Iranian Journal of Basic Medical Sciences
Year:2013 | Volume:16 | Issue:7|Papers Count:12

Objective(s): Adeno-associated virus type 2 (AAV2) vectors are widely used for both experimental and clinical gene therapy. A recent research has shown that the performance of these vectors can be greatly improved by substitution of specific surface-exposed tyrosine residues with phenylalanines. In this study, a fast and simple method is presented to generate AAV2 vector helper plasmids encoding capsid proteins with single, double or triple Y®F mutations.Materials and Methods: A one-step, high-fidelity polymerase chain reaction (PCR) cloning procedure involving the use of two partially overlapping primers to amplify a circular DNA template was applied to produce AAV2 cap genes encoding VP1 mutants with Y→F substitutions in residues 444, 500 or 730. The resulting constructs were used to make the different double and triple mutant by another round of PCR (Y444500F mutant), subcloning (Y444730F and Y500730F mutants) or a combination of both techniques (Y444500730F mutant).Results: Nucleotide sequence analysis revealed successful introduction of the desired mutations in the AAV2 cap gene and showed the absence of any unintended mutations in the DNA fragments used to assemble the final set of AAV2 vector helper plasmids. The correctness of these plasmids was further confirmed by restriction mapping.Conclusion: PCR-based, single-step site-directed mutagenesis of circular DNA templates is a highly efficient and cost-effective method to generate AAV2 vector helper plasmids encoding mutant Cap proteins for the production of vector particles with increased gene transfer efficiency.

Objective(s): Stem cell differentiation into different cell lineages depends upon several factors, cell cycle control elements and intracellular signaling elements, including P15INK4b and P16INK4a genes. Epigenetics may be regarded as a control mechanism which is affected by these factors with respect to their promoter structure.Materials and Methods: The CD34+ cord blood stem cells were purified, isolated and then expanded. The undifferentiated day genome was isolated from part of the cultured cells, and the seventh day differentiated genome was isolated from the other part after differentiation to erythroid lineage. The procedure was followed by a separate Real-Time PCR for the two genes using the obtained cDNA. The processed DNA of the former stages was used for MSP (Methylation Specific PCR) reaction. Finally, pre- and post differentiation results were compared.Results: After performing MSP for each gene, it became clear that P15INK4b gene has undergone methylation and expression in predifferentiation stage. In addition, its status has not been changed after differentiation. P15INK4b gene expression was reduced after the differentiation. The other gene, P16INK4a, showed no predifferentiation methylation. Itwas completely expressed methylated and underwent reduced expression after differentiation.Conclusion: Specific predifferentiation expression of P15INK4b and P16INK4a genes along with reduction in their expression after erythroid differentiation indicated animportant role for these two genes in biology of CD34+ cells in primary stages and before differentiation. In addition, both genes are capable of epigenetic modifications due to the structure of their promoters.

Objective(s): The purpose of this study was to investigate effects of short-term aerobic exercise on energy intake, appetite and energy-regulating hormones in free-living men and women.Materials and Methods: Sixteen (eight men, eight women) sedentary young normal weight subjects participated in two experimental conditions with two days apart: five days control with no exercise, and five days exercise (55% MHRR for 45 min/day). Subjects recorded dietary intake using a food diary and self-weighed intake during each five days. Appetite questionnaire (visual analogue scale) was completed each morning in the fasted state. Blood samples were taken in the morning on the 6th day in fasting status after control and exercise conditions.Results: No significant changes were found in absolute energy intake, appetite rate and level of acylated ghrelin and leptin between conditions in both sexes. In women, insulin concentration decreased significantly after exercise. Relative energy intake was significantly lower after exercise in men. On average, women compensated for about 23% of the exercise-induced energy deficit but men did not (-10%).Conclusion: Our findings show that low-intensity exercise for five consecutive days cannot create a negative energy balance in women. It seems that women are more resistant to exercise-induced energy deficit.

