Therapeutic efficacy of 211At-radiolabeled 2,6-diisopropylphenyl azide in mouse models of human lung cancer.

Targeted α-particle therapy (TAT) is an attractive alternative to conventional therapy for cancer treatment. Among the available radionuclides considered for TAT, astatine-211 (211At) attached to a cancer-targeting molecule appears very promising. Previously, we demonstrated that aryl azide derivatives could react selectively with the endogenous acrolein generated by cancer cells to give a diazo compound, which subsequently forms a covalent bond with the organelle of cancer cells in vivo. Herein, we synthesized 211At-radiolabeled 2,6-diisopropylphenyl azide (ADIPA), an α-emitting molecule that can selectively target the acrolein of cancer cells, and investigated its antitumor effect. Our results demonstrate that a single intratumor or intravenous administration of this simple α-emitting molecule to the A549 (human lung cancer) cell-bearing xenograft mouse model, at a low dose (70 kBq), could suppress tumor growth without inducing adverse effects. Furthermore, because acrolein is generally overproduced by most cancer cells, we believe ADIPA is a simple TAT compound that deserves further investigation for application in animal models and humans with various cancer types and stages.

x- and y-type thioredoxins maintain redox homeostasis on photosystem I acceptor side under fluctuating light.

Plants cope with sudden increases in light intensity through various photoprotective mechanisms. Redox regulation by thioredoxin (Trx) systems also contributes to this process. Whereas the functions of f- and m-type Trxs in response to such fluctuating light conditions have been extensively investigated, those of x- and y-type Trxs are largely unknown. Here, we analyzed the trx x single, trx y1 trx y2 double, and trx x trx y1 trx y2 triple mutants in Arabidopsis (Arabidopsis thaliana). A detailed analysis of photosynthesis revealed changes in photosystem I (PSI) parameters under low light in trx x and trx x trx y1 trx y2. The electron acceptor side of PSI was more reduced in these mutants than in the wild type. This mutant phenotype was more pronounced under fluctuating light conditions. During both low- and high-light phases, the PSI acceptor side was largely limited in trx x and trx x trx y1 trx y2. After fluctuating light treatment, we observed more severe PSI photoinhibition in trx x and trx x trx y1 trx y2 than in the wild type. Furthermore, when grown under fluctuating light conditions, trx x and trx x trx y1 trx y2 plants showed impaired growth and decreased level of PSI subunits. These results suggest that Trx x and Trx y prevent redox imbalance on the PSI acceptor side, which is required to protect PSI from photoinhibition, especially under fluctuating light. We also propose that Trx x and Trx y contribute to maintaining the redox balance even under constant low-light conditions to prepare for sudden increases in light intensity.

Predicting Rice Lodging Risk from the Distribution of Available Nitrogen in Soil Using UAS Images in a Paddy Field.

Rice lodging causes a loss of yield and leads to lower-quality rice. In Japan, Koshihikari is the most popular rice variety, and it has been widely cultivated for many years despite its susceptibility to lodging. Reducing basal fertilizer is recommended when the available nitrogen in soil (SAN) exceeds the optimum level (80-200 mg N kg-1). However, many commercial farmers prefer to simultaneously apply one-shot basal fertilizer at transplant time. This study investigated the relationship between the rice lodging and SAN content by assessing their spatial distributions from unmanned aircraft system (UAS) images in a Koshihikari paddy field where one-shot basal fertilizer was applied. We analyzed the severity of lodging using the canopy height model and spatially clarified a heavily lodged area and a non-lodged area. For the SAN assessment, we selected green and red band pixel digital numbers from multispectral images and developed a SAN estimating equation by regression analysis. The estimated SAN values were rasterized and compiled into a 1 m mesh to create a soil fertility map. The heavily lodged area roughly coincided with the higher SAN area. A negative correlation was observed between the rice inclination angle and the estimated SAN, and rice lodging occurred even within the optimum SAN level. These results show that the amount of one-shot basal fertilizer applied to Koshihikari should be reduced when absorbable nitrogen (SAN + fertilizer nitrogen) exceeds 200 mg N kg-1.

