Multiple Mutations on α, ß and γ Domains of Streptokinase Lead to the Generation of Highly Efficient Cysteine Analogues with Promising Features.

BACKGROUND: Streptokinase, one of the most widely used thrombolytic medicines, is a favorable protein for site-specific PEGylation as it lacks any cysteine residues in its amino acid sequence; however, any changes in the protein's structure should be carefully planned to avoid undesired changes in its function. OBJECTIVES: This study aimed to design and produce novel di/tri-cysteine variants of streptokinase from previously developed cysteine analogues, Arg45, Glu263, and Arg319, as candidates for multiple site-specific PEGylation. METHODS: Using bioinformatics tools and site-directed mutagenesis, we incorporated concurrent mutations at Arg45, Glu263, and Arg319 (carried out in our previous study) to create di/tri-cysteine variants of streptokinase proteins (SK45-319cys, SK263-319cys, and SK45-263-319cys) and evaluated their kinetic activity parameters by a colorimetric method, using H-D-Val-Leu-Lys-pNA.2HCl (S2251) as substrate. RESULTS: Based on the kinetic results, SK263-319cys with 44% enzyme efficiency increment compared to wild-type SK was the superior protein in terms of activity; as well, SK45-319cys and SK45-263-319cys showed 17 and 22% activity enhancement, respectively. Docking of the mutant streptokinase proteins with µ-plasmin demonstrated that changes in intermolecular interactions caused by amino acid substitution could be the reason for activity difference. CONCLUSION: The novel mutant proteins created in this study exhibit remarkable biological activity and may be uniquely suitable for simultaneous PEGylation on two/three domains. As well, PEGylated derivates of these variants might prove to be more proficient proteins, compared to the singlecysteine analogs of streptokinase; because of their more surface coverage and increased molecular weight.

Evaluation of recombinant toxin JFTX-23, an oral-effective anti-insect peptide from the spider Selenocosmia jiafu venom gland proteome.

Excessive utilization of chemical pesticides for pest control can lead to adverse consequences for the health of humans and other organisms and may also cause irreversible ecological changes; therefore, the use of biologically derived insecticides can be a safe alternate strategy. Transcriptomic studies have shown JFTX- 23,a small peptide from the spider Selenocosmia jiafuis highly similar to U1-TRTX-Sp1, a well-characterized oral-effective insecticide toxin from the Australian tarantula Selenotypus plumipes. First, we evaluated the JFTX-23 peptide sequence using bioinformatics tools and modeling studies. Preliminary results showed a high similarity of JFTX-23 to JZTX-58 (91.67%) and U1-TRTX-Sp1 (86.11%). Superimposition of the α-carbons of the modeled JFTX-23 and U1-TRTX-Sp1 demonstrated a very high similarity of the 3-D structure of the two peptides (RMSD of 0.02 Å).The injection assay of JFTX-23 in Helicoverpa armigera indicated an LD50 of 0.077 and 0.423 nmol/insect after 24 and 120 h, respectively. JFTX-23 was toxic to H. armigera via oral administration with an LC50 of 1.16 nmol/g food after 5 days, which was comparable to the toxicity of the oral-effective toxin U1-TRTX-Sp1. Our studies have shown that JFTX-23 is a potent oral-effective toxin that can be considered an attractive candidate for the biological control of insect pests.

Evaluation of immunological responses against outer membrane vesicles (OMV) of nontypeable Haemophilus influenzae using MPLA-CpG adjuvant as a vaccine candidate.

BACKGROUND AND OBJECTIVES: Nontypeable Haemophilus influenzae (NTHi) are major causes of non-invasive infections, including otitis media and sinusitis and it can also contribute to respiratory infections of all ages. Currently, there is no licensed vaccine against NTHi commercially available. Many studies have been conducted on the use of OMV as a vaccine against NTHi. The purpose of this study is to achieve an immunogenic vaccine against NTHi. MATERIALS AND METHODS: In this study, standard OMV Haemophilus (ATCC49766) with adjuncts CpG and MPLA was used and after infusion into BALB/c mice, the levels of antibodies and cytokines were measured on serum of immunized mice. RESULTS: The results showed that total IgG antibody and IgG1 and IgG2a isotypes in OMV immunized mice with mixture of CpG-MPLA adjuvant had a significant increase. Also, the results of cytokines (IL-10, IL-4 and IFN-γ) showed that IL-4 had the highest rate. CONCLUSION: These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.

Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Northwestern Iran by Using rDNA-ITS2.

Several mosquito species are vectors of disease; however, to understand their role in disease transmission, accurate species identification is of particular importance. Morphological identification is the main method used, but molecular techniques have emerged as a tool for the identification of closely related species. In this study, mosquitoes from the West Azerbaijan Province in northwestern Iran were characterized on the basis of their rDNA-ITS2 sequences. Nine populations of 6 species of mosquitoes belonging to the genera Anopheles, Culex, Culiseta, and Ochlerotatus were studied. To the best of our knowledge, ITS2 sequences of Culiseta longiareolata and Culex hortensis have been reported for the first time. In addition, ITS2 sequences of Culex theileri and Ochlerotatus caspius have been reported for the first time in Iran. Phylogenetic analysis based on ITS2 showed that subfamilies Anophelinae and Culicinae of the family Culicidae could be differentiated successfully and subgenera Anopheles and Cellia of the genus Anopheles were separated. The analysis showed that the genera Culex, Culiseta, and Ochlerotatus have diverged separately.

Towards a superior streptokinase for fibrinolytic therapy of vascular thrombosis.

Medical intervention with fibrinolytic drugs such as tissue plasminogen activator (tPA) and streptokinase (SK) is the principal treatment for life-treating thromboembolic disorders. Contrary to tPA, SK is a heterogenic and non-human (bacterial) protein produced by streptococci and its medical application may elicit sever immune and anaphylactic responses that restrict its utilization. Besides, human plasminogen (HPG) activation by SK is not blood-clot specific and associated with a risk of hemorrhage. Despite these limitations, comparative clinical trials on various thrombolytic agents suggested that SK is the most cost-effective fibrinolytic drug and almost as safe as its other counterparts such as tPA. Therefore, a number of studies were conducted to provide structurally modified SK with reduced immunogenicity, higher blood-clot specificity and half-lives. Although there are extensive overlaps in SK structural domains responsible for functionality, immunogenicity and stability that may limit its modifications, various strategies such as genetic manipulations (amino acid substitution /addition /deletion or domain fusions through production of chimeric SK proteins linked to HPG or hirudin) and chemical modification such as (homogenous/site-specific) PEGylation have been employed to develop a superior SK. In addition, data of the latest studies on SK screened from different streptococcal sources indicated the possibility of retrieving naturally occurring SKs with higher activities, less antigenicity and/or more fibrinspecificity. In the present review, after a survey on structure function relationships of SK domains and different strategies for SK improvement, recent advances and potential application of computer and matrix-based analyses for design and introduction of superior SKs will be presented.