Objective: The aim of the present study was to investigate whether the VKORC1 -1639 G>A polymorphisms could determine differences in the warfarin dose required to maintain a therapeutic INR in patients living in east of Turkish. Methods: A total of 80 patients (45 female and 35 male) aged between 17 and 85 years (mean age 58.96±16.59 years) using warfarin for at least one month and presenting at Atatürk University Medical Faculty Outpatient Clinic between May 2009 and May 2010 were included in the study. Patients underwent genotype investigations and carrier forms of VKORC1 -1639 variants; homozygous (AA), heterozygous (GA) and wild types (GG) were determined. Results: In AA and GA groups, daily warfarin maintenance doses were statistically significant lower than in GG (p=0.010 and p=0.016, respectively). There was no significant difference between AA and GA groups regarding to warfarin dose (p>0.05). INR values were higher in GA and AA groups than in GG group and differences were statistically significant (p=0.014 and p=0.016, respectively). Also there was no difference between GA and AA gropus in terms of INR. Conclusion: In conclusion, our study verified the interindividual variability in warfarin maintenance dose due to VKORC1-1639 G>A polymorphism in patients living in east of Turkish. In order to avoid serious bleeding events in warfarin therapy, prescription of warfarin should be done on an individual basis with consideration for the patient’s genetic background.
Objective: What we know about the relationship between oxidative stress parameters and ischemic stroke is still limited and controversial. Our study aimed to investigate the relationships among ischemic lesion volume, National Institutes of Health Stroke Scale (NIHSS) values, and oxidant and antioxidant levels to determine whether oxidative stress paramaters is effective on stroke severity in ischemic stroke patients. Methods: The study included 34 patients with ischemic stroke and 34 volunteers with no active diseases. Total Oxidant Status (TOS), Total Antioxidant Status (TAS), thiol, paraoxonase, stimulated paraoxonase (stparaoxonase) and arylesterase were measured in blood samples collected on admission from patients diagnosed with ischemic stroke. The Oxidative Stress Index (OSI) was calculated. The same oxidative stress parameters were measured in the control group and compared with the patient group. Correlation between the oxidative stress parameters, the infarct volume and the NIHSS was studied. NIHSS was calculated when patients were admitted to the emergency department. The infarct volume was calculated using diffusion-weighted magnetic resonance imaging performed in the first 72-96 hours. Results: TOS and OSI values were significantly higher in the case group than the control group. Paraoxonase, arylesterase, and thiol values were significantly lower in the case group than the control group. TAS and stparaoxonase values weren’t differed significantly between the case and control groups. There were significant negative correlations between the NIHSS value and both the paraoxonase value and stparaoxonase value. There were no significant correlations between the NIHSS value and the infarct volume and the TAS, TOS, OSI, arylesterase, and thiol values. Conclusion: We concluded that change in oxidative stress balance in favor of oxidants could be a cause in the pathogenesis of ischemic stroke but oxidative stress alone can’t be sufficient in predicting the severity of stroke.
Objective: Synthetic peptides are not sufficiently large or complex by themselves to induce immune system because of their small size. Synthetic peptides are usually conjugated to different carriers such as proteins and polyelectrolytes to enhance their immunogenic properties and antigen-specific antibody production (Abs) rate. Thus, the aim of this study is synthesis of peptide-protein covalent conjugates, and size and zeta potential analysis of these conjugates. Methods: In this study, synthetic peptide antigen of the 135-161 amino acids sequence of immunogenic VP1 capsid protein of “A” type foot-and-mouth disease virus (FMDV) was covalently conjugated to bovine serum albumin (BSA) with the carbodiimide method by using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) at different molar ratios of peptide (γ=n Peptide /n BSA ). Particle size and zeta potential analysis of peptide-protein bioconjugates have been performed by using dynamic and electrophoretic light-scattering methods. Results: The size and surface charge of bioconjugates are important factors in a synthetic peptide vaccine. Nevertheless, there virtually no research has been conducted on zeta potential and the size of peptide-protein bioconjugates detailed. Conclusion: Dynamic and electrophoretic light scattering analyses clearly demonstrated that zeta potential of the FMDV 135-161 synthetic peptide-BSA conjugates shifts to less negative potentials and particle sizes increase as the amount of peptide increased in conjugates. The data about peptide-carrier protein conjugates obtained by using these methods are very important for developing peptide-based vaccines.
Objective: The transformation system for the asparagus stem blight pathogen Phomopsis asparagi (Sacc.) Bubak has not yet been reported. In the present study, we intend to achieve and optimize the genetic transformation of P. asparagi. This study aims at establishing the foundation for understanding the pathogenic mechanism of P. asparagi, which will be of great theoretical and practical significance. Methods: The Agrobacterium tumefaciens-mediated transformation (ATMT) system for P. asparagi was constructed at three aspects, i.e. condition optimization, insertion verification and transformant stability. Results: The optimal conditions for this ATMT system were as follows: 8 h of pre-induction, 48 h of co-culture, 200 μmol/L AS in ISM, co-culture at 25-28°C and pH 5.6-5.8 of ISM at the co-culture phase. The PCR result of the hph gene revealed that an expected band of about 500 bp was amplified from all the 10 transformants selected at random, and the PCR result of the Vir gene revealed that an expected band of about 730 bp was amplified from the four strains of A. tumefacien as positive controls, whereas no corresponding DNA band could be amplified from the 10 transformants. The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate FJ2. The stability result revealed that transformants still displayed high resistance to hygromycin B and could grow normally after subculture for five generations. Conclusion: A stable and efficient ATMT transformation system for P. asparagus was constructed systematically, in which a high transformation rate was achieved. For improving this system, the trasformation conditions were optimized through gradient experiments, T-DNA insertion was verified through dual PCR and the insertion segment containing hph gene in the transformant was proved hereditary stable through subculture. This system layed a foundation for the research on pathogenic mechanism and pathogenicity-related genes of P. asparagi.
Objective: Kinetics of organic solvent tolerant and thermostable lipase production by recombinant E. coli in shake flask level and 2 L stirred tank bioreactor level was studied to observe the variations of important kinetic parameters at two different levels of bioprocess. Methods: Unstructured models based on Monod equation for growth and Luedeking-Piret equation for lipase production and glucose consumption were used to predict cell growth, lipase production and glucose utilization. The shake flask fermentation experiments were carried out at different initial glucose and yeast extract concentrations using recombinant bacterial strain E. coli BL21. Lipase production was also carried out using 2L stirred tank bioreactor for comparison. Results: In all cases, the data fitted well to the proposed models. The highest growth and lipase activity were obtained at 25 g/L glucose and 25 g/L yeast extract. Cell growth (6.42 g/L) and lipase production (65.32 IU/mL) in 2 L stirred tank bioreactor was comparable to those obtained in shake flask fermentations. The calculated value of growth associated constant (9.874 IU/g/h) was much higher than that of non-growth associated constant (0.022 IU/g/h) in bioreactor as well as in shake flasks. The values of maximum specific growth rate (μm) and glucose saturation constant (KS) for shake flask fermentations, calculated from Monod equation, were 0.476 h-1 and 5.237 g/L respectively. Conclusion: From the modelling exercise, it was concluded that the lipase production is dominantly growth associated process. The kinetic parameter values for fermentations in shake flask and 2L stirred tank bioreactor were comparable, indicating that the bioprocess could be transferred into larger scale.
Objective: Free radicals are generated during different reactions in cells and are potentially threats to macromolecules such as DNA and protein. Cells have established defense systems to remove these radicals. In some diseases, the systems are defective because of different damages and cells cannot remove the radicals. Plants have been used for a long time as sources of antioxidants for treatment of different diseases. Ferula species have also been used and investigated as a source of antioxidants in Iranian herbal medicine. Methods: Here, we have studied the antioxidants activity of catalase, superoxide dismutase and lipid peroxidation from aqueous and ethanolic extracts of Ferula flabelliloba and Ferula diversivitata plants. Results: Our results have shown that they have significant antioxidant activity in different parts at different stages. Conclusion: We can conclude that these plants extracts can be used for more studies as antioxidant sources.
Objective: Glucocorticoids have been used in the treatment of a number of diseases due to their widely therapeutic effects. But they have serious side effects. Although glucocorticoid therapy is an effective measure to prevent and treat many diseases in patients, it can cause adverse effects on the cardiovascular system secondary to oxidative stress. It has been suggested that melatonin is a potent antioxidant and that melatonin supplements may protect against such age-related diseases as atherosclerosis, cancer, and Alzheimer disease. In this study, we investigated whether such effects of a single high dose steroid has adverse consequences for the oxidant-antioxidant system of rabbit heart tissue and whether melatonin treatment is protective in the rabbit. For this purpose we measurument some biochemical parameters in the rabbit heart tissue. Methods: Level of malondialdehyde (MDA, a lipid peroxidation product), protein carbonyl (PC, a protein oxidation product) and nitric oxide (NO) were measured from heart tissue samples of all rabbits included in the study. Also antioxidant enzymes superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase activities were measured from heart tissues. In the study, 20 rabbits were used. The rabbits were separated into three groups. Group I: Control group, group II: Methylprednisolone group, group III: Methylprednisolone plus melatonin group. Results: The results of the study; PC levels significantly decreased in methylprednisolone plus melatoninin group compared to the control group (p=0.03). PC levels decreased in methlyprednisolone group compared to the control group and higher than methlyprednisolone + melatonin group but these changes aren’t significant statistically. No statistical difference was determined by MDA, NO levels and SOD, GSH-Px, catalase activities of all groups. Conclusion: According to the results of this study we can say that administration of a single high dose methylprednisolone doesn’t increase oxidative stress Also, melatonine is a potent antioxidant agent in the heart tissue.
