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. Proteins 2014; 82:1258–1271. © 2013 Wiley Periodicals, Inc.     

Epstein-Barr Virus�Cassociated Gastric Carcinoma: A Distinct Carcinoma of Gastric Phenotype by Claudin Expression Profiling

Epstein-Barr virus (EBV)-associated gastric carcinoma (GC) is a distinct subtype with characteristic clinicopathological features. To better characterize its cellular characteristics, 43 cases of EBV-associated GC, 68 cases of EBV-negative GC, and non-neoplastic gastric mucosa in adults and fetuses were examined immunohistochemically. We quantified the expression of the major tight-junction protein claudin (CLDN) -1, -3, -4, -7, and -18 together with gastric mucins (MUC5AC and MUC6), intestinal mucin (MUC2), and CD10. EBV-associated GC showed a high frequency of CLDN18 expression (84%) and a low frequency of CLDN3 expression (5%). This expression profile corresponded to that of normal gastric epithelium in adults and fetuses. Almost half of the EBV-associated GC cases demonstrated gastric mucin expression, whereas the other half lacked mucin or CD10 expression. In contrast, as demonstrated by the expression profiles of CLDN3 and CLDN18, EBV-negative GC comprised a heterogeneous group of four different CLDN phenotypes: gastric, intestinal, mixed, and an undifferentiated type with variable expression patterns of mucins. These results indicate that EBV-associated GC is considerably homogenous with regard to cellular differentiation and that it preserves well the nature of the cells of origin. EBV-associated GC may undergo distinct carcinogenic processes, which differ from those of EBV-negative GC. (J Histochem Cytochem 57:775–785, 2009)     

Unloading-induced bone loss was suppressed in gold-thioglucose treated mice

Loss of mechanical stress causes bone loss. However, the mechanisms underlying the unloading-induced bone loss are largely unknown. Here, we examined the effects of gold-thioglucose (GTG) treatment, which destroys ventromedial hypothalamus (VMH), on unloading-induced bone loss. Unloading reduced bone volume in control (saline-treated) mice. Treatment with GTG-reduced bone mass and in these GTG-treated mice, unloading-induced reduction in bone mass levels was not observed. Unloading reduced the levels of bone formation rate (BFR) and mineral apposition rate (MAR). GTG treatment also reduced these parameters and under this condition, unloading did not further reduce the levels of BFR and MAR. Unloading increased the levels of osteoclast number (Oc.N/BS) and osteoclast surface (Oc.S/BS). GTG treatment did not alter the basal levels of these bone resorption parameters. In contrast to control, GTG treatment suppressed unloading-induced increase in the levels of Oc.N/BS and Oc.S/BS. Unloading reduced the levels of mRNA expression of the genes encoding osteocalcin, type I collagen and Cbfa1 in bone. In contrast, GTG treatment suppressed such unloading-induced reduction of mRNA expression. Unloading also enhanced the levels of fat mass in bone marrow and mRNA expression of the genes encoding PPARgamma2, C/EBPalpha, and C/EBPbeta in bone. In GTG-treated mice, unloading did not increase fat mass and the levels of fat-related mRNA expression. These results indicated that GTG treatment suppressed unloading-induced alteration in bone loss.     

Site-specific incorporation of non-natural amino acids into proteins in mammalian cells with an expanded genetic code

We describe a detailed protocol for incorporating non-natural amino acids, 3-iodo-L-tyrosine (IY) and p-benzoyl-L-phenylalanine (pBpa), into proteins in response to the amber codon (the UAG stop codon) in mammalian cells. These amino acids, IY and pBpa, are applicable for structure determination and the analysis of a network of protein-protein interactions, respectively. This method involves (i) the mutagenesis of the gene encoding the protein of interest to create an amber codon at the desired site, (ii) the expression in mammalian cells of the bacterial pair of an amber suppressor tRNA and an aminoacyl-tRNA synthetase specific to IY or pBpa and (iii) the supplementation of the growth medium with these amino acids. The amber mutant gene, together with these bacterial tRNA and synthetase genes, is introduced into mammalian cells. Culturing these cells for 16-40 h allows the expression of the full-length product from the mutant gene, which contains the non-natural amino acid at the introduced amber position. This method is implemented using the conventional tools for molecular biology and treating cultured mammalian cells. This protocol takes 5-6 d for plasmid construction and 3-4 d for incorporating the non-natural amino acids into proteins.     

Effects of low-load resistance training with vascular occlusion on the mechanical properties of muscle and tendon

The present study aimed to investigate the effects of low-load resistance training with vascular occlusion on the specific tension and tendon properties by comparing with those of high-load training. Nine participants completed 12 weeks (3 days/week) of a unilateral isotonic training program on knee extensors. One leg was trained using low load (20% of 1 RM) with vascular occlusion (LLO) and other leg using high load (80% of 1 RM) without vascular occlusion (HL). Before and after training, maximal isometric knee extension torque (MVC) and muscle volume were measured. Specific tension of vastus lateralis muscle (VL) was calculated from MVC, muscle volume, and muscle architecture measurements. Stiffness of tendon-aponeurosis complex in VL was measured using ultrasonography during isometric knee extension. Both protocols significantly increased MVC and muscle volume of quadriceps femoris muscle. Specific tension of VL increased significantly 5.5% for HL, but not for LLO. The LLO protocol did not alter the stiffness of tendon-aponeurosis complex in knee extensors, while the HL protocol increased it significantly. The present study demonstrated that the specific tension and tendon properties were found to remain following low-load resistance training with vascular occlusion, whereas they increased significantly after high-load training.     

