Association of a GPI-anchored protein with detergent-resistant membranes facilitates its trafficking through the early secretory pathway

Membrane microdomains are implicated in the trafficking and sorting of several membrane proteins. In particular GPI-anchored proteins cluster into Triton X-100 resistant, cholesterol- and sphingolipid-rich membrane microdomains and are sorted to the apical membrane. A growing body of evidence has pointed to the existence of other types of microdomains that are insoluble in detergents, such as Lubrol WX and Tween-20. Here, we report on the role of detergent-resistant membranes formed at early stages in the biosynthesis of membrane dipeptidase (MDP), a GPI-anchored protein, on its trafficking and sorting. Pulse-chase experiments revealed a retarded maturation rate of the GPI-anchor deficient mutant (MDPΔGPI) as compared to the wild type protein (wtMDP). However, Golgi to cell surface delivery rate did not show a significant difference between the two variants. On the other hand, early biosynthetic forms of wtMDP were partially insoluble in Tween-20, while MDPΔGPI was completely soluble. The lack of association of MDPΔGPI with detergent-resistant membranes prior to maturation in the Golgi and the reduction in its trafficking rate strongly suggest the existence of an early trafficking control mechanisms for membrane proteins operating at a level between the endoplasmic reticulum and the cis-Golgi.     

Metabolic and evolutionary engineering research in Turkey and beyond

This review discusses metabolic engineering research with an emphasis on evolutionary (whole cell and protein) engineering, which is an inverse metabolic engineering approach. For each section on metabolic, inverse metabolic and evolutionary engineering research, a general review of the major global studies in the literature is made and research examples from Turkey are given and discussed. It is expected that with the rapid development in systems biology and the novel powerful analytical technologies to identify the genetic basis of cellular phenotypes, metabolic and evolutionary engineering research will become widespread and increasingly important in Turkey, following global scientific trends.     

Improved chromatographic method for purification of lactoperoxidase from different milk sources

Our previous studies showed that sulfanilamide is a new competitive inhibitor of and can be used in the purification of lactoperoxidase (LPO, EC1.11.1.7) from milk. However, this method has some disadvantages like a lower purification factor. The aim of the present study is to improve the purification process of milk LPO from different sources. For this purpose, 16 commercial sulfanilamide derivatives were selected for inhibition studies to determine the best inhibitor of bovine LPO by calculating kinetic parameters. A cyanogen bromide-activated Sepharose 4B affinity matrix was synthesized by coupling with each competitive inhibitor. Among the inhibitors, 5-amino-2-methylbenzenesulfonamide and 2-chloro-4-sulfamoylaniline were used as ligands for the purification of LPO from bovine, buffalo, cow, and goat milks with 1059.37, 509.09, 232.55, and 161.90, and 453.12-, 151.86-, 869.00-, and 447.57-fold, respectively. Our results show that 5-amino-2-methylbenzenesulfonamide, 2-chloro-4-sulfamoylaniline, and 5-amino-1-naphthalenesulfonamide are the best inhibitors for one-step purification of the enzyme.     

Assessment of the inhibitory effects and molecular docking of some sulfonamides on human serum paraoxonase 1

Paraoxonase‐1 (PON1) is an organophosphate hydrolyzer and antiatherogenic enzyme. Due to the PON1's crucial functions, inhibitors and activators of PON1 must be known for pharmacological applications. In this study, we investigated the in vitro effects of some sulfonamides compounds on human serum PON1 (hPON1). For this aim, we purified the hPON1 from human serum with high specific activity by using simple chromatographic methods, and after the purification processes, we investigated in vitro interactions between the enzyme and some sulfonamides (2‐amino‐5‐methyl‐1,3‐benzenedisulfonamide, 2‐chloro‐4‐sülfamoilaniline, 4‐amino‐3‐methylbenzenesulfanilamide, sulfisoxazole, sulfisomidine, and 5‐amino‐2‐methylbenzenesulfonamide). IC₅₀, K_i values, and inhibition types were calculated for each sulfonamide. 2‐amino‐5‐methyl‐1,3‐benzenedisulfonamide and 2‐chloro‐4‐sülfamoilaniline exhibited noncompetitive inhibition effect, whereas 4‐amino‐3‐methylbenzenesulfanilamide, sulfisoxazole, and sulfisomidine exhibited mixed type inhibition. On the other hand, 5‐amino‐2‐methylbenzenesulfonamide showed competitive inhibition and so molecular docking studies were performed for this compound in order to assess the probable binding mechanism into the active site of hPON1.     

