Yeni diterpen öncülleri geliştirmek amacıyla geranil difosfat C-metiltransferaz enzimin değiştirilmesi

Includes bibliographical references (leaves: 27-30)Text in English; Abstract: Turkish and Englishx, 30 leavesThesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2014Terpenoids constitute the most diverse family of natural products. They are involved in several biological functions and are used in medical and industrial applications. The key to their diverse biological activities is their structural diversity. Terpenoids are synthesized in three stages, all of which contribute to generation of structural diversity. In the terpenoid biosynthetic pathways, terpene synthases generate larger linear terpenoid precursors from smaller units via condensation reactions, terpene cyclases transform precursors via cyclization reactions, and then tailoring enzymes modify terpenoid products via addition of functional groups. Recently discovered geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is able to modify a linear monoterpenoid precursor, geranyl diphoshate (GPP), to produce a non-canonical terpenoid precursor, 2-methylgeranyl diphosphate. Modification of GPP by GPPMT is the first example of modification of a canonical linear isoprenoid precursor in nature. This study aims to achieve enzymatic synthesis of novel methylated non-canonical diterpenoid precursors, such as 2-methylgeranylgeranyl diphosphate (2MGGPP) by engineering GPPMT. The novel non-canonical precursors may later be utilized by cyclases to enhance the diversity of the terpenome. For example, taxadiene synthase could utilize 2MGGPP to generate variants of taxadiene, the precursor of the leading anti-cancer drug paclitaxel (Taxol®). Candidate mutants predicted to use GGPP as substrate were selected via in silico analysis of GPPMT structure. These mutations were introduced using the Quick-change site-directed mutagenesis. Mutant genes were expressed in E.coli strains. Mutant proteins were purified by Fast Protein Liquid Chromatography. Catalytic activities of mutants against canonical terpenoid precursors were determined by SAM methyltransferase assay

Yeni diterpen öncülleri geliştirmek amacıyla geranil difosfat C-metiltransferaz enzimin değiştirilmesi

Includes bibliographical references (leaves: 27-30)Text in English; Abstract: Turkish and Englishx, 30 leavesThesis (Master)--Izmir Institute of Technology, Molecular Biology and Genetics, Izmir, 2014Terpenoids constitute the most diverse family of natural products. They are involved in several biological functions and are used in medical and industrial applications. The key to their diverse biological activities is their structural diversity. Terpenoids are synthesized in three stages, all of which contribute to generation of structural diversity. In the terpenoid biosynthetic pathways, terpene synthases generate larger linear terpenoid precursors from smaller units via condensation reactions, terpene cyclases transform precursors via cyclization reactions, and then tailoring enzymes modify terpenoid products via addition of functional groups. Recently discovered geranyl diphosphate C-methyltransferase (GPPMT) from Streptomyces coelicolor A3(2) is able to modify a linear monoterpenoid precursor, geranyl diphoshate (GPP), to produce a non-canonical terpenoid precursor, 2-methylgeranyl diphosphate. Modification of GPP by GPPMT is the first example of modification of a canonical linear isoprenoid precursor in nature. This study aims to achieve enzymatic synthesis of novel methylated non-canonical diterpenoid precursors, such as 2-methylgeranylgeranyl diphosphate (2MGGPP) by engineering GPPMT. The novel non-canonical precursors may later be utilized by cyclases to enhance the diversity of the terpenome. For example, taxadiene synthase could utilize 2MGGPP to generate variants of taxadiene, the precursor of the leading anti-cancer drug paclitaxel (Taxol®). Candidate mutants predicted to use GGPP as substrate were selected via in silico analysis of GPPMT structure. These mutations were introduced using the Quick-change site-directed mutagenesis. Mutant genes were expressed in E.coli strains. Mutant proteins were purified by Fast Protein Liquid Chromatography. Catalytic activities of mutants against canonical terpenoid precursors were determined by SAM methyltransferase assay

Intravitreal bevacizumab as rescue therapy following treatment failure with laser photocoagulation in retinopathy of prematurity

Purpose: To evaluate the efficacy of intravitreal bevacizumab (IVB) in case of treatment failure with laser photocoagulation in retinopathy of prematurity (ROP). Methods: A retrospective review of infants treated with IVB injection due to treatment failure with laser photocoagulation was performed. The anatomical results and complications were evaluated after treatment. Results: 9 eyes of 6 premature infants with a mean gestational age of 26.8 ± 3.0 weeks (range, 23–32 w) and mean birth weight of 958 ± 319 g (range, 600–1400 g) were identified. Six of the eyes had zone 1 disease, and all of these eyes were classified as aggressive posterior ROP (APROP). Three of the eyes had zone 2 disease, and one of these eyes was classified as APROP, and the other two eyes were classified as high-risk pre-threshold disease. All of the eyes showed regression of the disease except one patient, both eyes of whom progressed to stage 4A. None of the patients developed ocular and systemic complications at the end of the six-month follow-up period. Conclusion: IVB could be an option in patients with ROP in whom laser photocoagulation failed

Structural and biochemical evidence for the emergence of a calcium-regulated actin cytoskeleton prior to eukaryogenesis

International audienceAbstract Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota