young Australian school children does not appear to have increased significantly over the last 30 years if one allows for the change in ethnic demographics. It is also proposed that an explanation for the large increase in prevalence of myopia reported in other countries must include questions relating to lifestyle in addition to genetic propensity.The author(s) declare that they have no competing interests.SC conceived and designed the study, jointly analysed the results and worked the drafts through to the final version. BJ coordinated the study, collated the clinic records, jointly analysed the results, researched the background for the paper, prepared the draft manuscript and was responsible for presentation. Both authors read and approved the manuscript.The pre-publication history for this paper can be accessed here:


After 2 weeks feeding period, OLETF rats revealed obesity and hyperlipidemia compared with their progenitor LETO rats. Feeding of the diet supplemented with 9% safflower oil and 1% pomegranate seed oil  did not affect abdominal white adipose tissue weights and serum lipid levels compared with the diet supplemented with 10% safflower oil (control diet) in OLETF rats. However, the accumulated hepatic triacylglycerol was markedly decreased by 9c, 11t, 13c-CLNA diet in OLETF rats. Activities of hepatic enzymes related to fatty acid synthesis and fatty acid β-oxidation were not altered by 9c, 11t, 13c-CLNA diet. Levels of monounsaturated fatty acid (MUFA), major storage form of fatty acid, in serum triacylglycerol were markedly higher in obese, hyperlipidemic OLETF rats than in lean LETO rats. In addition, 9c, 11t, 13c-CLNA diet significantly decreased MUFA levels in OLETF rats. This is the first study showing that 9c, 11t, 13c-CLNA suppresses delta-9 desaturation in vivo, and we suggest that the alleviation of hepatic triacylglycerol accumulation by 9c, 11t, 13c-CLNA diet was, at least in part, attributable to the suppression of delta-9 desaturation in OLETF rats.Conjugated fatty acid, the general term of positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds, has attracted considerable attention because of its potentially beneficial biological effects. In the present study, dietary effect of pomegranate seed oil rich in punicic acid (9 It has been reported that conjugated linoleic acid (CLA), the CFA form of linoleic acid, has favorable physiological effects, such as anti-atherosclerosis, anti-obesity, anti-tumor, and anti-hypertension -9. Thereseed oil . α-Eleosectively ,11. Catabout 33% ,15. Howebout 33% . Previoubout 33% .In the present study, we investigated the effects of pomegranate seed oil rich in 9c, 11t, 13c-CLNA on lipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats. OLETF rats develop a syndrome with multiple metabolic and hormonal disorders that shares many features with human obesity -21. OLETIn comparison with their progenitor Long-Evans Tokushima Otsuka (LETO) rats, OLETF rats had increased body weight gain with enhanced food intake during 2 weeks feeding period Table . In OLETThe effect of dietary 9c, 11t, 13c-CLNA on the accumulation of abdominal white adipose tissue (WAT) was investigated . Rats were housed individually in metal cages in temperature-controlled room (24°C) under a 12-hour light/dark cycle. After a 1-week adaptation period, OLETF rats were assigned to two groups (six rats each) that were fed with a semisynthetic diet supplemented with 10% safflower oil (the control group) or a semisynthetic diet supplemented with 9% safflower oil and 1% pomegranate seed oil rich in 9cis, 11trans, 13cis-CLNA . LETO rats were fed the same diet as the OLETF rats in the control group. The pomegranate seed oil rich in 9c, 11t, 13c-CLNA (69.0%) was prepared by Kaneka Co. . The semisynthetic diet were prepared according to recommendations of the AIN-76  and contSerum was separated by centrifuging the blood. Triacylglycerol, cholesterol, and phospholipids in serum were measured using enzyme assay kits from Wako Pure Chemicals . Liver lipids were extracted and purified according to the method of Folch et al . The conA piece of liver was homogenized in 6 volumes of a 0.25 M sucrose solution that contained 1 mM EDTA in a 10 mM tris Tris-HCL buffer (pH 7.4). Fractions of mitochondria, microsomes, and cytosol were obtained as previously