Genetic ablation of Tnfα demonstrates no detectable suppressive effect on inflammation-related mouse colon tumorigenesis

Colorectal cancer (CRC) is one of the most serious complications of inflammatory bowel disease. Tumor necrosis factor-α (Tnfα) is a major mediator of inflammation and there is increasing evidence that Tnfα/Tnf-receptor-1 (Tnfr1) signaling may act as an endogenous tumor promoter for colon carcinogenesis. In fact, a previous study revealed that mice lacking Tnfr1 develop significantly fewer colonic tumors in the inflammation-related CRC model. In addition, antibodies against Tnfα have been shown to inhibit the development of inflammation-related CRC. In the present study, Apc Min/+; Tnfα ?/? mice were treated with 2% dextran sodium sulfate (DSS) and the tumor development was compared with Apc Min/+; Tnfα +/+ control mice in order to investigate the role of Tnfα by itself in the inflammation-related CRC. Surprisingly, there were no detectable differences in either the severity of colonic inflammation or the expression of DSS-induced chemokines and cytokines (Ccl2, Cxcl1, Tnfβ, Il1β, Il6, and Cox-2) that relate to the colonic inflammation and tumorigenesis between these two groups. Furthermore, the genetic ablation of Tnfα did not suppress the colon tumorigenesis in comparison to the wild-type mice. Our observations suggest that intricate inflammatory responses promote the inflammation-related mouse colon tumorigenesis.     

Optimization of the Degenerated Interfacial ATP Binding Site Improves the Function of Disease-related Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Channels

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, an ATP binding cassette (ABC) protein whose defects cause the deadly genetic disease cystic fibrosis (CF), encompasses two nucleotide binding domains (NBD1 and NBD2). Recent studies indicate that in the presence of ATP, the two NBDs coalesce into a dimer, trapping an ATP molecule in each of the two interfacial composite ATP binding sites (site 1 and site 2). Experimental evidence also suggests that CFTR gating is mainly controlled by ATP binding and hydrolysis in site 2, whereas site 1, which harbors several non-canonical substitutions in ATP-interacting motifs, is considered degenerated. The CF-associated mutation G551D, by introducing a bulky and negatively charged side chain into site 2, completely abolishes ATP-induced openings of CFTR. Here, we report a strategy to optimize site 1 for ATP binding by converting two amino acid residues to ABC consensus (i.e. H1348G) or more commonly seen residues in other ABC proteins (i.e. W401Y,W401F). Introducing either one or both of these mutations into G551D-CFTR confers ATP responsiveness for this disease-associated mutant channel. We further showed that the same maneuver also improved the function of WT-CFTR and the most common CF-associated ΔF508 channels, both of which rely on site 2 for gating control. Thus, our results demonstrated that the degenerated site 1 can be rebuilt to complement or support site 2 for CFTR function. Possible approaches for developing CFTR potentiators targeting site 1 will be discussed.     

Survey of strontium in mineral waters sold in Japan

The concentrations of strontium, calcium, and magnesium in 33 brands of natural mineral waters commercially available in Japan were determined by inductively coupled plasma-atomic emission spectrometry. The geometric mean values were 94.4 microg/L for strontium, 19.1 mg/L for calcium, and 2.82 mg/L for magnesium. Wide confidence intervals of 1.96-4539 microg/L for strontium, 0.865-421 mg/L for calcium, and 0.064-123 mg/L for magnesium were observed. The significant linear relationships among the three elements over a wide distribution range suggest that the synchronized variations of these elements are regulated by the natural ecosystem and not from accidental contamination from human activities or exceptionally high natural sources. Using the results of multiple linear regression analysis, the strontium concentration can be predicted by that of calcium with the appropriate power function. The results of this study suggest that mineral water can be an important nutritional source of strontium. As trace elements imbalance is often found in older patients with chronic renal failure, we propose that close attention of trace elements intake from trendy foods or beverages is necessary to prevent this hidden problem of a rapidly aging society.     

An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF-kappaB activation increases susceptibility to tumor necrosis factor (TNF)alpha-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-kappaB activation-deficient, but not wild-type fibroblasts upon TNFalpha stimulation, indicating that NF-kappaB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFalpha-induced prolonged JNK activation and ROS accumulation in NF-kappaB activation-deficient fibroblasts. Conversely, TNFalpha induces prolonged JNK activation and ROS accumulation in c-Flip-/- fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFalpha-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFbeta-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.     

Spontaneous elicitation of potent antitumor immunity and eradication of established tumors by administration of DNA encoding soluble transforming growth factor-β II receptor without active antigen-sensitization

Since immunity is generally suppressed by immunoregulatory factors, such as transforming growth factor-beta (TGF-β), interleukin (IL)-10, and vascular endothelial growth factor (VEGF), produced by tumor cells or stromal cells surrounding tumor cells, various kinds of cancer immunotherapy mostly fail to elicit potent antitumor immunity. Herein, we tested whether neutralization of TGF-β can elicit strong antitumor immune responses and tumor regression in tumor-bearing mice. A plasmid DNA, pcDNA-sTGFβR/huIg, encoding a fusion protein consisting of the extracellular domain of TGF-β type II receptor (TGFβRII) and the Fc portion of human IgG heavy chain, was injected through different routes into B6 mice carrying established tumors of E.G7 cells, which consist of the poorly immunogenic tumor cells EL4, transfected with the ovalbumin (OVA) gene. The frequency of OVA-specific cytotoxic T lymphocytes (CTL), in the treated mice. increased resulting in the tumor eradication and relapse-free survival in around 70% of the E.G7-bearing mice. In contrast, administration of mock DNA into E.G7-bearing mice did not elicit tumor-specific immune responses. Therefore, administration of DNA encoding TGFβRII allowed tumor-bearing hosts to elicit sufficiently potent antitumor immune responses without requirement of further active antigen-immunization. This strategy seems to be applicable to clinical therapy against cancer, because it is low-cost, safe, and easy to manipulate.     