Objective(s): Helicobacter pylori, a human specific gastric pathogen is a causative agent of chronic active gastritis. The vacuolating cytotoxin (VacA) is an effective virulence factor involved in gastric injury. The aim of this study was to construct a recombinant protein containing antigenic region of VacA gene and determine its antigenicity.Materials and Methods: The antigenic region of VacA gene was detected by bioinformatics methods. The polymerase chain reaction method was used to amplify a highly antigenic region of VacA gene from chromosomal DNA of H. pylori. The eluted product was cloned into the prokaryotic expression vector pET32a. The target protein was expressed in the Escherichia coli BL21 (DE3) pLysS. The bacteria including pET32a-VacA plasmids were induced by IPTG. The antigenicity was finally studied by western blotting using sera of 15 H. pylori infected patients after purification.Results: Enzyme digestion analysis, PCR and DNA sequencing results showed that the target gene was inserted correctly into the recombinant vector. The expressed protein was purified successfully via affinity chromatography. Data indicated that antigenic region of VacA protein from Helicobacter pylori was recognized by all 15 patient’s sera.Conclusion: Our data showed that antigenic region of VacA protein can be expressed by in E. co.li. This protein was recognized by sera patients suffering from H. pylori infection. the recombinant protein has similar epitopes and close antigenic properties to the natural form of this antigen. Recombinant antigenic region of VacA protein also seems to be a promising antigen for protective and serologic diagnosis.

Objective (s): Apolipoprotein E genotype (APOE) polymorphism affects lipid levels and coronary artery disease (CAD) risk. The aim of this study was to study the association of the Apolipoprotein E genotypes with coronary artery disease in the Iranian population.Materials and Methods: The Apolipoprotein E genotype in DNA samples extracted from 66 CAD+ patients and 61 control subjects by restricting enzyme digestion of amplified exon 4 APOE gene was determined.Results: The ε3 allele was found at similar frequency in control subjects (88.5%) and atherosclerosis patients (83.3%) (P=0.314). Our results showed that the frequency of the e3/e3 and e3/e4 genotypes increased in three-vessel-disease patients and the frequency of e2/e2 genotype increased in one-vessel-disease patients.Conclusion: e3/e3 and e3/e4 genotypes are suggested to be predisposing factors, which, in combination with environmental factors, may trigger the degree of luminal narrowing. The possible mechanisms remain elusive and require further studies.

Objective (s): Fasting during the month of Ramadan is a religious obligation and belief for healthy adult Muslims. The aim of the present study was to determine the effects of Ramadan Fasting and physical activity on ‘Blood Hematological-Biochemical Parameters’.Materials and Methods: In this study, twenty-six healthy males in two experimental groups were compared in two different instances before and after the training period. The groups which were selected by convenience sampling method were divided into two non-active fasting (n=13) and active fasting (n=13) groups. For comparison purposes between groups, paired and independent sample t-test was performed, respectively, after ensuring their normality within a significance level of P£0.05.Results: HDL-C increased significantly in both active and non-active fasting groups, Moreover, amount of hematocrit (Hct), red blood cell count (RBC), TC, LDL, VLDL, LDL/HDL and TC/ HDL decreased significantly. Amount of hemoglobin (Hgb) and glucose reduced significantly in the active-fasting group. The variation of the means between the groups in the Hgb index and LDL/HDL were statistically significant.Conclusion: Fasting during the month of Ramadan by regular physical activity caused positive alterations in Hematological-Biochemical Index. These changes may be due to the alterations in diet, biology response of the body to the starving and physical activity during this month.

