HVCN1 modulates BCR signal strength via regulation of BCR-dependent generation of reactive oxygen species.

Voltage-gated proton currents regulate generation of reactive oxygen species (ROS) in phagocytic cells. In B cells, stimulation of the B cell antigen receptor (BCR) results in the production of ROS that participate in B cell activation, but the involvement of proton channels is unknown. We report here that the voltage-gated proton channel HVCN1 associated with the BCR complex and was internalized together with the BCR after activation. BCR-induced generation of ROS was lower in HVCN1-deficient B cells, which resulted in attenuated BCR signaling via impaired BCR-dependent oxidation of the tyrosine phosphatase SHP-1. This resulted in less activation of the kinases Syk and Akt, impaired mitochondrial respiration and glycolysis and diminished antibody responses in vivo. Our findings identify unanticipated functions for proton channels in B cells and demonstrate the importance of ROS in BCR signaling and downstream metabolism.

Targeting CD205 with the antibody drug conjugate MEN1309/OBT076 is an active new therapeutic strategy in lymphoma models.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

A death effector domain chain DISC model reveals a crucial role for caspase-8 chain assembly in mediating apoptotic cell death.

Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death.

Testing the joint effects hypothesis of elemental defense using Spodoptera exigua.

Metal hyperaccumulation may be an elemental defense, in which high concentrations of a metal in plant tissues decrease herbivore survival or growth rate. The Joint Effects Hypothesis suggests that a combination of metals, or a combination of a metal with an organic compound, may have an enhanced defensive effect. The enhancement may be additive or synergistic: in either case the concentration of a particular metal necessary to provide a defensive benefit for the plant is lowered. We tested the Joint Effects Hypothesis using Spodoptera exigua (beet armyworm) neonates fed artificial diets. Metal + metal experiments utilized diets amended with metal pairs, using four metals commonly hyperaccumulated by plants (Co, Cu, Ni, and Zn). We also conducted metal + organic compound experiments, pairing each metal with nicotine, mustard seed powder, or tannic acid. We tested for joint effects using both lethal (LC20 levels) and sublethal concentrations (10-25 % reduced larval weight) of the chemicals tested. For all experiments, either additive or synergistic effects were found. Of the metal + metal pairs tested, three (Co + Cu, Cu + Zn, and Ni + Zn) were synergistic in lethal concentration tests and only Co + Cu was synergistic in sublethal tests. For metal + organic combination lethal tests, synergism occurred for all combinations except for Co or Ni + nicotine, Ni + mustard seed powder, and Zn + nicotine. For sublethal tests, Zn + all three organic chemicals, Co + mustard seed powder or tannic acid, and Cu + nicotine, were synergistic. These results support the Joint Effects Hypothesis, suggesting that metals combined with other metals or organic compounds may be more effective against herbivores than individual metals.

Transfer of heavy metals through terrestrial food webs: a review.

Heavy metals are released into the environment by both anthropogenic and natural sources. Highly reactive and often toxic at low concentrations, they may enter soils and groundwater, bioaccumulate in food webs, and adversely affect biota. Heavy metals also may remain in the environment for years, posing long-term risks to life well after point sources of heavy metal pollution have been removed. In this review, we compile studies of the community-level effects of heavy metal pollution, including heavy metal transfer from soils to plants, microbes, invertebrates, and to both small and large mammals (including humans). Many factors contribute to heavy metal accumulation in animals including behavior, physiology, and diet. Biotic effects of heavy metals are often quite different for essential and non-essential heavy metals, and vary depending on the specific metal involved. They also differ for adapted organisms, including metallophyte plants and heavy metal-tolerant insects, which occur in naturally high-metal habitats (such as serpentine soils) and have adaptations that allow them to tolerate exposure to relatively high concentrations of some heavy metals. Some metallophyte plants are hyperaccumulators of certain heavy metals and new technologies using them to clean metal-contaminated soil (phytoextraction) may offer economically attractive solutions to some metal pollution challenges. These new technologies provide incentive to catalog and protect the unique biodiversity of habitats that have naturally high levels of heavy metals.

Exploring lower limits of plant elemental defense by cobalt, copper, nickel, and zinc.