Identification of TNF-alpha-responsive NF-kappaB p65-binding element in the distal promoter of the mouse serine protease inhibitor SerpinE2

Serine protease inhibitor SerpinE2 is known as a cytokine-inducible gene. Here, we investigated whether tumor necrosis factor alpha-(TNF-alpha)-induced expression of SerpinE2 is mediated by the nuclear factor-kappaB (NF-kappaB) p65 subunit. Both steady state and TNF-alpha-induced expression of SerpinE2 mRNA were abrogated in p65-/- murine embryonic fibroblasts (MEFs). Reconstitution with wild-type p65 rescued SerpinE2 mRNA expression in an IkappaB kinase beta-dependent manner. Electrophoresis mobility shift assay and ChIP assay demonstrated that p65 bound to the kappaB-like DNA sequence located at approximately -9 kbp in the SerpinE2 promoter. In addition, TNF-alpha stimulated luciferase gene expression driven by the kappaB-like element in the reconstituted MEFs, but not in p65-/- MEFs. These results indicated that activation of NF-kappaB p65 plays an important role in TNF-alpha-induced expression of SerpinE2.     

Evidence for redox cooperativity between c-type hemes of MauG which is likely coupled to oxygen activation during tryptophan tryptophylquinone biosynthesis

MauG is a novel 42 kDa diheme protein which is required for the biosynthesis of tryptophan tryptophylquinone, the prosthetic group of methylamine dehydrogenase. The visible absorption and resonance Raman spectroscopic properties of each of the two c-type hemes and the overall redox properties of MauG are described. The absorption maxima for the Soret peaks of the oxidized and reduced hemes are 403 and 418 nm for the low-spin heme and 389 and 427 nm for the high-spin heme, respectively. The resonance Raman spectrum of oxidized MauG exhibits a set of marker bands at 1503 and 1588 cm(-1) which exhibit frequencies similar to those of the nu3 and nu2 bands of c-type heme proteins with bis-histidine coordination. Another set of marker bands at 1478 and 1570 cm(-1) is characteristic of a high-spin heme. Two distinct oxidation-reduction midpoint potential (E(m)) values of -159 and -244 mV are obtained from spectrochemical titration of MauG. However, the two nu3 bands located at 1478 and 1503 cm(-1) shift together to 1467 and 1492 cm(-1), respectively, upon reduction, as do the Soret peaks of the low- and high-spin hemes in the absorption spectrum. Thus, the two hemes with distinct spectral properties are reduced and oxidized to approximately the same extent during redox titrations. This indicates that the high- and low-spin hemes have similar intrinsic E(m) values but exhibit negative redox cooperativity. After the first one-electron reduction of MauG, the electron equilibrates between hemes. This makes the second one-electron reduction of MauG more difficult. Thus, the two E(m) values do not describe redox properties of distinct hemes, but the first and second one-electron reductions of a diheme system with two equivalent hemes. The structural and mechanistic implications of these findings are discussed.     

Regulation of pharmacology by hetero-oligomerization between A1 adenosine receptor and P2Y2 receptor

Adenosine and ATP/UTP are main components of the purinergic system that modulate cellular and tissue functions via specific adenosine and P2 receptors, respectively. Here, we explored the possibility that A(1) adenosine receptor (A(1)R) and P2Y(2) receptor (P2Y(2)R) form heterodimers with novel pharmacological properties. Coimmunoprecipitation showed these receptors directly associate in A(1)R/P2Y(2)R-cotransfected HEK293T cells. Agonist binding by the A(1)R was significantly inhibited by P2Y(2)R agonists only in membranes from cotransfected cells. The functional activity of A(1)R, as indicated by the G(i/o)-mediated inhibition of adenylyl cyclase, in the cotransfected cells was attenuated by the simultaneous addition of A(1)R and P2Y(2)R agonists. The increase in intracellular Ca(2+) levels induced by P2Y(2)R activation of G(q/11) was synergistically enhanced by the simultaneous addition of an A(1)R agonist in the coexpressing cells. These results suggest that oligomerization of A(1)R and P2Y(2)R generates a unique complex in which the simultaneous activation of the two receptors induces a structural alteration that interferes signaling via G(i/o) but enhances signaling via G(q/11).     

Improving effect of ethyl eicosapentanoate on statin-induced rhabdomyolysis in Eisai hyperbilirubinemic rats

The effect of ethyl eicosapentanoate (EPA-E) on statin-induced rhabdomyolysis was investigated by co-administration of EPA-E and pravastatin (PV), as a typical statin, to Eisai hyperbilirubinemic rats (EHBR). It was confirmed that the plasma PV concentration was not affected by simultaneous administration of EPA-E, and there was no cumulative increase of PV during prolonged co-administration of EPA-E and PV. Muscular degeneration was prominent (incidence 5/5; average grade 3.5 (range 2-4)) in EHBR treated with PV alone at 200 mg/kg/day for 14 days, but co-administration of EPA-E at doses of 100, 300, and 1000 mg/kg/day decreased the average grades to 1.4 (range 0.3-3.0), 0.5 (0.2-1.0), and 0.6 (0.0-1.7), respectively. Creatine phosphokinase (CPK) and myoglobin levels in plasma were well correlated with the grade of skeletal muscle degeneration. Thus, EPA-E appears to reduce the severity of statin-induced rhabdomyolysis.