Synthesis and biological evaluations of putative metabolically stable analogs of VN/124-1 (TOK-001): head to head anti-tumor efficacy evaluation of VN/124-1 (TOK-001) and abiraterone in LAPC-4 human prostate cancer xenograft model

In a continuing study of our clinical candidate 5 VN/124-1 (TOK-001) and analogs as potential agents for prostate cancer therapy, putative metabolites (10, 15 and 18) of compound 5 were rationally designed and synthesized. However, none of these agents were as efficacious as 5 in several in vitro studies. Using western blot analysis, we have generated a preliminary structure–activity relationship (SAR) of 5 and related analogs as androgen receptor ablative agents (ARAAs). In vivo using the androgen-dependent LAPC-4 prostate cancer xenograft model, we demonstrated for the first time that 5 is more efficacious than the 17-lyase inhibitor 3 (abiraterone)/4 (abiraterone acetate) that is currently in phase III clinical trials. In our desire to optimize the potency of 5, compounds 6 (3ξ-fluoro-) and 9 (3β-sulfamate-) designed to increase the stability and oral bioavailability of 5, respectively were evaluated in vivo. We showed, that on equimolar basis, compound 6 was ∼2-fold more efficacious versus LAPC-4 xenografts than 5, but the toxicity observed with 6 is of concern. These studies further demonstrate the efficacy of 5 in a clinically relevant prostate cancer model and justify its current clinical development as a potential treatment of prostate cancer.     

Effect of TNF-α and IL-1β genetic variants on the development of myocardial infarction in Turkish population

Tumor necrosis factor-alpha (TNF-α) and interleukin 1 beta (IL-1β) genetic variants which resulting in TNF-α and IL-1 overproduction may increase susceptibility to autoimmune diseases such as atherosclerosis. We have studied the association of TNF-α G308A and IL-1β (+3953) C/T polymorphism with myocardial infarction in Turkish population. 143 patients with myocardial infarction and 213 age-matched healthy controls were included in the study. In univariant analysis, the frequencies of IL-1β, TNF-α genotype or allele, and haplotype of C:A and T:A were significantly elevated in patients when compared with those of controls. GA genotype of TNF-α, T allele of IL-1β and A of TNF-α allele seem to be risk factors for myocardial infarction. In contrast, CC genotype of IL-1β and GG genotype of TNF-α have protective effect against myocardial infarction. In multivariate logistic regression analysis, TNF-α A allele, gender and smoking were associated with myocardial infarction. In conclusion, we can state that TNF-α A allele might be associated with myocardial infarction.     

Gastroprotective,cytoprotective and antioxidant effects of Oleum cinnamomi on ethanol induced damage

Peptic ulcer disease is a gastrointestinal disorder defined by mucosal damage and free oxygen radicals associated with peptic ulcer and gastritis. Cinnamon is a traditional herb used for many diseases and it has also effects as an antioxidant, anti-inflammatory, antispasmodic and anti-ulcerative. Our research is based on oxidative stress and effects of Oleum cinnamomi on stomach, liver and kidney disorders induced by ethanol. In our experiment, 2–3 month old male Sprague–Dawley rats were used. One hour before the mucosal damage induced by 70 % ethanol, O. cinnamomi (2.5 ml/kg) was added into the groups. Gastric pH, analysis of gastric mucus and ulcer index were calculated from samples obtained from the stomach. Superoxide dismutase (SOD), malondialdehyde and catalase (CAT) levels were determined in stomach, liver and kidney homogenates and erythrocyte hemolysate. Histopathological examination of stomach, liver and kidney were determined with H&E staining. The non-treated ulcerative group showed higher scores than the control group which was treated with O. cinnamomi, when ulcer scores, gastric mucus and pH level of stomach are compared. Increased lipid peroxidation levels were observed in the liver, kidney and erythrocyte hemolysate. SOD activity was decreased in liver whereas increased in stomach of ethanol treated ulcerative groups. CAT levels were increased in stomach and liver of ethanol treated rats. Histopathological findings showed that ethanol treatment cause multiply organ damage such as stomach, liver and kidney injury. O. cinnamomi treatment protected these tissues from ethanol-induced damage. Consequently, the current investigation shows that O. cinnamomi has protective effects on ethanol-induced oxidative and mucosal damage.