Mechanism of Isoxaben Tolerance in Agrostis palustris var. Penncross

Previous work has demonstrated that isoxaben tolerant mutantsof Arabidopsis thaliana var. Columbia are most likely alteredat the site of isoxaben binding. The salient question becomeswhether or not species selectivity to this herbicide might alsobe a result of differential target site binding. Grasses aregenerally more tolerant to isoxaben than dicots. In this communicationwe show that Agrostis palustris var. Penncross, a grass, is83-fold more tolerant in a soil incorporation test and 170-foldmore tolerant to inhibition of glucose incorporation into cellulosethan is Arabidopsis, a dicot. Cell wall fractionation of Agrostisshows a specific effect on cellulose biosynthesis. At most,5-fold of the 170-fold tolerance exhibited by Agrostis in termsof cellulose biosynthesis can be attributed to decreased isoxabenuptake under the test conditions. Furthermore, Agrostis is unableto metabolize isoxaben to any significant degree. Therefore,we suggest that the major portion of the tolerance in Agrostismight be due to differences in isoxaben binding. Key words: Isoxaben, cellulose, Arabidopsis, Agrostis, herbicide tolerance     

An Experimental Investigation into the Response of Some New Zealand Sand Dune Species to Salt Spray

The tolerance to salt spray of 29 species, mainly from New Zealandsand dunes, was investigated. Plants were grown in water culturein a glasshouse and subjected to overhead salt spraying at intervals.Growth rates in many species were reduced by salt spray buta significant decrease occurred only in six native herbs. However,many species showed sensitivity in leaf necrosis. Tolerant speciesincluded Scirpoides nodosa, Elymus farctus and Desmoschoenusspiralis. Ammophila arenaria, tolerant of spray as an adult,was less so when younger. There was little correlation between tolerance to salt sprayand tolerance to root salinity. Some species were tolerant toboth, e.g. S. nodosa and E. farctus, and some intolerant toboth, e.g. Wahlenbergia congesta. One species, Lupinus arboreus,was glycophytic in respect to root salt but tolerant of aerialsalt. Other species, such as Senecio elegans L. and Austrofestucalittoralis, were intolerant of salt spray but tolerant of mediumroot salinities. For some species salt spray tolerance correlated well with fielddistribution, e.g. D. spiralis and Bromus diandrus. However,some species present in semi-fixed dunes close to the sea havemuch lower tolerance than would be expected from their fieldsituation, e.g. W. congesta. This apparent inconsistency couldbe explained by the ameliorating high rainfall on the West Coast,or protection by ridges. One environmental variable alone, suchas salt spray, could not explain the field distribution formany species. Salt spray, growth rate, live leaf area, New Zealand, dune species, root salinity     

Antigen-specific T contrasuppressor factor in cell-mediated immunity: interactions leading to eradication of the tolerant state

Interactions between a T cell-derived, antigen-specific, contrasuppressor factor (TcsF) and immune T cells that block the action of T suppressor factors and allow the transfer of cellular immunity into tolerant recipients are described. Immune T cells from contact-sensitized donors are capable of transferring specific immunity into normal recipients but not into animals rendered tolerant to the specific antigen. Brief exposure of the immune cells to the TcsF enables the effective transfer of immunity into such tolerant recipients. In addition, treated immune cells become resistant to subsequent exposure to T suppressor factor (capable of inhibiting transfer of immunity to normal recipients). A cyclophosphamide-sensitive, I-J+, Ly-2 T transducer cell is required in the immune donor cell population for contrasuppression to be induced by the TcsF plus specific antigen. These cells release an antigen-non-specific contrasuppressive factor capable of rendering immune targets, depleted of transducer cells, resistant to suppression (either by suppressor factor or in the tolerant recipient). The results indicate that contrasuppression in contact sensitivity is antigen specific and that the balance of suppression and contrasuppression determines tolerance vs responsiveness in this system. The symmetrical resemblance of the contrasuppressive interactions to those of suppression in contact sensitivity are discussed.     

T cell tolerance in the chicken. I. Parameters affecting tolerance induction to human gamma-globulin in agammaglobulinemic and normal chickens.

T cell-mediated delayed hypersensitivity (DH) to human gamma-globulin (HGG) can be induced in chickens by subcutaneous injection of the antigen in complete Freund's adjuvant (CFA). In the present work, it has been demonstrated that specific tolerance of the cells mediating this DH can readily be induced in both normal and bursectomized (BX) FP strain chickens by simple i.v. injection of soluble antigen, regardless of the presence of antibody production to the tolerogen. A significant degree of tolerance at the DH and helper T cell levels could be generated in BX birds by injection of as little as 0.5 mg of HGG; such a dose only induced tolerance in normal birds when it had been previously deagregated by ultracentrifugation. Regular, nondeaggregated antigen could produce tolerance in normal animals, but only at doses of greater than 5 mg. The tolerizing injection induced a primary antibody response in normal birds in all cases, but a secondary response could not be obtained in animals rendered tolerant at the T cell level. Establishment of tolerance appeared to be very rapid, and animals remained refractory to induction of DH for at least 3 weeks after the tolerizing injection. The mode in which the antigen was presented to the animals appeared to be crucial in determining whether tolerance or sensitivity would be established.     

Tolerance and contact sensitivity to DNFB in mice. VI. Inhibition of afferent sensitivity by suppressor T cells in adoptive tolerance.

