Effect of tempol on the passive avoidance and novel object recognition task in diabetic rats.

Diabetes mellitus (DM) has several effects, including cognitive impairment. Oxidative stress is associated with complications from diabetes. It seems that antioxidants can reduce some complications of the diabetes induced by oxidative stress. The objective of this study was to evaluate the effect of synthetic antioxidant, tempol on the passive avoidance (PA) memory and novel object recognition (NOR) tests in the diabetic rats. Forty male Wistar rats randomly divided into the control, diabetic, diabetic receiving tempol and healthy receiving tempol groups. Diabetes was induced by injection of streptozotocin (STZ) (60 mg/kg, i.p.). Then, the rats received saline or tempol (30 mg/kg) orally by gavages for 60 days. After that, they were assessed using the PA memory and NOR tests. The results of NOR test showed that the discrimination index (DI) in the healthy receiving tempol group and diabetic control group was significantly lower than control group. Also the amount of this index in diabetic receiving tempol group was significantly higher than diabetic group. The results of PA test indicated that the number of trials to acquisition in the diabetic rats is significantly more than control and diabetic tempol treated groups. Also, the time spent in the dark compartment (TDC) in the control and diabetic receiving tempol groups was less than diabetic group. TDC in the healthy receiving tempol group was more than control group. It can be concluded that although use of tempol is restricted as a cognitive enhancer in non-diabetic subjects but long-term administration of synthetic antioxidant, tempol, is able to dramatically improve diabetes-induced learning and memory deficit in both PA and NOR tests.

Aminoguanidine partially prevents the reduction in liver pyruvate kinase activity in diabetic rats.

BACKGROUND: Low molecular weight aldehydes and carbonyl compounds which are derived from glucose metabolism are prevalent in diabetic plasma. These compounds react to amino groups of Lys and Arg and lead to the formation of advanced glycation end products (AGEs). This modification changes the function of the proteins. The present study aimed to survey the effect of diabetes on rat liver pyruvate kinase activity and to show the inhibitory effect of aminoguanidine (AG). MATERIALS AND METHODS: Male Wistar rats (n = 18, 6 to 8 weeks old) were divided randomly in three groups: the first group as control; second and third groups were induced diabetes using streptozocin. Third group received AG orally for 8 weeks after diabetes induction. Liver cell homogenate was prepared from all studied groups and L-type pyruvate kinase was separated from the homogenate. Pyruvate kinase activity was determined in both liver cell homogenate and extracted L-type PK. The PK activity was compared in all samples between groups. RESULTS: PK activity in isolated form and in liver cell homogenate was lower in diabetic rats as compared to control group. AG-treated group showed higher PK activity compared to untreated diabetic group; however, the difference was not significant. Non-significant difference in PK activity between AG-treated diabetic and non-diabetic (control) group indicated the inhibitory effect of AG in glycation of PK. CONCLUSION: The obtained results showed PK activity decreased in diabetic rats and AG can partially prevent the reduction in PK activity.

Improved real-time rt-PCR assays of two colorectal cancer peripheral blood mRNA biomarkers: a pilot study.

BACKGROUND: Efficient screening for detection of colorectal cancer (CRC) at earlier stages reduces its mortality. The purpose of this study was to investigate expression of carcinoembryonic antigen (CEA) and human telomerase reverse transcriptase (hTERT) mRNA in peripheral blood of CRC patients and to present strategies for early detection screen test. METHODS: Twenty seven patients in non-metastatic stage and 27 healthy individuals were studied. Expression of CEA, hTERT mRNA and 18srRNA (18s subunit of ribosomal RNA, as reference gene) were determined based on real-time RT-PCR on 3 µg of total RNA from blood in 3 separate vials (1 µg per vial). RESULTS: Positive expression rate of CEA mRNA (78%) and hTERT mRNA (81%) were higher in patient group (P<0.001). These rates were meaningfully higher than the results of individual vials containing only 1 µg of total RNA. Difference between Ct values of markers with 18srRNA ΔCt) was higher in healthy group than patient one. Therefore, a ΔCt cut-off value was determined for distinguishing between true- and false-positive results. Concurrent expression of both markers was found in 67% of the patients, which was higher than healthy cases (11%). Combination of concurrent marker expression with cut-off point strategy increased specificity to 100%. CONCLUSION: These results showed that concurrent evaluation of marker expression and performing the test on 3 µg of samples in 3 separate vials may increase specificity and sensitivity of real-time RT-PCR for early detection of non-metastatic CRC. However, more investigations with larger numbers of samples are needed to verify these results.

Association between leptin gene G-2548A polymorphism with metabolic syndrome.

