Feasibility study of anti-neurofilament antibodies detection by indirect quenching fluoroimmunoassay

Neurofilaments [NFs] are the main constitutes of intermediate filaments in neurons. They are composed of three subunits with heavey, medium and low molecular weight. Anti-neurofilament antibodies exist in serum of patients with some neurodegenerative diseases. A fluoroimmunoassay has been developed for determining of antibodies against neurofilaments, using an anti-fluorescein serum and fluorescein-labeled NFs. Antibodies raised against bovine spinal cord NFs in rabbit and the labeled NFs are incubated with anti-fluorescein serum at room temperature. At high levels, binding of anti-neurofilaments [anti-NFs] to labeled NFs prevented subsequent binding of the anti-fluorescein to fluorescein groups, resulting in little change in the signals of the label. Conversely, at low level of anti-NFs the free fraction of the labeled NFs is available to be bound by anti-fluorescein, which markedly reduced fluorescence intensity of label. Thus, the fluorescence intensity of assay mixture directly reflects the amount of anti-NFs antibodies in the serum. It is concluded that the availability of fluorescein-labeled NFs and antibody directed against fluorescein group permit measurement of anti-NFs antibodies in serum of neurodegenerative patients

Aberrant promoter methylation profile of niemann-pick type C1 gene in cardiovascular disease

Background: the protein of Niemann-pick type C1 [NPC1] gene promotes the egress of cholesterol from late endosomes and lysosomes to other cellular compartments and contributes to a process known as reverse cholesterol transport. This study aimed to examine whether promoter methylation of NPC1 is associated with risk of cardiovascular disease [CVD]

Methods: fifty CVD patients and 50 healthy subjects as the control group were recruited in this study. Promoter methylation of NPC1 gene was defined using a nested-methylation specific polymerase chain reaction method. Statistical analyses were done using the chi-square, t-test or ANOVA tests

Results: our study showed that the frequency of semi-methylated promoter [methylated/unmethylated status] was significantly higher in CVD patients than that in controls [OR = 6.521, 95% CI = 2.211-19.215, P = 0.008]. However, a completely methylated promoter [methylated/methylated status] was not detected in any subjects in either of the two groups tested. Additionally, the analysis of clinical data according to the methylation status of NPC1 gene demonstrated that serum levels of total cholesterol, total triglycerides, high low-density lipoprotein cholesterol [LDL-C] and low high-density lipoprotein cholesterol [HDL-C] are influenced by NPC1 methylation, so that subjects with a completely unmethylated promoter [Unmethylated/unmethylated status] held lower levels of total triglycerides, total cholesterol, LDL-C and higher levels of HDL-C

Conclusion: our findings propose that the NPC1 promoter methylation is a probable mechanism that can result in reduced/impaired NPC1 expression/activity and may thus contribute to progression of CVD. Iran. Biomed. J. 17 [2]: 77-83, 2013

[Markers of oxidative damage of proteins and lipids in patients with senile cataract]

Senile cataract is the most prevalent cause of blindness in the world. Various factors are involved in the pathogenesis of senile cataract. The most important factor is oxidative stress. Oxidative stress is due to increased production of free radicals or reduction of antioxidant capacity in the body. Free radicals cause oxidative damage to cellular components and produce oxidative stress markers. The aim of the present study was to compare markers of oxidative stress associated with proteins and lipids in patients with senile cataract and healthy individuals. In this case-control study that was conducted on 45 patients with senile cataract and 45 healthy peoples, oxidative stress markers of proteins and lipids were measured in serum. Protein carbonyl and reduced total thiol as markers of protein oxidation and malondialdehyde as marker of lipid oxidation were investigated. SPSS 18 and appropriate statistical test were used for comparing average serum level makers of oxidative stress between patient and control groups. Serum protein carbonyl level in patient and control groups was 0.233 +/- 0.096 and 0.248 +/- 0.104, respectively, and there was no statistically significant difference [P=0.265], but serum malondialdehyde levels was found significantly higher in patient group [10.32 +/- 2.53] than that measured in the control group [6.45 +/- 1.16] [P=0.001]. The level of serum reduced total thiol level in patient and control group was 277.5 +/- 104.2 and 392.5 +/- 121.3, respectively. Serum total thiol was significantly lower in the senile cataract group compared with control group [P<0.001]. Oxidation of lens proteins and lipids is a reason for cataract formation. Increased production of free radicals with aging and subsequently creation of oxidative stress conditions lead to oxidation lipids and thiol groups of proteins and can cause increased senile cataract risk. In present study decreased level of serum reduced total thiol and increased level of malondialdehyde in patient group in comparison with controls suggest involvement of oxidative stress in the development of senile cataract formation

