N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

The Use of Fungal Biomass Agaricus bisporus Immobilized on Amberlite XAD-4 Resin for the Solid-Phase Preconcentration of Thorium

Solid-phase extraction method was developed for the preconcentration of thorium (Th). Fungal biomass Agaricus bisporus was immobilized to Amberlite XAD-4 as solid-phase sorbent. The critical parameters such as pH of the sample solution, flow rate of the sample, volume of the sample, and the effect of major ions that affect the preconcentration of thorium in this system were evaluated. The optimum pH for the sorption of Th is 6.0, and quantitative elution occurs with 1.0 mol L^?1HCl. The loading capacity was determined as 0.079 mmol g^?1. The optimized method was validated through analysis of the certified reference material of tea leaves (NCS ZC73014) and successfully applied to the determination of Th in a real ore sample with satisfactory results.     

Evidence of natural Wolbachia infections and molecular identification of field populations of Culex pipiens complex (Diptera: Culicidae) mosquitoes in western Turkey

Establishing reliable risk projection information about the distribution pattern of members of the Culex pipiens complex is of particular interest, as these mosquitoes are competent vectors for certain disease‐causing pathogens. Wolbachia, a maternally inherited bacterial symbiont, are distributed in various arthropod species and can induce cytoplasmic incompatibility, i.e., reduced egg hatch, in certain crosses. It is being considered as a tool for population control of mosquito disease vectors. The Aegean region is characterized by highly populated, rural, and agricultural areas and is also on the route of the migratory birds. In this study, a fragment of the 658 bp of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene, which includes the barcode region, was employed to differentiate Cx. pipiens complex species found in this region. Moreover, for the first time, the prevalence of Wolbachia endobacteria in these natural populations was examined using PCR amplification of a specific wsp gene. Our results revealed a widespread (more than 90%, n=121) presence of the highly efficient West Nile virus vector Cx. quinquefasciatus in the region. We also found that Wolbachia infection is widespread; the average prevalence was 62% in populations throughout the region. This study provided valuable information about the composition of Cx. pipiens complex mosquitoes and the prevalence of Wolbachia infection in these populations in the Aegean region. This information will be helpful in tracking mosquito‐borne diseases and designing and implementing Wolbachia‐based control strategies in the region.     

Effects of Continuous and Intermittent Magnetic Fields on Oxidative Parameters In vivo

Continuous and intermittent 50 Hz, 1.5 mT magnetic field with the exposure period of 4 h/day for 4 days was used to investigate its possible effect on adult guinea pigs. Tissues and plasma specimens were assessed by biochemical parameters. Malondialdehyde (MDA), glutathione (GSH), nitric oxide (NO) levels and myeloperoxidase activity (MPO) were examined in plasma, liver and brain tissues. All parameters were determined by spectrophotometer. While intermittent magnetic field was effective on plasma lipid peroxidation, continuous magnetic field was found to be effective on plasma MPO activity and NO levels. Augmentation of lipid peroxidation was also observed in liver tissue both intermittent and continuous magnetic field exposures. These results indicate that both the intermittent and continuous magnetic field exposures affect various tissues in a distinct manner because of having different tissue antioxidant status and responses.     

Characterization and properties of catalase immobilized onto controlled pore glass and its application in batch and plug-flow type reactors

Bovine liver catalase was covalently immobilized onto controlled pore glass (CPG) beads modified with 3-aminopropyltriethoxysilane (3-APTES) followed by treatment with glutaraldehyde. Coupling of catalase onto CPG was optimized to improve the efficiency of the overall immobilization procedure. The optimum coupling conditions: pore diameter of CPG, pH, buffer concentration, temperature, coupling time and initial catalase amount per grams of carrier were determined as 70 nm, 6.0, 75 mM, 5 °C, 7 h and 6 mg catalase, respectively. Catalytic efficiencies (k_cat/K_m) and thermal inactivation rate constants (k_i) of ICPG1 were determined and compared with that of free catalase. Suitability of ICPG1 was also investigated by using it in batch and plug-flow type reactors. When the remaining activity of ICPG1 retained was about 50% of its initial activity the highest total productivity of ICPG1 was determined as 7.6 × 10⁶ U g immobilized catalase⁻¹ in plug-flow type reactor. However, the highest total productivity of ICPG1 was 6.2 × 10⁵ U g immobilized catalase⁻¹ in batch type reactor. ICPG1 may have great potentials as biocatalyst for the application in decomposition of hydrogen peroxide in plug-flow type reactor.     