Objective: To clone a full-length cDNA sequence of xynB, encoding endo-1,4-β-xylanase of Aspergillus niger C71 and express in Escherichia coli BL21(DE3). Methods: The xynB was cloned using rapid amplification of cDNA ends (RACE) methods. The sequenced DNA was compared with the available sequences from GenBank using the BLASTX program, and the phylogenetic tree of the xylanases was then constructed using MEGA version 5.0. The amino acid sequences of XYNB were submitted to the ESyPred3D Web Server 1.0 for Homology modeling. The XYNB was purified by MonoQ an ion exchange chromatography and analyzed by SDS-PAGE. Results: The results showed that xynB is 678 bp in length, and encodes a 18 amino acid signal peptide as well as a 22 amino acid mature peptide with a calculated molecular weight of 24.127 kDa. Phylogenetic analysis of xynB, three-dimensional structure and overlap analysis of XYNB demonstrated that XYNB has a β-jelly-roll architecture, which is more conversation in the catalytic domain of GH11 xylanases, belonging to the family 11 of glycosyl hydrolases. A maximum enzyme activity of 62,242.33 U•ml -1 was obtained from XYNB. The XYNB with MW of 41 kDa demonstrated a broad pH stability from pH 3.0 to pH 12. 0.5 mM of Fe 2+ could greatly enhance activity of XYNB to 567.02%. The mainly hydrolysis products of beechwood xylan were xylobiose. The hydrolysis products of XYNB effecting on beechwood xylan were analysed by HPLC. Conclusion: The xynB had been successfully expressed in Escherichia coli BL21, with high enzyme activity and a broad pH stability, and it would be a very good application prospect as the feed enzyme.
Objective: In this study, investigating the effects of inhibition of the enzyme activity of some antitumor drugs and the Cancer-Related Human Carbonic Anhydrase IX (hCA IX) isoenzyme expressing as a SUMO fusion protein in an Escherichia coli expression system were aimed. Methods: hCA IX isoenzyme was expressed using SUMO fusion technology. The fusion protein was expressed in a totally soluble form and the expression was verified by SDS-PAGE analysis. Affinity chromatography was used in the purification processes. The effects of certain antitumor drugs on enzyme activity were investigated in vitro conditions by using esterase activity. IC 50 values of drugs showing the inhibitory effect were calculated. Inhibition types and K i values for antitumor drugs, which inhibit the enzyme, were determined by separately plotting Lineweaver- Burk plots. Results: The molecular weight of the fusion protein was approximately 85kDa. The optimal induction concentration of IPTG and the growth temperature were found to be 1.0mM and 30°C. The fusion protein was purified at approximately 3.07-fold with a yield of 92.58%, and a specific activity of 43707EU/mg proteins by nickel nitrilo-triacetic acid resin chromatography. Conclusion: Our work is extremely important because CA IX plays a clinical role as a biomarker in cancer diagnosis and the use of specific inhibitors of the CA IX enzyme will be useful in the fight against cancer. In vitro inhibition studies on the recombinant hCA IX enzyme can shed light on the development of anticancer drugs for cancers overexpressing CA IX.
Objective: Measurements of electrolyte levels made by blood gas analyzers or routine methods based on membrane and selective electrodes are expected similar performance characteristics that do not show significant differences. It is clinically important that those measurements should give equivalent results and confirm the closeness to the absolute value. In this study, it was aimed to compare sodium (Na), potassium (K), chlorine (Cl), hemoglobin (Hb) and hematocrit (Hct) values measured with blood gas analyzer and standard automatic devices in the laboratory (lab) from venous blood samples. Methods: A prospective, randomized study was conducted in simultaneous venous samples from antecubital regions of patients admitted to emergency department for various reasons, were analyzed for Na, K, Cl, Hb and Hct values on blood gas analyzer and standard automatic devices in the lab. The obtained data were compared statistically. Results: A total of 100 patients were included in the study. A statistically significant correlation was found between laboratory and venous blood gas (vbg) Na, K, Cl, Hb and Hct values (p<0.001). The average difference between them was -7.9 mEq/L (r=0.720) for Na, 0.5 mEq/L (r=0.785) for K, -5.1mEq/L (r=0.790) for Cl, -0.14 grams/dL (r=0.757) for Hb and -% 1.54 (r=0.749) for Hct, respectively. Their correlation was formulated (Lab Na=vkg Nax0.621+46.081; Lab K=vkg Kx0.23+1.977; Lab Cl=vkg Clx0.847+11.050; Lab Hb=vkg Hbx0.583+5.015; Lab Hct=vkg Hctx0.565+15.024). Conclusion: In our study, it was detected that there is a statistically significant positive correlation between venous blood Na, K, Cl, Hb and Hct values of blood gas analyzer and standard automated devices in lab, and closeness of the desired performance is provided.
