Retinal guanilat siklaz aktivitesi üzerine atp, amp-pnp ve all-trans retinal etkisinin incelenmesi

TEZ6397Tez (Yüksek Lisans) -- Çukurova Üniversitesi, Adana, 2007.Kaynakça (s.51-55) var.x, 56 s. : res. ; 29 cm.Retinal guanylate cyclases, retGC-1 and retGC-2, are a subclass of the membrane dependent guanylate cyclase family. Synthesize cGMP, the intracelular second messenger of phototransduction, they play a crucial role in photoreceptor physiology. In recent work, it is intended to investigate the effect of adenine nucleotides and all-trans retinal on the retinal guanylate cyclase activity. Crosslinking experiments indicate, protein phosphorylation might be essential for physiological activity of cyclases.Retinal guanilat siklazlar, retGC-1 ve retGC-2, membrana bağlı guanilat siklaz ailesinin bir alt kolunu oluştururlar. Fototransdüksiyonunun intraselüler mesajcısı olan cGMP'yi sentezledikleri için fotoreseptör fizyolojisinde önemli rol oynarlar. Bu çalışmada, adenin nükleotidlerinin retinal guanilat siklazların fizyolojisine etkilerinin incelenmesi amaçlanmıştır. Çapraz bağlama çalışmaları protein fosforilasyonunun siklazın fizyolojik aktivitesinin ortaya çıkmasında önemli rol oynadığını göstermiştir

Improving Recombinant Membrane Protein Expression in Rhodobacter sp. via Utilization of MISTIC Protein Fusion

Being located at the interface of intracellular and extracellular regions of biological cells, membrane proteins (MPs) mediate many vital events including signal transduction, energy generation, selective transport of solutes etc.. Because of these regulatory properties, nearly 70% of currently FDA approved drugs on the market target membrane proteins. Despite their importance, low abundance of MPs in natural resources is one of the major bottlenecks in studying their structure and functionality. Moreover, MPs have been employed in miscellaneous applications such as sensor technology, optogenetics, biomimetic separation membranes, in-vitro photosynthesis leads to an ever increasing demand for avaliability of MPs. Recombinant expression of MPs from heterologous expresion hosts was proven to be challenging especially for mammalian MPs due to improper folding, inclusion body formation and toxic effects on host organism. Therefore, development of alternative expression host systems for functional MP expression is of critical importance. Purple non-sulphur bacteria from Rhodobacter genus are known for their diverse metabolic pathways and intensively studied as model organisms in photosynthetis research. Under anaerobic, photosynthetic growth mode, plasma membrane surface area of Rhodobacter ssp immensely increase to accomodate the photosynthetic apparatus. An order of magnitude higher membrane surface area with inducible expression vectors imparts Rhodobacter sp as an attractive recombinant MP expression host. In this study, we employed codon optimized MISTIC chaperonin protein gene as N-terminal and mBanana fluorescent protein gene as C-terminal fusion partner for human AQP6 and AQP9 genes for co-expression in Rhodobacter sp. Cloning procedures were carried out in E. coli Top10 and expression vectors were transfered in Rhodobacter by biparental mating using E. coli S17 -pir. Protein expression was investigated under semi-aerobic and photosynthetic growth modes using two different promoters. A significant improvement in expression titers was observed with MISTIC fusions as monitored by SDS-PAGE.</p

Isolation, Characterization and Antimicrobial Activity of Bacteriocin Producing Lactic Acid Bacteria from Raw Water Buffalo Milk

Antibiotic resistance is a globally emerging health care concern. In recent years, a major difficulty in treatment of diseases such as tuberculosis, malaria, pneumonia and certain nosocomial infections is the presence of multi drug-resistant pathogens. In this regard, availability of the alternative antimicrobial materials is of paramount importance. Bacteriocins are ribosome-derived antimicrobial peptides generated by vast majority of bacteria to gain advantage over their competition. Bacteriocins from lactic acid bacteria (LAB) are often employed as natural food preservatives in food industry to increase the shelf-life of products. Their utilization as antimicrobial and anti-cancer agents in medical and veterinary treatments have been proposed. Therefore, discovery of new bacteriocin-producer LAB strains will benefit health and food industry applications. In this study, bacteriocin producer LAB strains were isolated from water buffalo milk samples collected from dairy farms located in Sorgun and Akdağmağdeni districts of Yozgat Province. Selective culture media were used for isolation of LAB genera including Lactobacillus, Enterococcus and Bifidobacterium. Screening of bacteriocin producer strains were carried out by reverse-side agar spot test using E. coli and S. aureus as indicator bacteria. A further verification of bacteriocin activity was carried out using neutralized cell-free supernatants via agar well diffusion assay. LAB isolates were characterized using miscellaneous biochemical methods including TSI, MR-VP, indole and catalase tests. Majority of the bacteriocin producer strains formed some degree of inhibition zone against selected indicator bacteria implying a broad bacteriocin antimicrobial activity spectrum, which might enable their utilization as novel antimicrobial agents.</p