The role of the ADRA2A C1291G genetic polymorphism in response to dexmedetomidine on patients undergoing coronary artery surgery

The existence of interindividual drug response variability has been known for a long time. Individual susceptibility which might cause toxicity or inadequate treatment is important in drug therapy. Genetic polymorphisms in genes responsible for drug response are expected to be useful in keeping track of differences among individuals. Dexmedetomidine is a sedative drug, whose use in intensive care unit patients was confirmed by USA-Food Drug Administration (FDA) by the end of 1999. It was proven that dexmedetomidine shows its clinic effect via the α₂-AR. However, to the best of our knowledge, to date, there is no investigation in clinic indicating the relation between dexmedetomidine and α_2A-AR gene polymorphism. The aim of our study was to investigate the association between the effect of α_2A-Adrenergic Receptor (ADRA2A) C-1291G gene polymorphism in the promoter region of the candidate gene and clinical effects (sedative and haemodynamics effects) of dexmedetomidine. One hundred and ten patients undergoing coronary artery surgery were prospectively studied. Anesthetic technique was standardized with fentanyl, midazolam and rocuronium bromide. Patients were monitorized with Bispectral Index (BIS) monitor in addition to the routine invasive haemodynamic monitorization in the operation room. The Ramsay Sedation Scale was also used in order to determine the sedation level just arriving to Intensive Care Unit (ICU). The genotyping of ADRA2A C1291G was done by Restriction Fragment Length Polymorphism-Polymerase Chain Reaction (RFLP-PCR). We found the frequencies of C1291C, C1291G and G1291G genotypes, as 43.6, 45.5 and 10.9%, respectively. Patients who carry variant genotype had higher BIS and Ramsay Sedation Scores, indicating a longer period for falling asleep. The results of our study are promising, considering the association between ADRA2A polymorphism and response to dexmedetomidine. However, further investigations on other ADRA2A locus or haplotypes might be useful to clarify the relation between this gene and dexmedetomidine activity.     

Tauroursodeoxycholic acid reduces endoplasmic reticulum stress, acinar cell damage, and systemic inflammation in acute pancreatitis

In acute pancreatitis, endoplasmic reticulum (ER) stress prompts an accumulation of malfolded proteins inside the ER, initiating the unfolded protein response (UPR). Because the ER chaperone tauroursodeoxycholic acid (TUDCA) is known to inhibit the UPR in vitro, this study examined the in vivo effects of TUDCA in an acute experimental pancreatitis model. Acute pancreatitis was induced in Wistar rats using caerulein, with or without prior TUDCA treatment. UPR components were analyzed, including chaperone binding protein (BiP), phosphorylated protein kinase-like ER kinase (pPERK), X-box binding protein (XBP)-1, phosphorylated c-Jun NH(2)-terminal kinase (pJNK), CCAAT/enhancer binding protein homologues protein, and caspase 12 and 3 activation. In addition, pancreatitis biomarkers were measured, such as serum amylase, trypsin activation, edema formation, histology, and the inflammatory reaction in pancreatic and lung tissue. TUDCA treatment reduced intracellular trypsin activation, edema formation, and cell damage, while leaving amylase levels unaltered. The activation of myeloperoxidase was clearly reduced in pancreas and lung. Furthermore, TUDCA prevented caerulein-induced BiP upregulation, reduced XBP-1 splicing, and caspase 12 and 3 activation. It accelerated the downregulation of pJNK. In controls without pancreatitis, TUDCA showed cytoprotective effects including pPERK signaling and activation of downstream targets. We concluded that ER stress responses activated in acute pancreatitis are grossly attenuated by TUDCA. The chaperone reduced the UPR and inhibited ER stress-associated proapoptotic pathways. TUDCA has a cytoprotective potential in the exocrine pancreas. These data hint at new perspectives for an employment of chemical chaperones, such as TUDCA, in prevention of acute pancreatitis.