Identification and characterization of sperm motility-initiating substance-2 gene in internally fertilizing Cynops species.

Sperm motility-initiating substance (SMIS) is an oviductal protein critical for internal fertilization in urodeles. It contributes to the establishment of various reproductive modes in amphibians and is thus a unique research model for the gene evolution of gamete-recognizing ligands that have diversified among animal species. In this study, a paralogous SMIS gene, smis2, was identified via the RNA sequencing of the oviduct of the newt, Cynops pyrrhogaster. The base sequence of the smis2 gene was homologous (˃90%) to that of the original smis gene (smis1), and deduced amino acid sequences of both genes conserved six cysteine residues essential for the cysteine knot motif. Furthermore, smis2 complementary DNA was identified in the oviduct of Cynops ensicauda, and the base substitution patterns also suggested that the smis gene was duplicated in the Salamandridae. Nonsynonymous/synonymous substitution ratios of smis1 and smis2 genes were 0.79 and 2.6, respectively, suggesting that smis2 gene evolution was independently driven by positive selection. Amino acid substitutions were concentrated in the cysteine knot motif of SMIS2. The smis2 gene was expressed in some organs in addition to the oviduct; in contrast, SMIS1 was only expressed in the oviduct. The SMIS2 protein was suggested to be produced and secreted at least in the oviduct and redundantly act in sperm. These results suggest that smis1 plays the original role in the oviduct, whereas smis2 may undergo neofunctionalization, which rarely occurs in gene evolution.

Tumor Targeting of 211At-Labeled Antibody under Sodium Ascorbate Protection against Radiolysis.

Astatine-211 (211At) is an alpha emitter applicable to radioimmunotherapy (RIT), a cancer treatment that utilizes radioactive antibodies to target tumors. In the preparation of 211At-labeled monoclonal antibodies (211At-mAbs), the possibility of radionuclide-induced antibody denaturation (radiolysis) is of concern. Our previous study showed that this 211At-induced radiochemical reaction disrupts the cellular binding activity of an astatinated mAb, resulting in attenuation of in vivo antitumor effects, whereas sodium ascorbate (SA), a free radical scavenger, prevents antibody denaturation, contributing to the maintenance of binding and antitumor activity. However, the influence of antibody denaturation on the pharmacokinetics of 211At-mAbs relating to tumor accumulation, blood circulation time, and distribution to normal organs remains unclear. In this study, we use a radioactive anti-human epidermal growth factor receptor 2 (anti-HER2) mAb to demonstrate that an 211At-induced radiochemical reaction disrupts active targeting via an antigen-antibody interaction, whereas SA helps to maintain targeting. In contrast, there was no difference in blood circulation time as well as distribution to normal organs between the stabilized and denatured immunoconjugates, indicating that antibody denaturation may not affect tumor accumulation via passive targeting based on the enhanced permeability and retention effect. In a high-HER2-expressing xenograft model treated with 1 MBq of 211At-anti-HER2 mAbs, SA-dependent maintenance of active targeting contributed to a significantly better response. In treatment with 0.5 or 0.2 MBq, the stabilized radioactive mAb significantly reduced tumor growth compared to the denatured immunoconjugate. Additionally, through a comparison between a stabilized 211At-anti-HER2 mAb and radioactive nontargeted control mAb, we demonstrate that active targeting significantly enhances tumor accumulation of radioactivity and in vivo antitumor effect. In RIT with 211At, active targeting contributes to efficient tumor accumulation of radioactivity, resulting in a potent antitumor effect. SA-dependent protection that successfully maintains tumor targeting will facilitate the clinical application of alpha-RIT.

Romosozumab successfully regulated progressive osteoporosis in a patient with autosomal dominant polycystic kidney disease undergoing hemodialysis.

