Iranian Biomedical Journal
Volume:14 Issue: 1, Jan - Apr 2010

RNA interference (RNAi) is the mechanism of gene silencing-mediated messenger RNA degradation by small interference RNA (siRNA), which becomes a powerful tool for in vivo research, especially in the areas of cancer. In this research, the potential use of an expression vector as a specific siRNA producing tool for silencing of Bcr-abl in K562 cell line has been investigated. siRNA specific for Bcr-abl as short hairpin RNA (shRNA) was designed and cloned in expression vector (pRNAH1.1/Neo). K562 cells were cultured in RPMI media and transfected with shRNA expressing vector using lipofectamin 2000. Successful transfection was confirmed by significant increase of enhanced green fluorescent protein (EGFP) levels in K562-treated cells with expression vector (pEGFP-C1). In vitro studies in human K562 cell line entailed modulation of endogenous Bcr-abl mRNA levels which induced apoptosis. Effects of siRNA treatment on K562 cells were measured by ELISA. Successful expression of siRNA was confirmed by significant reduction of Bcr-abl mRNA levels in K562 cells treated with expression vector (pRNAH1.1/Neo). siRNA directed against Bcr-abl effectively induced apoptosis and reduced viability in human K562 cell lines. Expression vector of siRNA can be used in vitro to target specific RNA and to reduce the levels of the specific gene product in the targeted cells. Results of this work suggest that RNAi has potential application for the treatment of a variety of diseases, including those involving abnormal gene expression and viral contamination.

Zinc (Zn) as an important trace element is essential for testicular development and spermatogenesis. Molecular mechanism of Zn action in the reproductive system may be related to metal binding low-molecular weight proteins, metallothioneins (MT). Our objective was to determine the effect of Zn on two important isoforms of MT, MT1M and MT1G genes expression on testicular sertoli cells. Cultured sertoli TM4 cells were exposed to different concentrations of Zn at different time points. Cellular uptake of Zn was tested using flame atomic absorption spectrometry. The cellular viability and gene expression were assessed by MTT and real-time PCR methods, respectively. The treated cells resulted in higher Zn concentration and cellular viability. The expression of MT1M and MT1G genes in the treated cells were greater than those of the untreated cells (P<0.05). In the high dosage treated group (100 and 500 μM), Zn concentration and expression of MT1M and MT1G genes increased three h after treatment; MT1G gene expression increased more at sixth h. At 18th h of treatment, the expression of both genes especially MT1G, increased dramatically while Zn concentration decreased. Since the increase of MT1G mRNA was coincident with cellular Zn level, it seems that MT1G has a more prominent role than MT1M in the homeostasis of Zn. In addition, Zn at dosage of 50 μM (pharmacologic concentration) may protect cells by increasing the expression of MT genes at longer periods.

Vasopressin type 2 receptor (V2R) plays an important role in the water reabsorption in the kidney collecting ducts. V2R is a G protein coupled receptor (GPCR) and the triplet of amino acids aspartate-arginine-histidine (DRH) in this receptor might significantly influence its activity similar to other GPCR. However, the role of this motif has not been fully confirmed. Therefore, the present study attempted to shed some more light on the role of DRH motif in G protein coupling and V2R function with the use of site-directed mutagenesis. Nested PCR using specific primers was used to produce DNA fragments containing aspartate-lysine-isoleucine and aspartate-arginine-tyrosine mutations with replacements of the arginine to lysine and histidine to tyrosine, respectively. After digestion, these inserts were ligated into the pcDNA3 vector and transformation into E. coli HB101 was performed using heat shock method. The obtained colonies were analyzed for the presence and orientation of the inserts using proper restriction enzymes. After transient transfection of COS-7 cells using diethylaminoethyl-dextran method, the adenylyl cyclase activity assay was performed for functional study. The cell surface expression was analyzed by indirect ELISA method. The functional assay indicated that none of these mutations significantly altered cAMP production and cell surface expression of V2R in these cells. Since some substitutions in arginine residue have shown to lead to the inactive V2 receptor, further studies are required to define the role of this residue more precisely. However, it seems that the role of the histidine residue is not critical in the V2 receptor function.

We aimed at evaluating the toxicity effects of low (subtoxic) concentrations of silver nanoparticles (AgNP, 5-10 nm) in human hepatoblastoma (HepG2) cell line after and during a period of about one month. XTT and MTT assays were used to draw a dose-response curve; IC50 (half maximal inhibitory concentration) value of the AgNP on HepG2 cells was calculated to be 2.75-3.0 mg/l. The cells were exposed to concentrations of 0% (control), 1%, 4% and 8% IC50 of AgNP (corresponding to 0.00, 0.03, 0.12 and 0.24 mg/l of AgNP, respectively) for four consecutive passages. The treated cells were compared to the control group with respect to morphology and proliferation at the end of the period. The biochemical studies revealed significant increases of lactate dehydrogenase and alanine aminotransferase enzyme activity in the culture media of cells receiving 4% and 8% IC50; the increases in the aspartate aminotransferase enzyme activity and nitric oxide concentration became significant at 8% IC50. In the cell extracts, the average total protein and activity of glutathione peroxidase enzyme remained unchanged; the decrease in the average content of glutathione (GSH) and superoxide dismutase (SOD) activity became significant at 4% and 8% IC50. There were increases in lipid peroxidation (significant at 4% and 8% IC50) and cytochrome c content (significant at 8% IC50). The accumulations of the effects, during the experiment from one generation to the next, were not statistically remarkable except in cases of GSH and SOD. The results indicate clearly the involvement of oxidative changes in the cells after exposure to low doses of AgNP. The results might help specify a safer amount of AgNP for use in different applications.

