Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Incubation of purified C57BL/6 murine CD4(+) T lymphocytes with anti-CD3 mAb serves as a model of TCR-mediated activation and results in increased IFN-gamma production and cell surface expression of CD25 and CD69. We demonstrate here that signaling through the TCR causes a rapid (4-h) 5-fold increase in A(2A) adenosine receptor (AR) mRNA, which is correlated with a significant increase in the efficacy of A(2A)AR-mediated cAMP accumulation in these cells. A(2A)AR activation reduces TCR-mediated production of IFN-gamma by 98% with a potency order of 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}cyclohexanecarboxylic acid methyl ester (ATL146e; EC(50) = 0.19 +/- 0.03 nM) > 4-{3-[6-amino-9-(5-cyclopropyl-carbamoyl-3,4-dihydroxytetrahydrofuran-2-yl)-9H-purin-2-yl]prop-2-ynyl}piperidine-1-carboxylic acid methyl ester (ATL313; 0.43 +/- 0.06 nM) > 5'-N-ethylcarboxamidoadenosine (3.5 +/- 0.77 nM) > 2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamidoadenosine (CGS21680; 7.2 +/- 1.4 nM) >> N(6)-cyclohexyladenosine (110 +/- 33 nM) > 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide (390 +/- 160 nM), similar to the potency order to compete for radioligand binding to the recombinant murine A(2A)AR but not the A(3)AR. The selective A(2A)AR antagonist, 4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385), inhibits the effect of ATL146e with a pA(2) of 0.34 nM and also inhibits the effects of N(6)-cyclohexyl-adenosine and 2-chloro-N(6)-(3-iodobenzyl)-5'-N-methylcarboxamide. In CD4(+) T cells derived from A(2A)AR(-/-) and A(2A)AR(+/-) mice, the IFN-gamma release response to ATL146e is reduced by 100 and 50%, respectively, indicative of a gene dose effect. The response of T cells to the phosphodiesterase inhibitor, 4-(3'-cyclopentyloxy-4'-methoxyphenyl)-2-pyrrolidone (rolipram), is not affected by A(2A)AR deletion. We conclude that the rapid induction of the A(2A)AR mRNA in T cells provides a mechanism for limiting T cell activation and secondary macrophage activation in inflamed tissues.

A2A adenosine receptors (A2AAR) inhibit inflammation, although the mechanisms through which adenosine exerts its effects remain unclear. Although the transfer of regulatory Th cells blocks colitis induced by pathogenic CD45RBhigh Th cells, we show that CD45RBlow or CD25+ Th cells from A2AAR-deficient mice do not prevent disease. Moreover, CD45RBhigh Th cells from A2AAR-deficient mice were not suppressed by control CD45RBlow Th cells. A2AAR agonists suppressed the production of proinflammatory cytokines by CD45RBhigh and CD45RBlow T cells in association with a loss of mRNA stability. In contrast, anti-inflammatory cytokines, including IL-10 and TGF-β, were minimally affected. Oral administration of the A2AAR agonist ATL313 attenuated disease in mice receiving CD45RBhigh Th cells. These data suggest that A2AAR play a novel role in the control of T cell-mediated colitis by suppressing the expression of proinflammatory cytokines while sparing anti-inflammatory activity mediated by IL-10 and TGF-β.

Ischemia reperfusion injury results from tissue damage during ischemia and ongoing inflammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A2A receptors (A2ARs) with the selective agonist 4- {3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]- prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce interferon (IFN)-γ by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1d-mediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-γ elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purified from WT or A2AR KO mice but not IFN-γ KO mice. Additionally, animals with transferred A2AR−/− NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD3 and α-galactosylceramide–triggered production of IFN-γ by NKT cells. These findings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A2AR activation.

Extracellular adenosine binds specifically to a family of four G protein-coupled cell-surface adenosine receptors (ARs). As the activation of the A2AAR modulates the activity of multiple inflammatory cells including neutrophils, macrophages and T lymphocytes, the receptor is considered to be a promising pharmacological target for the treatment of inflammatory disorders. Although adenosine binds nonselectively to all four AR subtypes, A2AAR selective agonists have been developed and shown to inhibit multiple manifestations of inflammatory cell activation including superoxide anion generation, cytokine production and adhesion molecule expression. A2AAR agonists are also vasodilators, but the inhibition of inflammation occurs at low doses that produce few or no cardiovascular side effects. Therefore, the selective activation of the A2AAR by these compounds holds significant potential in the treatment of inflammation.

