Research in Pharmaceutical Sciences
Volume:10 Issue: 4, Aug 2015

Lentiviral vectors are among the promising viral based-vectors in gene therapy applications, but the efficiency of their targeting needs to be improved. (Strept)avidin-biotin adaptor system is a novel approach to modify the lentiviral envelope for better targeting properties. Herein, we describe utilization of this adaptor system by designing a candidate envelope protein-bearing biotin acceptor peptide (BAP) and evaluation of its expression in 293T cells. To this end, a DNA sequence containing flexible linkers, a 15-aminoacids BAP and specific membrane regions of a viral protein was designed and synthesized in tandem. The synthesized gene was amplified with polymerase chain reaction to include BglII and SalI restriction sites and subcloned into the same sites of pDisplay vector in frame with HA-tag and myc epitope to construct the pDis-GS-BAP. 293T cells were transfected with pDis-GS-BAP and expression of resulting protein (dis-GS-BAP) was evaluated by Western blotting using anti-HA tag antibody. Efficiency of transfection procedure was evaluated by pEGFP-N1 vector and tracking for green fluorescent protein expression via fluorescence microscopy. Restriction analysis and DNA sequencing confirmed the precision of cloning steps. Fluorescence microscopy indicated above 70% transfection efficiency and Western blot analysis of pDis-GS-BAP-transfected 293T cells showed a protein band of approximately 17 kDa corresponding to the predicted size of dis-GS-BAP protein. These promising results indicate the possibility of cell surface expression and further biotinylation of dis-GS-BAP protein in ongoing studies.

The prevalence of non-alcoholic fatty liver disease (NAFLD) has markedly increased, especially in patients exhibit one or more features of the metabolic syndrome. This study investigates the effect of rosuvastatin (RSV) and/or b-carotene (bC) in NAFLD-induced rats. Rats were classified into nine groups; normal (I), NAFLD-induced with high-fat diet (HFD; II), NAFLD switched to regular diet (RD; III), NAFLD-HFD or NAFLD-RD treated with RSV (IV, V), bC (VI, VII) or both RSV+bC (VIII, IX), respectively. After four weeks, rats were sacrificed to obtain serum samples and liver tissues. Liver histology, lipid profile, liver oxidative stress markers, and adipocytokines were measured. Liver sections of rats with NAFLD-HFD revealed steatosis, lose of hepatic architecture, inflammation and hepatocyte vacuolation with high percentage of cell fibrosis. Serum levels of ALT, AST, ALP, gamma glutamyl transferase (GGT) and lipid profile (triglycerides, cholesterol, LDL and VLDL) were significantly increased (P<0.05) compared with normal. Also, hepatic malondialdehyde level and serum leptin, tumor necrosis factor-alpha (TNF-a) and transforming growth factor-b1 (TGF-b1) were increased. Meanwhile, superoxide dismutase (SOD) activity, GSH content in liver, serum HDL and adiponectin were decreased (P<0.05) vs normal. These changes were observed to a lesser extent in NAFLD-RD group. Administration of RSV or/and bC almost improved all previously mentioned parameters. Moreover, hepatic steatosis was decreased and inflammation was markedly ameliorated with reduction of TNF-a and TGF-b. These results were more pronounced in the groups VIII and IX vs each drug alone. In conclusion RSV and βC could be beneficial for the treatment and prevention of NAFLD. Combined RSV with bC is more effective than RSV alone.

The antidiuretic effect of arginine vasopressin (AVP) is mediated by the vasopressin V2 receptor. The docking study of AVP as a ligand to V2 receptor helps in identifying important amino acid residues that might be involved in AVP binding for predicting the lowest free energy state of the protein complex. Whereas previous researchers were not able to detect the exact site of the ligand-receptor binding, we designed the current study to identify the vasopressin V2 receptor hormone binding site using bioinformatic methods. The 3D structure of nonapeptide hormone vasopressin was extracted from Protein Data Bank. Since no suitable template resembling V2 receptor was found, an ab initio approach was chosen to model the protein receptor. Using protein docking methods such as Hex protein-protein docking, the model of V2 receptor was docked to the peptide ligand AVP to identify possible binding sites. The residues that involved in binding site are W293, W296, D297, A300, and P301. The lowest free energy state of the protein complex was predicted after mutation in the above residues. The amount of gained energies permits us to compare the mutant forms with native forms and help to asses critical changes such as positive and negative mutations followed by ranking the best mutations. Based on the mutation/docking predictions, we found some mutants such as W293D and A300E possess positively inducing effect in ligand binding and some of them such as A300R present negatively inducing effect in ligand binding.

