We investigated the transport of cationic neurotoxins and neurotransmitters by the potential-sensitive organic transporter OCT3 and its steroid sensitivity using heterologous expression systems and also analyzed the expression of OCT3 in the brain. When expressed in mammalian cells, OCT3 mediates the uptake of the neurotoxin 1-methyl-4-phenylpyridinium (MPP ؉ ) and the neurotransmitter dopamine. Competition experiments show that several cationic neuroactive agents including amphetamines interact with OCT3. When expressed in Xenopus laevis oocytes, OCT3-mediated MPP ؉ uptake is associated with inward currents under voltage-clamp conditions. The MPP ؉ -induced currents are saturable with respect to MPP ؉ concentration, and half-maximal saturation (K 0.5 ) occurs at about 25 M MPP ؉ with membrane potential clamped at ؊50 mV. The K 0.5 for MPP ؉ is markedly influenced by membrane potential. OCT3 is inhibited by several steroids, and ␤-estradiol is the most potent inhibitor (K i ϳ1 M). The pattern of steroid sensitivity of OCT3 is different from that of OCT1 and OCT2 but correlates significantly with that of the extraneuronal monoamine transporter (uptake 2 ). The transport characteristics and steroid sensitivity provide strong evidence for the molecular identity of OCT3 as uptake 2 . OCT3 is expressed in the brain as evidenced from Northern blot analysis, reverse transcription-polymerase chain reaction, and in situ hybridization using OCT3-specific probes. The molecular identity of the transcript hybridizing to the probe has been established by sequencing the reverse transcription-polymerase chain reaction product and also by the isolation of the OCT3 cDNA from a brain cDNA library. Regional distribution studies with in situ hybridization show that OCT3 is expressed widely in different brain regions, especially in the hippocampus, cerebellum, and cerebral cortex. OCT3 is likely to play a significant role in the disposition of cationic neurotoxins and neurotransmitters in the brain.

This report provides definitive evidence that the protein 1-Cys peroxiredoxin is a bifunctional ("moonlighting") enzyme with two distinct active sites. We have previously shown that human, rat, and bovine lungs contain an acidic Ca 2؉ -independent phospholipase A 2 (aiPLA 2 ). The cDNA encoding aiPLA 2 was found to be identical to that of a non-selenium glutathione peroxidase (NSGPx). Protein expressed using a previously reported E. coli construct which has a His-tag and 50 additional amino acids at the NH 2 terminus, did not exhibit aiPLA 2 activity. A new construct which contains the His-tag plus two extra amino acids at the COOH terminus when expressed in Escherichia coli generated a protein that hydrolyzed the sn-2 acyl chain of phospholipids at pH 4, and exhibited NSGPx activity with H 2 O 2 at pH 8. The expressed 1-Cys peroxiredoxin has identical functional properties to the native lung enzyme: aiPLA 2 activity is inhibited by the serine protease inhibitor, diethyl p-nitrophenyl phosphate, by the tetrahedral mimic 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), and by 1-Cys peroxiredoxin monoclonal antibody (mAb) 8H11 but these agents have no effect on NSGPx activity; NSGPx activity is inhibited by mercaptosuccinate and by 1-Cys peroxiredoxin mAb 8B3 antibody which have no effect on aiPLA 2 activity. Mutation of Ser 32 to Ala abolishes aiPLA 2 activity, yet the NSGPx activity remains unaffected; a Cys 47 to Ser mutant is devoid of peroxidase activity but aiPLA 2 activity remains intact. These results suggest that Ser 32 in the GDSWG consensus sequence provides the catalytic nucleophile for the hydrolase activity of aiPLA 2 , while Cys 47 in the PVCTTE consensus sequence is at the active site for peroxidase activity. The bifunctional catalytic properties of 1-Cys peroxiredoxin are compatible with a simultaneous role for the protein in the regulation of phospholipid turnover as well as in protection against oxidative injury.