Objective (s): Pulmonary fibrosis (PF) is the most common outcome of a collection of diverse lung disorders known as interstitial lung diseases. It is proposed that alterations in the levels of fibrogenic mediators and the profibrotic/antifibrotic imbalance play a substantial role in the progression of PF in animal models and possibly in humans. Caffeic acid phenethyl ester (CAPE), an active component of propolis, has numerous biological effects. In the present study, the main objective was to investigate the effects of CAPE on some key mediators including TGF-b1, TNF-a and prostaglandin E 2 (PGE2) involved in profibrotic/ antifibrotic balance and pathogenesis of idiopathic pulmonary fibrosis (IPF).Materials and Methods: In this study, forty male Sprague- Dawley rats were divided into 5 groups (n=8). (1) “Bleomycin (BLM) -treated (Model) group”: BLM (5 mg/kg, single intratracheal dose), (2) “Saline-treated group”: the rats were given only saline, (3) “Treatment-1 group”: BLM+CAPE (5 mmol/kg/day, 28 days, IP), (4) “Treatment-2 group”: BLM+CAPE (10 mmol/kg/day, 28 days, IP) and (5) “Vehicle+CAPE group”: CAPE (10 mmol/kg/day, 28 days, IP).Results: BLM could significantly increase the levels of TNF-α and TGF-β 1 and decrease the PGE 2 concentration compared to the saline control group. CAPE could considerably improve these values almost close to normal levels.Conclusion: Briefly, CAPE can be suggested as a novel, attractive and effective agent for prevention and treatment of pulmonary fibrosis.

Objective (s): Brain edema is one of the most serious causes of death within the first few days after trauma brain injury (TBI). In this study we have investigated the role of Shilajit on brain edema, blood-brain barrier (BBB) permeability, intracranial pressure (ICP) and neurologic outcomes following brain trauma.Materials and Methods: Diffuse traumatic brain trauma was induced in rats by drop of a 250 g weight from a 2 m high (Marmarou’s methods). Animals were randomly divided into 5 groups including sham, TBI, TBI-vehicle, TBI-Shi150 group and TBI-Shi250 group. Rats were undergone intraperitoneal injection of Shilajit and vehicle at 1, 24, 48 and 72 hr after trauma. Brain water content, BBB permeability, ICP and neurologic outcomes were finally measured.Results: Brain water and Evans blue dye contents showed significant decrease in Shilajit-treated groups compared to the TBI-vehicle and TBI groups. Intracranial pressure at 24, 48 and 72 hr after trauma had significant reduction in Shilajit-treated groups as compared to TBI-vehicle and TBI groups (P<0.001). The rate of neurologic outcomes improvement at 4, 24, 48 and 72 hr after trauma showed significant increase in Shilajit-treated groups in comparison to theTBI- vehicle and TBI groups (P<0.001).Conclusion: The present results indicated that Shilajit may cause in improvement of neurologic outcomes through decreasing brain edema, disrupting of BBB, and ICP after the TBI.

Objective (s): The objective of this study was to design a topical microemulsion of Vit B12 and to study the correlation between internal structure and physicochemical properties of the microemulsions. Microemulsions are thermodynamically stable mixtures of water, oil, surfactants and usually cosurfactants with several advantages for topical and transdermal drug delivery. The formulation of microemulsions for pharmaceutical use requires a clear understanding of the properties and microstructures of the microemulsions.Materials and Methods: In this study, phase behavior and microstructure of traditional and novel microemulsions of Vit B12 have been investigated by Small-angle X-ray (SAXS), differential scanning calorimetery (DSC) and measuring density, particle size, conductivity and surface tension.Results: WO and bicontinuous microemulsion with different microstructures were found in novel and traditional formulations. In this study, amount of water, surfactant concentration, oil/ surfactant ratio and physicochemical properties of cosurfactants influenced the microstructures. In both formulations, water behavior was affected by the concentration of the surfactant. Water Solubilization capacity and enthalpy of exothermic peak of interfacial and free water of traditional formulations were more than novel ones. This means that the affinity of water to interfacial film is dependent on the surfactant properties.Conclusion: This study showed that both microemulsions provided good solubility of Vit B12 with a wide range of internal structure. Low water solubilization capacity is a common property of microemulsions that can affect drug release and permeability through the skin. Based on Vit B12 properties, specially, intermediate oil and water solubility, better drug partitioning into the skin may be obtained by traditional formulations with wide range of structure and high amount of free and bounded water.

