Pathobiology Research
Year:2009 | Volume:12 | Issue:1|Papers Count:10

Objective: Angiogenesis a process that results in neo-vascularization is an essential stage in growth of solid tumors and the formation of metastases. Vascular endothelial growth factor (VEGF) and its receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1), are the major regulators for tumor angiogenesis. Recent studies showed that second domain of VEGFR-1is a key factor for VEGF/VEGFR-1 interaction. Materials and Methods: In this study, after RNA purification and cDNA synthesis, the second domain of VEGFR-1 (VEGFR-1-II) was amplified by PCR and cloned in T/A cloning vector. In order to increase the expression of the protein, we sub-cloned the gene into pET22b(+) and transformed the construct in Rosseta-gami 2, an efficient host for expression. The expression was induced by IPTG and confirmed by SDS-PAGE and Western Blotting. The recombinant protein was purified by IMAC column and the growth inhibition of human umbilical vein endothelial cells (HUVEC) was analyzed by the recombinant protein. Result: The results of SDS-PAGE and Blotting confirmed the protein purification accuracy. The recombinant protein concentration was determined by Bradford protocol. The results showed that nearly 300 mg/L VEGFR-1-II protein was produced. The function of this protein was confirmed by inhibition of HUVEC cells growth. Conclusion: Since this protein inhibited the angiogenesis in vitro it may be consider as an efficient anti-angiogenesis factor.

Objective: The present study investigated the role in transmission of M. tuberculosis strains isolated from tuberculosis patients residing in Northwest (East and West Azarbaijan) of Iran.Materials and Methods: We performed restriction fragment length polymorphism analysis on IS6110 of M. tuberculosis isolated from Northwest Iran. Total of 165 isolates of M. tuberculosis were analyzed by RFLP method.Results: The 5 copies and more IS6110 isolates comprised 30.52% of the total isolates. They formed 16 clustered groups consisting of 2 to 10 cases each. 69.48% of patients had a unique RFLP patterns. Cases from male patients were more clustered than female patients but statistically was not significant (P>0.05). In this study patients with 56 and older age were strongly associated with clustering (59.6%), which were significantly more than younger patients (P<0.05). Conclusion: In the present study we found old age as a major risk factor in contact dependent transmission of TB compared to disease recurrence. Unemployment and poor living condition were also among the risk factors in transmission of tuberculosis.

Objective: Vancomycin-resistant enterococci (VRE) have emerged worldwide and have become an increasing problem in clinical settings. Acquired glycopeptide resistance in Enterococcus species is due to the acquisition of van A, van B, van D, van C and van E genes, resulting in the production of peptidoglycan precursors with reduced affinity for glycopeptide antibiotics. The origin of these van genes is still unknown, but recent studies have indicated that van B resistance in enterococci might arise from gene transfer from the human bowel flora.In this study, we investigated the presence of Enterococcus-associated van A, van B, van C, van D, van E genes in the feces of hospitalized patients.Materials and Methods: To determine the prevalence of vancomycin-resistant enterococci (VRE) fecal colonization of hospitalized patients, 422 Enterococcus spp. isolated from stool of patients in Amiralam hospital. Disk diffusion method was used to detect resistance to vancomycin. The MICs of vancomycin were determined by the agar dilution method. The presence of van A, B, C, D and E genes were assassed by PCR analysis. Results: PCR was positive for van A for 6 out of 10 (60%) and van B for 4 out of 10 (40%) of VRE strains. Among the van positive enterococci, two (20%) specimens contained both van A and van B gene, whereas no van C, D and E positive enterococcal isolates were identified from these specimens. The MIC of VRE isolates were between 512- 1024 mg/ml.Conclusion: Our results showed that most glycopeptide resistant Enterococcus isolated from stool of hospitalized patients carried van A and van B. It is also possible that frequency of infections caused by glycopeptide-resistant enterococci will increase in our geographical area.

Objective: It is a firm belief that blood transfusion is life-saving in many situations, but at the same time transfusion complications could be life-threatening.The possible effects of blood Transfusion Related Immunomodulatory (TRIM) and its related mechanisms are one of the important debatable subjects in the field of blood transfusion medicine. One of the mechanisms through which transfusion can induced TRIM effects in recipient is apoptosis induction. Aim of this study was to investigate the apoptotic effects of stored blood in an in vitro model.Materials and Methods: To evaluate the apoptotic effects of blood storage, we studied the effect of the plasma (from whole blood) during storage on days 3, 10, 21 and 35 on Jurkat cells, which are sensitive to apoptosis .The plasma of whole blood was separated by centrifugation on different days. Then, Jurkat cells were cultured with plasma for 24 hours. Finally apoptosis level was studied by using flowcytometry for analyzing Annexin V on Jurkat cells (by SeroTec Annexin V: FITC Assay Kit).Results: The percentages of apoptosis on days 3, 10, 21 and 35 were 3.85±1.52, 5.27±2.12, 8.44±1.90, 12.01±2.32, respectively. The percentages of apoptotic cells in negative and positive control group was 3.85±1.94 and 65.80±2.28, respectively.Conclusion: The results of this study showed that plasma of whole blood have the apoptotic effects which enhanced during the storage of plasma. This in vitro model is also suitable for studying other TRIM related mechanisms. 

