Comparative Large-Scale Analysis of Interactions between Several Crop Species and the Effector Repertoires from Multiple Pathovars of Pseudomonas and Ralstonia

Bacterial plant pathogens manipulate their hosts by injection of numerous effector proteins into host cells via type III secretion systems. Recognition of these effectors by the host plant leads to the induction of a defense reaction that often culminates in a hypersensitive response manifested as cell death. Genes encoding effector proteins can be exchanged between different strains of bacteria via horizontal transfer, and often individual strains are capable of infecting multiple hosts. Host plant species express diverse repertoires of resistance proteins that mediate direct or indirect recognition of bacterial effectors. As a result, plants and their bacterial pathogens should be considered as two extensive coevolving groups rather than as individual host species coevolving with single pathovars. To dissect the complexity of this coevolution, we cloned 171 effector-encoding genes from several pathovars of Pseudomonas and Ralstonia. We used Agrobacterium tumefaciens-mediated transient assays to test the ability of each effector to induce a necrotic phenotype on 59 plant genotypes belonging to four plant families, including numerous diverse accessions of lettuce (Lactuca sativa) and tomato (Solanum lycopersicum). Known defense-inducing effectors (avirulence factors) and their homologs commonly induced extensive necrosis in many different plant species. Nonhost species reacted to multiple effector proteins from an individual pathovar more frequently and more intensely than host species. Both homologous and sequence-unrelated effectors could elicit necrosis in a similar spectrum of plants, suggesting common effector targets or targeting of the same pathways in the plant cell.     

Longitudinal impact of physical activity on lipid profiles in middle-aged adults: the Atherosclerosis Risk in Communities Study

Evidence exists that increased levels of physical activity decrease the population burden of cardiovascular disease (CVD). Although risk factors for CVD, including plasma lipids and lipoproteins, have been associated with physical activity, studies including a sizeable number of minority participants are lacking. Our purpose was to interrogate the longitudinal effect of physical activity on plasma lipids and lipoproteins in the African American and white participants of the Atherosclerosis Risk in Communities (ARIC) Study. Nine years of follow-up data on 8,764 individuals aged 45–64 years at baseline were used in linear mixed-effects models to estimate the association between increases in baseline physical activity on mean change in HDL, LDL, total cholesterol, and triglyceride levels. Increases in the level of activity were associated with increases in HDL in all strata and decreases in triglycerides among white participants. Physical activity was associated with LDL in all women, while the association with total cholesterol was limited to African American women. This study is one of the few to investigate the effect of physical activity on lipids and lipoproteins in a race- and sex-specific manner. Overall our results highlight the importance of physical activity on plasma lipid profiles and provide evidence for novel differential associations.     

An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.Polyunsaturated lipids in biological membranes are particularly reactive targets for oxygen radicals (1–3). Lipid peroxidation, the chain reaction of peroxyl radicals that is a consequence of oxidative stress, is thought to be involved in human diseases such as cancer, atherosclerosis, and neurodegenerative disorders (4–8). A variety of electrophilic compounds are byproducts of lipid peroxidation, 4-hydroxynon-2-enal (HNE)¹ being a particularly toxic electrophile (9–12) that forms mutagenic DNA adducts (13–15). HNE and other lipid-derived electrophiles also form protein modifications, and some of these adducts have been characterized on a limited number of proteins and peptides by mass spectrometry (MS) and in tissues by antibody-based methods (16). Until recently, relatively little was known about the target selectivity of oxidant-derived electrophiles in proteins, the relative reactivities of different amino acid targets, and the properties of the adducts. We recently described the application of a post-labeling strategy in which biotin hydrazide was used to biotinylate carbonyl-containing adducts formed by HNE in RKO cells (17). When combined with shotgun proteome analysis of the captured proteins, this approach provided a global perspective on patterns of protein damage by a prototypical lipid electrophile. However, biotin hydrazide labels many carbonyls, thus generating a background inventory derived from endogenous carbonyls, which is difficult to characterize and may mask more subtle patterns of selectivity in protein adduction. Moreover, the biotin hydrazide approach can only capture adducts with a reactive carbonyl group.To deal with these limitations, we have explored labeled electrophile probes and selective adduct capture chemistries (18). We recently reported that 4-hydroxynon-2-en-8-ynal, alkynyl-HNE (aHNE), can be used as an HNE surrogate in whole cells to isolate proteins that are adducted by this electrophile (19). aHNE displays similar toxicity in RKO cells as does HNE, and studies with model peptides and isolated proteins show that HNE and the alkynyl surrogate display similar chemistry in reactions with protein nucleophiles. For example, reaction of aHNE with proteins or peptides followed by sodium borohydride reduction gives Michael and imine adducts as shown in structures 1 and 2. This same chemistry is observed for HNE itself.Reaction of cellular aHNE protein adducts with an azido-biotin reagent followed by capture of the triazole cycloadducts on streptavidin beads permitted a number of adducted proteins to be identified by shotgun proteomics (19). Thus, tryptic digestion of the proteins pulled down by means of the alkyne affinity tag generates mixtures that include adducted peptides such as 3 as well as unmodified peptides. The chemistry associated with the alkynyl electrophile works as planned, but the strategy suffers from two significant drawbacks. First, nonspecific protein binding to the streptavidin beads complicates the identification of adducted proteins and second, biotinylated peptides such as 3 generated in the sequence have MS/MS fragmentation patterns that do not permit the ready identification of the amino acid adduction site on the peptide. The biotin appendage is a major site of positive charge localization in the MS/MS experiment, and the formation of characteristic b and y ions is frequently not sufficient for peptide identification.Open in a separate windowWe report here a strategy that couples the alkynyl electrophile azido-biotin capture for the isolation of adducted protein with a photochemical release of the adduct from streptavidin. This approach reduces the protein nonspecific binding problem because release from the bead requires only a photochemical event, and it permits the identification of specific nucleophilic sites on proteins that are modified by reactive electrophiles. By the application of this strategy to capture both adducted proteins and peptides, we have identified plasma protein targets of the probes and also mapped several nucleophilic sites on the plasma protein ApoA1 that are modified by aHNE.     

