Zahra Zamani

Activation of Nrf2-Antioxidant Response Element Mediated Glutamate Cysteine Ligase Expression in Hepatoma Cell line by Homocysteine.

Background Homocysteine is a sulfur-containing amino acid which formed (mainly in the liver) during the metabolism of methionine. Prior studies indicated the important role of hyperhomocysteinemia in pathogenesis and progression of alcoholic liver disease, liver steatosis and cirrhosis. One of the most important mechanisms by which homocysteine promote the development of hepatic injury is oxidative stress. Transcription factor Nrf2-mediated antioxidant response, represents critical cellular defense mechanism that serves to maintain intracellular redox homeostasis and limit oxidative stress. Glutamate cysteine ligase catalytic (GCLc) is rate limiting enzyme in the synthesis of glutathione, an important endogenous antioxidant. Objectives This study was conducted to investigate whether homocysteine induces the Nrf2 dependent expression of GCLc in hepatoma cell line (HepG2) and whether this induction is mediated by antioxidant response element (ARE) which present within its promoter. Materials and Methods Glutathione (GSH) content was measured by flow cytometry. Using electro mobility shift assay (EMSA) and western blotting, ARE-binding activity of Nrf2 for GCLc was demonstrated. Real time RT-PCR and western blotting were performed to evaluate whether homocysteine was able to induce mRNA and protein expression of GCL. Results Exposure of HepG2 cells to 50 µMD/L homocysteine and western blotting of nuclear extracts revealed that Nrf2 is strongly stabilized and became detectable in nuclear extracts. EMSA demonstrated increased binding of Nrf2 to oligomers containing GCL promoter - specific ARE -binding site.A time- dependent increase in the gene and protein expression of GCL was observed. Additionally, GSH, which is prime scavenger of free radicals in cells, decreased initially. Elevation of GSH, following the initial decline, closely correlated with gene expression profile of GCLc, which is a rate-limiting enzyme in GSH synthesis. Conclusions Altogether, we provide direct evidence that homocysteine activates Nrf2-mediated antioxidant response, which protects HepG2 cells from oxidative damage.

Nuclear magnetic resonance-based screening of thalassemia and quantification of some hematological parameters using chemometric methods.

High-resolution (1)H NMR spectroscopy of biofluids is a good representation of metabolic pattern and offers a high potential noninvasive technique for pathological diagnosis. Diagnosis of thalassemia and quantification of some blood parameters can be performed by using (1)H NMR spectra of human blood serum in parallel with chemometric techniques. Spectra of 28 samples were collected from 15 adult male and female thalassemia patients as experimental set and 13 healthy volunteers as control set. Principal component analysis (PCA) as a dimension reduction tool was used for transforming spectra to abstract factors. The abstract factors were introduced to linear discriminant analysis (LDA), which is a common technique for classification, in order to establish adequate model for discrimination of healthy and unhealthy samples. In addition, these abstract factors were used for calibration of some blood parameters using radial basis function neural network (RBFNN) as an artificial intelligence modeling method. Different test sets (left out samples in training algorithm) were used for evaluating the quality and robustness of the built models. PCA abstract factors were employed as input for LDA model and successfully classified all the members of the test sets except one member of third test set. RBFNN also has a good capability for modeling the most of blood parameters according to proposed network parameters optimization procedure. We conclude that (1)H NMR spectroscopy, LDA and RBFNN assisted by PCA provide a powerful method for thalassemia diagnosis and prediction of some blood variants.

A Metabolic Study on Colon Cancer Using 1H Nuclear Magnetic Resonance Spectroscopy

Background. Colorectal carcinoma is the third cause of cancer deaths in the world. For diagnosis, invasive methods like colonoscopy and sigmoidoscopy are used, and noninvasive screening tests are not very accurate. We decided to study the potential of 1HNMR spectroscopy with metabolomics and chemometrics as a preliminary noninvasive test. We obtained a distinguishing pattern of metabolites and metabolic pathways between colon cancer patient and normal. Methods. Sera were obtained from confirmed colon cancer patients and the same number of healthy controls. Samples were sent for 1HNMR spectroscopy and analysis was carried out Chenomex and MATLAB software. Metabolites were identified using Human Metabolic Data Base (HDMB) and the main metabolic cycles were identified using Metaboanalyst software. Results. 15 metabolites were identified such as pyridoxine, orotidine, and taurocholic acid. Main metabolic cycles involved were the bile acid biosynthesis, vitamin B6 metabolism, methane metabolism, and glutathione metabolism. Discussion. The main detected metabolic cycles were also reported earlier in different cancers. Our observations corroborated earlier studies that suggest the importance of lowering serum LCA/DCA and increasing vitamin B6 intake to help prevent colon cancer. This work can be looked upon as a preliminary step in using 1HNMR analysis as a screening test before invasive procedures.

A metabolomic study on the effect of intravascular laser blood irradiation on type 2 diabetic patients.

Intravenous laser blood irradiation (ILBI) is widely applied in the treatment of different pathologies including diabetes mellitus. The aim of this study is to evaluate the effects of ILBI on the metabolites of blood in diabetic type 2 patients using metabolomics. We compared blood samples of nine diabetic type 2 patients, using metabolomics, before and after ILBI with blue light laser. The results showed significant decrease in glucose, glucose 6 phosphate, dehydroascorbic acid, R-3-hydroxybutyric acid, l-histidine, and l-alanine and significant increase in l-arginine level in blood and blood sugar in the patients have reduced significantly (p < 0.05). This study clearly demonstrated a significant positive effect of ILBI on metabolites of blood in diabetic type 2 patients. These findings support the therapeutic potential of ILBI in diabetic patients.

CREB in long‐term potentiation in hippocampus: Role of post‐translational modifications‐studies In silico

The multifunctionality of proteins is dictated by post‐translational modifications (PTMs) which involve the attachment of small functional groups such as phosphate and acetate, as well as carbohydrate moieties. These functional groups make the protein perform various functions in different environments. PTMs play a crucial role in memory and learning. Phosphorylation of synaptic proteins and transcription factors regulate the generation and storage of memory. Among these is the cAMP‐regulated element binding protein CREB that regulates CRE containing genes like c‐fos. Both phosphorylation and acetylation control the function of CREB as a transcription factor. CREB is also susceptible to O‐GlcNAc modification, which inhibits its activity. O‐GlcNAc modification occurs on the same or neighboring Ser/Thr residues akin to phosphorylation. An interplay between these modifications was shown to operate in nuclear and cytoplasmic proteins. In this study computational methods were utilized to predict different modification sites in CREB. These in silico results suggest that phosphorylation, O‐GlcNAc modification and acetylation modulate the transcriptional activity of CREB and thus dictate its contribution to synaptic plasticity. J. Cell. Biochem. 112: 138–146, 2011.

Anti-leishmanial Effects of Trinitroglycerin in BALB/C Mice Infected with Leishmania major via Nitric Oxide Pathway

This study investigated whether trinitroglycerine (TNG) as nitric oxide (NO) releasing agent had anti-leishmanial effects and mediated pathology in BALB/c mice infected with Leishmania major. Cutaneous leishmaniasis (CL), a zoonotic infection caused by leishmania protozoa is still one of the health problems in the world and in Iran. NO is involved in host immune responses against intracellular L. major, and leishmania killing by macrophages is mediated by this substance. Moreover, application of CL treatment with NO-donors has been recently indicated. In our study, TNG was used for its ability to increase NO and to modify CL infection in mice, in order to evaluate NO effects on lesion size and formation, parasite proliferation inside macrophages, amastigote visceralization in target organs, and NO induction in plasma and organ suspensions. Data obtained in this study indicated that TNG increased plasma and liver-NO, reduced lesion sizes, removed amastigotes from lesions, livers, spleens, and lymph nodes, declined proliferation of amastigotes, hepatomegaly, and increased survival rate. However, TNG reduced spleen-NO and had no significant effects on spelenomegaly. The results show that TNG therapy reduced leishmaniasis and pathology in association with raised NO levels. TNG had some antiparasitic activity by reduction of positive smears from lesions, livers, spleens, and lymph nodes, which could emphasize the role of TNG to inhibit visceralization of L. major in target organs.

NMR-based metabonomics survey in rats envenomed by Hemiscorpius lepturus venom.