Objective: To study the mechanisms involved in amyloid formation processes, we made a mammalian cell culture model of amylin aggregation and characterized its properties.Materials and Methods: Amyloid fibrils were extracted from CHO cells and binding affinity to Thioflavin T and Congo red was investigated. Then the apple-green birefringence of extracted fibrils was detected under polarized light to confirm the presence of aggregated protein in the extracts. To better investigate amyloid formation in CHO cells, we decided to overexpress an amyloidogenic protein in these cells. To do so, we amplified ProIAPP gene and subcloned it in EGFP-N1 expression vector. Then, CHO cells were transfected with EGFP-N1-ProIAPP and EGFP-N1 as a control. The phenotypes of around 100 transfected cells were characterized for several days after the transfection, using fluorescence microscopy. Additionally, the presence of amyloid structure in these cells was detected by Congo red staining under exposure to polarized light. Cell viability assay was performed using Trypan blue staining.Results: Low level natural amyloid was detected in CHO cells. In addition, the ProIAPP-EGFP transfected cells exhibited aggregated phenotype in which the cells with round morphology had far more aggregates than oval ones.Conclusion: We found amyloid fibrils in CHO cells at low level. Overexpression of amylin protein in CHO cells caused aggregation phenotypes. These cells can be used as a model of cell culture for studying protein aggregation. Amyloidogenic properties of this protein could immensely help us to study the mechanisms that are involved in amyloid formation in mammalians including humans.

Objective: In about 50% of infertile couples a male factor can be found to be present either alone or in addition to other female factors. A new molecular mechanism recently revealed to be involved in infertility is epigenetic pattern modification like as aberrant methylation. This mechanism can change or even silence gene expression.On the other hand, GSTM1 (glutathione S-transferase mu 1) is shown to be important in transport of hormones and sexual steroids. Recent studies are in favor of implication of GSTM1 in male infertility. Based on the role of this gene in spermatogenesis, and protection of sperms, proving its epigenetic modification in male infertility and its implication in other diseases were reported. We explored its implication in Iranian men with infertility.Materials and Methods: This study is performed on peripheral blood samples of 50 fertile and 50 infertile men as well as 32 testicular tissue samples of infertile men with non obstructive azoospermia and 5 infertile men with obstructive azoospermia. Methylation detection method used in this study is Methylation Specific PCR.Results: Regarding GSTM1 gene in both cases (infertile men with non obstructive azoospermia) and normal control (fertile men) blood samples showed both methylated and unmethylated alleles. In tissue samples, 2 out of 32 testicular tissues showed only methylated and other samples showed both methylated and unmethylated alleles.Conclusion: This result shows a trend to methylation in Iranian men with infertility. However, statistically this difference is not significant (P-value=0.3). This study, in contrast to a previous report in another population, does not confirm the implication of methylation modification of GSTM1 5’UTR region in Iranian patients with infertility.

Objective: Listeria monocytogenes is a facultative, gram-positive bacterium which is found in soil, water, decaying vegetables, raw milk, and contaminated dairies. Listeria monocytogenes causes listeriosis. Listeriosis is a zoonosis disease, which transfers from animals to humans by animal feces and contaminated dairies. Listeriosis causes the flu like disease or self-limited enteritis, but it leads to serious disease in elderlies, new-borns, pregnant women and immunocompromised persons.If pregnant women are infected by Listeria monocytogenes, the newborn probably will be miscarried, prematured or stillborn.For the importance of the bacteria on pregnant women’s and newborn’s health, there are so much concentration and studies on it.Culturing of the bacteria is so difficult and time-consuming and it needs at least 5 days to confirm. Our goal in this survey was to develop an PCR based molecular method for fast detection of the bacteria from the vaginal samples.Materials and Methods: In this survey 100 vaginal samples were examined. All of the samples were cultured and assayed with PCR method.Results: Among them, 7 samples for culture and 36 samples by PCR were positive for Listeria monocytogenes.Conclusion: In this study we showed that the PCR is a faster, more accurate and sensitive than culture method for the detection of Listeria monocytogenes in vaginal samples.

Objective: Methicillin-resistant Staphylococcus aureus is a major cause of nosocomial and community-acquired infections. Aminoglycosides are potent bactericidal agents that are often used in combination with either a b-lactam or a glycopeptide, especially in the treatment of staphylococcal endocarditis. The main mechanism of aminoglycoside resistance in staphylococci is drug inactivation by cellular aminoglycoside-modifying enzymes.The main aim of the present study is determining the prevalence of ant(4’)-Ia gene encoding one of the most important aminoglycoside-modifying enzymes and simultaneous detection of mecA gene responsible for methicillin resistance in clinical isolates of Staphylococcus aureus by Multiplex-PCR method.Materials and Methods: A total of 100 clinical S. aureus isolates were collected from Shariati and Baqiatollah hospitals in Tehran, then antibiotic susceptibility pattern of strains were determined by disk diffusion method using penicillin, oxacillin, vancomycin, tetracycline, erythromycin, gentamicin, tobramycin, amikacin, netilmicin and kanamycin disks, considering CLSI principles. Using agar dilution method the MIC for oxacillin, gentamicin, tobramycin and amikacin were also determined. In order to detect resistance genes, ant(4’)-Ia and mecA, two pairs of specific primers were used and their prevalence was determined by using a Multiplex-PCR method.Results: All strains were resistant to penicillin (100%) and after that the highest rate of resistance was observed against kanamycin (68%), tetracycline (61%), erythromycin (56%), tobramycin (53%), gentamicin (52%), amikacin and oxacillin (48%) and netilmicin (22%), respectively. All of the strains were also susceptible to vancomycin. In agar dilution method 50% of strains were oxacillin resistant and 49%, 45% and 51% of the strains showed resistance toward gentamicin, amikacin and tobramycin, respectively. Thirty-seven percent of the strains also showed high-level gentamicin resistance with MIC of ³128mg/ml. In Multiplex-PCR method 53% of the strains possessed mecA gene and 58% of the strains were ant(4’)-Ia positive.Conclusion: Results obtained by phenotypic and genotypic antibiotic susceptibility determination tests show that there is a statistically meaningful relationship between methicillin resistance and aminoglycoside resistance in MRSA strains (P<0.05).