BACKGROUND: Metabolic syndrome (MetS) is a serious public health problem. It is an important risk factor of cardiovascular disease in developed countries. Adipose tissue considered as an organ that releases a variety of molecules referred to adipocytokines such as leptin. Polymorphism of their related genes may play an important role in development of MetS. The aim of this study was to determine the association of leptin gene-2548G/A (LEP-2548G/A) polymorphism with lipid profile in subjects with and without Mets. MATERIALS AND METHODS: In this case/control study a frequency of LEP-2548G/A single nucleotide polymorphism was determined between 200 patients (142 women and 58 men) and 200 controls (122 women and 78 men). Both groups were selected randomly from Hamadan city, Iran. Blood samples were collected then followed by routine biochemical analysis, DNA extraction and serum leptin measurements. Polymerase chain reaction-restriction fragment length polymorphism was applied to identify LEP-2548G/A genotypes. Statistical analyses were applied using SPSS software version 10. Continuous variables were presented as means± SD and compared by independent sample t-test. Variables without normal distribution compared through Mann-Whitney U test. RESULTS: In both groups, a significant difference was observed between biochemical factors and leptin concentration. Serum leptin concentration was more in females than males. No statistical significant difference was detected in the frequency of LEP-2548G/A polymorphism between both MetS and healthy groups. CONCLUSION: In summary, it is concluded that frequency of LEP G-2548A polymorphism in Metabolic syndrome (MetS) and healthy subjects was not significantly different and more research with large sample size is needed in this area.

Construction of Plasmid Insulin Gene Vector Containing Metallothionein IIA (pcDNAMTChIns) and Carbohydrate Response Element (ChoRE), and Its Expression in NIH3T3 Cell Line.

BACKGROUND: Type 1 diabetes mellitus is one of the metabolic diseases that cause insulin-producing pancreatic ß cells be destroyed by immune system self-reactive T cells. Recent-ly, new treatment methods have been developed including use of the stem cells, ß islet cells transplantation and gene therapy by viral and non-viral gene constructs. OBJECTIVES: The aim of this project was preparing the non-viral vector containing the glucose inducible insulin gene and using it in the NIH3T3 cell line. MATERIALS AND METHODS: Cloning was carried out by standard methods. Total RNA was extracted from pancreatic tissue, RNA was converted to cDNA using RT-PCR reaction and preproinsulin gene was amplified using specific primers. PNMTCH plasmid was extract-ed and digested by NotI, HindIII, and MTIIA and ChoRE genes were purified and cloned into pcDNA3.1 (-) plasmid and named pcDNAMTCh. Finally, the preproinsulin genes were cloned into pcDNA3.1 (-) plasmid and pcDNAMTChIns was built. RESULTS: The cloned gene constructs were evaluated by restriction enzyme digestion and RT-PCR. The NIH3T3 cells were transfected by plasmid naked DNA containing preproinsu-lin gene and expression was confirmed by Reverse Transcriptase PCR and Western Blot-ting Techniques. CONCLUSIONS: Gel electrophoresis of PCR products confirmed that cloning was per-formed correctly. The expression of preproinsulin gene in recombinant plasmid in NI-H3T3 cell line was observed for the first time. The findings in this study can be the basis of further research on diabetes mellitus type 1 gene therapy on animals.

Taq1B polymorphism of cholesteryl ester transfer protein (CETP) gene in primary combined hyperlipidaemia.

BACKGROUND & OBJECTIVE: Cholesteryl ester transfer protein (CETP) gene polymorphism is known to be associated with changes in lipid profiles. Primary hyperlipidaemia is considered to be a major risk factor for pancreatitis, atherosclerosis and coronary heart disease. We investigated the association of one common polymorphism in the CETP gene (Taq1B) with plasma lipid levels and CETP activity in Iranian subjects with and without primary combined hyperlipidaemia. METHODS: The study included 102 patients with primary combined hyperlipidaemia and 214 health individuals. Polymerase chain reaction and restriction fragment length polymorphisms were used for genotype detection. To determine the relationship between Taq1B polymorphism and lipid levels, lipids and CETP activity were measured in primary combined hyperlipidaemic and normolipidaemic subjects, with and without Taq1B polymorphism. RESULTS: Plasma CETP activity was significantly (P<0.001) higher in primary combined hyperlipidaemic individuals than in controls. Plasma HDL-C was higher in both groups, in the B(2)B(2) genotype than in the B(1)B(1) and B(1)B(2) genotypes, whereas the serum TG concentrations and CETP activity were lower in B(2)B(2) genotype compared with other genotypes (B(1)B(1) and B(1)B(2)). The genotype and allelic frequencies for this polymorphism differed significantly between hyperlipidaemic and nonmolipidaemic individuals (P<0.05). In both groups, CETP Taq 1B polymorphism (presence of B(2) allele) correlated significantly with HDL-cholesterol (HDL-C) (r=0.201 and r=0.452 in control and patient groups respectively) and CETP activity (r= -0.123 for controls and r= -0.192 for patients). INTERPRETATION & CONCLUSION: The results showed that Taq 1B polymorphism of CETP gene was associated with changes in lipids profile and plasma CETP activity in the selected population and might have a role in contributing to genetic risk of developing coronary artery disease.

Activity of antioxidant enzymes in seminal plasma and their relationship with lipid peroxidation of spermatozoa.

