An optical dynamic mass redistribution assay reveals biased signaling of dualsteric GPCR activators

Increasing attention is paid in basic science and in drug discovery to pathway selective intracellular signaling as a novel approach to achieve precise control of cell function via G protein-coupled receptors (GPCRs). With respect to signaling, GPCRs are often promiscuous in that more than one intracellular biochemical pathway is activated upon receptor stimulation by the endogenous transmitter or by exogenous drugs. We studied signaling by a novel class of GPCR activators that were designed to bind simultaneously to the orthosteric transmitter-binding site and the allosteric site of muscarinic acetylcholine receptors. An optical biosensor technique was applied to measure activation-induced dynamic mass redistribution (DMR) in CHO cells stably expressing the muscarinic receptor subtype of interest. The use of tools to modulate signaling and measuring G protein activation directly proved that DMR is a valid and comfortable approach to gain real-time insight into intracellular signaling pathway activation and to identify signaling pathway-selective drugs.     

Ectonucleotidases CD39 and CD73 on OvCA cells are potent adenosine-generating enzymes responsible for adenosine receptor 2A-dependent suppression of T cell function and NK cell cytotoxicity

The ectonucleotidases CD39 and CD73 degrade immune stimulatory ATP to adenosine that inhibits T and NK cell responses via the A(2A) adenosine receptor (ADORA2A). This mechanism is used by regulatory T cells (T(reg)) that are associated with increased mortality in OvCA. Immunohistochemical staining of human OvCA tissue specimens revealed further aberrant expression of CD39 in 29/36 OvCA samples, whereas only 1/9 benign ovaries showed weak stromal CD39 expression. CD73 could be detected on 31/34 OvCA samples. While 8/9 benign ovaries also showed CD73 immunoreactivity, expression levels were lower than in tumour specimens. Infiltration by CD4(+) and CD8(+) T cells was enhanced in tumour specimens and significantly correlated with CD39 and CD73 levels on stromal, but not on tumour cells. In vitro, human OvCA cell lines SK-OV-3 and OaW42 as well as 11/15 ascites-derived primary OvCA cell cultures expressed both functional CD39 and CD73 leading to more efficient depletion of extracellular ATP and enhanced generation of adenosine as compared to activated T(reg). Functional assays using siRNAs against CD39 and CD73 or pharmacological inhibitors of CD39, CD73 and ADORA2A revealed that tumour-derived adenosine inhibits the proliferation of allogeneic human CD4(+) T cells in co-culture with OvCA cells as well as cytotoxic T cell priming and NK cell cytotoxicity against SK-OV3 or OAW42 cells. Thus, both the ectonucleotidases CD39 and CD73 and ADORA2A appear as possible targets for novel treatments in OvCA, which may not only affect the function of T(reg) but also relieve intrinsic immunosuppressive properties of tumour and stromal cells.     

Lack of glyphosate resistance gene transfer from Roundup Ready soybean to Bradyrhizobium japonicum under field and laboratory conditions

A field study was conducted at the Russell E. Larson Agricultural Research Center to determine the effect of transgenic glyphosate-resistant soybean in combination with herbicide (Roundup) application on its endosymbiont Bradyrhizobium japonicum. DNA of bacteroids from isolated nodules was analysed for the presence of the transgenic 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) DNA sequence using polymerase chain reaction (PCR). To further assess the likelihood that the EPSPS gene may be transferred from the Roundup Ready (RR) soybean to B. japonicum, we have examined the natural transformation efficiency of B. japonicum strain 110spc4. Analyses of nodules showed the presence of the transgenic EPSPS DNA sequence. In bacteroids that were isolated from nodules of transgenic soybean plants and then cultivated in the presence of glyphosate this sequence could not be detected. This indicates that no stable horizontal gene transfer (HGT) of the EPSPS gene had occurred under field conditions. Under laboratory conditions, no natural transformation was detected in B. japonicum strain 110spc4 in the presence of various amounts of recombinant plasmid DNA. Our results indicate that no natural competence state exists in B. japonicum 110spc4. Results from field and laboratory studies indicate the lack of functional transfer of the CP4-EPSPS gene from glyphosate-tolerant soybean treated with glyphosate to root-associated B. japonicum.     

