Crystal structure of mitochondrial quinol-fumarate reductase from the parasitic nematode Ascaris suum

In the anaerobic respiratory chain of the parasitic nematode Ascaris suum, complex II couples the reduction of fumarate to the oxidation of rhodoquinol, a reverse reaction catalyzed by mammalian complex II. In this study, the first structure of anaerobic complex II of mitochondria was determined. The structure, composed of four subunits and five co-factors, is similar to that of aerobic complex II, except for an extra peptide found in the smallest anchor subunit of the A. suum enzyme. We discuss herein the structure-function relationship of the enzyme and the critical role of the low redox potential of rhodoquinol in the fumarate reduction of A. suum complex II.     

A novel serum metabolomics-based diagnostic approach for colorectal cancer

Background

To improve the quality of life of colorectal cancer patients, it is important to establish new screening methods for early diagnosis of colorectal cancer.

Methodology/Principal Findings

We performed serum metabolome analysis using gas-chromatography/mass-spectrometry (GC/MS). First, the accuracy of our GC/MS-based serum metabolomic analytical method was evaluated by calculating the RSD% values of serum levels of various metabolites. Second, the intra-day (morning, daytime, and night) and inter-day (among 3 days) variances of serum metabolite levels were examined. Then, serum metabolite levels were compared between colorectal cancer patients (N = 60; N = 12 for each stage from 0 to 4) and age- and sex-matched healthy volunteers (N = 60) as a training set. The metabolites whose levels displayed significant changes were subjected to multiple logistic regression analysis using the stepwise variable selection method, and a colorectal cancer prediction model was established. The prediction model was composed of 2-hydroxybutyrate, aspartic acid, kynurenine, and cystamine, and its AUC, sensitivity, specificity, and accuracy were 0.9097, 85.0%, 85.0%, and 85.0%, respectively, according to the training set data. In contrast, the sensitivity, specificity, and accuracy of CEA were 35.0%, 96.7%, and 65.8%, respectively, and those of CA19-9 were 16.7%, 100%, and 58.3%, respectively. The validity of the prediction model was confirmed using colorectal cancer patients (N = 59) and healthy volunteers (N = 63) as a validation set. At the validation set, the sensitivity, specificity, and accuracy of the prediction model were 83.1%, 81.0%, and 82.0%, respectively, and these values were almost the same as those obtained with the training set. In addition, the model displayed high sensitivity for detecting stage 0–2 colorectal cancer (82.8%).

Conclusions/Significance

Our prediction model established via GC/MS-based serum metabolomic analysis is valuable for early detection of colorectal cancer and has the potential to become a novel screening test for colorectal cancer.     

Thermotomaculum hydrothermale gen. nov., sp. nov., a novel heterotrophic thermophile within the phylum Acidobacteria from a deep-sea hydrothermal vent chimney in the Southern Okinawa Trough

A novel heterotrophic, thermophilic bacterium, designated strain AC55^T, was isolated from a deep-sea hydrothermal vent chimney at the Hatoma Knoll in the Okinawa Trough, Japan. Cells of strain AC55^T were non-motile, long rods (2.0- to 6.8-μm long and 0.3- to 0.6-μm wide). The strain was an obligatory anaerobic heterotroph capable of fermentative growth on complex proteinaceous substances. Elemental sulfur was reduced to hydrogen sulfide but did not stimulate growth. Growth was observed between 37 and 60°C (optimum 55°C), pH 5.5 and 8.5 (optimum pH 6.6), and in the presence of 1.5–4.5% (w/v) NaCl (optimum 2.5%, w/v). Menaquinone-7 and -8 were the major respiratory quinones. The G + C content of the genomic DNA from strain AC55^T was 51.6 mol%. The 16S rRNA gene sequence analysis revealed that strain AC55^T was the first cultivated representative of Acidobacteria subdivision 10. Based on the physiological and phylogenetic features of the novel isolate, the genus name Thermotomaculum gen. nov. is proposed, with Thermotomaculum hydrothermale sp. nov. as the type species. The type strain is AC55^T (=JCM 17643^T = DSM 24660^T = NBRC 107904^T).     

Structure-activity relationship of α hormones, the mating factors of phytopathogen Phytophthora

The mating hormones α1 and α2 induce sexual reproduction of the phytopathogenic genus Phytophthora. To demonstrate the structural elements responsible to hormonal activity, 17 derivatives of α1 and α2 were synthesized and their hormonal activity (oospore-inducing activity) was evaluated. The terminal ester derivatives of α1 (diacetate and dibenzoate) retained the hormonal activity, whereas a dicarbamate derivative completely suppressed the activity. Even monocarbamates showed weak activities; among them the 1-O-carbamate was less active than 16-O-carbamate, suggesting that the 1-OH group is a little more important than 16-OH. Dihydro, dehydro, and demethyl derivatives exhibited the minimum level of activity. Surviving activity of 15-epi-α1 suggested a less importance of this stereochemistry. Contrary to α1, not only the terminal diacetate derivative but also monoacetates of α2 exhibited no or little activity. Among the monoacetates, 1-O-acetyl-α2 exhibited little yet relatively better activity than the others. No activity was observed for mono- and dicarbamoyl derivatives of α2. Dihydro α2 with the saturated double bond lost most of the activity. These findings suggest that both the mating hormones α1 and α2 require most of the functional (hydroxyl, keto, and olefinic) groups they possess in their natural form for inducing the sexual reproduction of Phytophthora.     

