Pathobiology Research
Year:2012 | Volume:15 | Issue:3|Papers Count:10

Objective: Group A rotaviruses (GARV) are responsible for the vast majority of severe diarrhea worldwide that kills an estimated 600, 000-870, 000 children annually. Since infantile gastroenteritis is a main health problem, therefore diagnosis and treatment of this disease is crucial. Gene rearrangements have been detected in vitro during serial passages of the virus at a high multiplicity of infection (MOI) in cell culture, as well as in chronically infected immunodeficient individuals. In this study, we developed an RT-PCR method to detect and diagnose the standard and gene rearranged bovine rotavirus.Methods: Rotavirus RNA was extracted from confluent monolayers of infected MA-104 cells, stained with silver nitrate, and then electrophoresed in a 10% polyacrylamide gel. The full-length gene products that encoded the NSP1, 2, and 3 genes of the standard and rearranged rotavirus were amplified by RT-PCR using specific primers.Results: We observed rearranged NSP1 and NSP3 genes that had different migration patterns seen with polyacrylamide gel electrophoresis. NSP1, 2, and 3 gene segments from standard and rearranged rotaviruses were amplified by RT-PCR, then the complete nucleotide sequence of each gene was subjected to sequencing. The results showed the generation of gene rearrangement through serial passages of the bovine rotavirus RF strain.Conclusion: Serial passage of rotavirus in cell culture at a high MOI and chronic infection in immunodeficient target groups might alter rotavirus evolution. The methods utilized for detection and characterization of rotaviruses are continually evolving and being refined. Data collection is necessary to understand the molecular and antigenic features of the rotavirus in order to have a successful implementation of rotavirus studies and the development of a rotavirus vaccine. This study shows the importance of genetic variation and can provide valuable information about the amplification, diversity, biology, and evolution of rotaviruses.

Objective: Due to recent advances in nanotechnology it is now possible to accumulate high atomic-number nanomaterial such as gold nanoparticles (GNPs) in cancerous cells and take advantage of their absorbed dose enhancement property as radiosensitizing agents. This study aimed to investigate the absorbed dose enhancement factor due to the presence of PEGylated GNPs under the irradiation of an MCF-7 cancerous cell line using orthovoltage X-ray beams.Methods: We synthesized GNPs with an average diameter of 47 nm and joined them to polyethylene glycol. A total of 50μg/mL of the pegylated GNPs were incubated with MCF-7 cells for 1, 2, 6, 12, and 24 hours, after which we compared their cytotoxicities. Then, PEGylated GNPs (50 mg/mL) were incubated with MCF-7 cells for 12 and 24 hours and their radiosensitizing effect during 2Gy delivery of 120, 180 and 200 kVp X-ray beams were compared by the MTT assay.Results: Cytotoxicity studies showed no significant effect of GNPs on cell viability. Significant differences in cell survival were observed between the groups irradiated with and without GNPs, which lead to an average absorbed dose enhancement factor of 1.22±0.06. According to the results, there was no radiosensitization difference due to the usage of 120, 180 and 200 kVp X-ray beams. However increased incubation time increased the dose enhancement factor.Conclusion: By using PEGylated GNPs we can decrease the prescribed X-ray dose, yet maintain the same level of cancerous cell killing.

Objective: Escherichia coli (E.coli) O157: H7 is one of the most important pathogenic causes of hemorrhagic colitis in humans. Cattle are the main reservoirs of this bacteria and vaccination is a key mechanism for its control. The intimin, translocated intimin receptor (tir), and EspA proteins are virulence factors expressed by the LEE locus of enterohemorrhagicE. coli. EspA protein is a member of the type III secretion system (TTSS) needle complexes that delivers the tir protein into the host cell. Surface arrayed intimin docks the bacterium to the translocated intimin receptor (Tir). This intimate linkage is the starting point for attachment and effacing lesions. We hypothesize that the chimeric recombinant forms of two of these three effectors, as edible-based immunogens, would reduce colonization of E. coli O157: H7 in the mice model.Methods: We constructed a synthetic gene (it) composed of eae (i) and tir (t) attached together by a peptide linker. The synthetic gene (it) was codon optimized based on the tobacco (Nicotiana tobbacum) plant and cloned into plant expression vectors adjacent to CaMV35S promoters for expression in transgenic tobacco plants. The antigen produced in this plant was orally fed to mice.Results: Immunization of the mice model by the transgenic plant that contained the divalent immunogen showed the presence of IgG antibodies against E. coli O157: H7.Conclusion: This method could be an effective tool for protecting against E. coli O157: H7 hemorrhagic colitis.