described. The proThe enzyme activities of ME (EC 1.1.1.40) , G6PDH (P<0.05.All values are expressed as means ± SE. Data were analyzed by one-way ANOVA, and all differences were inspected by Duncan's new multiple-range test . DiffereCFA, conjugated fatty acid; CLA, conjugated linoleic acid; CLNA, conjugated linolenic acid; OLETF rat, Otsuka Long-Evans Tokushima fatty rat; LETO rat, Long-Evans Tokushima Otsuka rat; WAT, white adipose tissue; G6PDH, glucose-6-phosphate dehydrogenase; ME, malic enzyme; FAS, fatty acid synthase; CPT, carnitine palmitoyltransferase; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; SCD, stearoyl-CoA desaturase


Granulosa cell tumors (GCTs) are relatively rare and are subtypes of the sex-cord stromal neoplasms. Methylation induced silencing in the promoters of genes such as tumor suppressor genes, DNA repair genes and pro-apoptotic genes is recognised as a critical factor in cancer development.We examined the role of promoter hypermethylation, an epigenetic alteration that is associated with the silencing tumor suppressor genes in human cancer, by studying 5 gene promoters in 25 GCTs cases by methylation specific PCR and RT-PCR. In addition, the compatible tissues  from three non-astrocytoma patients were also included as the control.FHIT), 6/25 (24% for FNACF), 3/25 (12% for Cyclin D2), 1/25 (4% for BRCA2) and 14/25 (56%) in RUNX3 genes. Correlation of promoter methylation with clinical characteristics and other genetic changes revealed that overall promoter methylation was higher in more advanced stage of the disease. Promoter methylation was associated with gene silencing in GCT cell lines. Treatment with methylation or histone deacetylation-inhibiting agents resulted in profound reactivation of gene expression.Frequencies of methylation in GCTs were 7/25 (28 % for These results may have implications in better understanding the underlying epigenetic mechanisms in GCT development, provide prognostic indicators, and identify important gene targets for treatment. Ovarian cancer is one of the most common cancers in women of all age groups. Among ovarian neoplasms, granulosa cell tumors (GCTs) are relatively rare, accounting for approximately 3% of all ovarian cancers. One common DNA modification is promoter hypermethylation associated with loss of expression of a tumor suppressor gene. During cancer development, there is a shift in methylation patterns and some promoter region CpG islands become methylated leading to silencing of the adjacent genes  and thisFHIT gene is a member of the histidine triad gene family [FHIT gene has been shown to be hypermethylated in oesophageal, lung, breast, prostate, bladder, cervical, and oral cancers [cyclin D2 was also demonstrated in human ovarian granulosa cell tumors and testicular germ cell tumor cell lines [cyclin D2 gene promoter region. Several studies have indicated that methylation of cyclin D2 and its mRNA and protein were absent in most breast cancer cell lines examined and in primary breast cancers although normal breast epithelial cells had abundant expression [BRCA2 in breast cancers cell lines and other normal human breast, bladder, colon, and liver tissues [The human e family , the fun cancers -9. Recen cancers . Methylall lines . In breapression -14. BRCA tissues . RUNX3 i tissues ,17.FHIT, FANCF and RUNX3 gene promoters were frequently methylated. Methylation status was correlated with histologic characteristics. We also found evidence that promoter methylation inactivates gene expression in GCTs and exposure to methylation and/or histone deacetylase (HDAC)-inhibiting agents reactivate the gene expression.The role of epigenetic gene inactivation in GCTs of ovarian origin is yet not fully understood. Previously published reports on GCTs and its precursor lesions showed varying degree of promoter methylation of many tumour suppressor genes -20. To iFHIT, FANCF, Cyclin-D2, BRCA2 and RUNX3 in 25 ovarian GCTs and ovarian cell line DNAs using the MSP assay . Hypermethylation of FANCF was significantly more frequent in stage IC . Cyclin-D2 methylation was found only in patients with IC type of cancer . No FANCF mRNA in TOV-21G, Cyclin-D2 in C13* and OV-90, and RUNX3 in C13* and KGN was detected in untreated cell lines  studied extensively in these tumours are in fact localised on these