RAP1 Protects from Obesity through Its Extratelomeric Role Regulating Gene Expression

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PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2

Prostaglandin H(1) (PGH(1)) is the cyclo-oxygenase metabolite of dihomo-γ-linolenic acid (DGLA) and the precursor for the 1-series of prostaglandins which are often viewed as "anti-inflammatory". Herein we present evidence that PGH(1) is a potent activator of the pro-inflammatory PGD(2) receptor CRTH2, an attractive therapeutic target to treat allergic diseases such as asthma and atopic dermatitis. Non-invasive, real time dynamic mass redistribution analysis of living human CRTH2 transfectants and Ca(2+) flux studies reveal that PGH(1) activates CRTH2 as PGH(2), PGD(2) or PGD(1) do. The PGH(1) precursor DGLA and the other PGH(1) metabolites did not display such effect. PGH(1) specifically internalizes CRTH2 in stable CRTH2 transfectants as assessed by antibody feeding assays. Physiological relevance of CRTH2 ligation by PGH(1) is demonstrated in several primary human hematopoietic lineages, which endogenously express CRTH2: PGH(1) mediates migration of and Ca(2+) flux in Th2 lymphocytes, shape change of eosinophils, and their adhesion to human pulmonary microvascular endothelial cells under physiological flow conditions. All these effects are abrogated in the presence of the CRTH2 specific antagonist TM30089. Together, our results identify PGH(1) as an important lipid intermediate and novel CRTH2 agonist which may trigger CRTH2 activation in vivo in the absence of functional prostaglandin D synthase.     

Neutrophil-Derived Myeloperoxidase Aggravates Non-Alcoholic Steatohepatitis in Low-Density Lipoprotein Receptor-Deficient Mice

Background

Chronic inflammation and oxidative stress play fundamental roles in the pathogenesis of non-alcoholic steatohepatitis (NASH). Previously, we reported that myeloperoxidase (MPO), an aggressive oxidant-generating neutrophil enzyme, is associated with NASH severity in man. We now investigated the hypothesis that MPO contributes to the development and progression of NASH.

Methodology

Low-density lipoprotein receptor-deficient mice with an MPO-deficient hematopoietic system (LDLR^−/−/MPO^−/−tp mice) were generated and compared with LDLR^−/−/MPO^+/+tp mice after induction of NASH by high-fat feeding.

Results

High-fat feeding caused a ∼4-fold induction of liver MPO in LDLR^−/−/MPO^+/+ mice which was associated with hepatic sequestration of MPO-positive neutrophils and high levels of nitrotyrosine, a marker of MPO activity. Importantly, LDLR^−/−/MPO^−/−tp mice displayed markedly reduced hepatic neutrophil and T-lymphocyte infiltration (p<0.05), and strong down regulation of pro-inflammatory genes such as TNF-α and IL-6 (p<0.05, p<0.01) in comparison with LDLR^−/−/MPO^+/+tp mice. Next to the generalized reduction of inflammation, liver cholesterol accumulation was significantly diminished in LDLR^−/−/MPO^−/−tp mice (p = 0.01). Moreover, MPO deficiency appeared to attenuate the development of hepatic fibrosis as evident from reduced hydroxyproline levels (p<0.01). Interestingly, visceral adipose tissue inflammation was markedly reduced in LDLR^−/−/MPO^−/−tp mice, with a complete lack of macrophage crown-like structures. In conclusion, MPO deficiency attenuates the development of NASH and diminishes adipose tissue inflammation in response to a high fat diet, supporting an important role for neutrophils in the pathogenesis of metabolic disease.     

Analysis of the antibody repertoire of lymphoma patients

Cancer testis or cancer germline antigens (CGA) are promising vaccine candidates because they are expressed only in malignant but not in normal tissues, except for germ cells in the testis. Since non-Hodgkin's lymphomas (NHL) express the known CGA at low frequencies, we aimed at increasing the number of CGA with frequent expression in NHL by screening a cDNA expression library derived from normal testis for reactivity with high-titered IgG antibodies in the sera of lymphoma patients using SEREX, the serological identification of antigens by recombinant cDNA expression cloning. The analysis of 1.6x10(6) clones with the sera of 25 lymphoma patients revealed 42 clones which coded for 23 antigens, 12 of which had already been included in the SEREX databank. Four cDNA clones coded for unknown and 19 for known genes. Three antigens reacted only with the serum by which they had been detected, 9 antigens reacted with the sera of several NHL patients, but not with that of healthy controls, and 11 antigens reacted with both normal and NHL sera. Most of the antigens were ubiquitously expressed. Only HOM-NHL-6, HOM-NHL-8, HOM-NHL-21 and HOM-NHL-23 showed a restricted expression pattern. HOM-NHL-6 and HOM-NHL-8 were homologous to the previously described CGA NY-ESO-1 and HOM-TES-14/SCP-1, respectively. HOM-NHL-21 was expressed in rare cases of lymphomas, but not in normal tissues except for testis and brain, while HOM-NHL-23 appeared to be a testis-specific antigen. In summary, using the antibody repertoire of these 25 NHL patients, no new CGA were detected. The number of CGA detectable by the classical SEREX approach appears to be limited, and novel strategies are necessary to identify antigens that can serve as a vaccine target in a broad spectrum of NHL patients.     

