Thioredoxin-related Protein 32 (TRP32) Specifically Reduces Oxidized Phosphatase of Regenerating Liver (PRL)

PRL family constitutes a unique class of phosphatases associated with metastasis. The phosphatase activity of PRL has been reported to be important for promoting metastasis, and it is inactivated by reversible oxidation of its catalytic cysteine. Here, we show that TRP32 specifically reduces PRL. Reduction of oxidized PRL in cells is inhibited by 2,4-dinitro-1-chlorobenzene, an inhibitor of TRX reductase. In vitro assays for the reduction of PRL show that only TRP32 can potently reduce oxidized PRL, whereas other TRX-related proteins linked to TRX reductase show little or no reducing activity. Indeed, TRP32 knockdown significantly prolongs the H₂O₂-induced oxidation of PRL. Binding analyses reveal that the unique C-terminal domain of TRP32 is required and sufficient for its direct interaction with PRL. These results suggest that TRP32 maintains the reduced state of PRL and thus regulates the biological function of PRL.     

Emergent chemodiversity: The case of stereoisomerism in acyclic alkanes

The objective of this pen‐and‐paper study is to witness the emergence of stereoisomeric properties when comparing lower to higher families of homologs. Specifically, the study compares all acyclic hexanes (five constitutional isomers, a.k.a. regioisomers), all nine heptanes, all 18 octanes, all 35 nonanes, and all 75 decanes. The first part of the work examines the nature and number of stereoisomeric properties seen to emerge in chemical structures featuring one chiral center (i.e., enantiomerism) or two such centers, in which case more complex stereoisomeric features emerge (enantiomerism, diastereoisomerism, pseudoasymmetry, and meso ‐isomers). The first emergence of chirality (i.e., enantiomerism) occurred in some heptanes. Diastereoisomerism and meso ‐isomers appear with some octanes, while a pseudoasymmetric center exists in a decane regioisomer. The second part of the work is an attempt to rationalize the numbers of regioisomers, chiral centers, and stereoisomers as these numbers grow from one family of regioisomers to the higher ones. Far from being random, such increases prove regular and ordered.     

Intramembranal disulfide cross-linking elucidates the super-quaternary structure of mammalian CatSpers

CatSper is a voltage-dependent calcium channel located in the plasma membrane of the sperm flagellum and is responsible for triggering hyperactive motility. A homology model for the transmembrane region was built in which the arrangement of the subunits around the pseudo-four-fold symmetry axis was deduced by the pairing of conserved transmembranal cysteines across mammals. Directly emergent of the predicted quaternary structure is an architecture in which tetramers polymerize through additional, highly conserved cysteines, creating one or more double-rows channels extending the length of the principal piece of the mammalian sperm tail. The few species that are missing these cysteines are eusocial or otherwise monogamous, suggesting that sperm competition is selective for a disulfide-crosslinked macromolecular architecture. The model suggests testable hypotheses for how CatSper channel opening might behave in response to pH, 2-arachidonoylglycerol, and mechanical force. A flippase function is hypothesized, and a source of the concomitant disulfide isomerase activity is found in CatSper-associated proteins β, δ and ε.     

Comparison of in vitro and in vivo oligomeric states of a wild type and mutant trimeric inner membrane multidrug transporter

Many membrane proteins exist and function as oligomers or protein complexes. Routine analytical methods involve extraction and solubilization of the proteins with detergents, which could disturb their actual oligomeric state. AcrB is a trimeric inner membrane multidrug transporter in E. coli. In previous studies, we created a mutant AcrB_P223G, which behaves like a monomer when extracted from the cell membrane. However, the actual oligomeric state of AcrB_P223G in cell membranes remained unclear, which complicated the interpretation of the mechanism by which the mutation affects function. Here we used several complementary methods to determine the oligomeric state of AcrB_P223G in E. coli cell membranes. Two sets of quantitative fluorescent techniques were exploited. For these, we created fluorescent tagged AcrB, AcrB-CFP and AcrB-YPet. Fluorescence resonance energy transfer (FRET) and fluorescence recovery after photobleaching (FRAP) were employed to characterize independently the efficiency of energy transfer between co-expressed AcrB-CFP and AcrB-YPet, and the diffusion coefficient of AcrB-YPet and AcrB_P223G-YPet in live E. coli cells. Second, we introduced Cys pairs at the inter-subunit interface and used controlled oxidation to probe inter-subunit distances. The results from all studies converge on the conclusion that AcrB_P223G exists as a trimer in cell membranes, which dissociates during the purification steps. The small change in trimer affinity and structure leads to a significant loss of AcrB activity. In addition, throughout this study we developed protocols and established benchmark values, useful for further studies on membrane protein associations in cell membranes.     