Objective: Biodegradable polycaprolactone/starch composites can be used for bone tissue engineering applications. The effect of the ratio of components on composite properties is of tremendous importance.Methods: Polycaprolactone/starch composite of 80.20 and 70.30 ratios were fabricated by dissolving them in chloroform followed by evaporation of the solvent.Results: The composites were characterized by fourier transform infrared spectroscopy. Their bioactivity was evaluated by studying the apatite formation ability after immersing the specimens in simulated body fluid. The results of compressive test on samples showed that the composite’s modulus and strength approximated that of human trabecular bone. Mass loss in distilled water and starch degradation rate in PBS was evaluated, which showed that the starch ratio was effective in composite degradation. MTT analysis and alkaline phosphatase levels showed that this composite had no toxicity and could increase G-299 cell line osteoblastic activities.Conclusion: The increase in cellular osteoblastic activities and the ability for apatite formation on the composite surface, in addition to the polycaprolactone/starch samples’ mechanical properties shows their capability to be used as substitutes for bone. Because this composite degradation rate is controlled by changing the starch ratio, it has the potential for use in bone tissue engineering applications. Samples that have a 70.30 ratio are considered optimal due to their enhanced cellular response and better mechanical properties.

Objective: Environmental pollution is of major concern today and lead is considered to be one of the most important environmental pollutants. Long-term contact with lead causes harmful effects to humans. This study seeks to determine the effects of Curcuma longa(turmeric extract) consumption and exercise training on glutathione peroxidase and protein carbonyl in kidney and spleen tissues from rats exposed to lead.Methods: We randomly classified 60 male rats into the following six groups of 10 rats per group: 1) control; 2) sham (turmeric extract solvent); 3) lead; 4) training+lead; 5) turmeric extract+lead; and 6) training+lead+turmeric extract. The training program for groups 3 and 6 consisted of running on a level treadmill for 40 sessions (eight weeks at five sessions per week) at a speed of 22 to 15 m/min for 26 to 64 minutes. Turmeric extract (30 mg/kg) was injected three times per week for eight weeks. Amounts of glutathione peroxidase and protein carbonyl were measured by ELISA.Results: The amount of protein carbonyl in the kidney and spleen tissues of the lead group increased compared to the sham, training, combined and extract groups. Rats in the combined, extract and practice groups (F=4.787; P=0.002) had lower levels of protein carbonyl in their kidney and spleen tissues compared to the sham group (F=6.970, P=0.000). Glutathione peroxidase levels in the kidney and spleen tissues were less in the lead group compared to the sham group. However these levels in the training, extract, and combined groups increased compared with the sham group (respectively, in kidney and spleen P=0.051, F=2.466 and P=0.086, F=2.11).Conclusion: Intake of turmeric extract and exercise alone did not cause complete inhibition of the oxidative effects in kidney and spleen tissues. However, exercise and consumption of turmeric extract can be effective in reducing the harmful effects of lead.