Osteoporosis is a crucial complication in patients with chronic kidney disease (CKD), similar to that in the general population. Although romosozumab, a monoclonal antibody targeting sclerostin, has been administered for patients with CKD, its clinical effectiveness in these patients, especially in patients on hemodialysis (HD), remains to be studied. Herein, we report the case of a 42-year-old man on HD who developed severe osteoporosis. Serum calcium levels were extremely high, bone metabolic markers were abnormal, and the patient had pathological fractures. The bone biopsy indicated a bone metabolism disorder and high bone turnover. We administered romosozumab once a month as an intervention for bone alteration. Through the 10-month usage, bone metabolic markers improved, and the decrease in bone mineral density was ameliorated. We hypothesized that romosozumab could be a therapeutic option for osteoporosis in patients undergoing HD, especially in those with bone mineralization disorders.

Analysis of Molecular Disordering Processes in the Phase Transition of Liquid Crystals Observed by Patterned-Illumination Time-Resolved Phase Microscopy.

The initial processes of the phase transition dynamics of liquid crystals (LCs) subject to UV pulse irradiation were clarified using a nanosecond time-resolved imaging technique called pattern-illumination time-resolved phase microscopy (PI-PM). Two types of LCs were studied: a photo-responsive LC and dye-doped LCs. We found two steps of molecular disordering processes in the phase transition, namely local disordering proceeding anisotropically, followed by the spreading of the isotropic phase. These two processes were separated for a photo-responsive LC while being simultaneously observed for the dye-doped LCs. It was found that the photomechanical dyes induced the phase transition process faster than the photothermal dyes.

Evaluation of CBSX Proteins as Regulators of the Chloroplast Thioredoxin System.

The chloroplast-localized cystathionine ß-synthase X (CBSX) proteins CBSX1 and CBSX2 have been proposed as modulators of thioredoxins (Trxs). In this study, the contribution of CBSX proteins to the redox regulation of thiol enzymes in the chloroplast Trx system was evaluated both in vitro and in vivo. The in vitro biochemical studies evaluated whether CBSX proteins alter the specificities of classical chloroplastic Trx f and Trx m for their target proteins. However, addition of CBSX proteins did not alter the specificities of Trx f and Trx m for disulfide bond reduction of the photosynthesis-related major thiol enzymes, FBPase, SBPase, and NADP-MDH. In vivo analysis showed that CBSX-deficient mutants grew similarly to wild type plants under continuous normal light conditions and that CBSX deficiency did not affect photo-reduction of photosynthesis-related thiol enzymes by Trx system at several light intensities. Although CBSX proteins have been suggested as modulators in the chloroplast Trx system, our results did not support this model, at least in the cases of FBPase, SBPase, and NADP-MDH in leaves. However, fresh weights of the cbsx2 mutants were decreased under short day. Since Trxs regulate many proteins participating in various metabolic reactions in the chloroplast, CBSX proteins may function to regulate other chloroplast Trx target proteins, or serve as modulators in non-photosynthetic plastids of flowers. As a next stage, further investigations are required to understand the modulation of Trx-dependent redox regulation by plastidal CBSX proteins.

Critical Role of NdhA in the Incorporation of the Peripheral Arm into the Membrane-Embedded Part of the Chloroplast NADH Dehydrogenase-Like Complex.

The chloroplast NADH dehydrogenase-like (NDH) complex mediates ferredoxin-dependent plastoquinone reduction in the thylakoid membrane. In angiosperms, chloroplast NDH is composed of five subcomplexes and further forms a supercomplex with photosystem I (PSI). Subcomplex A (SubA) mediates the electron transport and consists of eight subunits encoded by both plastid and nuclear genomes. The assembly of SubA in the stroma has been extensively studied, but it is unclear how SubA is incorporated into the membrane-embedded part of the NDH complex. Here, we isolated a novel Arabidopsis mutant chlororespiratory reduction 16 (crr16) defective in NDH activity. CRR16 encodes a chloroplast-localized P-class pentatricopeptide repeat protein conserved in angiosperms. Transcript analysis of plastid-encoded ndh genes indicated that CRR16 was responsible for the efficient splicing of the group II intron in the ndhA transcript, which encodes a membrane-embedded subunit localized to the connecting site between SubA and the membrane subcomplex (SubM). To analyze the roles of NdhA in the assembly and stability of the NDH complex, the homoplastomic knockout plant of ndhA (ΔndhA) was generated in tobacco (Nicotiana tabacum). Biochemical analyses of crr16 and ΔndhA plants indicated that NdhA was essential for stabilizing SubA and SubE but not for the accumulation of the other three subcomplexes. Furthermore, the crr16 mutant accumulated the SubA assembly intermediates in the stroma more than that in the wild type. These results suggest that NdhA biosynthesis is essential for the incorporation of SubA into the membrane-embedded part of the NDH complex at the final assembly step of the NDH-PSI supercomplex.