Adana ve Körfez : savaş, korku ve göç

Ankara : İhsan Doğramacı Bilkent Üniversitesi İktisadi, İdari ve Sosyal Bilimler Fakültesi, Tarih Bölümü, 2016.This work is a student project of the The Department of History, Faculty of Economics, Administrative and Social Sciences, İhsan Doğramacı Bilkent University.by Özer, Abdurrahim

Marker-assisted selection and validation of DNA markers associated with cadmium content in durum wheat germplasm

Cadmium (Cd) is a non-essential heavy metal having toxic effects on all living organisms. Durum wheat (Triticum durum Desf.) is widely used in human diets but has the potential to accumulate Cd. It also has a high level of genetic diversity, which may be exploited to develop cultivars with low Cd content. We aimed to perform marker-assisted selection and validate previously identified Cd markers in durum wheat germplasm for use in the investigation of accessions that accumulate low grain Cd content. We assessed 130 durum wheat accessions phenotypically and using three different molecular markers. Grain Cd contents of the studied germplasm varied 4.91-fold (26.2-128.7 pg/kg) with an average of 58.2 mu g/kg. Landraces showed lower average values of grain Cd content than cultivars. Three molecular markers (usw47, Cad-5B and KASP marker Cad-5B) were used to differentiate high and low Cd accumulating lines. Results showed high correlation and successfully classified the accessions to the expected high or low Cd level; 87 accessions showed the low Cd alleles, and 43 accessions the high Cd alleles, except for five accessions with the usw47 marker that showed heterozygous status. A significant correlation coefficient (r = 0.944*) was observed among the three molecular markers. Based on molecular markers, 96.2% of the accessions were classified accurately. The KASP assay was highly effective in successfully separating low from high Cd content accessions and could be used as a molecular tool in durum wheat breeding programs, with less cost and time, targeting reduced grain Cd levels. The results of this study will allow durum wheat breeders to accelerate their progress to select suitable genotypes with the desired alleles

Cloning and verification of RsAqpZ expression.

<p>a) Verification of the transfer of RsAqpZ into <i>Rhodobacter sphaeroides 17023</i> by colony PCR using primers directed to <i>pRKPLHT7</i> vector backbone. Lanes 1: negative control, 2: positive control (<i>E. coli</i> cloning vector), 3–9: <i>Rhodobacter</i> exconjugants (expected band 864 bp), L: NEB 100 bp ladder; b) SDS-PAGE of RsAqpZ overexpressed and purified from <i>Rhodobacter</i>. M:Marker, P:Pellet, FT: Flow-through, W3∶3rd Wash fraction, E1: First Elution from IMAC column, E2: Second elution; c) Western-blot image of the gel in part b); RsAqpZ in monomeric and tetrameric form were detected by anti-His antibody.</p

Comparison of water channel activity for RsAqpZ and RsAqpZ-mBanana in phospholipid vesicles.

<p>mBanana fluorescent protein was expressed as a C-terminal fusion partner along with RsAqpZ. The water permeability did not show a significant change, which enabled assessment of the protein properties at the single molecule level.</p

Water permeability of ABA55 vesicles embedded with EcAqpZ and RsAqpZ-mBanana.

<p>ABA55 polymersomes embedded with EcAqpZ and RsAqpZ-mBanana were prepared using film rehydration method and dialyzed for insertion of the proteins in polymer. Unilamellar polymersomes with a low polydispersity (PDI <0.25) were obtained using consecutive extrusions through 400 nm and 200 nm track-etched filters and were then used to measure water permeability by stopped-flow light scattering. EcAqpZ and RsAqpZ-mBanana remained active in ABA55 polymer vesicles. RsAqpZ-mBanana showed higher water permeability in part due to higher incorporation efficiency.</p

Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from <i>Rhodobacter sphaeroides</i>

<div><p>Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, <i>Rhodobacter sphaeroide</i>s <i>ATCC 17023.</i> The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10⁻¹² cm³/s or 4.17 (±1.38)×10¹⁰ H₂O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.</p></div