Diabetes mellitus in some clinical cases is accompanied with hyperalgesia. In this study, we evaluated the possible beneficial effect of chronic pelargonidin (PG) treatment on hyperalgesia in streptozotocin (STZ)-diabetic neuropathic rat. Male Wistar rats (n = 56) were divided into seven groups, i.e. control, diabetic, PG-treated control, PG (single- and multiple-dose)-treated diabetic, and sodium salicylate-treated control and diabetics. For induction of diabetes, STZ was injected i.p. at a single dose of 60 mg/kg. PG was orally administered at a dose of 10 mg/kg once and/or on alternate days for 8 weeks; 1 week after diabetes induction. After two months, hyperalgesia was assessed using standard formalin and hot tail immersion tests. Meanwhile, markers of oxidative stress in brain were measured. One-way analysis of variance was used for statistical analysis of the data. Diabetic rats showed a marked chemical and thermal hyperalgesia, indicating that development of diabetic neuropathy and PG treatment (especially multiple-doses) significantly ameliorated the alteration in hyperalgesia (P<0.05-0.01) in diabetic rats as compared to untreated diabetics. PG (multiple doses) also significantly decreased diabetes-induced thiobarbituric acid reactive substances formation and non-significantly reversed elevation of nitrite level and reduction of antioxidant defensive enzyme superoxide dismutase. These results clearly suggest that PG prevents diabetic neuropathic hyperalgesia through attenuation of oxidative stress.

Use of hormone replacement therapy (HRT) may increase the risk of adult-onset asthma in women. Various in vitro studies have reported that estradiol stimulates human mast cell lines causing release of allergic mediators which was not observed in estrogen receptor-α (ER-α) knockout mice. Thus, estrogen might be a key element in occurrence of asthma. In the present study, we proposed to determine the role of ER-α in an experimental model of bronchial asthma. Trypsin and egg albumin induced chronic model of asthma were used. On the 28th day, various parameters such as pO2 level, serum bicarbonate level, tidal volume, respiratory rate, air flow rate, differential white blood cells count in the bronchoalveolar lavage (BAL) fluid and serum cholesterol level were measured as well as lung histopathological examination and uterine weight measurement were carried out. Estradiol treatment resulted in lower pO2 level, tidal volume and air flow rate. Also, serum bicarbonate level, respiratory rate and eosinophil rate and eosinophil count in BAL fluid were higher as compared to asthmatic control group. These effects were not observed in methyl-piperidino-pyrazole (MPP) co-treated group. Histopathological data suggested that the destruction of alveolar and muscular layers was more prominent in estradiol-treated group than asthmatic control and MPP co-treated groups. Estradiol-treated group showed lower total serum cholesterol levels and higher uterine weight as compared to asthmatic control group which was not observed in MPP co-treated group; indicating antagonism of estradiol by MPP at ER-α receptor. Estrogen seems to have a strong promoting effect on pathogenesis of bronchial asthma via ER-α receptors. Iran. Biomed.

The involvement of water-soluble carotenoids, crocins, as the main and active components of Crocus sativus L. extract in learning and memory processes has been proposed. In the present study, the effect of crocins on sporadic Alzheimer's disease induced by intracerebroventricular (icv) streptozocin (STZ) in male rats was investigated. Male adult Wistar rats (n = 90 and 260-290 g) were divided into 1, control; 2 and 3, crocins (15 and 30 mg/kg); 4, STZ; 5 and 6, STZ + crocins (15 and 30 mg/kg) groups. In Alzheimer's disease groups, rats were injected with STZ-icv bilaterally (3 mg/kg) in first day and 3 days later, a similar STZ-icv application was repeated. In STZ + crocin animal groups, crocin was applied in doses of 15 and 30 mg/kg, i.p., one day pre-surgery and continued for three weeks. Prescription of crocin in each dose was repeated once for two days. However, the learning and memory performance was assessed using passive avoidance paradigm, and for spatial cognition evaluation, Y-maze task was used. It was found out that crocin (30 mg/kg)-treated STZ-injected rats show higher correct choices and lower errors in Y-maze than vehicle-treated STZ-injected rats. In addition, crocin in the mentioned dose could significantly attenuated learning and memory impairment in treated STZ-injected group in passive avoidance test. Therefore, these results demonstrate the effectiveness of crocin (30 mg/kg) in antagonizing the cognitive deficits caused by STZ-icv in rats and its potential in the treatment of neurodegenerative diseases such as Alzheimer's disease.