Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) levels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern and microarray hybridizations. MPK4 kinase activity is required to repress SAR, as an inactive MPK4 form failed to complement mpk4. Analysis of mpk4 expressing the SA hydroxylase NahG and of mpk4/npr1 double mutants indicated that SAR expression in mpk4 is dependent upon elevated SA levels but is independent of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression.

Animal SGT1 is a component of Skp1-Cullin-F-box protein (SCF) ubiquitin ligases that target regulatory proteins for degradation. Mutations in one (SGT1b) of two highly homologous Arabidopsis SGT1 genes disable early plant defenses conferred by multiple resistance (R) genes. Loss of SGT1b function in resistance is not compensated for by SGT1a. R genes differ in their requirements for SGT1b and a second resistance signaling gene, RAR1, that was previously implicated as an SGT1 interactor. Moreover, SGT1b and RAR1 contribute additively to RPP5-mediated pathogen recognition. These data imply both operationally distinct and cooperative functions of SGT1 and RAR1 in plant disease resistance.

We have identified the Arabidopsis ortholog of barley RAR1 as a component of resistance specified by multiple nucleotide binding/Leu-rich repeat resistance ( R ) genes recognizing different bacterial and oomycete pathogen isolates. Characterization of partially and fully defective rar1 mutations revealed that wild-type RAR1 acts as a rate-limiting regulator of early R gene-triggered defenses, determining the extent of pathogen containment, hypersensitive plant cell death, and an oxidative burst at primary infection sites. We conclude that RAR1 defense signaling function is conserved between plant species that are separated evolutionarily by 150 million years. RAR1 encodes a protein with two zinc binding (CHORD) domains that are highly conserved across eukaryotic phyla, and the single nematode CHORDcontaining homolog, Chp , was found previously to be essential for embryo viability. An absence of obvious developmental defects in null Arabidopsis rar1 mutants favors the notion that, in contrast, RAR1 does not play a fundamental role in plant development. INTRODUCTIONIn countering attack by microbial pathogens or insects, plants have evolved resistance ( R ) genes that specifically recognize corresponding pathogen avirulence ( avr ) genes to trigger plant defenses (Dangl and Jones, 2001). Two plant R gene-encoded proteins, tomato Pto and rice Pi-ta, have been shown to interact physically with their pathogen Avr counterparts, AvrPto and Avr-Pita, respectively, in in vitro assays (Scofield et al., 1996;Tang et al., 1996;Jia et al., 2000). Other plant R proteins may associate with pathogen Avr proteins indirectly within a protein complex . In the absence of a corresponding R gene, the pathogen is able to colonize its host. Some Avr proteins are virulence factors that facilitate pathogen growth or interfere with basal plant defenses (Nimchuk et al., 2000;Staskawicz et al., 2001). R-Avr protein recognition commonly involves localized programmed plant cell death (the hypersensitive response [HR]), an oxidative burst producing reactive oxygen intermediates (ROI), and the accumulation of salicylic acid (SA), a phenolic molecule necessary for the induction of systemic immunity (systemic acquired resistance) (Feys and Parker, 2000).Plant R proteins share a limited repertoire of motifs with animal proteins that control innate immunity (Staskawicz et al., 2001). The most prevalent R gene class encodes predicted cytosolic proteins with a central nucleotide binding (NB) domain and C-terminal Leu-rich repeats (LRRs) (Dangl and Jones, 2001). At least one NB-LRR-type protein, Arabidopsis RPM1, is tethered to the plasma membrane, where it may encounter bacterial Avr proteins that are secreted into the plant cell (Boyes et al., 1998;Nimchuk et al., 2000). NB-LRR proteins fall into two subclasses based on their different N-terminal motifs. One group possesses an N-terminal coiled-coil (CC) domain. The second group has N-terminal similarity to the cytoplasmic Toll Interleukin-1 Receptor (TIR) domains of human and Drosophila Toll-like r...

