Many snake venoms comprise different factors, which can either promote or inhibit the blood coagulation pathway. Coagulation disorders and hemorrhage belong to the most prominent features of bites of the many vipers. A number of these factors interact with components of the human blood coagulation. This study is focused on the effect of Echis carinatus snake venom on blood coagulation pathway. Anticoagulant factors were purified from the Iranian Echis carinatus venom by two steps: gel filtration (Sephadex G-75) and ion-exchange (DEAE-Sephadex) chromatography, in order to study the anticoagulant effect of crude venom and their fractions. The prothrombin time was estimated on human plasma for each fraction. Our results showed that protrombin time value was increase from 13.4 s to 170 s for F2C and to 280 s for F2D. Our study showed that these fractions of the venom delay the prothrombine time and thus can be considered as anticoagulant factors. They were shown to exhibit proteolytic activity. The molecular weights of these anticoagulants (F2C, F2D) were estimated by SDS/PAGE electrophoresis. F2C comprises two protein bands with molecular weights of 50 and 79 kDa and F2D a single band with a molecular weight of 42 kDa.
Tobacco use is a leading cause of preventable mortality worldwide. New cost-effective smoking cessation treatments are needed especially in some low-to-middle income countries where smoking rates are rising, and current pharmacotherapy treatments remain cost-prohibitive. Since the 1960’s, cytisine has been used as an effective nicotine substitution agent to aid in smoking cessation albeit limited to a selected few Eastern/Central Europe and Central Asian countries. Cytisine is a biologically active alkaloid of plant origin and is known to be a ligand of nicotinic acetylcholinergic receptors (nAChRs). For several decades, the properties of cytisine have been investigated and reported in the biomedical and pharmaceutical literature. Due to the beneficial impact of cytisine on smoking cessation and its costly multistep synthesis, there is a growing interest in extraction from natural sources as well as in analytical identification and quantification for clinical medicine and forensic toxicology. In this paper, we present several current analytical approaches to cytisine extraction and identification from biological samples of plant and human origin. The development of extraction techniques will allow for the widespread use of the drug in experimental and clinical pharmacology, toxicology and forensic medicine.
Gas chromatography, coupled with the temperature controlled pyrolysis technique, can be used as a quick method of identification of polymers such as acrylates, methacrylates and polyurethanes. Polymers based on alkyl methacrylates are widely used as construction materials and coatings. Polyurethanes are widely used as self-adhesives, sealants and electrical products (due to polyurethane's low glass transition temperature Tg). The aim of this work is to investigate which products can be obtained from polymethacrylates and polyurethanes.
Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.
Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.
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