Recognition of an avirulent pathogen stimulates an oxidative burst generating O2- and H2O2, and these reactive oxygen intermediates (ROIs) cue the induction of defense genes and cell death in the development of a restricted lesion. This localized hypersensitive response (HR) is accompanied by the development of systemic acquired resistance to virulent pathogens. Here we show that inoculation of Arabidopsis leaves with avirulent Pseudomonas syringae induces secondary oxidative bursts in discrete cells in distant tissues, leading to low-frequency systemic micro-HRs. The primary oxidative burst induces these systemic responses, and both the primary burst and the secondary microbursts are required for systemic immunity. Hence, ROIs mediate a reiterative signal network underlying systemic as well as local resistance responses.

Programmed cell death (PCD) occurs in plants during development and defense, but the processes and mechanisms are not yet defined. Culture of carrot single cells at a cell density of <104 cells ml−1 activates a cell death process involving condensation and shrinkage of the cytoplasm and nucleus and fragmentation of the DNA. Modest abiotic stress treatments also cause cell condensation and shrinkage and the formation of DNA fragments, but the same abiotic stresses at high levels cause rapid necrosis with cell swelling and lysis. The common morphological features of cells dying at low cell density and following modest abiotic stress treatments suggest that these features reveal a PCD pathway in carrot. The addition of a cell‐conditioned growth medium allows cells at low cell density to remain alive, demonstrating that cell‐derived signal molecules suppress a pathway that is otherwise induced by default. Differences in the morphology of the dead cells suggest that proteolysis during PCD differs in detail in plants and animals; however, these findings show that plants, like animals, can control PCD by social signaling, and imply that the mechanism of PCD in plants and animals may be similar. Consistent with this, manipulation of signal pathway intermediates that regulate PCD in animals shows that Ca2+ and protein phosphorylation events are PCD pathway intermediates in carrot.

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(2011) Rational identification of an optimal antibody mixture for targeting the epidermal growth factor receptor, mAbs, 3:6, 584-595,

Due to technical limitations, little knowledge exists on the composition of Ag-specific polyclonal Ab responses. Hence, we here present a molecular analysis of two representative human Ab repertoires isolated by using a novel single-cell cloning approach. The observed genetic diversity among tetanus toxoid-specific plasma cells indicate that human polyclonal repertoires are limited to the order of 100 B cell clones and hypermutated variants thereof. Affinity and kinetic binding constants are log-normally distributed, and median values are close to the proposed affinity ceilings for positive selection. Abs varied a million-fold in affinity but were restricted in their off-rates with an upper limit of 2 × 10−3 s−1. Identification of Abs of high affinity without hypermutations in combination with a modest effect of hypermutations on observed affinity increases indicate that Abs selected from the naive repertoire are not only of low affinity but cover a relatively large span in affinity, reaching into the subnanomolar range.

HER2 plays an important role in the development and maintenance of the malignant phenotype of several human cancers. As such, it is a frequently pursued therapeutic target and two antibodies targeting HER2 have been clinically approved, trastuzumab and pertuzumab. It has been suggested that optimal inhibition of HER2 is achieved when utilizing two or more antibodies targeting nonoverlapping epitopes. Superior clinical activity of the trastuzumab plus pertuzumab combination in metastatic breast cancer supports this hypothesis. Because trastuzumab and pertuzumab were not codeveloped, there may be potential for further optimizing HER2 targeting. The study herein evaluated functional activity of anti-HER2 antibody combinations identifying optimal epitope combinations that provide efficacious HER2 inhibition. High-affinity antibodies to all four extracellular domains on HER2 were identified and tested for ability to inhibit growth of different HER2-dependent tumor cell lines. An antibody mixture targeting three HER2 subdomains proved to be superior to trastuzumab, pertuzumab, or a combination in vitro and to trastuzumab in two in vivo models. Specifically, the tripartite antibody mixture induced efficient HER2 internalization and degradation demonstrating increased sensitivity in cell lines with HER2 amplification and high EGFR levels. When compared with individual and clinically approved mAbs, the synergistic tripartite antibody targeting HER2 subdomains I, II, and IV demonstrates superior anticancer activity. Mol Cancer Ther; 14(3); 669-80. Ó2015 AACR.

In this study, a respiration-deficient Chinese hamster cell line with a defect in succinate dehydrogenase activity is shown to result from a single base change in a codon in the coding sequence for the membrane anchor protein C II-3 (also referred to as QPs-1). A premature translation stop results in the truncation of 33 amino acids from the C terminus. Bovine cDNA encoding this peptide complements the mutation. There is about 82% identity between these two mammalian proteins. The gene for C II-3 was mapped on human chromosome 1, and because it is also found on minichromosomes characterized by our laboratory, we can localize it on the short arm within 1-2 megabases from the centromere.A series of respiration-deficient Chinese hamster cell mutants isolated by our laboratory can grow normally in tissue culture as long as an adequate supply of glucose is available for glycolysis (1-3). Depletion of glucose leads to rapid cessation of growth and cell death. The mutants were grouped into seven complementation groups by somatic cell fusions (1), and one, represented by mutant CCL16-B9, was characterized to be almost completely deficient in succinate dehydrogenase activity (SDH) 1 (4,5). This enzyme, which links the reactions of the Krebs cycle to the electron transport chain, is part of a complex of four polypeptides (complex II) in the inner mitochondrial membrane. The active site for the substrate is on the flavoprotein (Fp, 70 kDa), while the iron-sulfur protein (Ip, 27 kDa) is believed to link it to two small integral membrane proteins (C II-3 and C II-4 , 15 and 7-9 kDa, respectively). A b-type heme group is associated with the membrane proteins. Electrons from the oxidation of the substrate in the mitochondrial matrix pass from the Fp via three non-heme iron-sulfur centers in the Ip , , ) to the integral membrane proteins and from there to ubiquinone. The function of the heme group associated with the membrane proteins is not completely clear (see Refs. 6 -9 for reviews).All four peptides are encoded by nuclear genes in eukaryotic organisms. Thus, the precursor polypeptides are synthesized in the cytosol and subsequently or concurrently imported into mitochondria. Following processing to their mature forms, the biogenesis of a functional complex II requires covalent attachment of flavin to the largest subunit (Fp) (10), formation of the three non-heme iron-sulfur clusters in the Ip subunit, and assembly of the heme with the membrane anchor proteins. Although this complex is the smallest and least intricate of the electron transport complexes in the inner mitochondrial membrane, much remains to be learned about the mechanism or pathway of its assembly.With the Fp-Ip complex dissociated from the membrane by chaotropic ions, SDH activity can be assayed with artificial electron acceptors such as tetrazolium ions, phenazine methosulfate and dichlorophenol-indophenol, or ferricyanide. Based on the absence of activity in the mutant CCL16-B9, we hypothesized that the defective protein was either the Ip or the Fp subun...

Symplex is an antibody discovery technology that identifies fully human antigen-specific antibody repertoires directly from plasma cells. The technology utilizes reverse transcription and overlap extension polymerase chain reaction performed on single-cell-sorted plasma cells, whereby the heavy- and light-chain cognate pairing of the antibodies is maintained. The isolated antibodies from a plasma cell donor reflect the diversity, affinity, and selectivity of the donor antibody repertoire, making the technology an ideal tool for identifying drug leads and studying the development of human antibody repertoires.