Objective: Aflatoxin is important in the food industry, in animal husbandry and the medical area; there are enormous negative economic impacts due to this toxin. Numerous studies have researched extracts and plant compounds with the intent to reduce the growth of aflatoxin-producing organisms, inhibit toxin production and suppress the major toxin encoded genes (i.e., aflR) in these organisms. Licorice is an important plant in traditional medicine that possesses numerous antimicrobial activities. There is no report regarding the effects of licorice or its mechanism of action on the aflatoxin-producingAspergillus species. The present study focuses on the inhibitory effects of licorice extract on aflR gene expression and the growth and survival of Aspergillus parasiticus (A. parasiticus).Methods: After the culture of A. parasiticus in toxin-inducer medium, we measured the minimal inhibitory concentration (MIC) for licorice extract. The aflatoxin concentration in the control and treated media was determined by HPLC. After harvesting the fungi from the toxin-inducing medium, its mRNA was extracted and cDNA synthesized by universal primers. The quantitative change in the aflR expression was analyzed via realtime PCR. Statistical analysis was performed by SPSS (v16).Results: The production of fungal mycelium decreased with increasing concentrations of licorice extract. The highest inhibitory concentration observed was 500 mg/ml of the extract. HPLC analyses revealed that the 10 mg/ml concentration of licorice extract inhibited toxin production by 99.9%. At this concentration, aflR gene expression was suppressed up to 40% as documented by quantitative RT-PCR analysis.Conclusion: Overall we concluded that the Licorice extract could inhibit the aflR gene expression and consequently the aflatoxin production efficiently in the A. parasiticus.

Objective: The goals of the study are evaluation the effect (s) of food deprivation as a social stress on testis structure. We also investigated the effects of melatonin treatment as an antioxidant component and inequality on the effect (s) of food deprivation.Methods: We investigated the improving effects of melatonin and social stress (food deprivation) on 42 male rats in 7 groups including control, sham, melatonin received (M), food deprivation (1/3 of control daily food) plus observation (FD), FD+melatonin (FDM), isolated FD (FDi), and FDi+melatonin (FDMi) groups. After 14 days, rats’ testes were studied using immuno histochemistry and TUNEL assays to determine the number of apoptotic cells. Biochemical evaluation was taken on malodialdehide (MDA) and glutathione (GSH). ANOVA and Tukey’s tests were done to analyse the data. P<0.05 was considered statistically significant.Results: The results of sham group was declined for similarity to results of control group. In FD group, MDA was increased significantly (P<0.01), GSH was decreased and the number of apoptotic cells was increased, significantly (P<0.01). In FDi group, there was no effect on the ratio of oxidative stress compared to the control group. Melatonin treatment could decrease apoptotic cells (P<0.05) and MDA concentration (P<0.05) in the FD group.Conclusion: Food deprivation can induce oxidative stress which is associated with increasment of apoptotic cells in testis. Isolation can compensate these effects. These results refer to inequality. Since melatonin is recognized for its anti-oxidative and improving effects, we have shown involvement of oxidative stress mechanisms on the stress of food deprivation with inequality.

Objective: Bone morphogenetic protein-7 (BMP-7) is a multifunctional growth factor predominantly recognized for its osteoinductive properties. Due to the high cost of this protein, the availability of BMP-7 for treatment is limited. The heterologous production of recombinant hBMP-7 has been performed in a number of expression systems. In this study a novel form of BMP-7 was expressed in eukaryotic and prokaryotic hosts.Methods: For expression in the prokaryotic system, the novel protein was secreted to the periplasmic space of Escherichia coli using a pel B signal sequence followed by single step purification by Ni2+-charged column chromatography. In the mammalian cell expression system, we transferred a full-length cDNA encoding precursor of the novel protein to CHO cells then selected stable clones by using the appropriate antibiotic concentration. Expressions in both systems were confirmed by Western blot analysis.Results: The novel recombinant protein was produced as a 36-38 kDa dimer in the CHO cell line and a 16 kDa monomer in the Escherichia coli system. Quantitative analysis according to ELISA showed that the expression levels of the mutant protein in the eukaryotic and prokaryotic expression systems were 40 ng/ml and 135 ng/ml of the culture media, respectively.Conclusion: In this study, the expression level in Escherichia coli was at least three times more than observed in the CHO cells. However, further optimization is required to obtain a dimer protein in Escherichia coli. The results show that periplasmic expression may be suitable for the production of complex proteins such as BMPs.