Psychological disorders such as depression have more prevalence in inflammatory bowel disease patients and can exacerbate the clinical course of the disease, so anti-depressant therapy may have a potential to positively impact the disease course. On the other hand several antidepressant drugs have shown anti-inflammatory and immunomodulatory properties. Thus, this study aimed to explore the beneficial effects of venlafaxine on experimental colitis in normal and reserpinised depressed rats. Acetic acid colitis was induced in both reserpinised and non-reserpinised rats. Reserpinised groups received reserpine at dose of 6 mg/kg i.p.1 h prior to colitis induction and then treated with venlafaxine at doses of 10, 20, 40 mg/kg given i.p. 2 h after induction of colitis and daily for 4 consecutive days. Non-reserpinised groups treated with 10, 20, 40 mg/kg venlafaxine i.p. 2 h after the induction of colitis and daily for 4 successive days. Dexamethasone (1 mg/kg, i.p.) was used as reference drug. Colonic inflammation was evaluated using macroscopic, histological and myeloperoxidase activity measurements. Results showed that reserpine at dose of 6 mg/kg exacerbated the colitis damage. Compared to acetic acid control, venlafaxine at dose of 40 mg/kg as well as dexamethasone significantly improved colitis parameters in both reserpinised and non-reserpinised animals. Venlafaxine reduced inflammatory injury in this animal model of induced ulcerative colitis. These effects are probably mediated first through depressive behavioral changes that could be mediated through the brain-gut axis and second for the anti-inflammatory effect of the drug.

P2X7 antagonist activity for a set of 49 molecules of the P2X7 receptor antagonists, derivatives of purine, was modeled with the aid of chemometric and artificial intelligence techniques. The activity of these compounds was estimated by means of combination of principal component analysis (PCA), as a well-known data reduction method, genetic algorithm (GA), as a variable selection technique, and artificial neural network (ANN), as a non-linear modeling method. First, a linear regression, combined with PCA, (principal component regression) was operated to model the structure–activity relationships, and afterwards a combination of PCA and ANN algorithm was employed to accurately predict the biological activity of the P2X7 antagonist. PCA preserves as much of the information as possible contained in the original data set. Seven most important PC’s to the studied activity were selected as the inputs of ANN box by an efficient variable selection method, GA. The best computational neural network model was a fully-connected, feed-forward model with 7−7−1 architecture. The developed ANN model was fully evaluated by different validation techniques, including internal and external validation, and chemical applicability domain. All validations showed that the constructed quantitative structure–activity relationship model suggested is robust and satisfactory.

Ferula foetida (Bunge) Regel. is one of the most widespread and important Ferula species with nutritional and medicinal applications. Some phytochemicals with helpful cardiovascular effects have been isolated from Ferula species. The present study was designed to evaluate the effects of hydroalcoholic extract of the stems of F. foetida in dexamethasone (Dex)-induced hypertension in rats. Hypertension was induced by subcutaneous injection of Dex (30 µg/kg) for 14 days. In a prevention study, rats received oral F. foetida extract (200, 400 and 800 mg/kg) for 4 days prior to Dex administration and during the test period (Days 1-18). In a treatment study, F. foetida extract was administered from day 8 to 14. Systolic blood pressure (SBP) was evaluated using tail-cuff method. The thymus weight was measured as an indicator of glucocorticoid activity. The hydrogen peroxide (H2O2) concentration and ferric reducing antioxidant power (FRAP) were measured in plasma samples. Dex-induced hypertensive rats showed significant increases in SBP and in plasma H2O2 and decreases in the body and thymus weights and in FRAP value (P<0.001). Administration of F. foetida extract significantly prevented and reversed hypertension at all doses. It also increased plasma FRAP value (P<0.001) but failed to decrease plasma H2O2 concentration. These results suggest antihypertensive and antioxidant effects of F. foetida stem extract in Dex-induced hypertension. More investigations are needed to elucidate the exact mechanism of antihypertensive effect of this traditional phytomedicine.

Artemisia is an important genus of Iranian flora whose potent anti-proliferative effect has been demonstrated previously on human cancerous cell lines. In the current study, further fractionation was carried out on the petroleum ether extract of A. aucheri and their cytotoxic effects were evaluated on three human cancer cell lines. Cell viability was determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Real time polymerase chain reaction (RT-PCR) was used to evaluate the expression of apoptotic related genes. Activation of caspases and detection of intracellular doxorubicin (DOX) accumulation were evaluated using a spectrophotometer. Mitochondrial membrane potential (MMP) was measured using flow cytometry. The fraction NO-7 (F7) of petroleum ether extract showed the highest anti-proliferative effect, especially against SKNMC cells. Therefore, we focused on a description of the cytotoxic mechanism of the most potent fraction on SKNMC cells. The results indicated that F7 was able to induce apoptosis through MMP disruption, activation of caspases and increament of proapoptotic genes Bax and Smac/DIABLO. Moreover, our observation indicated that F7 is able to increase the cytotoxicity of DOX in SKNMC cells. The combination of F7+DOX significantly increased the intracellular accumulation of DOX. These results indicated that F7 induces apoptosis in SKNMC cells. Moreover, it might enhance the antitumor activity of DOX, through modulating the activity of multidrug resistant cancer cells and inducing apoptosis.