The lymphotoxin pathway regulates Aire-independent expression of ectopic genes and chemokines in thymic stromal cells

Medullary thymic epithelial cells (mTEC) play an important and unique role in central tolerance, expressing tissue-restricted Ags (TRA) which delete thymocytes autoreactive to peripheral organs. Since deficiencies in this cell type or activity can lead to devastating autoimmune diseases, it is important to understand the factors which regulate mTEC differentiation and function. Lymphotoxin (LT) ligands and the LTbetaR have been recently shown to be important regulators of mTEC biology; however, the precise role of this pathway in the thymus is not clear. In this study, we have investigated the impact of this signaling pathway in greater detail, focusing not only on mTEC but also on other thymic stromal cell subsets. LTbetaR expression was found in all TEC subsets, but the highest levels were detected in MTS-15(+) thymic fibroblasts. Rather than directing the expression of the autoimmune regulator Aire in mTEC, we found LTbetaR signals were important for TRA expression in a distinct population of mTEC characterized by low levels of MHC class II (mTEC(low)), as well as maintenance of MTS-15(+) fibroblasts. In addition, thymic stromal cell subsets from LT-deficient mice exhibit defects in chemokine production similar to that found in peripheral lymphoid organs of Lta(-/-) and Ltbr(-/-) mice. Thus, we propose a broader role for LTalpha1beta2-LTbetaR signaling in the maintenance of the thymic microenvironments, specifically by regulating TRA and chemokine expression in mTEC(low) for efficient induction of central tolerance.     

Energetic heavy ions accelerate differentiation in the descendants of irradiated normal human diploid fibroblasts

Ionizing radiation-induced genomic instability has been demonstrated in a variety of endpoints such as delayed reproductive death, chromosome instability and mutations, which occurs in the progeny of survivors many generations after the initial insult. Dependence of these effects on the linear energy transfer (LET) of the radiation is incompletely characterized; however, our previous work has shown that delayed reductions in clonogenicity can be most pronounced at LET of 108 keV/microm. To gain insight into potential cellular mechanisms involved in LET-dependent delayed loss of clonogenicity, we investigated morphological changes in colonies arising from normal human diploid fibroblasts exposed to gamma-rays or energetic carbon ions (108 keV/microm). Exposure of confluent cultures to carbon ions was 4-fold more effective at inactivating cellular clonogenic potential and produced more abortive colonies containing reduced number of cells per colony than gamma-rays. Second, colonies were assessed for clonal morphotypic heterogeneity. The yield of differentiated cells was elevated in a dose- and LET-dependent fashion in clonogenic colonies, whereas differentiated cells predominated to a comparable extent irrespective of radiation type or dose in abortive colonies. The incidence of giant or multinucleated cells was also increased but much less frequent than that of differentiated cells. Collectively, our results indicate that carbon ions facilitate differentiation more effectively than gamma-rays as a major response in the progeny of irradiated fibroblasts. Accelerated differentiation may account, at least in part, for dose- and LET-dependent delayed loss of clonogenicity in normal human diploid cells, and could be a defensive mechanism that minimizes further expansion of aberrant cells.     

Dual-gradient high-performance liquid chromatography for identification of cytosolic high-mannose-type free glycans

It has been shown that free oligosaccharides derived from N-linked glycans accumulate in the cytosol of animal cells. Most of the glycans have only a single GlcNAc at their reducing termini (Gn1 glycans), whereas the original N-glycans retain N,N′-diacetylchitobiose at their reducing termini (Gn2 glycans). Under the conditions of high-performance liquid chromatography (HPLC) mapping established for pyridylamine (PA)-labeled Gn2 N-glycans, Gn1 glycans are not well retained on reversed-phase HPLC, making simultaneous analysis of Gn1 and Gn2 glycans problematic. We introduced a dual gradient (i.e., pH and butanol gradient) for the separation of Gn1 and Gn2 glycans in a single reversed-phase HPLC. Determination of elution time for various standard Gn2 high-mannose-type glycans, as well as Gn1 glycans found in the cytosol of animal cells, showed that elution of Gn1 and Gn2 glycans could be separated. Sufficient separation for most of the structural isomers could be achieved for Gn1 and Gn2 glycans. This HPLC, therefore, is a powerful method for identification of the structures of PA-labeled glycans, especially Gn1-type glycans, isolated from the cytosol of animal cells.     