chromosomes.Cancer cells especially of ovarian origin keep on accumulating genetic changes that allow them to evade various chemotherapeutic drugs and hence become increasingly dangerous. We try to answer some of the questions by exploring the role of methylation mediated gene silencing in five tumour suppressor gene in the present study. We asked a question whether hypermethylation of comatous . Taken tFANCF gene disrupts the FA-BRCA pathway, resulting in cisplastin resistance. We found FANCF promoter hypermethylation in 24% of the tumours. This is in line with the previous findings in squamous cell carcinomas of lung and oral cavity and, in cervical and ovarian cancer [BRCA2 after treatment with DAC. DNA hypermethylation-mediated gene silencing is closely associated with histone modifications such as methyl-H3-K9. In this regard, DNA-demethylating agents 5'-aza-2'deoxycytidine (DAC) and trichostatin (TSA) reactivates expression of epigenetically silenced genes [FHIT, cyclin-D2 and RUNX3 in C13*, FANCF in TOV-21G and cyclin-D2 in OV-90 cell line after exposure to DAC in the absence of promoter methylation suggests that key histone modifications, either by direct or indirect involvement of promoter methylation, also play a role in down-regulating FANCF gene expression in this cancer type. It is therefore assumed that FANCF silencing might be considered a candidate-mechanism underlying the state of genomic instability may be considered to be a rate-limiting in the origin of GCTs of ovarian origin. FANCF gene silencing has been shown to revert in vitro as a result of demethylation in some ovarian cell lines as seen in the present study also [FANCF methylation. The present finding along with the results from other cancer types suggests the presence of genomic instability due to the methylation mediated FANCF gene silencing. Cyclin D2 gene promoter is hypermethylated in 12% of GCTs, a number that is consistent with previous findings in other cancers [Cyclin D2 methylation is found only in IC type tumors.FANC genes are essential in DNA repair pathways and recently, it has been shown that promoter hypermethylation of n cancer -25. To ted genes . Althouged genes ,28. In tudy also . These r cancers ,29,30. WRUNX3 gene promoter frequently occurred in ovarian GCTs. In human gastric cancers it has been shown that promoter hypermethylation and hemizygous deletion of the RUNX3 gene correlated with a significant reduction in expression, and the tumorigenicity of cell lines in nude mice was inversely related to their level of RUNX3 expression, indicating that RUNX3 is a tumor suppressor involved in the development of gastric cancers [RUNX3 CpG island hypermethylation in GCTs, but not in normal ovarian tissue or peripheral blood suggests that RUNX3 hypermethylation might be associated with the genesis of this cancer type and this frequent methylation might serve as a biological marker. Loss of expression in RUNX3 gene in patient 11 which also lacks any PCR product with either of the unmethylated and methylated primers indicates that it could be due to the large deletion in the gene. However, studies involving the markers D1S234 and D1S199 showed the loss of heterozygosity which could to some extent explain the loss of expression in this patient. We could not confirm this due to the lack of further sample. However, the sample quality was good as indicated by the presence of band in GAPDH1 which was used as an internal control.The present study showed that hypermethylation of the  cancers . The preThe histological examination of granulosa cells revealed that these are small, usually round to polygonal, but may be spindle-shaped with scanty amphophilic cytoplasm, containing only occasional small lipid droplets, and having indistinct cell borders. Granulosa cells regularly express inhibin and contain vimentin and smooth muscle actin intermediate filaments and, less commonly, cytokeratins. However, during their malignant transformation, theses cells exhibit one or more of so-called 'microfollicular', 'macrofollicular', 'trabecular' or 'insular' patterns. The microfollicular variant is characterized by multiple small rounded spaces formed by cystic degeneration in small aggregates of granulosa cells and often fragments of nuclear debris or pyknotic nuclei. These spaces, known as Call-Exner