Purification and characterization of a new hyperthermostable,allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus

A new chitinase (1,4-beta-D-N-acetyl-glucosaminidase, EC 3.2.1.14) was detected and purified to homogeneity in its native form from the chitinolytic enzyme system of the extremely thermophilic archaeon Thermococcus chitonophagus. This is the first nonrecombinant chitinase purified and characterized from archaea and also constitutes the first case of a membrane-associated chitinase isolated from archaea. The enzyme is a monomer with an apparent molecular weight of 70 kDa [therefore named chitinase 70 (Chi70)] and pI of 5.9; it is hydrophobic and appears to be associated with the outer side of the cell membrane. Chi70 is optimally active at 70 degrees C and pH 7.0 and exhibits remarkable thermostability, maintaining 50% activity even after 1 h at 120 degrees C, and therefore the enzyme is the most thermostable chitinase so far isolated. The enzyme was not inhibited by allosamidin, the natural inhibitor of chitinolytic activity, and was also resistant to denaturation by urea and SDS. On the other hand, guanidine hydrochloride significantly reduced enzymatic activity, indicating that, apart from the hydrophobic interactions, ion pairs located on the surface of the protein could be playing an important role in maintaining the protein's fold and enzyme activity. Chi70 showed broad substrate specificity for several chitinous substrates and derivatives. The lowest K(m) and highest K(cat) values were found for pNP(NAG)(2) as substrate and were determined to be 0.14 mM and 23 min(-1), respectively. The hydrolysis pattern was similar for oligomers and polymers, with N, N'-diacetylchitobiose [(NAG)(2)] being the final, major hydrolysis product. Chi70 was classified as an endochitinase due to its ability to release chitobiose from colloidal chitin. Additionally, the enzyme presented considerable cellulolytic activity. Analysis of the NH(2)-terminal amino acid sequence showed no detectable homology with other known sequences, suggesting that Chi70 is a new protein.     

Characterization of a globin-coupled oxygen sensor with a gene-regulating function

Globin-coupled sensors (GCSs) are multiple-domain transducers, consisting of a regulatory globin-like heme-binding domain and a linked transducer domain(s). GCSs have been described in both Archaea and bacteria. They are generally assumed to bind O(2) (and perhaps other gaseous ligands) and to transmit a conformational change signal through the transducer domain in response to fluctuating O(2) levels. In this study, the heme-binding domain, AvGReg178, and the full protein, AvGReg of the Azotobacter vinelandii GCS, were cloned, expressed, and purified. After purification, the heme iron of AvGReg178 was found to bind O(2). This form was stable over many hours. In contrast, the predominant presence of a bis-histidine coordinate heme in ferric AvGReg was revealed. Differences in the heme pocket structure were also observed for the deoxygenated ferrous state of these proteins. The spectra showed that the deoxygenated ferrous derivatives of AvGReg178 and AvGReg are characterized by a penta-coordinate and hexa-coordinate heme iron, respectively. O(2) binding isotherms indicate that AvGReg178 and AvGReg show a high affinity for O(2) with P(50) values at 20 degrees C of 0.04 and 0.15 torr, respectively. Kinetics of CO binding indicate that AvGReg178 carbonylation conforms to a monophasic process, comparable with that of myoglobin, whereas AvGReg carbonylation conforms to a three-phasic reaction, as observed for several proteins with bis-histidine heme iron coordination. Besides sensing ligands, in vitro data suggest that AvGReg(178) may have a role in O(2)-mediated NO-detoxification, yielding metAvGReg(178) and nitrate.