Pathobiology Research
Year:2007 | Volume:10 | Issue:2|Papers Count:11

Objective: The ITPA gene is responsible to remove free deaminated purine nucleotides of ITP, dITP and XTP from nucleotide pool of the cells. It seems that dysfunction in its activity, not only can increas the base substitution mutations frequency but also can works as a contrived factor to creating instability in genetic materials of the cells. There are several reports about the existence of structural and numerical genetic instability in the K562 cell genome. In this research, we examined the expression of ITPA gene as a possible contrived factor in observed genetic instability of this cell line. Materials and Methods: To evaluate the expression of target gene semi-quantitative RT-PCR technique was used. Then to examine the functionality of gene products, its cDNAs were cloned and their sequences were determined. Their proteins products were predicted using available bioinformatics soft wares and the results were compared.Results: The result of structural prediction of second mRNA showed that it has ability to encode a protein which has inability in substrate binding and also in its normal enzymatic activity. With regard to the fact that enzymatic activity of protein is dependent on the dimer formation, the function of hetero-dimer enzyme is changed. Therefore the catalytic activity of ITPase is predicted to be abnormal and it can be considered as a contrived factor for creating genetic instability in K562 cell line.Conclusion: The study of gene expression showed that ITPA gene is expressed in moderate level compared to GAPDH expression as an internal control in K562 cell. Two types of transcripts were detected in this line. One of them was the normal product of splicing process of primary hnRNA, but the second one contained a 51 nucleotides deletion in the mRNA coding region. It seems, this transcript is the product of a rare splicing process in this line.

Objective: Recent studies suggest that intermittent and prolonged normobaric hyperoxia (HO) results in ischemic tolerance to preventing ischemia brain injury. In this research attempts were made to see the changes in excitatory amio-acid transporter 3 (EAAT3), TNF-a levels, and NF-kB activity following prolonged and intermittent NBHO preconditioning.Materials and Methods: Rats were divided into four experimental groups, each with 21 animals. The first two groups were exposed to 95% inspired HO for 4h/day for 6 consecutive days (intermittent HO; InHO) or for 24 continuous hours (prolonged HO; PrHO). The second two groups acted as controls, and were exposed to 21% oxygen in the same chamber (normobaric normoxia, RA; room air) continuously for six days (intermittent RA, InRA) or for 24 hours (prolonged RA; PrRA). Each main group was subdivided to MCAOoperated (middle cerebral artery occlusion), sham-operated (without MCAO), and intact (without any surgery) subgroups. After 24h, MCAO-operated subgroups were subjected to 60min of right MCAO. After 24h reperfusion, neurologic deficit score (NDS) were assessed in MCAO-operated subgroups. Immediately and 48h after pretreatment, blood sampling for assessment of serum TNF-a levels were purformed. Then, the effect of InHO and PrHO on serum TNF-a levels, NF-kB activity and EAAT3 expression were measured. Results: Preconditioning with InHO and PrHO decreased NDS and upregulate EAAT3 and increase serum TNF-a level and NF-kB activity significantly.Conclusion: Although further studies are needed to clarify the mechanisms of ischemic tolerance, InHO and PrHO seems to partly exert their effects via increase in serum TNF-a levels, NF-kB activity and upregulation of glutamate transporters.

Objective: Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 (Invitrogen) could lead us to generate tolerogenic dendritic cells. Materials and Methods: In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and housekeeping gene (GADPH) was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit (Qiagen) was used for RNA extraction and the Purity and integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD40 using IQ sybergreen (Biorad) were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed.After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Results: Using gradient real time PCR, optimum annealing temperature, Ct and DRn for CD40 and GADPH were determined, annealing temperature was 59.5°c and melting temperature was 84°c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48 h after transfection.Conclusion: Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit (IQ-sybergreen) and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64.

Objective: Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary.Materials and Methods: In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5'UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT-Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study.Results: 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation.Conclusion: According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period.

Objective: In this study, a SYBR Green real-time RT-PCR assay for quantification of HIV-1 viral RNA was developed.Materials and Methods: This assay was performed based on amplification of the pol region of HIV-1 and product analysis by an ABI 7500 system. We quantified HIV-1 viral load in 26 seropositive patients by this system and the data were subsequently compared with results obtained with a reference technique represented by COBAS AMPLICOR HIV-1 Monitor test.Results: The results demonstrated that this technique could detecte up to 500 HIV-1 RNA copies/ml of plasma. The linearity of this approach was conserved over a wide range of HIV-1 copy numbers (5×102- 5×109). Since no positive signal was observed in seronegative volunteers, the specificity of the test was calculated as 100%. Comparison of the results with those obtained by the reference quantification method, revealed a significant correlation between the results (R2= 0.95).Conclusion: On the basis of the most recent recorded cases for HIV-1 infection and AIDS in Iran, the prevalence of this disease is rising rapidly and the situation has been called to be alarming by national health representatives. Determination of HIV-1 viral load in plasma has been considered as the most effective single prediction tool of clinical outcome. Indeed, the development and stabilization of HIV-1 RNA assays have given physicians a unique tool for monitoring HIV-1 patients treated with antiviral drugs.In this study, we have developed a SYBR-Green Real Time RT-PCR assay for quantitative analysis of HIV- 1 in infected patients. Since a synthetic RNA standard was used in this assay, the upper limit of detection was detected to be higher than the standard test (5×109 versus 7.5×105). This can be important in patients with acute high viral load infections. Reproducibility was assessed by Intra assay and Inter assay analysis.Coefficient of variations Ct, in reproducibility tests for Intra assay and Inter assay variability were less than 3% and 4.5% accordingly. The above results, indicates that the new developed test can be a used in substitution of the commercial assay for quantitative analysis of HIV-1.