A completely in?vitro system for obtaining scFv using mRNA display, PCR, direct sequencing, and wheat embryo cell-free translation

Using mRNA display followed by in vitro sequencing and translation, a complete in vitro system for obtaining scFv has been developed. An mRNA display library for synthetic scFv was panned against human TNF receptor (TNFR). The nucleotide portion of the enriched molecules was subjected to limiting dilution, and PCR-amplified. Three of the proteins encoded by the amplified fragments were synthesized in a wheat embryo (WE) cell-free system using a batch method. They were shown to bind TNFR by ELISA. One of their sequences was identified in vitro. The identified clone was further synthesized at approx. 0.5 mg/ml reaction mixture in a WE system with dialysis as a totally soluble protein.     

Crustacean molt-inhibiting hormone: Structure,function, and cellular mode of action

In Crustacea, secretion of ecdysteroid molting hormones by Y-organs is regulated, at least in part, by molt-inhibiting hormone (MIH), a polypeptide neurohormone produced by neurosecretory cells of the eyestalks. This article reviews current knowledge of MIH, with particular emphasis on recent findings regarding the (a) structure of the MIH peptide and gene, (b) levels of MIH in eyestalks and hemolymph, (c) cellular mechanism of action of MIH, and (d) responsiveness of Y-organs to MIH. At least 26 MIH/MIH-like sequences have been directly determined by protein sequencing or deduced from cloned cDNA. Recent studies reveal the existence of multiple forms of MIH/MIH-like molecules among penaeids and raise the possibility that molecular polymorphism may exist more generally among MIH (type II) peptides. The hemolymphatic MIH titer has been determined for two species, a crayfish (Procambarus clarkii) and a crab (Carcinus maenas). The data are dissimilar and additional studies are needed. Composite data indicate cellular signaling pathways involving cGMP, cAMP, or both may play a role in MIH-induced suppression of ecdysteroidogenesis. Data from the two species studied in our laboratories (P. clarkii and Callinectes sapidus) strongly favor cGMP as the physiologically relevant second messenger. Ligand-binding studies show an MIH receptor exists in Y-organ plasma membranes, but the MIH receptor has not been isolated or fully characterized for any species. Such studies are critical to understanding the cellular mechanism by which MIH regulates ecdysteroidogenesis. Rates of ecdysteroid synthesis appear also to be influenced by stage-specific changes in the responsiveness of Y-organs to MIH. The changes in responsiveness result, at least in part, from changes in glandular phosphodiesterase (PDE) activity. The PDE isotype (PDE1) present in Y-organs of C. sapidus is calcium/calmodulin dependent. Thus, calcium may regulate ecdysteroidogenesis through activation of glandular PDE.     

Knockdown of COPA,Identified by Loss-of-Function Screen,Induces Apoptosis and Suppresses Tumor Growth in Mesothelioma Mouse Model

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes—COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A—would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.     

NUDT16 and ITPA play a dual protective role in maintaining chromosome stability and cell growth by eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals

Mammalian inosine triphosphatase encoded by ITPA gene hydrolyzes ITP and dITP to monophosphates, avoiding their deleterious effects. Itpa⁻ mice exhibited perinatal lethality, and significantly higher levels of inosine in cellular RNA and deoxyinosine in nuclear DNA were detected in Itpa⁻ embryos than in wild-type embryos. Therefore, we examined the effects of ITPA deficiency on mouse embryonic fibroblasts (MEFs). Itpa⁻ primary MEFs lacking ITP-hydrolyzing activity exhibited a prolonged doubling time, increased chromosome abnormalities and accumulation of single-strand breaks in nuclear DNA, compared with primary MEFs prepared from wild-type embryos. However, immortalized Itpa⁻ MEFs had neither of these phenotypes and had a significantly higher ITP/IDP-hydrolyzing activity than Itpa⁻ embryos or primary MEFs. Mammalian NUDT16 proteins exhibit strong dIDP/IDP-hydrolyzing activity and similarly low levels of Nudt16 mRNA and protein were detected in primary MEFs derived from both wild-type and Itpa⁻ embryos. However, immortalized Itpa⁻ MEFs expressed significantly higher levels of Nudt16 than the wild type. Moreover, introduction of silencing RNAs against Nudt16 into immortalized Itpa⁻ MEFs reproduced ITPA-deficient phenotypes. We thus conclude that NUDT16 and ITPA play a dual protective role for eliminating dIDP/IDP and dITP/ITP from nucleotide pools in mammals.