Localization of sperm intracellular Ca2+ keeps fertilizability in the newt vas deferens.

Sperm intracellular Ca2+ is crucial for the induction of sperm-egg interaction, but little is known about the significance of Ca2+ maintenance prior to induction. In sperm of the newt Cynops pyrrhogaster, intracellular Ca2+ is localized to the midpiece during storage in the vas deferens, while extracellular Ca2+ is influxed in modified Steinberg's salt solution to promote a spontaneous acrosome reaction related to the decline of sperm quality. In the present study, sperm from the vas deferens were loaded with the Ca2+ indicator Fluo8H, and changes in Ca2+ localization in modified Steinberg's salt solution were examined. Calcium ions expanded from the cytoplasmic area of the midpiece to the entire tail in most sperm during a 1-h incubation and localized to the principal piece in some sperm within 24 h. Similar changes in Ca2+ localization were observed in reconstructed vas deferens solution that included ions and pH at equivalent levels to those in the vas deferens fluid. Sperm with Ca2+ localization in the entire tail or the principal piece weakened or lost responsiveness to sperm motility-initiating substances, which trigger sperm motility for fertilization, but responded to a trigger for acrosome reaction. The change in Ca2+ localization was delayed and transiently reversed by ethylene glycol tetraacetic acid or a mixture of Ca2+ channel blockers including Ni2+ and diltiazem. These results suggest that C. pyrrhogaster sperm localize intracellular Ca2+ to the midpiece through Ca2+ transport in the vas deferens to allow for responses to sperm motility-initiating substances.

211At-labeled immunoconjugate via a one-pot three-component double click strategy: practical access to α-emission cancer radiotherapeutics.

α-Emission radiotherapeutics has potential to be one of most effective cancer therapeutics. Herein, we report a facile synthesis of an 211At-labeled immunoconjugate for use as an α-emission molecular targeting therapy. We synthesized a tetrazine probe modified with closo-decaborate(2-), a prosthetic group that forms a bioavailable stable complex with 211At. Our one-pot three-component double-click labeling method was used to attach decaborate to trastuzumab (anti-HER2 antibody) using decaborate-tetrazine and TCO-aldehyde probes without reducing the antibody binding affinity. Labeling the decaborate-attached trastuzumab with 211At produced in the cyclotron at the RIKEN Nishina Center, at which highly radioactive 211At can be produced, readily furnished the 211At-labeled trastuzumab with a maximum specific activity of 15 MBq µg-1 and retention of the native binding affinity. Intratumor injection of the 211At-labeled trastuzumab in BALB/c nude mice implanted with HER2-expressing epidermoid cancer cells yielded efficient accumulation at the targeted tumor site as well as effective suppression of tumor growth.

Cancer stem-like properties of hormonal therapy-resistant breast cancer cells.

BACKGROUND: Presently, hormonal therapy targeting estrogen receptors is the most effective treatment available for luminal breast cancer. However, many patients relapse after the therapy. It has been suggested that cancer stem-like cells are involved with hormonal therapy resistance; in the present study, we evaluated this hypothesis. METHODS: In the present study, we used our previously established hormonal therapy-resistant cell lines, including aromatase inhibitor (AI)-resistant cells (Type 1 and Type 2) and fulvestrant-resistant cells (MFR). RESULTS: AI-resistant cell lines expressing ER (Type 1 V1 and V2) showed high cancer stemness in terms of their CD44/CD24 expression and side populations, which were stimulated by the addition of estrogen and inhibited by fulvestrant. However, ALDH activity was lower than in the ER-negative resistant cells, suggesting that the stemness of luminal cells is distinct from that of basal-like breast cancer cells. The migration and invasion activity of the ER-positive Type 1 V1 and V2 cells were higher than in the ER-negative cell lines, Type 2 and MFR. CONCLUSIONS: Fractionation of parental cells based on CD44/CD24 expression and colony formation assay indicated that CD44+/CD24+ cells might be the origin of hormonal therapy-resistant cells. This population reconstituted various other subpopulations under estrogen deprivation. These results indicate that hormonal therapy resistance is closely related to the cancer stem cell-like properties of luminal breast cancer.