Structures of Trypanosoma cruzi dihydroorotate dehydrogenase complexed with substrates and products: atomic resolution insights into mechanisms of dihydroorotate oxidation and fumarate reduction

Dihydroorotate dehydrogenase (DHOD) from Trypanosoma cruzi (TcDHOD) is a member of family 1A DHOD that catalyzes the oxidation of dihydroorotate to orotate (first half-reaction) and then the reduction of fumarate to succinate (second half-reaction) in the de novo pyrimidine biosynthesis pathway. The oxidation of dihydroorotate is coupled with the reduction of FMN, and the reduced FMN converts fumarate to succinate in the second half-reaction. TcDHOD are known to be essential for survival and growth of T. cruzi and a validated drug target. The first-half reaction mechanism of the family 1A DHOD from Lactococcus lactis has been extensively investigated on the basis of kinetic isotope effects, mutagenesis and X-ray structures determined for ligand-free form and in complex with orotate, the product of the first half-reaction. In this report, we present crystal structures of TcDHOD in the ligand-free form and in complexes with an inhibitor, physiological substrates and products of the first and second half-reactions. These ligands bind to the same active site of TcDHOD, which is consistent with the one-site ping-pong Bi-Bi mechanism demonstrated by kinetic studies for family 1A DHODs. The binding of ligands to TcDHOD does not cause any significant structural changes to TcDHOD, and both reduced and oxidized FMN cofactors are in planar conformation, which indicates that the reduction of the FMN cofactor with dihydroorotate produces anionic reduced FMN. Therefore, they should be good models for the enzymatic reaction pathway of TcDHOD, although orotate and fumarate bind to TcDHOD with the oxidized FMN and dihydroorotate with the reduced FMN in the structures determined here. Cys130, which was identified as the active site base for family 1A DHOD (Fagan, R. L., Jensen, K. F., Bjornberg, O., and Palfey, B. A. (2007) Biochemistry 46, 4028-4036.), is well located for abstracting a proton from dihydroorotate C5 and transferring it to outside water molecules. The bound fumarate is in a twisted conformation, which induces partial charge separation represented as C 2 (delta-) and C 3 (delta+). Because of this partial charge separation, the thermodynamically favorable reduction of fumarate with reduced FMN seems to proceed in the way that C 2 (delta-) accepts a proton from Cys130 and C 3 (delta+) a hydride (or a hydride equivalent) from reduced FMN N 5 in TcDHOD.     

Excitation and deexcitation processes of Eu(III) benzo-15-crown-5 complex studied on comparing with its first coordination sphere

The coordination sphere and the deexcitation mechanism of the Eu(III) benzo-15-crown-5 complex, Eu(B15C5), were studied with references of the Eu(III) complexes with a similar coordination sphere; the dibenzo-18-crown-6 complex, Eu₃(B₂18C6)₂, and the cryptand[2.2.1] complex, Eu([2.2.1]). NMR spectroscopy reveals that the Eu(B15C5) complex is quite stable in acetonitrile solution whereas only 40% of the Eu(III) ion forms the complex in the equimolar Eu(NO₃)₃ and B₂18C6 acetonitrile solution. The coordination sphere of the Eu(III) complexes in acetonitrile solutions were also discussed by the degenerate ⁷F₀→⁵D₀ transition energy levels. The Eu(B15C5) have a negative shift compared with the europium(III) nitrate in acetonitrile and it is explained by the coordination of both nitrate ions and the crown ether ligand. Energy transfer from the n–π* excited state located in the catechol structure to the central europium ion was first observed as the sensitized luminescence of ⁵D₀→⁷F_J. The excited state lifetime of the Eu(B15C5) complex was first determined as 201 μs in the present study.     

Production of recombinant granulocyte colony-stimulating factor by knocking into the active immunoglobulin heavy chain gene locus in the hybridoma cell line

The hybridoma cell line KM50 originally produces a monoclonal antibody at a concentration of ∼40 mg ml^-1 in ascites. To investigate the possibility to apply this expression system to the production of useful proteins, the cDNA encoding human granulocyte colony-stimulating factor was inserted by homologous recombination into just downstream of the promoter of the active immunoglobulin heavy chain gene of KM50. Site directed integration of targeting DNAs resulted in the disruption of expression of the immunoglobulin heavy chain proteins with a frequency of 1 in 10 ∼ 100 G418-resistance transfectants. One of the monoclonal antibody-deficient transfectants produced25 ng ml^-1 of granulocyte colony-stimulating factor in the supernatant of its cell culture the number of molecules of which corresponds to that of the monoclonal antibody originally produced by KM50. However, when this transfectant was injected intraperitoneally, it produced only a 9 μg ml^-1 concentration of granulocyte colony-stimulating factor in ascites, which is approximately 3 orders of magnitude less than the monoclonal antibody. This method may be applicable to production of other recombinant proteins, although further optimization in the conditions of production would be needed in order to reach much higher yields. This revised version was published online in August 2006 with corrections to the Cover Date.