bodies, are found in only 30–50% of tumors. The granulosa cell nuclei are oriented somewhat radially around these structures. The nuclei are typically bland and often grooved in granulosa cell tumors and have few mitoses, but show variation in size and shape, and marked hyperchromatism.There exist two different pathways that can contribute to the development of cancers; genome-wide hypomethylation may lead to the loss of chromosomes (as seen in these tumours) leading to chromosomal instability, whereas promoter methylation in tumour suppressor genes, which are responsible for gene silencing, can lead to the development of cancers in somatic cells. Therefore, the balance in DNA methylation is very important, and alteration in these may be protective in one pathway but deleterious in the other.In view of the high mortality rates associated with ovarian cancer, a better understanding of the molecular mechanisms underlying tumor progression in the disease could reveal novel pathways of high clinical relevance. It can therefore be concluded that promoter hypermethylation of these genes, and loss of expression in ovarian cancers (GCTs) is relatively common and this may also be useful as a tumour marker for early diagnosis and subsequent disease monitoring. Hence, these epigenetic signatures could play a decisive role in designing treatment options for this category of ovarian cancer. These results may also have implications in better understanding the underlying epigenetic mechanisms in GCT development thus can provide prognostic indicators, and identify important gene targets for treatment of this type of cancer in females.The subjects were 25 patients affected with GCTs of ovarian origin without a positive family history. The study was approved by the Health Research Ethics Board of the Faculty of Natural Sciences. All of them were untreated at the time of study. Fifty normal individuals ranging in age from 20–60 years were studied simultaneously under similar experimental conditions. The tumor grading is as follows: 11 FIGO stage IA, 10 FIGO stage IB and 4 FIGO stage IC. All these diagnoses were reviewed by a gynaecologic pathologist and the tumor were assessed using standard criteria. An informed consent was taken from all the subjects prior the study. Fresh cancer tissue specimens were received from all the patient and these were cultured as per standard protocol. A part of the fresh tissue was used to isolate DNA and RNA for further analysis where as the cultured cells were used for the 5'-Aza-2'-deoxycytidine experiments. Microscopically, GCTs are composed of granulosa cells, theca cells, and fibroblasts in varying amounts and combinations. The term granulosa-theca cell tumor had been applied to all tumors in which both cell types were identified, regardless of the amounts present.2 [Five human ovarian cancer cell lines  from the American Type Culture Collection and European Collection of Cell Culture were maintained in RPMI 1640 supplemented with 10% fetal bovine serum  and were grown at 37°C in 5% CO2 . DNA wasFHIT gene, methylated CpG site, forward 5'-ttggggcgcgggtttgggtttttacgc-3' and reverse 5'-cgtaaacgacgccgaccccacta-3', unmethylated CpG site, forward 5'-ttggggtgtgggtttgggtttttatg-3', and reverse 5'-cataaacaacaccaaccccacta-3'; 189–262 bp relative to transcription start site, FANCF gene, methylated CpG site, forward 5'-tttttgcgtttgttggagaatcgggttttc-3' and reverse 5'-atacaccgcaaaccgccgacgaacaaaacg-3', unmethylated CpG site, forward 5'-tttttgtgtttgttggagaattgggttttt-3' and reverse 5'-atacaccacaaaccaccaacaaacaaaaca-3'; the primers corresponds to the position +280 to +432, BRCA2 gene, methylated CpG site, forward 5'-gacggttgggatgtttgataagg-3' and reverse 5'-aatctatcccctcacgcttctcc-3', unmethylated CpG site, forward 5'-agggtggtttgggatttttaagg-3' and reverse 5'-tcacacttctcccaacaacaacc-3'; these primers are 135 and 211 bp upstream of transcription start site and cyclin-D2 gene, methylated, forward 5'-tacgtgttagggtcgatcg-3' (-1427 to -1409) and reverse 5'-cgaaatatctacgctaaacg-3' (-1152 to -1171) and unmethylated, forward 5'-gttatgttatgtttgttgtatg-3' (-1616 to -1594) and reverse 5'-taaaatccaccaacacaatca-3' (-1394 to -1414). Each PCR reaction generated 74 bp products both with methylated and unmethylated primers for FHIT, 153 bp for FANCF both with methylated and unmethylated