Mesenchymal stromal cells alone or expressing interferon-β suppress pancreatic tumors in vivo,an effect countered by anti-inflammatory treatment

Background aimsBecause of the inflammatory nature and extensive stromal compartment in pancreatic tumors, we investigated the role of mesenchymal stromal cells (MSC) to engraft selectively in pancreatic carcinomas and serve as anti-tumor drug delivery vehicles to control pancreatic cancer progression.MethodsHuman pancreatic carcinoma cells, PANC-1, expressing renilla luciferase were orthotopically implanted into SCID mice and allowed to develop for 10 days. Firefly luciferase-transduced MSC or MSC expressing interferon (IFN)-β were then injected intraperitoneally weekly for 3 weeks. Mice were monitored by bioluminescent imaging for expression of renilla (PANC-1) and firefly (MSC) luciferase.ResultsMSC selectively homed to sites of primary and metastatic pancreatic tumors and inhibited tumor growth (P = 0.032). The production of IFN-β within the tumor site by MSC–IFN-β further suppressed tumor growth (P = 0.0000083). Prior studies indicated that MSC home to sites of inflammation; therefore, we sought to alter the tumor microenvironment through treatment with a potent anti-inflammatory agent. After treatment, inflammation-associated mediators were effectively down-regulated, including NFκB, vascular endothelial growth factor (VEGF) and interleukin (IL)-6 as well as chemokines involved in MSC migration (CCL3 and CCL25). Treatment with the anti-inflammatory agent CDDO-Me before and after MSC–IFN-β injections resulted in reduction of MSC in the tumors and reversed the positive effect of tumor inhibition by MSC–IFN-β alone (P = 0.041).ConclusionsThese results suggest that MSC exhibit innate anti-tumor effects against PANC-1 cells and can serve as delivery vehicles for IFN-β for the treatment of pancreatic cancer. However, these beneficial effects may be lost in therapies combining MSC with anti-inflammatory agents.     

Role of the p38 mitogen-activated protein kinase pathway in the generation of the effects of imatinib mesylate (STI571) in BCR-ABL-expressing cells

Imatinib mesylate (STI571), a specific inhibitor of the BCR-ABL tyrosine kinase, exhibits potent antileukemic effects in vitro and in vivo. Despite the well established role of STI571 in the treatment of chronic myelogenous leukemia, the precise mechanisms by which inhibition of BCR-ABL tyrosine kinase activity results in generation of antileukemic responses remain unknown. In the present study we provide evidence that treatment of CML-derived BCR-ABL-expressing leukemia cells with STI571 results in activation of the p38 mitogen-activated protein (MAP) kinase signaling pathway. Our data indicate that STI571 induces phosphorylation of the p38 and activation of its kinase domain, in KT-1 cells and other BCR-ABL-expressing cell lines. We also identify the kinases MAP kinase-activated protein kinase-2 and Msk1 as two downstream effectors of p38, activated during inhibition of BCR-ABL activity by STI571. Importantly, pharmacological inhibition of p38 reverses the growth inhibitory effects of STI571 on primary leukemic colony-forming unit granulocyte/macrophage progenitors from patients with CML. Altogether, our data establish that activation of the p38 MAP kinase signaling cascade plays an important role in the generation of the effects of STI571 on BCR-ABL-expressing cells. They also suggest that, in addition to activation of mitogenic pathways, BCR-ABL promotes leukemogenesis by suppressing the function of growth inhibitory signaling cascades.     

Thermotolerant guard cell protoplasts of tree tobacco do not require exogenous hormones to survive in culture and are blocked from reentering the cell cycle at the G1-to-S transition

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.     

Two cDNAs from the purple sea urchin,<Emphasis Type="Italic"> Strongylocentrotus purpuratus</Emphasis>, encoding mosaic proteins with domains found in factor H,factor I,and complement components C6 and C7

The vertebrate complement system is composed of about 30 serum and cell surface proteins that make up three activation pathways, a lytic pathway, and a set of proteins that regulate complement. Regulatory proteins are required for host protection against autologous complement attack and to control the amplification feedback loop of the alternative pathway. Purple sea urchin, Strongylocentrotus purpuratus, homologues of complement C3 (SpC3) and factor B (SpBf) have been identified, suggesting the presence of an alternative complement pathway. This implies that echinoderms require a complement regulatory system for the same reasons that it is required in higher vertebrates. Two cDNAs, Sp5 and Sp5013, have been characterized from coelomocytes and the deduced structures of the encoded mosaic proteins, SpCRL (S. purpuratus complement related protein, long form) and SpCRS (short form), have domains that are also found in regulatory proteins such as factor H and factor I and the terminal pathway components C6 and C7. These domains include multiple short consensus repeats, a fucolectin domain, Ser/Thr/Pro-rich regions, a Cys-rich region, and a factor I-membrane attack complex domain. The genes are constitutively expressed in all tissues of the sea urchin and are not induced in response to immune challenge. Multiple bands of varying intensity on both genome blots and RNA blots suggest that Sp5 and Sp5013 are members of a small gene family and that they might undergo alternative splicing. Based on the domains present in SpCRL and SpCRS, they might be either examples of complement regulatory proteins or members of the terminal pathway of complement.