Elemental defense is a relatively newly recognized phenomenon in which plants use elements present in their tissue to reduce damage by herbivores or pathogens. In the present study, neonates of the generalist herbivore, Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae), were fed artificial diets amended with varying concentrations of Co, Cu, Ni, and Zn that are hyperaccumulated by plants to determine minimum lethal concentrations (MLC) and minimum sublethal concentrations (MSC) for each metal. MLC values (dry mass) for Co (45 µg/g), Ni (230 µg/g), and Zn (280 µg/g) were below published minimum hyperaccumulator levels. MSC levels (dry mass) for Co (15 µg/g), Ni (140 µg/g), and Zn (200 µg/g) were at concentrations lower than published minimum accumulator levels. Furthermore, both MLC and MSC values for Zn were within normal tissue concentrations. These results indicate that elemental defense for Co, Ni, and Zn may be effective at concentrations lower than hyperaccumulator levels and so may be more widespread than previously believed.

Biotransfer, bioaccumulation and effects of herbivore dietary Co, Cu, Ni, and Zn on growth and development of the insect predator Podisus maculiventris (Say).

Increased metal availability in the environment can be detrimental for the growth and development of all organisms in a food web. In part, this toxicity is due to biotransfer or bioaccumulation of metals between trophic levels. We evaluated the survival, growth, and development of a generalist Hemipteran predator (Podisus maculiventris) when fed herbivorous prey (Spodoptera exigua) reared on artificial diet amended with Cu, Zn, Ni, and Co. Predator nymphs were fed S. exigua larvae raised on diet amended with sublethal (Minimum Sublethal Concentration or MSC) or lethal (Minimum Lethal Concentration or MLC) concentrations of each metal, as well as control diet. We determined if metals were biotransferred or bioaccumulated from the diet to herbivore and predator, as well as if predator growth or survival was affected by herbivore diet. Podisus maculiventris fed herbivores raised on MLC levels of both Cu and Zn took significantly longer to mature to adults, whereas their overall survival was not affected by prey diet metal concentration for any metal. Adult weights were significantly reduced for predators raised on herbivores reared on diets amended with the MLC of Cu and Zn. Copper and Zn were bioaccumulated from diet to herbivore and from herbivore to predator, whereas Ni was biotransferred (although concentrations decreased as trophic level increased). The pattern for Co was more complex, with biotransfer the main outcome. Our results show that availability of metals in a food web can affect growth and development of a hemipteran predator, and that metals are transferred between trophic levels, with metal-specific biotransfer and bioaccumulation outcomes.

Protein profiling of plasma membranes defines aberrant signaling pathways in mantle cell lymphoma.

We used shotgun proteomics to identify plasma membrane and lipid raft proteins purified from B cells obtained from mantle cell lymphoma (MCL) patients in leukemic phase. Bioinformatics identified 111 transmembrane proteins, some of which were profiled in primary MCL cases, MCL-derived cell lines, and normal B cells using RT-PCR and Western blotting. Several transmembrane proteins, including CD27, CD70, and CD31 (PECAM-1), were overexpressed when compared with normal B cells. CD70 was up-regulated (>10-fold) in three of five MCL patients along with its cognate receptor CD27, which was up-regulated (4-9-fold) in five of five patients, suggesting that MCL cells may undergo autocrine stimulation via this signaling pathway. Activated calpain I and protein kinase C betaII were also detected in the plasma membranes, suggesting that these proteins are constitutively active in MCL. Protein kinase C betaII has been associated with lipid rafts, and shotgun proteomics/protein profiling revealed that key lipid raft proteins, raftlin (four of five patients) and CSK (C-terminal Src kinase)-binding protein (Cbp)/phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG) (four of four patients) were down-regulated in MCL. Levels of other known lipid raft proteins, such as Lyn kinase and flotillin 1, were similar to normal B cells. However, 5-lipoxygenase (5-LO), a key enzyme in leukotriene biosynthesis, was associated with lipid rafts and was up-regulated approximately 7-fold in MCL compared with normal B cells. Significantly inhibitors of 5-LO activity (AA861) and 5-LO-activating protein (FLAP) (MK886, its activating enzyme) induced apoptosis in MCL cell lines and primary chronic lymphocytic leukemia cells, indicating an important role for the leukotriene biosynthetic pathway in MCL and other B cell malignancies. Thus, using shotgun proteomics and mRNA and protein expression profiling we identified a subset of known and unknown transmembrane proteins with aberrant expression in MCL plasma membranes. These proteins may play a role in the pathology of the disease and are potential therapeutic targets in MCL.

Heavy metal pollutants and chemical ecology: exploring new frontiers.