About 1% of scorpion stings in Iran have been reported in cities and more than 5% in rural areas. The genus Hemiscorpous lepturus belongs to the family Hemiscorpiidae that is endemic in the south-western province of Khoozestan and other parts of western Iran. Although this species is responsible for only 10% of the reported stings, it is also responsible for 95% of mortalities. The heavy cytotoxic and neurotoxic venomous effects of scorpion sting may cause serious disorders such as erythema, purpuric changes, bulla, necrosis and ulcer, either alone or in combination. The toxic effects of H. lepturus venom cause vast changes in both primary and secondary metabolites of the victim, which finally lead to death if not treated early enough. Metabolomics is the systematic study of chemical fingerprints resulting from cytotoxic and pathogenic reactions of cells. We studied the toxic mechanism of H. lepturus venom on metabolome profiling of the victims along with biochemical pathways, organs and physiological detectable effects of this venom by the help of (1)H NMR. Our results showed that pyrimidine, histidine and tyrosine metabolisms, and steroid hormone biosynthesis were the most affected pathways. The major action of the crude venom is on the pancreas, and also on the nerve cells, spleen and mitochondria, causing acute seizures, which resemble the early markers of myocardial injury and seizure disorders.

A Metabonomic Study on Samples of Cutaneous Leishmaniasis and its Correlation with the Selenium Level in the Blood Serum

This paper presents the result of extensive experiments on the blood serum samples of 20 leishmaniasis patients and 44 healthy individuals using nuclear magnetic resonance (NMR) spectroscopy and the Chenomx software. The concentrations of selenium in all 64 serum samples were also measured using an atomic absorption device. The healthy and patient groups were completely differentiated using the partial least square method (PLS). In addition, important variables that could highly infl uence the group of patients were detected by the PLS loading plot. This paper also presents the results corresponding to linear and non-linear modeling of selenium concentration in serum. Stepwise multiple linear regression (MLR) was used to select the fi ve most important descriptors. In the multiple linear regression modeling approach the results obtained for R2 training, test and validation sets were 0.98, 0.97 and 0.94 respectively. Employing the same descriptors in the MLR modeling approach, a non-linear artifi cial neural network (ANN) with a 5-3- 1 structure was constructed; the results obtained from this model showed no signifi cant improvement compared with those of MLR. There was good agreement between the experimental values of selenium concentration and the values from the two models.

Purification of periplasmic flagellar antigen from Borrelia microtti.

Borrelia microtti and Borrelia persica are 2 Iranian strains of Borrelia found in western Asia and responsible for relapsing fever. The outer surface antigens of Borrelia undergo variations which are responsible for the relapsing phenomenon. The fixed flagellar antigen is required for diagnosis as the variant antigens cannot be used in serological methods of diagnosis. The fixed flagellar antigen was purified for the first time from the Iranian strain of Borrelia microtti using detergent treatment and shearing in an omnimixer. Periplasmic flagella were extracted, as confirmed by electron microscopy. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed a band corresponding to 42 kDa. Indirect haemagglutination kits were designed using the pure flagella and the complete sonicate of Borrelia and showed 98% sensitivity and 95% specificity.

The Effect of Aqueous Extract of Cinnamon on the Metabolome of Plasmodium falciparum Using 1 HNMR Spectroscopy

Malaria is responsible for estimated 584,000 deaths in 2013. Researchers are working on new drugs and medicinal herbs due to drug resistance that is a major problem facing them; the search is on for new medicinal herbs. Cinnamon is the bark of a tree with reported antiparasitic effects. Metabonomics is the simultaneous study of all the metabolites in biological fluids, cells, and tissues detected by high throughput technology. It was decided to determine the mechanism of the effect of aqueous extract of cinnamon on the metabolome of Plasmodium falciparum in vitro using 1HNMR spectroscopy. Prepared aqueous extract of cinnamon was added to a culture of Plasmodium falciparum 3D7 and its 50% inhibitory concentration determined, and, after collection, their metabolites were extracted and 1HNMR spectroscopy by NOESY method was done. The spectra were analyzed by chemometric methods. The differentiating metabolites were identified using Human Metabolome Database and the metabolic cycles identified by Metaboanalyst. 50% inhibitory concentration of cinnamon on Plasmodium falciparum was 1.25 mg/mL with p < 0.001. The metabolites were identified as succinic acid, glutathione, L-aspartic acid, beta-alanine, and 2-methylbutyryl glycine. The main metabolic cycles detected were alanine and aspartame and glutamate pathway and pantothenate and coenzyme A biosynthesis and lysine biosynthesis and glutathione metabolism, which are all important as drug targets.