Endothelial nitric oxide synthase gene Glu298Asp polymorphism in patients with coronary artery disease

Endo-derived nitric oxide [NO] is synthesized from L-arginine by endothelial nitric oxide synthase [NOS3]. Since reduced NO synthesis in endothelial cells has been implicated in the development of coronary atherosclerosis, we investigated the association of NOS3 gene polymorphisms and coronary artery disease [CAD] in an Iranian population. We studied the NOS3 gene Glu298Asp polymorphism in 241 CAD patients with positive coronary angiograms [i.e., >50% stenosis affecting at least one coronary vessel] in Shahid Rajaee Heart Hospital and 261 control subjects without a history of symptomatic CAD. The NOS3 gene polymorphism was analyzed by polymerase chain reaction and restriction fragment length polymorphism. Lipid profile and other risk factors were also determined. The genotype frequencies of Glu298Asp polymorphism for Glu/Glu, Glu/Asp, and Asp/Asp were 61.3%, 32.2%, and 6.5%, respectively, in control subjects, and 46.5%, 42.7%, and 10.8% in CAD patients, respectively. The genotype frequencies differed significantly between the two groups [P=.003]. The frequencies of the Asp alleles were 32.2% and 22.6% for CAD patients and control subjects, respectively; the difference between the two groups was statistically significant [P=.001; odds ratio=1.6]. Plasma lipids, except HDL-C, were also significantly increased in the CAD groups. These results suggest that CAD is associated with Glu298Asp polymorphism of the NOS3 gene in our population and that this polymorphism is an independent risk factor for CAD

Eucalyptus globulus [Eucalyptus] treatment of candidiasis in normal and diabetic rats

The leaves of Eucalyptus globulus [eucalyptus] are used for treatment of diabetes mellitus in traditional medicine. The aim of this study was to evaluate the effects of eucalyptus in treatment of established systemic infection with Candida albicans in normal and streptozotocin-induced diabetic rats. Sixty normoglycemic male Wistar rats, weighing 200-250 g, were selected and randomly divided into six groups [n= 10]: normal control, control + C. albicans, control + eucalyptus + C. albicans, diabetic control, diabetic + C. albicans, diabetic + eucalyptus + C. albicans. Diabetes was induced after a single intraperitoneal injection of streptozotocin [60 mg/kg body weight] and eucalyptus was added to the diet [62.5 g/kg] and drinking water [2.5 g/L] of treated animals for 4 weeks. The concerned groups were inoculated with C albicans 15 days after diabetes induction. At the end of one month experiment, fasted rats were killed by cervical decapitation. Blood was collected from neck vein for estimation of glucose. C. albicans concentrations were estimated in liver and kidneys using serial dilution culture of tissue homogenates. Eucalyptus administration significantly improved the hyperglycemia, polydipsia, polyphagia, and it also compensated weight loss of diabetic rats [P<0.05]. Moreover, eucalyptus caused a significant reduction in C. albicans concentration in liver and kidney homogenates [P<0.01]. The results revealed that eucalyptus improves Candidia infection in normal and diabetic rats that in some ways validates the traditional use of this plant in treatment of diabetic patients