Heavy metals are an important class of pollutants with both lethal and sublethal effects on organisms. The latter are receiving increased attention, as these may have harmful ecological outcomes. For example, recent explorations of heavy metals in freshwater habitats reveal that they can modify chemical communication between individuals, resulting in "info-disruption" that can impact ecological relationships within and between species. Info-disruption can affect animal behavior and social structure, which in turn can modify both intraspecies and interspecies interactions. In terrestrial habitats, info-disruption by metals is not well studied, but recent demonstrations of chemical signaling between plants via both roots and volatile organic molecules provide potential opportunities for info-disruption. Metals in terrestrial habitats also can form elemental plant defenses, in which they can defend a plant against natural enemies. For example, hyperaccumulation of metals by terrestrial plants has been shown to provide defensive benefits, although in almost all known cases the metals are not anthropogenic pollutants but are naturally present in soils inhabited by these plants. Info-disruption among microbes is another arena in which metal pollutants may have ecological effects, as recent discoveries regarding quorum sensing in bacteria provide an avenue for metals to affect interactions among bacteria or between bacteria and other organisms. Metal pollutants also may influence immune responses of organisms, and thus affect pathogen/host relationships. Immunomodulation (modification of immune system function) has been tied to some metal pollutants, although specific metals may boost or reduce immune system function depending on dose. Finally, the study of metal pollutants is complicated by their frequent occurrence as mixtures, either with other metals or with organic pollutants. Most studies of metal pollutants focus on single metals and therefore oversimplify complex field conditions. Study of pollutant impacts on chemical ecology also are difficult due to the necessity of studying effects at varying ecological scales: "dynamic scaling" of chemical ecology studies is rarely done completely. It is clear that much remains to be learned about how heavy metal pollution impacts organisms, and that exciting new research frontiers are available for experimental exploration.

Proteomic analysis of cell surface membrane proteins in leukemic cells.

Plasma membrane proteins play a key role in cellular processes such as migration, adhesion, and cell survival. The comprehensive annotation of the leukemic cell plasma membrane proteome allows the identification of proteins that may be involved in the pathogenesis of disease and may provide novel therapeutic targets. The identification of known adhesion molecules or novel proteins with similar attributes to adhesion molecules provides the starting point for the generation of hypothesis on the role of these proteins in adhesion processes. In order to identify these novel proteins, we have developed a proteomics methodology using purified plasma membranes prepared from human leukemic cells.

Dynamics of Ni-based defence and organic defences in the Ni hyperaccumulator, Streptanthus polygaloides (Brassicaceae).

Plants use chemical defences to reduce damage from herbivores and the effectiveness of these defences can be altered by biotic and abiotic factors, such as herbivory and soil resource availability. Streptanthus polygaloides, a nickel (Ni) hyperaccumulator, possesses both Ni-based defences and organic defences (glucosinolates), but the extent to which these defences interact and respond to environmental conditions is unknown. S. polygaloides plants were grown on high-Ni and low-Ni soil and concentrations of Ni and glucosinolates were compared with those of the congeneric non-hyperaccumulator, S. insignus spp. insignus, grown under the same conditions. Ni contents were highest (4000 µg g-1 dry tissue) in S. polygaloides plants grown on high-Ni soil. Glucosinolate content was significantly higher in S. insignus than in S. polygaloides suggesting that plants defended by Ni produce a lower concentration of organic defences. In a separate experiment, high-Ni S. polygaloides plants were exposed to simulated herbivory or live folivores to determine the inducibility of Ni-based and organic defences. Contents of Ni were not affected by either herbivory treatment, whereas glucosinolate concentrations were >30% higher in damaged plants. We concluded that the Ni-based defence of S. polygaloides is not induced by herbivory.

The ecological significance of nickel hyperaccumulation: a plant chemical defense.

Nickel hyperaccumulating plants have more than 1000 mg Ni kg-1 dry weight when grown on nickel-bearing soils. We hypothesized that Ni hyperaccumulation could serve as a chemical defense against herbivores In feeding experiments with potential insect herbivores and Ni hyperaccumulating plants, only those inseets fed leaves from plants grown on non-nickel-bearing soil survived or showed a weight gain. Among chemical parameters measured, only Ni content of plants was sufficient to explain this result. When subjected to herbivory by lepidopteran larvae, plants grown on Ni-amended soil showed greater survival and yield than plants on unamended soil. Ni hyperaccumulation may be an effective plant chemical defense against herbivores because of its high lethality, apparent low cost, and broad spectrum of toxicity.