PURPOSE: To determine the activity of seminal plasma catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) and their relationship with malondialdehyde (MDA), as a marker of lipid peroxidation, content of spermatozoa and seminal plasma in normozoospermic and asthenozoospermic males. MATERIALS AND METHODS: Semen samples were obtained from 15 normozoospermic and 30 asthenozoospermic men. RESULTS: We observed inverse correlations between activities of CAT (k/mL) and SOD (U/mL) in seminal plasma with MDA content of spermatozoa from normozoospermic samples (r =- 0.43, p < 0.05 and r =- 0.5, p < 0.05, respectively). Significant correlations were observed between total activity CAT (k/total seminal plasma) with total SOD (U/total seminal plasma) and GPX activity (mU/total seminal plasma) in seminal plasma from normozoospermic samples (r = 0.67, p = 0.008 and r = 0.455, p = 0.047, respectively). Furthermore, we found positive correlations between total activities of CAT, SOD and GPX with total content of MDA in seminal plasma (nmoL/total seminal plasma) from normozoospermic samples (r = 0.67, p = 0.003; r = 0.73, p = 0.003; r = 0.74, p = 0.004, respectively). In asthenozoospermic samples, there were no significant correlations observed between activities of CAT (k/mL), SOD (U/mL) and GPX (mU/mL) of seminal plasma with MDA content of spermatozoa. However, we found significant correlations between total activities of CAT (k/total seminal plasma) and SOD (U/total seminal plasma) with total content of MDA in seminal plasma (r = 0.4, p = 0.018 and r = 0.34, p = 0.03, respectively). CONCLUSION: These findings indicate a protective role for antioxidant enzymes of seminal plasma against lipid peroxidation of spermatozoa in normozoospermic samples.

Activity of antioxidant enzymes in seminal plasma and their relationship with lipid peroxidation of spermatozoa

PURPOSE: To determine the activity of seminal plasma catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPX) and their relationship with malondialdehyde (MDA), as a marker of lipid peroxidation, content of spermatozoa and seminal plasma in normozoospermic and asthenozoospermic males. MATERIALS AND METHODS: Semen samples were obtained from 15 normozoospermic and 30 asthenozoospermic men. RESULTS: We observed inverse correlations between activities of CAT (k/mL) and SOD (U/mL) in seminal plasma with MDA content of spermatozoa from normozoospermic samples (r =- 0.43, p < 0.05 and r =- 0.5, p < 0.05, respectively). Significant correlations were observed between total activity CAT (k/total seminal plasma) with total SOD (U/total seminal plasma) and GPX activity (mU/total seminal plasma) in seminal plasma from normozoospermic samples (r = 0.67, p = 0.008 and r = 0.455, p = 0.047, respectively). Furthermore, we found positive correlations between total activities of CAT, SOD and GPX with total content of MDA in seminal plasma (nmoL/total seminal plasma) from normozoospermic samples (r = 0.67, p = 0.003; r = 0.73, p = 0.003; r = 0.74, p = 0.004, respectively). In asthenozoospermic samples, there were no significant correlations observed between activities of CAT (k/mL), SOD (U/mL) and GPX (mU/mL) of seminal plasma with MDA content of spermatozoa. However, we found significant correlations between total activities of CAT (k/total seminal plasma) and SOD (U/total seminal plasma) with total content of MDA in seminal plasma (r = 0.4, p = 0.018 and r = 0.34, p = 0.03, respectively). CONCLUSION: These findings indicate a protective role for antioxidant enzymes of seminal plasma against lipid peroxidation of spermatozoa in normozoospermic samples.

Relationship between seminal antioxidant enzymes and the phospholipid and fatty acid composition of spermatozoa.

Sperm cell membranes are susceptible to peroxidative damage through an excess of reactive oxygen species. The objective of this study was to determine seminal plasma glutathione peroxidase (GPX) and superoxide dismutase (SOD) activity and relate these to phospholipid profiles and phospholipid-esterified fatty acid composition of spermatozoa. Seminal plasma GPX and SOD activity, phospholipid, phospholipid-esterified fatty acid composition and malondialdehyde (MDA) of spermatozoa were assayed in 10 normozoospermic and 25 asthenozoospermic subjects. Mean seminal GPX and SOD activity in normozoospermic men were not significantly different from asthenozoospermic men. A significant positive correlation was observed between seminal plasma GPX activity and phosphatidylcholine content (r = +0.77, P = 0.037) and there was a significant negative correlation with lysophosphatidylcholine content (r = -0.89, P = 0.02) in normozoospermic sperm samples. Positive correlations were found between SOD activity and polyunsaturated fatty acid composition of spermatozoa. MDA content in the spermatozoa of asthenozoospermic subjects was significantly higher than in normozoospermic males (P < 0.05). Negative correlations were found between MDA content and seminal SOD activity and arachidonic acid content of spermatozoa from normozoospermic samples (r = -0.5; P = 0.046, r = -0.9; P = 0.001 respectively). Seminal plasma GPX and SOD provide protection against lipid peroxidation of phospholipid and phospholipid-bound fatty acids in normozoospermic samples.