primers, 337 and 250 bp product with BRCA2 primers specific for methylated and unmethylated primers and 276 and 222 bp product for cyclin-D2 primers specific for methylated and unmethylated PCR reactions respectively. For the PCR reaction of 25 μl, 50 ng sodium bisulfite treated DNA was added to reaction buffer containing 0.2 mM dNTP, 16.6 mM (NH4)2SO4, 67 mM Tris pH 8.8, 10 mM β-mercaptoethanol, 1.5 mM MgCl2, 10 pmol of forward and reverse primers specific to the methylated and methylated DNA sequences and 1.25 units of AmpliTaq Gold . The PCR reactions were cycled in a GeneAmp 9600 thermal cycler (Applied Biosystems) under the following conditions: preheat at 94°C for 3 min. followed by 40 cycles  methylation status using the primers: forward, 5'-ataatagcggtcgttagggcgtcg-3', and reverse, 5'-gcttctactttcccgcttctcgcg-3' , for methylated DNA of RUNX3; and forward, 5'-ataatagtggttgttagggtgttg-3', and reverse, 5'-acttctactttcccacttctcaca-3' , for unmethylated DNA of RUNX3 with annealing temperature of 55°C for 20 seconds. For each PCR set, DNA isolated from normal peripheral lymphocytes of healthy individuals served as a negative methylation control. Human placental DNA was treated in vitro with SssI methyltransferase  to create completely methylated DNA at all CpG-rich regions and served as positive methylation control. Methylation-specific PCR products were analysed on 3% agarose gel electrophoresis with ethidium bromide staining. A positive control and a negative control were included in each amplification reaction.DNA methylation patterns in CpG islands of tumor suppressor genes FHIT, FNACF and BRCA2 were determined by chemical modification with sodium bisulphite as described previously . BrieflyFHIT, FNACF and cyclin-D2 mRNA levels in cancer patients were compared to their expression in normal cells using semi-quantitative reverse transcriptase polymerase chain reaction. In brief, cDNA was synthesized using AMV reverse transcriptase  and amplified in duplex reactions in a total volume of 25 μl containing 150 μM of each dNTP, 1.5 mM MgCl2, 10 pmol of each primer pair and 1.0 units of Taq polymerase. After initial denaturation at 95°C (94°C) for 5 min, 30 cycles of 30 sec (45 sec) at 95°C (94°C), 30 sec (50 sec) at 58°C (55°C) and 45 sec (50 sec) at 72°C were performed followed by a final 10 min elongation step at 72°C. The values in the brackets correspond to FANCF. The following primers were used to amplify FHIT: forward 5'-gctcttgtgaataggaaacc-3' and reverse 5'-tcactggttgaagaatacagg-3' which yields 532 bp product spanning within exon 5 to exon 10, FANCF: forward 5'-ttcggaagtctttgctgcct-3' and reverse 5'-agtaataacacacgattgcc-3' which yields 413 bp product spanning from +733 to +1144. RT-PCR was performed for Cyclin D2 using the primers 5'-catggagctgctgtgccacg-3' (forward) and 5'-ccgacctacctccagcatcc-3' (reverse) with PCR conditions being: 1 cycle of 94°C for 3 min and 35 cycles at 94°C for 20 s, 55°C for 30 s, 72°C for 45 s followed by 72°C for 5 min. RT-PCR was also carried out for RUNX3 gene using the primers: forward 5'-aggcattgcgcagctcagcggagta-3' and reverse 5'-tctgctccgtgctgccctcgcactg-3' (152 bp). GAPDH1 was used as internal control. Each reaction was performed in triplicate. PCR products were electrophoresed on 2% agarose gels and quantified using densitometer (Molecular Dynamics). Fold increases in expression in cancer cells were calculated with respect to the levels of the transcripts in normal cells.Cell lines , normal ovarian cells and tumour cells of ovarian GCTs were treated with demethylating agent 5-Aza-2' deoxycytidine (Sigma) for five days at a concentration of 2.5 μM, HDAC-inhibiting agent trichostatin (TSA) at a final concentration of 5 μM for the last 24 hours or a combination of both. Total RNA isolated from treated, untreated cell lines and cell of the cancerous tissue was reverse transcribed using random primers and the Pro-STAR first strand RT-PCR kit . A semi-quantitative analysis of gene expression was performed in replicate experiments using 30 cycles of RT-PCR as described above.The authors declare that they have no competing interests.VSD for executing the MSP and RT-PCR experiments; completing manuscript; MS, for carrying out cell culturing; and SAH conceived and coordinated the study. All authors read and approved the final manuscript.