The defensive function of Ni in plants: response of the polyphagous herbivore Spodoptera exigua (Lepidoptera: Noctuidae) to hyperaccumulator and accumulator species of Streptanthus (Brassicaceae).

Metals sequestered by plants may defend them against herbivores and/or pathogens. We explored the effect of plant metal content on a polyphagous herbivore, Spodoptera exigua. Plant experiments used a Ni hyperaccumulator (Streptanthus polygaloides) and two Ni accumulator species (S. breweri and S. tortuosus). High- and low-Ni plants of each species were produced by growing plants on either Ni-amended or unamended soil. Mean leaf Ni contents for plants grown on Ni-amended soil and control soil, respectively, were: 1500 and 20 mg Ni kg-1 for S. polygaloides, 40 and 9 mg kg-1 for S. breweri, and 93 and 0.5 mg kg-1 for S. tortuosus. Neonate or second-instar Sp. exigua larvae were fed high- or low-metal leaves of each plant species, and survival and other parameters were monitored. High-Ni leaves of S. polygaloides were acutely toxic, resulting in 96% mortality within 10 days, whereas only 48% of larvae fed low-Ni leaves died. Low- and high-Ni leaves of S. breweri did not differ in their effects on larval survival, larval weight, adult weight, and duration of pupation. Leaves of S. tortuosus from high-Ni soil did not significantly affect larval survival relative to low-Ni leaves. However, larvae eating high-Ni leaves weighed significantly less and pupation was significantly delayed. Larval feeding experiments using artificial diet amended with Ni demonstrated a toxic threshold at 963 mg Ni kg-1 and a sublethal threshold at 535 mg Ni kg-1. Because plant material containing less Ni had detectable sublethal effects, we suggest that Ni interacts with other plant qualities (including secondary defensive compounds) to produce those effects. We conclude that hyperaccumulated Ni is a potent defense against polyphagous folivorous insects, but suggest that the sublethal impacts of the lesser Ni levels found in accumulator plant species may play only a minor defensive role against herbivores.

Plant defense using toxic inorganic ions: conceptual models of the defensive enhancement and joint effects hypotheses.

The concept of plant defense using toxic mineral elements originated as an explanation for extremely elevated concentrations of some elements (termed hyperaccumulation) in some plant tissues. The Defensive Enhancement Hypothesis suggests that hyperaccumulation evolved because, after an initial defensive benefit accrued from a relatively low initial concentration, increased concentration of an element provided increased plant fitness and drove evolution of higher element concentrations until hyperaccumulation was achieved. The Joint Effects Hypothesis postulates that additive or synergistic effects between element-based defenses, or between toxic element and organic chemical defenses, may have contributed to the evolution of hyperaccumulation. By lessening the concentration of an element necessary to provide an initial defensive benefit to a plant, joint effects could decrease the level of an element that provides an initial defensive benefit, allowing additive or synergistic defensive enhancement to take effect. Recent experimental tests have demonstrated defense at relatively low element concentrations, and tests of metal/metal and metal/organic compound combinations have shown joint effects. These hypotheses suggest how hyperaccumulator plants may have evolved in response to plant-herbivore interactions, and suggest that toxic element levels below those used to define hyperaccumulation may be ecologically effective.

Proteomic analysis of B-cell malignancies.

The identification of proteins aberrantly expressed in malignant B-cells can potentially be used to develop new diagnostic, prognostic or therapeutic targets. Proteomic studies of B-cell malignancies have made significant progress, but further studies are needed to increase our coverage of the B-cell malignant proteome. To achieve this goal we stress the advantages of using sub-cellular fractionation, protein separation, quantitation and affinity purification techniques to identify hitherto unidentified signalling and regulatory proteins. For example, proteomic analysis of B-cell plasma membranes isolated from patients with mantle cell lymphoma (MCL) identified the voltage-gated proton channel (HVCN1,[1]). This protein has now been characterised as a key modulator of B-cell receptor (BCR) signalling and abrogation of HVCN1 function could have a role in the treatment of B-cell malignancies dependent on maintained BCR signalling [2]. Similarly, proteomic studies on cell lysates from prognostic subtypes of CLL, distinguished by the absence (UM-CLL) or presence (M-CLL) of somatic hypermutation of the immunoglobulin heavy chain locus identified nucleophosmin 1 (NMP1) as a potential prognostic marker [3,4]. Thus, targeted proteomic analysis on selected organelles or sub-cellular compartments can identify novel proteins with unexpected localisation or function in malignant B-cells that could be developed for clinical purposes.