Ischemia reperfusion injury results from tissue damage during ischemia and ongoing infl ammation and injury during reperfusion. Liver reperfusion injury is reduced by lymphocyte depletion or activation of adenosine A 2A receptors (A 2A Rs) with the selective agonist 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester (ATL146e). We show that NKT cells are stimulated to produce interferon (IFN)-by 2 h after the initiation of reperfusion, and the use of antibodies to deplete NK1.1-positive cells (NK and NKT) or to block CD1dmediated glycolipid presentation to NKT cells replicates, but is not additive to, the protection afforded by ATL146e, as assessed by serum alanine aminotransferase elevation, histological necrosis, neutrophil accumulation, and serum IFN-elevation. Reduced reperfusion injury observed in RAG-1 knockout (KO) mice is restored to the wild-type (WT) level by adoptive transfer of NKT cells purifi ed from WT or A 2A R KO mice but not IFN-KO mice. Additionally, animals with transferred A 2A R −/− NKT cells are not protected from hepatic reperfusion injury by ATL146e. In vitro, ATL146e potently inhibits both anti-CD3 and -galactosylceramide-triggered production of IFN-by NKT cells. These fi ndings suggest that hepatic reperfusion injury is initiated by the CD1d-dependent activation of NKT cells, and the activation of these cells is inhibited by A 2A R activation.

Research indicates that there is a preference for natural v. synthetic products, but the influence of this preference on drug choice in the medical domain is largely unknown. We present 5 studies in which participants were asked to consider a hypothetical situation in which they had a medical issue requiring pharmacological therapy. Participants ( N = 1223) were asked to select a natural, plant-derived, or synthetic drug. In studies 1a and 1b, approximately 79% of participants selected the natural v. synthetic drug, even though the safety and efficacy of the drugs were identical. Furthermore, participants rated the natural drug as safer than the synthetic drug, and as that difference increased, the odds of choosing the natural over synthetic drug increased. In studies 2 and 3, approximately 20% of participants selected the natural drug even when they were informed that it was less safe (study 2) or less effective (study 3) than the synthetic drug. Finally, in study 4, approximately 65% of participants chose a natural over synthetic drug regardless of the severity of a specific medical condition (mild v. severe hypertension), and this choice was predicted by perceived safety and efficacy differences. Overall, these data indicate that there is a bias for natural over synthetic drugs. This bias could have implications for drug choice and usage.

The alpha 4/beta 1 integrin very late antigen-4 (CD49d/CD29) is up-regulated on circulating neutrophils of septic patients. Although no individual agent mimics this effect of sepsis, we now report that following priming of human neutrophils with lipopolysaccharide or tumor necrosis factor alpha (TNF-alpha), addition of formyl-Met-Leu-Phe (fMLP) results in a "stimulated", sepsis-like, four- to fivefold rise in CD49d expression. TNF/fMLP stimulation also produced a similar increase in CD49d-mediated adhesion of neutrophils to a vascular cell adhesion molecule-1 (VCAM-1)-coated surface. Adenosine is a naturally occurring, anti-inflammatory mediator released from injured or inflamed tissues. We observed that stimulated neutrophil CD49d expression was decreased by activation of A(2A) adenosine receptors (A(2A)AR) with the selective agonist 4-[3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2-yl)-9H-purin-2-yl]-prop-2-ynyl]-cyclohexanecarboxylicacid methyl ester (ATL146e; EC(50)=6.4 nM). ATL146e (100 nM) also reduced the fraction of stimulated neutrophils that adhered to VCAM-1 from 38 +/- 6% to 27 +/- 5%. Inhibition of CD49d expression was equally inhibited by ATL146e, added before or after TNF priming, and was reversed by incubation with the A(2A)AR-selective antagonist 4-[2-[7-amino-2-(2-furyl) (1, 2, 4)triazolo(2,3-a) (1, 3, 5)triazin-5-yl-amino]ethyl]-phenol (ZM241385; 100 nM). A suboptimal ATL146e concentration (1 nM) combined with the type IV phosphodiesterase inhibitor rolipram (100 nM) synergistically decreased stimulated CD49d expression by >50%. The cyclic adenosine monophosphate (cAMP)-dependent kinase [protein kinase A (PKA)] inhibitor H-89 (10 microM) reversed the effect of ATL146e on stimulated CD49d expression. Other means of increasing cAMP in neutrophils also decreased stimulated CD49d expression. We conclude that adenosine binding to A(2A)AR counteracts stimulation of neutrophil CD49d integrin expression and neutrophil binding to VCAM-1 via a cAMP/PKA-mediated pathway.