Plastid translation is essential for lateral root stem cell patterning in Arabidopsis thaliana.

The plastid evolved from a symbiotic cyanobacterial ancestor and is an essential organelle for plant life, but its developmental roles in roots have been largely overlooked. Here, we show that plastid translation is connected to the stem cell patterning in lateral root primordia. The RFC3 gene encodes a plastid-localized protein that is a conserved bacterial ribosomal protein S6 of ß/γ proteobacterial origin. The rfc3 mutant developed lateral roots with disrupted stem cell patterning and associated with decreased leaf photosynthetic activity, reduced accumulation of plastid rRNAs in roots, altered root plastid gene expression, and changes in expression of several root stem cell regulators. These results suggest that deficiencies in plastid function affect lateral root stem cells. Treatment with the plastid translation inhibitor spectinomycin phenocopied the defective stem cell patterning in lateral roots and altered plastid gene expression observed in the rfc3 mutant. Additionally, when prps17 defective in a plastid ribosomal protein was treated with low concentrations of spectinomycin, it also phenocopied the lateral root phenotypes of rfc3 The spectinomycin treatment and rfc3 mutation also negatively affected symplasmic connectivity between primary root and lateral root primordia. This study highlights previously unrecognized functions of plastid translation in the stem cell patterning in lateral roots.

A case of Ménétrier's disease seemingly caused by hilar cholangiocarcinoma.

A 54-year-old man presented to our department with abdominal discomfort and anorexia and was diagnosed as having Ménétrier's disease (MD) with hilar cholangiocarcinoma. Based on his clinical examination, there was no evidence of Helicobacter pylori or cytomegalovirus (CMV) infection. Although we administered proton pump inhibitor and high-calorie enteral nutrition, hypoproteinemia did not improve, and the refractory protein-losing enteropathy persisted. However, interestingly, MD improved immediately after resection of the hilar cholangiocarcinoma. Generally, the etiology of MD is unknown, but H. pylori and CMV infections have been implicated. To our knowledge, there has been no previous report indicating that a malignant tumor could be involved in the etiology of MD. Thus, we report an extremely rare case of MD which is seemingly caused by malignancy.

CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6(-/-) mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6(-/-) mice and C1qtnf6(-/-) embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H2O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases.

Thrombomodulin in the management of acute cholangitis-induced disseminated intravascular coagulation.

AIM: To evaluate the need for thrombomodulin (rTM) therapy for disseminated intravascular coagulation (DIC) in patients with acute cholangitis (AC)-induced DIC. METHODS: Sixty-six patients who were diagnosed with AC-induced DIC and who were treated at our hospital were enrolled in this study. The diagnoses of AC and DIC were made based on the 2013 Tokyo Guidelines and the DIC diagnostic criteria as defined by the Japanese Association for Acute Medicine, respectively. Thirty consecutive patients who were treated with rTM between April 2010 and September 2013 (rTM group) were compared to 36 patients who were treated without rTM (before the introduction of rTM therapy at our hospital) between January 2005 and January 2010 (control group). The two groups were compared in terms of patient characteristics at the time of DIC diagnosis (including age, sex, primary disease, severity of cholangitis, DIC score, biliary drainage, and anti-DIC drugs), the DIC resolution rate, DIC score, the systemic inflammatory response syndrome (SIRS) score, hematological values, and outcomes. Using logistic regression analysis based on multivariate analyses, we also examined factors that contributed to persistent DIC. RESULTS: There were no differences between the rTM group and the control group in terms of the patients' backgrounds other than administration. DIC resolution rates on day 9 were higher in the rTM group than in the control group (83.3% vs 52.8%, P < 0.01). The mean DIC scores on day 7 were lower in the rTM group than in the control group (2.1 ± 2.1 vs 3.5 ± 2.3, P = 0.02). The mean SIRS scores on day 3 were significantly lower in the rTM group than in the control group (1.1 ± 1.1 vs 1.8 ± 1.1, P = 0.03). Mortality on day 28 was 13.3% in the rTM group and 27.8% in the control group; these rates were not significantly different (P = 0.26). Multivariate analysis identified only the absence of biliary drainage as significantly associated with persistent DIC (P < 0.01, OR = 12, 95%CI: 2.3-60). Although the difference did not reach statistical significance, primary diseases (malignancies) (P = 0.055, OR = 3.9, 95%CI: 0.97-16) and the non-use of rTM had a tendency to be associated with persistent DIC (P = 0.08, OR = 4.3, 95%CI: 0.84-22). CONCLUSION: The add-on effects of rTM are anticipated in the treatment of AC-induced DIC, although biliary drainage for AC remains crucial.

