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"nBackground: Recombinant tissue plasminogen activator (rt-PA) is one of the most important thrombolytic agents used in patients with vascular occlusions such as acute ischemic stroke or myocardial infarction. A variety of recombinant protein expression systems have been developed for heterologous gene expression in prokaryotic and eukaryotic hosts. In recent years, Leishmania tarentolae (L. tarentolae), a non-pathogenic trypanosomatid protozoa, has come under consideration because of its safety and immunogenicity as a vaccine vector and special attributes in the expression of complex proteins. This study was done to improve rt-PA expression in this protozoon and create the opportunity for the replacement of rt-PA gene with other genes for the production of other complex proteins."n "n Methods: Two expression cassettes were used for the integration of two copies of t-PA cDNA, one copy in each cassette, into the parasite genome by electroporation. The transformed clones were selected by antibiotic resistancy. The expression of active secreted rt-PA was confirmed by Western blot analysis and Chromolize assay."n "n Results: Appearance of a 64 kD band in nitrocellulose membrane in the Western blot analysis confirmed the presence of full-length rt-PA in the culture media. Chromolize assay showed the expression levels of active recombinant t-PA in single and double transfected L. tarentolae clones- 375 IU/ml and 480 IU/ml of the culture media, respectively."n "n Conclusion: The produced rt-PA in the culture media containing the transfected cells was at least seven times higher than what has been reported in previous studies on L. tarentolae or on some other eukaryotic systems.

Introduction: This study compared the efficacy of Aryoseven with Novoseven to control bleeding episodes in patients with hemophilia A with inhibitors. Methods: Sixty-six patients were randomized into 2 groups, with 4 consecutive block randomization. These groups received Aryoseven and Novoseven dosages of 90 to 120 μg/kg intravenously every 2 hours. Results: Median (interquartile range) level of factor VIII (FVIII) inhibitor in groups A and B was 15.0 and 19.0 Bethesda Unit (BU) preadministration. Bleeding onset in group A was 1246 ± 1104 minutes and in group B was 2301 ± 1693 minutes (P = .311). The Kavakli global response scores and treatment success rate was comparable in both the groups. The side effects in groups A (9.7%) and B (2.9%) were comparable. Conclusion: Biosimilar recombinant activated FVII is found to be as effective as Novoseven in the treatment of acute joint bleeding in patients with hemophilia with inhibitors. Its usage will decrease the gaps in hemophilia. [ABSTRACT FROM AUTHOR]

The worldwide demand for pro-insulin increases continuously as new medical applications are developed. Lettuce (Lactuca sativa L.) has the potential to help supply this rising demand as a "green bio-reactor". A DNA construct containing the immunoglobulin G-binding protein A gene of Staphylococcus aureus (ProA) genetically fused to a nucleotide sequence encoding pro-insulin (Pins) was produced and transformed into lettuce. Integration of the ProA-Pins fusion gene into the chromosomes of transgenic lettuce, and copy numbers were verified by PCR amplification of genomic DNA and Southern blotting, respectively. Expression of the ProA-Pins fusion protein gene in transgenic lettuce was confirmed by reverse transcription-PCR analysis. Western immunobloting analysis of the proteins extracted from the leaves of transgenic lettuce plants revealed the presence of an immuno-reactive band with a molecular weight of approx. 20 kDa using rabbit polyclonal antibody against insulin. The plant-produced ProA-Pins fusion protein gave significant readings for indirect enzyme-linked immunosorbent assays (ELISA) using rabbit polyclonal antibody against insulin. The highest level of ProA-Pins expression in transgenic lettuce plants was 0.13% (w/w) of total soluble protein. This work demonstrated that plant-based systems expressing pro-insulin may provide an effective, low-cost approach to produce insulin for the treatment of Types I and II diabetes. [ABSTRACT FROM AUTHOR]

Background and Objectives Breast cancer cells can be hidden from immune cells by shedding tumor markers such as HER2. The increased soluble HER2(sHER2) concentrations are associated with the outcome of HER2-positive breast cancer and sensitivity to trastuzumab treatment. To this end, camelid single domain antibodies (VHH) with unique properties and binding ability are very striking targeting agents to detect sHER2. Materials and Methods By panning on HER2 negative and positive cells, an immune camel library was enriched against HER2 antigen. Affinity and specificity of four selected VHHs to HER2, detection of sHER2 and binding of selected VHHs to HER2 on breast cancer cells were investigated by ELISA. Results After cell panning and ELISA, four VHHs that recognized HER2 antigen better than negative control were identified. High affinity HER2 specific VHHs (1012-1013 M-1) were able to detect sHER2 (2 μg/ml). When the mixture of VHH and sMUC1 was added to HER2 coated-well, the VHH bound to HER2. RR4 and RR6 VHHs showed the highest binding to SKBR3 (HER2 expressing cell) (2±0.33 and 2.5±0.15, respectively) compared to HER2 negative cell (0.38 ±0.17 and 0.4±0.12, respectively). Conclusions The HER2 status of a primary tumor and responses to treatment can be evaluated by measuring sHER2 as a biomarker by HER2 specific VHHs. [ABSTRACT FROM AUTHOR]

Background: 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. Methods: 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 μg 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. Results: The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Background: Influenza is a contagious respiratory infectious disease out breaking in cold seasons of the year. The outbreak of the new influenza A (H1N1) virus in 2009 involved large populations of the world with considerable mortality. Hemagglutinin (HA) molecule, the main surface glycoprotein of the influenza virus, is one of the key factors for serological diagnostic kits and vaccine development. Thus establishment of HA gene bank of the circulating influenza viruses is essential in gaining quick access to large amounts of protein. Materials and Methods: The first step in providing such a bank is detection and isolation of HA full genome and its subunits by using specific primers and cloning them in proper vectors. For this purpose, using standard virus genome (A/New Caledonia/20/99(H1N1)) cultured on MDCK cell, HA coding gene was proliferated by RT-PCR using specific primers. Results: Isolation and cloning of the HA gene was verified by RT-PCR, enzyme digestion and determining nucleotide synonymy. Through the use of specific cloning primers, different HA gene constructs were propagated for expression of the gene in insect cells and E.coli bacteria. Conclusion: The results indicated the complete compatibility of the extracted HA gene with the influenza (A/New Caledonia/20/99(H1N1)) hemagglutinin. It makes it possible to use the gene as a source of cloning in a variety of eukaryotic and prokaryotic expression systems. [ABSTRACT FROM AUTHOR]