Colitis is an inflammatory disease of the intestine with unknown etiology involving multiple immune, genetic and environmental factors. We were interested to examine the effect of total extract from Pycnocycla spinosa Boiss. on the treatment of experimental colitis. Mediators involved in colonic inflammation are prostaglandins, interleukins, leukotriene as well as an increase in myeloperoxidase (MPO) activity. Therefore, MPO activity was also determined in this research. P. spinosa hydroalcoholic extract (5, 10 and 20 mg/kg) or isoacetovanillone (2, 5 and 10 mg/kg) were administered orally, started 2 h before induction of colitis by intrarectal administration of acetic acid (3%) in rats. Prednisolone (4 m/kg) was used as the standard drug for comparison. Biochemical evaluation of inflamed colon was done using assay of MPO activity. After 5 days treatments, mucosal ulceration was evaluated. Intrarectal instillation of acetic acid caused significant inflammatory reactions as indicated by macroscopic and microscopic changes. The activity of MPO increased in vehicle treated groups while recovered to normal level by pretreatment of animals with P. spinosa extract, isoacetovanillone and prednisolone. P. spinosa and isoacetovanillone-treated groups showed significantly lower score values of macroscopic and microscopic characters when compared with the vehicle treated negative control group. The beneficial effect of P. spinosa was comparable with that of prednisolone. This research has shown the anti-inflammatory potential of P. spinosa extract and isoacetovanillone in experimentally induced colitis.

Discoidin domain receptor (DDR) is a new member of the receptor tyrosine kinase family. There are two isoforms of discoidin domain receptor (DDR), DDR1 and DDR2. These receptors play a major role in the adhesion, motility and cell proliferation. Due to the important role of DDR2 in the development of tumor extension, this receptor is pivotal in the field of carcinogenesis. The aim of this study was to investigate the mRNA and protein expression of DDR2, in the malignant, benign prostatic hyperplasia (BPH) and normal tissues of patients with prostate cancer. In this study the gene and protein expression of DDR2 in adjacent normal (n=40), BPH (n=40), and malignant (n=40) prostate tissue were measured using real-time PCR and Western blotting. Then, the correlation of DDR2 gene and protein expression with prognostic factors such as age, tumor grade, tumor stage, lymph node involvement, and serum prostate-specific antigen (PSA) concentration were evaluated. The relative mRNA and protein expression level of DDR2 in malignant and benign prostate tissue was significantly higher than those of adjacent normal tissues (P<0.01). This expression was found to increase approximately 3.5 and 2.1 fold for mRNA and protein levels, respectively. Spearman test indicated a significant correlation between DDR2 mRNA and protein expression with prognostic factors such as tumor grade, stage, lymph node involvement, and serum PSA concentration. However, significant correlation with age was not observed. These findings suggest that DDR2 is a cancer-related gene associated with the aggressive progression of prostate cancer patients.

Iron overload is a clinical problem which can be prevented by using iron chelating agents. An alternative method of relieving iron overload is to reduce iron absorption from the intestine by administering specific iron chelating agents, which can bind iron to form nonabsorbable complexes. Based on this strategy, a series of polymeric ligands containing the chelating moiety 3-hydroxypyridin-4-ones (HPOs) were synthesized. The synthetic route involves the benzylation of hydroxyl group of (2-methyl-3-hydroxypyran-4-one (maltol) and conversion of benzylated maltol to 3-benzyloxypyridin-4-one derivatives by using three suitable primary amines (2,6-diaminohexanoic acid (lysine) and 1,6-diaminohexane and 5-aminopentanol). The resulted compounds incorporated into polymer by copolymerization with acryloyl chloride using 2, 2''-azobisisobutyronitrile (AIBN) as the initiator. Finally, the benzyl groups of polymers were removed by catalytic hydrogenation (Pd/C). In this work, three final polymers of HPO derivatives namely poly-2-propylamido-6-(3- hydroxy -1,4-dihydro-2-methy-4-oxopyrid-1-yl) hexanoic acid, 6-(3-hydroxy-1, 4-dihydro-2-methyl-4-oxopyrid-1-yl) hexyl-1-polypropylamide and 5-(3-hydroxy-1-,4-dihydro-2-methyl-4-oxopyrid-1-yl)-1-polyacrylate pentane were synthesized. Identification and structural elucidation of compounds were achieved by proton nuclear magnetic resonance (1H NMR), carbon nuclear magnetic resonance (13C NMR) and infrared (IR) spectroscopy.