Objective (s): It has been suggested that the efficiency of dry poder inhaler (DPI) is generally low. Therefore, the aim of the present research work was to use the solvent treatment of the carrier in DPIformulations to see the possibility of inducing desirable characteristics.Materials and Methods: Lactose sieve fractions of 63-90 mm were submerged in ethanol or 80% v/v ethanol, methanol or propanol. Lactose crystals were then blended with either 1% w/w or 4% w/w salbutamol sulphate using a Turbula mixer. Drug detachment was studied using a multistage liquid. Laser particle size analyzer, DSC, and pycnometer were used to characterize the treated lactose and mannitol samples. SEM was used to study surface morphologies. In case of mannitol as a carrier only ethanol was used as a solvent.Results: SEM images displayed less rugosities and increased surface smoothness after submersion. Although the tomahawk like shape remained fairly constant in most lactose samples, the solvent treatment changed the shape of mannitol particles which was proved by SEM results. Deposition results showed that the type of solvent had an influence on fine particle fraction. In addition, the payload had also a big impact on fine particle fraction values. Generally, treated samples showed better performance compared to untreated samples. Solid state analysis by DSC showed that no major changes occurred in the treated samples compared to untreated samples.Conclusion: The solvent treated method can be used as an approach to improve the performance of carriers such as lactose and mannitol in dry powder inhaler.

Objective (s): The ongoing increasing antibiotic resistance is one of the biggest challenges faced by global public health. The perennial need for new antimicrobials against a background of increasing antibiotic resistance in pathogenic and opportunistic microorganisms obliges the scientific community to constantly develop new drugs and antimicrobial agents. Lichens are known prolific sources of natural antimicrobial drugs and biologically active natural products. This study was aimed to explore in vitro antimicrobial activity of lichen Parmotrema sp.Material and Methods: The methanol and aqueous extracts of lichen Parmotrema sp. was extracted using Soxhlet extractor. Antibiotic assessment of methanol and aqueous extracts was done against eight bacterial (Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Salmonella sp., Shigella sp., Enterococci faecalis, Pseudomonas aeruginosa, Klebsiella pneumoniae,) clinical pathogens and five plant pathogenic fungal strains (Aspergillus terreus strain JAS1, Scedosporium sp. JAS1, Ganoderma sp. JAS4, Candida tropicalis and Fusarium sp.) by Kirby-Bauer method.Results: The methanol lichen Parmotrema sp. extract inhibited all the test organisms. The highest antibacterial activity was found against Pseudomonas aeruginosa and Staphylococcus aureus. The weakest activity was manifested in Salmonella sp. and Scedosporium sp. JAS1. Strong antifungal effect was found against Ganoderma sp. JAS4 and Fusarium sp. The aqueous lichen Parmotrema sp. extract revealed neither antibacterial nor antifungal activity.Conclusion: The present study shows that tested lichen Parmotrema sp. extracts demonstrated a strong antimicrobial effect. That suggests the active components from methanol extracts of the investigated lichen Parmotrema sp. can be used as natural antimicrobial agent against pathogens.

Objective (s): Scar formation in injured peripheral nerve bed causes several consequences which impede the process of nerve regeneration. Several animal models are used for scar induction in preclinical studies which target prevention and/or suppression of perineural scar. This study evaluates the translational capacity of four of physical injury models to induce scar formation around the sciatic nerve of rat: laceration, crush, mince and burn.Materials and Methods: Functional (Toe out angle), macroscopic, and microscopic evaluations were performed weekly for four weeks and correlation of findings were analyzed.Result: While macroscopic and microscopic findings suggested a well-developed and adhesive fibrosis surrounding the sciatic nerve, functional assessment did not reveal any significant difference between control and experimental groups (P>0.05).Conclusion: Our study suggests that none of the applied animal models reproduce all essential features of clinical perineural scar formation. Therefore, more studies are needed to develop optimal animal models for translating preclinical investigations.