Differences in stemness properties associated with the heterogeneity of luminal-type breast cancer.

BACKGROUND: Luminal-type breast cancers are the most abundant subtype. Endocrine therapies targeting estrogen receptor (ER) or estradiol (E2) synthesis have achieved marked improvement in disease-free and overall survival of ER-positive cancers. However, approximately one-third of these cancers are poorly responsive to endocrine therapies, suggesting nonuniform tumor cell characteristics of this subtype. Recently, the tumorigenesis theory which states that CSCs are capable of self-renewal, tumorigenicity, and therapeutic resistance, became widely accepted. We investigated the relationship between the heterogeneity of luminal-type breast cancer and stemness properties. MATERIALS AND METHODS: CSC surface markers and expression of stemness-related genes, including Octamer-binding transcription factor 4 (OCT4), Nanog homeobox (NANOG), and Kruppel-like factor 4 (KLF4), were analyzed in clinical samples. ER activities were analyzed using the adenovirus vector carrying the ERE-green fluorescent protein (GFP). We separated the luminal-type breast cancers into 2 groups according to stemness-related gene expression patterns in mammospheres. RESULTS: The group that predominantly expressed NANOG mRNA showed a high percentage of the cells that were positive for CD44 and negative for CD24 and Hoechst (possessing high-stemness properties), younger patient age, higher p53 expression, and tended to show higher histological grade and higher topoisomerase IIα expression. The ERE-GFP assay revealed that the luminal-type breast cancer mammospheres were heterogeneous. Mammospheres from several specimens lacked ER activity and responsiveness to E2 but some retained ER activities. CONCLUSION: ERE activity differences might be associated with endocrine therapy effectiveness. Mammosphere stemness properties could be a useful and novel criterion for subclassification of luminal-type breast cancers.

Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: enzymatic activity and active site structure.

Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238-246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

CTRP3 plays an important role in the development of collagen-induced arthritis in mice.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease exhibited most commonly in joints. We found that the expression of C1qtnf3, which encodes C1q/TNF-related protein 3 (CTRP3), was highly increased in two mouse RA models with different etiology. To elucidate the pathogenic roles of CTRP3 in the development of arthritis, we generated C1qtnf3(-/-) mice and examined the development of collagen-induced arthritis in these mice. We found that the incidence and severity score was higher in C1qtnf3(-/-) mice compared with wild-type (WT) mice. Histopathology of the joints was also more severe in C1qtnf3(-/-) mice. The levels of antibodies against type II collagen and pro-inflammatory cytokine mRNAs in C1qtnf3(-/-) mice were higher than WT mice. These observations indicate that CTRP3 plays an important role in the development of autoimmune arthritis, suggesting CTRP3 as a possible medicine to treat RA.

Structures of reduced and ligand-bound nitric oxide reductase provide insights into functional differences in respiratory enzymes.

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3-CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ∼0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (∼4.5 Å) than the heme/Cu distance in CCO (∼5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2O.