Effectiveness of metal-metal and metal-organic compound combinations against Plutella xylostella: implications for plant elemental defense.

Plants that contain elevated foliar metal concentrations can be categorized as accumulators or, if the accumulation is extreme, hyperaccumulators. The defense hypothesis suggests that these plants may be defended against folivore attack, and recent research has indicated that metal concentrations at or below the accumulator range may be defensively effective. This experiment explored the toxicity of four metals hyper-accumulated by plants (Cd, Ni, Pb, and Zn) and asked if combinations of metals, or metals and organic chemicals, might broaden the defensive effectiveness of metals. Metals were used alone and in certain metal + metal (Zn plus Ni, Pb, or Cd) and metal + organic defensive chemical (Ni plus tannic acid, atropine, or nicotine) combinations. Artificial diet amended with these treatments was fed to larvae of the crucifer specialist herbivore Plutella xylostella. Combinations of metals and metals + organic chemicals significantly decreased survival and pupation rates, compared to single treatments, for at least some concentrations in every experiment. Effects of combinations were additive rather than synergistic or antagonistic. Because Zn enhanced the toxicity of other metals and Ni enhanced the toxicity of organic defensive chemicals, our findings suggest that the defensive effects of metals are more widespread among plants than previously believed. They also support the hypothesis that herbivore defense may have led to the evolution of metal hyper-accumulation by increasing the preexisting defensive effects of metals at accumulator levels in plants.

Nickel hyperaccumulation as an elemental defense of Streptanthus polygaloides (Brassicaceae): influence of herbivore feeding mode.

No study of a single nickel (Ni) hyperaccumulator species has investigated the impact of hyperaccumulation on herbivores representing a variety of feeding modes. Streptanthus polygaloides plants were grown on high- or low-Ni soils and a series of no-choice and choice feeding experiments was conducted using eight arthropod herbivores. Herbivores used were two leaf-chewing folivores (the grasshopper Melanoplus femurrubrum and the lepidopteran Evergestis rimosalis), a dipteran rhizovore (the cabbage maggot Delia radicum), a xylem-feeder (the spittlebug Philaenus spumarius), two phloem-feeders (the aphid, Lipaphis erysimi and the spidermite Trialeurodes vaporariorum) and two cell-disruptors (the bug Lygus lineolaris and the whitefly Tetranychus urticae). Hyperaccumulated Ni significantly decreased survival of the leaf-chewers and rhizovore, and significantly reduced population growth of the whitefly cell-disruptor. However, vascular tissue-feeding insects were unaffected by hyperaccumulated Ni, as was the bug cell-disruptor. We conclude that Ni can defend against tissue-chewing herbivores but is ineffective against vascular tissue-feeding herbivores. The effects of Ni on cell-disruptors varies, as a result of either variation of insect Ni sensitivity or the location of Ni in S. polygaloides cells and tissues.

Extending the elemental defense hypothesis: dietary metal concentrations below hyperaccumulator levels could harm herbivores.

Previous work has shown that hyperaccumulator levels of some metals can defend plants against herbivores, but the possibility of defense by metal concentrations at accumulator or normal levels is unexplored. This study tested the hypothesis that metals can defend plants at low concentrations. We determined the relative toxicities of eight metals commonly acquired by plants: Cd, Co, Cr, Cu, Mn, Ni, Pb, and Zn. Larvae of the diamondback moth (Plutella xylostella), a representative crucifer specialist, were fed with artificial diet amended with concentrations of metal varying from 2 to 3,000 microg/g. Different concentration ranges were used for each of the eight metals, and larval survival at 10-14 days was calculated for each concentration. All metals were toxic to diamondback moth larvae at hyperaccumulator levels. All metals, however, were also toxic to larvae at accumulator concentrations, far below those found in hyperaccumulating plants. Five metals (Cd, Mn, Ni, Pb, and Zn) were toxic below accumulator levels, Cd and Pb were toxic near the concentration ranges of normal plants, and Zn was toxic at a concentration within the normal range. Our results indicate that uptake of certain metals may provide a defensive benefit for plants, and that elemental defenses may be effective at concentrations far lower than previously hypothesized. This study implies that elemental defenses are more widespread in plants than previously believed, and that the ecological consequences of even low levels of metal accumulation need to be explored.