• In the clinic, all oral antiplatelet medicines have a risk of bleeding complications.• We present an antidote for ticagrelor that reverses its antiplatelet effect in human platelet-rich plasma and its bleeding effect in mice.Ticagrelor is a direct-acting reversibly binding P2Y 12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y 12 . Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 59-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures. (Blood. 2015;125(22):3484-3490)

Highlights d ''Re-epitoping'' of an existing antibody by in silico design d Engineering a functional antibody to IL-17A d Crystal structure of the antibody-antigen complex confirms the targeted epitope

The RAF-mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 (RAF-MEK1/2-ERK1/2) pathway is activated in many human tumours and can protect cells against growth factor deprivation; however, most such studies have relied upon overexpression of RAF or MEK constructs that are not found in tumours. Here we show that expression of the endogenous BRAFV600E allele in mouse embryonic fibroblasts from conditional knock-in transgenic mice activates ERK1/2, represses the BH3-only protein BIM and protects cells from growth factor withdrawal. Human colorectal cancer (CRC) cell lines harbouring BRAFV600E are growth factor independent for the activation of ERK1/2 and survival. However, treatment with the MEK1/2 inhibitors U0126, PD184352 or the novel clinical candidate AZD6244 (ARRY-142886) overcomes growth factor independence, causing CRC cell death. BIM is de-phosphorylated and upregulated following MEK1/2 inhibition in all CRC cell lines studied and knockdown of BIM reduces cell death, indicating that repression of BIM is a major part of the ability of BRAFV600E to confer growth factor-independent survival. We conclude that a single endogenous BRAFV600E allele is sufficient to repress BIM and prevent death arising from growth factor withdrawal, and CRC cells with BRAFV600E mutations are addicted to the ERK1/2 pathway for repression of BIM and growth factor-independent survival.

The cytotoxic T-lymphocyte (CTL) response plays an important role in controlling the severity and duration of viral infections. Immunization by direct in vivo administration of retroviral vector particles represents an efficient means of introducing and expressing genes and, subsequently, the proteins they encode in vivo in mammalian cells. In this manner foreign proteins can be provided to the endogenous, class I major histocompatibility complex antigen presentation pathway leading to CTL activation. A nonreplicating recombinant retroviral vector, encoding the human immunodeficiency virus type 1 (HIV-1) IIIB envelope and rev proteins, has been developed and examined for stimulation of immune responses in mouse, rhesus macaque, and baboon models. Animals were immunized by direct intramuscular injection of the retroviral vector particles. Vector-immunized mice, macaques, and baboons generated long-lived CD8+, major histocompatibility complex-restricted CTL responses that were HIV-1 protein specific. The CTL responses were found to be dependent on the ability of the retroviral vector to transduce cells. The vector also elicited HIV-1 envelope-specific antibody responses in mice and baboons. These studies demonstrate the ability of a retroviral vector encoding HIV-1 proteins to stimulate cellular and humoral immune responses and suggest that retrovector immunization may provide an effective means of inducing or augmenting CTL responses in

Molecular genetic approaches were adopted in the model crucifer, Arabidopsis thaliana, to unravel components of RPP5- and RPP1-mediated disease resistance to the oomycete pathogen, Peronospora parasitica. The products of RPP5 and three genes comprising the RPP1 complex locus belong to a major subclass of nucleotide-binding/leucine-rich repeat (NB-LRR) resistance (R) protein that has amino-terminal homology to the cytoplasmic domains of Drosophila and mammalian Toll and interleukin-1 family receptors (the so called 'TIR' domain). Similarities in the domain architecture of these proteins and animal regulators of programmed cell death have also been observed. Mutational screens revealed a number of genes that are required for RPP5-conditioned resistance. Among these are EDS1 and PAD4. Both EDS1 and PAD4 precede the function of salicylic acid-mediated plant responses. The EDS1 and PAD4 genes were cloned and found to encode proteins with similarity to the catalytic site of eukaryotic lipases, suggesting that they may function by hydrolysing a lipid-based substrate.