Two closely linked genes encoding thioredoxin and thioredoxin reductase in Clostridium litorale

The genes encoding thioredoxin and thioredoxin reductase of Clostridium litorale were cloned and sequenced. The thioredoxin reductase gene (trxB) encoded a protein of 33.9 kDa, and the deduced amino acid sequence showed 44% identity to the corresponding protein from Escherichia coli. The gene encoding thioredoxin (trxA) was located immediately downstream of trxB. TrxA and TrxB were each encoded by two gene copies, both copies presumably located on the chromosome. Like other thioredoxins from anaerobic, amino-acid-degrading bacteria investigated to date by N-terminal amino acid sequencing, thioredoxin from C. litorale exhibited characteristic deviations from the consensus sequence, e.g., GCVPC instead of WCGPC at the redox-active center. Using heterologous enzyme assays, neither thioredoxin nor thioredoxin reductase were interchangeable with the corresponding proteins of the thioredoxin system from E. coli. To elucidate the molecular basis of that incompatibility, Gly-31 in C. litorale thioredoxin was substituted with Trp (the W in the consensus sequence) by site-directed mutagenesis. The mutant protein was expressed in E. coli and was purified to homogeneity. Enzyme assays using the G31W thioredoxin revealed that Gly-31 was not responsible for the observed incompatibility with the E. coli thioredoxin reductase, but it was essential for activity of the thioredoxin system in C. litorale. Received: 19 September 1996 / Accepted: 21 May 1997     

Enzymatic fractionation of conjugated linoleic acid isomers by selective esterification

Attempt was done to prepare food supplements with high content of c9, t11-CLA or t10, c12-CLA. A free acid mixture containing CLA isomers was esterified with ethanol by enzyme catalysis. Novozyme 435 and Lipase AY30 were screened, and Lipase AY30 was employed to catalyze esterification reaction because of its high fractionation efficiency. Effect of reaction conditions on total esterification was investigated, and the optimal reaction conditions were: 140 U of lipase amount, reaction temperature at 50 °C, a pH of 6.5, and molar ratio of FFA–CLA to ethanol at 1:1. Based on the studies above, experiments of esterification and purification were done, and the best fractionation efficiency was obtained when the total esterification was 37%, and the corresponding purity and recovery of c9, t11-CLA were 75.50 and 49.85%, and that of t10, c12-CLA were 72.02 and 62.32%.     

香芹酮和薄荷醇手性异构体对淡色库蚊熏蒸及驱避活性

【目的】比较香芹酮和薄荷醇各手性异构体对淡色库蚊Culex pipiens pallens成蚊的熏蒸及驱避活性,为其应用提供理论依据。【方法】以3日龄淡色库蚊雌成蚊为供试昆虫,采用三角瓶熏蒸法比较测定香芹酮和薄荷醇的5种手性异构体的击倒与熏蒸致死活性;用Y-型嗅觉仪法比较测定驱避作用;将活性较好单体制成电热蚊香片,用方箱法进一步评价药效。【结果】5种异构体对淡色库蚊均具有一定的击倒和致死作用,其中L-香芹酮的击倒速度最快,KT50为9.75 min;D-香芹酮的致死作用最强,LC50为0.26μL/L。L-香芹酮和D-香芹酮对淡色库蚊均具较好的驱避作用,在0.5μL剂量下,处理30 min后的驱避率仍达100%。方箱法实验表明,以200μL的L-香芹酮和D-香芹酮为有效成分的电热蚊香片加热1 h后,对淡色库蚊雌成蚊的KT50分别为13.70和17.90 min。【结论】香芹酮对淡色库蚊雌成蚊的熏杀活性优于薄荷醇,且不同异构体的活性存在显著差异,L-香芹酮具备被开发为植物源卫生害虫防控剂的潜力。     

Two new structured intermediates in the oxidative folding of RNase A

Two new three-disulfide intermediates have been found to be populated in the oxidative folding pathway of bovine pancreatic ribonuclease A at a low temperature (15 degrees C). These intermediates, des-[26-84] and des-[58-110], possess all but one of the four native disulfide bonds and have a stable tertiary structure, similar to the two previously observed intermediates, des-[65-72] and des-[40-95]. While the latter two des species each lack one surface-exposed disulfide bond, the newly discovered intermediates each lack one buried disulfide bond. The possible involvement of these species in the rate-determining steps during the oxidative folding of RNase A is discussed and a specific role for such species during oxidative folding is suggested.