Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Inhibition of leishmania major PTR1 gene expression by antisense in escherichia coli

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase [DHFR]. Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

Isolation of leishmania major from paraffinembedded skin biopsies of patients with sarcoidal type granuloma, using kinetoplast DNA-PCR [K-DNA PCR] technique

In the past, different causative agents were identified for sarcoidosis. However, recently new evidence points to an association between sarcoidosis and leishmaniasis. To further investigate this relationship, we performed a crosssectional study on paraffinembedded skin biopsy samples obtained from patients with clinical and histopathological diagnosis of cutaneous sarcoidosis referring to Skin Research Center of Shahid Beheshti University of Medical Sciences, in Tehran, from January 2001 to March 2005. In order to isolate leishmania parasite from their skin lesions, we employed conventional diagnostic criteria to detect infectious agents including leishmania parasites, we reexamined twentyfive paraffinembedded skin biopsies, diagnosed as naked sarcoidal granuloma, with no positive results obtained. Furthermore, DNA extracted from all specimens was analyzed by the commercially available polymerase chain reaction [PCR] kits. Out of twentyfive, eight samples were positive for leishmania major [L. major], whereas L. tropica was detected in none. In fact, from histopathological point of view, cutaneous leishmaniasis [CL] may be misinterpreted as sarcoidosis, all the more so in cases in which conventional methods fail to detect leishmania parasite. In endemic areas for leishmaniasis, PCR for leishmaniaspecific DNA should be utilized in any granulomatous skin disease compatible with sarcoidosis, regardless of both the clinical presentation and histopathological interpretation

[Construction of a novel recombinant vector carrying herpes simplex virus thimidine kinase and yeast cytosine deaminase suicide genes to manipulate leishmania genetically]

Leishmaniasis is a group of diseases with various clinical pictures, which is caused by Leishmania spp. This parasite causes disease in human and about one hundred animal species. The disease is widely distributed in Iran and also across the world. One of the best approaches in the development of vaccine against Leishmania is genetically modification of the parasite. Therefore, the aim of this study was to design a novel genetic construct in order to insert Herpes Simplex Virus thimidine kinase [HSV-tk] and Yeast cytosine deaminase [Yeast-cd] suicide genes into the Leishmania genome. In this work, at the first step, HSV-tk and Yeast-cd fragments along with a gene of Leishmani, alpha-tubuline were cloned into pBluescript vector and the arrangement of tk-aplha tub-cd was created. Subsequently, these fragments were cut off from the plasmid and sub cloned into the plasmid pF4X1.4.4sat. The final construct was confirmed by digestion with relevant restriction enzymes. The fragments tk-alpha tub-cd was cloned successfully in the plasmid pBluescript and its authenticity was confirmed. Subsequently, this genetic collection was inserted into the plasmid pF4X1.4sat and finally, the orientation of it was checked. The construct designed in this study was able to insert two cellular suicide genes HSV-tk and Yeast-cd into the Leishmanai genome. Thus, this is an approach in achievement of vaccine against Leishmanai in the coming up researches

[Evaluation the immunogenesity structure of HIV recombinant protein gp41-p24]

HIV recombinant proteins can be used as immunogen and inducer of immune system. The gag and env proteins are among the most important ones that are located on internal and surface sections, respectively. In this study, we considered the immunogenisity structure of HIV recombinant protein gp41-p24, which has not yet been studied in detail before. In the present study, the HIV recombinant protein gp41-p24 was prepared by cloning methods and kept as unfolded. Using the dilution procedure, unfolded protein was changed to refolded state. The molecular weight and concentration of protein [refolded and unfolded] was measured by electrophoresis and spectrophotometer methods. The measurement of refolded protein can be estimated by native gel and circular dichroism method [CD] based on secondary structure of the protein. Immunological activity and immunogenic structure of these two proteins, based on protein type and optical density was recorded by ELISA and western blot methods. Our results showed that molecular weight of each protein was 32 KD and also they were pure. The refolded protein was observed by native gel method. In the above protein compared with the unfolded one, increased content of helix and beta strand structures and decreased random form was shown. Immune reaction with the antibodies in the serum of HIV positive control patients was observed in the standard and refolded proteins. There was no significant difference based on the protein type. Our research indicated that the HIV recombinant protein gp41-p24, after refolding, has immunogenic activity and we suggest its application as an immunogen in immunization and stimulation of immune system

Pre-symptomatic human cytomegalovirus disease diagnosis in renal transplant recipients by the virus DNA PCR

Human cytomegalovirus [HCMV, CMV] is a major infectious complication of renal transplantation. The objective of this survey was to optimize and establish a polymerase chain reaction [PCR] technique for rapid and early detection of CMV disease in renal transplant recipients. In a cross sectional study, a total of eighty-one EDTA-blood samples were collected as simple nonrandomized [sequential] weekly from thirty-seven renal transplant recipients during a 1-6 months period after their transplantation in Kidney Transplant Center of Shaheed Labbafinejad Hospital of Tehran. Peripheral blood leukocytes [PBLs] were isolated and DNA was extracted. HCMV DNA in PBLs was detected by PCR using a conserved set of primers. Amplified fragment was confirmed by restriction fragment length polymorphism [RFLP] and sequencing. Correlation between PCR results and patients' data was analyzed. Twelve patients from thirty-seven renal transplant recipients had positive samples containing HCMV DNA in PBLs [32.4%], whereas, five of them showed symptomatic CMV disease [13.5%] and seven of them did not show symptomatic CMV disease, but had some signs of pre-symptomatic CMV disease. Twenty-five patients had negative PCR results, and all of them did not have symptomatic CMV disease. Considering type one error [alpha = 0.05], a nonparametric Fisher's exact test showed a good correlation between two variables of positive PCR results and symptomatic CMV disease in renal transplant recipients [P=0.002]. In conclusion, establishing methods for early detection of HCMV DNA, even prior to showing symptomatic CMV disease, has been shown to be an effective way for starting antiviral therapy, prior to patients' experience of symptomatic CMV disease