This study investigated phospholipid hydroperoxides as substrates for non-selenium GSH peroxidase (NSGPx), an enzyme also called 1-Cys peroxiredoxin. Recombinant human NSGPx expressed in Escherichia coli from a human cDNA clone (HA0683) showed GSH peroxidase activity with sn-2-linolenoyl-or sn-2-arachidonoylphosphatidylcholine hydroperoxides as substrate; NADPH or thioredoxin could not substitute for GSH. Activity did not saturate with GSH, and kinetics were compatible with a ping-pong mechanism; kinetic constants (mM ؊1 min ؊1 ) were k 1 ‫؍‬ 1-3 ؋ 10 5 and k 2 ‫؍‬ 4 -11 ؋ 10 4 . In the presence of 0.36 mM GSH, apparent K m was 120 -130 M and apparent V max was 1.5-1.6 mol/min/mg of protein. Assays with H 2 O 2 and organic hydroperoxides as substrate indicated activity similar to that with phospholipid hydroperoxides. Maximal enzymatic activity was at pH 7-8. Activity with phospholipid hydroperoxide substrate was inhibited noncompetitively by mercaptosuccinate with K i 4 M. The enzyme had no GSH S-transferase activity. Bovine cDNA encoding NSGPx, isolated from a lung expression library using a polymerase chain reaction probe, showed >95% similarity to previously published human, rat, and mouse sequences and does not contain the TGA stop codon, which is translated as selenocysteine in selenium-containing peroxidases. The molecular mass of bovine NSGPx deduced from the cDNA is 25,047 Da. These results identify a new GSH peroxidase that is not a selenoenzyme and can reduce phospholipid hydroperoxides. Thus, this enzyme may be an important component of cellular antioxidant defense systems.

Recombinant human erythropoietin (rhEPO) and darbepoetin alpha (DPO) are protein-based drugs for the treatment of anemia by stimulating red blood cell production. Consequently, they are abused in human and equine sports. To deter their abuse in the horse racing industry, a sensitive and reliable method for confirmation of these agents in equine plasma has been in urgent need. Such a method by LC-MS/MS is described in this paper. The method involved analyte enrichment by immunoaffinity separation using anti-rhEPO antibody linked to magnetic beads, digestion by trypsin, and analysis by LC-MS/MS. Two specific proteotypic peptides, 46VNFYAWK52 and 144VYSNFLR150 from rhEPO and DPO were employed for confirmation of the analytes based on chromatographic retention times and major product ions. The limit of confirmation of this method was 0.2 ng/mL, and the limit of detection was 0.1 ng/mL for rhEPO and DPO in equine plasma. This method was successful in confirming the presence of rhEPO and DPO in plasma samples collected from research horses to which rhEPO or DPO was administered and from racehorses following competition and in noncompetition samples in North America. To our knowledge, this is the first LC-MS method with adequate sensitivity and specificity in providing unequivocal confirmation of rhEPO and DPO in equine plasma samples. This method provides a powerful enforcement tool that was lacking in the fight against the abuse of rhEPO and DPO in the horse racing industry.

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Objective: Several mouse models of cardiac neural crest cell (NCC)-associated conotruncal heart defects exist, but the specific cellular and molecular defects within cardiac NCC morphogenesis remain largely unknown. Our objective was to investigate the underlying 2 H mechanisms resulting in outflow tract defects and why insufficient cardiac NCC reach the heart of the Splotch (Sp ) mouse mutant embryo. Methods: For this study we used in vitro cell culture techniques, in vivo mouse-chick chimeras, BrdU cell proliferation labeling, TUNEL labeling to visualize apoptosis and the molecular markers AP-2, Wnt-1 and Wnt-3a to characterize NCC morphogenesis in vivo. Results: Expression of the NCC marker AP-2 revealed an extensive reduction in migratory NCC, however the rates of cell proliferation 2 H and apoptosis were unaffected, and do not account for the Sp NCC-associated heart defects. Further expression analysis revealed that 2 H Wnt-1, but not Wnt-3a, is expressed at decreased levels within Sp and that the cardiac NCC fail to undergo normal NC stem cell proliferative expansion prior to migration while still in the neural folds. However, when placed into a wild-type matrix or a tissue culture all these data indicate that the Sp defect is intrinsic to the NC stem cells themselves and that there is a decrease in the number of pre-migratory cardiac NCC that form. It appears that this decrease in NCC number is the primary defect that ultimately leads to a lack of 2 H a cardiac NCC-derived Sp outflow tract septum.

To reduce the carbon footprint and greenhouse gas (GHG) emissions associated with heavy crude oil/bitumen upgrading and refining in the production of clean transportation fuels, researchers are targeting the production of fuels from renewable energy resources. These resources are mainly biomass-derived oils, which include oils produced by biomass pyrolysis (bio-oil), edible and inedible vegetable oils, and animal fats. Over the past 2 decades, research has focused on the evaluation of biomass-derived oil processing using conventional fluid catalytic cracking (FCC), a technology responsible for producing the majority of gasoline in a petroleum refinery. The present review summarizes research associated with the FCC of various biomass-derived oil feedstocks as well as studies related to the co-processing of these oils with conventional petroleum feedstocks. The objective of this review is to present a comprehensive perspective of the effects of renewable oil processing on existing FCC technology, operation, catalysts, and product quality and quantity.

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