Objective: Because of the necessity of more effective treatments for the nervous system injuries and considering the role of survivin in cellular proliferation and apoptotic cell death, we have monitored survivin gene expression changes during the course of regeneration in injured sciatic nerves and also L4-L6 segments of spinal cord.Materials and Methods: We used adult male NMRI mice as a model. After anesthetizing the animals, the right sciatic nerve was transected and at the indicated times (3, 6, 12, 24, 48, 96 and 144 hours) the animals were sacrificed and both distal and proximal segments of the transected sciatic nerve, intact left sciatic nerve and L4-L6 segments of spinal cord were dissected. The total RNA was extracted from each sample and semiquantitative RT-PCR with specific primers for survivin and also b2-microglobulin genes, as an internal control, was performed. To determine cellular distribution of survivin protein, 6 days (144 hours) after the axotomy, survivin protein expression was evaluated using immunohistochemistry technique. Results: Our results demonstrated the expression of both survivin140 and survivin40 in distal and proximal segments of sciatic nerve with different intensity, where the expression of survivin140 was higher than survivin40. In spinal cord segments, only survivin140 expression was detected. In Immunohistochemistry analysis of spinal cord segments, both the nuclear and cytoplasmic distribution of survivin protein was observed. In contrast, survivin protein has not been detected in either distal or proximal segments of sciatic nerve.Conclusion: Our data suggest that survivin is differentially expressed and spliced during the course of regeneration in damaged nerve and spinal cord. It seems that manipulation of expression and/or splicing of survivin could potentially affect the process of regeneration in nerve and/or spinal cord injuries.

Objective: Inhibition of apoptosis may favor the onset and progression of cancer. Survivin is an inhibitor of apoptosis that has been considered as a potential marker for diagnostic and/or prognostic of bladder cancer. The survivin protein regulates both cell division and cell death and is overexpressed in the vast majority of human cancers. In this study, the expression pattern and potential prognostic value of survivin was assessed in Formalin-Fixed Paraffin-Embedded (FFPE) samples of bladder tumor.Materials and Methods: FFPE samples, from patients with a well-known five-year survival record, were assessed by semi-quantitative RT-PCR technique. 51 samples from 30 patients were analyzed on the basis of Survivin expression. Tissue distribution and subcellular localization of survivin protein in tumor tissues was also examined by immunohistochemistry (IHC).Results: The expression of survivin was detected in 66.6% of the samples, with an increase of expression in higher grades of tumor. Furthermore, survivin was overexpressed in 2nd and 3rd recurrences of the same patients. Also, with the increased malignancy and accordingly increased expression of surviving, the overall 5-year survival rate of patients was significantly declined (P=0.036). IHC results also localized a nuclear localization for Survivin protein in tumor tissues.Conclusion: In conclusion, we were able to detect the expression of survivin in FFPE samples of bladder tissues, at the level of mRNA and protein and find a correlation between the level of Survivin expression and the degree of malignancy of the tumors. Our findings introduce Survivin as a suitable prognostic marker for predicting the bladder tumors.

Objective: 22q11.2 chromosomal region is a hot spot for many cytogenetic rearrangements especially microdeletions which are responsible for DiGeorge and VeloCardioFacial syndromes. The most characteristic sign in these patients is congenital cardiac conotruncal anomalies. The gold standard diagnostic test for these microdeletions is FISH (Fluorescent In Situ Hybridization). However this diagnostic technique has some drawbacks such as high final cost and low sensitivity in smaller and uncommon microdeletions found in this region. The aim of this study was to introduce a less expensive and a priori more sensitive molecular method to help small and peripheral laboratories to find genetic causes of congenital heart diseases and DiGeorge syndrome.Materials and Methods: 10 patients with congenital conotruncal anomalies and symptoms of DiGeorge syndrome were included in this study. These patients had been analyzed by FISH probe TUPLE1 before the inclusion. 3 normal persons were included as normal controls for microdeletion region. Semi Quantitative Multiplex PCRs were designed based on known markers in and out of the region of intrest. Results were analyzed by TotalLab software.Results: 4 patients showed a decrease in gene dosage more than 60% compared to normal persons. FISH analysis found only one patient with microdeletion.Conclusion: The designed method based on semi quantitative PCR was able to find 4 patients (40%) with microdeletion in a population of 10 patients with congenital cardiac anomalies. This technique was also able to find microdeletions in three FISH negative patients. Molecular diagnosis of microdeletions is supposed to be more sensitive than FISH in small microdeletions. This study confirms the presence of atypical deletions in Iranian patients and shows that the applied technique can detect some FISH negative patients. However further studies are needed to determine the sensitivity and specificity of the mentioned molecular diagnosis. It seems that this can be used at least for the patients with typical phenotypic features of 22q11DS and negative FISH results.