Tolerance in contact sensitivity to DNFB can be adoptively transferred to normal mice with lymph node cells from tolerant donors. This tolerance is antigen specific and is mediated by T cells, i.e., "suppressor" T cells. Experiments were carried out to investigate the mechanism(s) by which the suppressor T cells induce tolerance to DNFB contact sensitivity. The suppressor cells were effective only if they were present during the early stages of the afferent limb of sensitization. As measured by DNA synthesis, cell proliferation in the draining lymph nodes of recipients of suppressor cells was found to be significantly less than in control animals indicating that the suppressor cells acted, at least in part, by limiting or inhibiting DNFB-induced cell proliferation. This inhibition was shown to be antigen specific since the DNFB suppressor cells did not inhibit cell proliferation induced by oxazolone, an unrelated contact sensitizer. The ability to DNFB tolerant cells to block afferent sensitization pathways differs from the mechanism of tolerance to picryl chloride, reported by others, where efferent pathways are blocked.     

Interleukin-15 rescues tolerant CD8+ T cells for use in adoptive immunotherapy of established tumors

CD8+ T cells can mediate eradication of established tumors, and strategies to amplify tumor-reactive T-cell numbers by immunization or ex vivo expansion followed by adoptive transfer are currently being explored in individuals with cancer. Generating effective CD8+ T cell-mediated responses to tumors is often impeded by T-cell tolerance to relevant tumor antigens, as most of these antigens are also expressed in normal tissues. We examined whether such tolerant T cells could be rescued and functionally restored for use in therapy of established tumors. We used a transgenic T-cell receptor (TCR) mouse model in which peripheral CD8+ T cells specific for a candidate tumor antigen also expressed in liver are tolerant, failing to proliferate or secrete interleukin (IL)-2 in response to antigen. Molecular and cellular analysis showed that these tolerant T cells expressed the IL-15 receptor alpha chain, and could be induced to proliferate in vitro in response to exogenous IL-15. Such proliferation abrogated tolerance and the rescued cells became effective in treating leukemia. Therefore, high-affinity CD8+ T cells are not necessarily deleted by encounter with self-antigen in the periphery, and can potentially be rescued and expanded for use in tumor immunotherapy.     

Acclimation of Trees to Pollution Stress: Cellular Metal Tolerance Traits

Cell suspension cultures were established from shoot explantsof mature trees of Acer pseudoplatanus L. (sycamore) at a sitecontaminated by aerial deposition of copper and cadmium frommetal processing industry, and from the same species at uncontaminatedsites. The responses of cell cultures to elevated metal concentrationsin growth media differed markedly according to site of origin.Both Cu and Cd, applied singly at concentrations of 10–15mg l^–1, inhibited growth and were toxic to cultures originatingfrom the uncontaminated sites, but not to cultures from thecontaminated site. This metal tolerance trait in the culturesfrom the contaminated site was stable through repeated sub-culturing.It could also be induced in one culture originating from thereference uncontaminated site, by gradually exposing the cultureto increasing concentrations of Cu. A reduced level of metalremoval from the media was found in tolerant cultures, comparedto non-tolerant cultures. The results of these experiments demonstratethe occurrence of an alteration of gene expression in responseto pollution stress, suggesting that metal tolerance may beinduced within shoot meristems in vivo. It also represents thefirst example of non-mycorrhizal adaptation to metal toxicityidentified in woody plants. Trees, pollution, metal tolerance, acclimation, plant tissue culture, Acer pseudoplaianus L., sycamore     

Small Heat Shock Proteins Protect Electron Transport in Chloroplasts and Mitochondria During Stress

Evidence indicates that small heat-shock proteins (Hsps) areinvolved in stress tolerance, but the specific cell componentsor functions that small Hsps protect or repair are mostly unidentified.We recently showed that the chloroplast small Hsps of higherplants (1) are produced in response to many environmental stresses(e.g., heat, oxidative, and high-light stress); and (2) protect(but do not repair) photosynthetic electron transport in vitroduring stress, specifically by interacting with the oxygen-evolving-complexproteins of Photosystem II (PSII) within the thylakoid lumen.However, in vivo evidence of the importance of these Hsps tophotosynthetic stress tolerance is lacking. Here we report positiverelationships between chloroplast small Hsp production and PSIIthermotolerance in (1) a heattolerant genotype of Agrostis palustris(bentgrass) and a heat sensitive genotype which lacks one ormore chloroplast small Hsps produced by the tolerant genotype;(2) ecotypes of Chenopodium album (lambs quarters) from thenorthern vs. southern U.S. (New York vs. Georgia); and (3) nineLycopersicon (tomato) cultivars/species differing in heat tolerance.These in vivo results are consistent with our previous in vitroobservations and indicate that genetic variation in productionof the chloroplast small Hsp is an important determinant ofphotosynthetic and, thereby, whole-plant thermotolerance. Recently,we showed that the mitochondrial small Hsp of plants protectsrespiratory (specifically Complex I) electron transport in vitroduring heat stress, and here we present evidence for previouslyunidentified small Hsps in mitochondria of mammal (rat) cellswhich also protect Complex I during heat stress. These resultssuggest that the mitochondrial small Hsps, like the small chloroplastHsps, are general stress proteins that contribute significantlyto cell and organismal stress tolerance.     

Induction of “Low Dose” Tolerance to a Bacterial Somatic Antigen in Neonatal Mice

THE concept of “low dose” tolerance rests on the observation that specific immunological unresponsiveness can be induced by extremely small doses of antigen, sometimes in quantities too small to provoke antibody formation as such^1–3. The initial observations were made with soluble serum proteins, which are generally not considered “strong” antigens^4,5. Low dose tolerance can be induced also to the highly immunogenic protein antigen derived from Salmonella flagella, as assessed at the level of serum antibody^6,7. Furthermore, recent studies at the level of antibody forming cells indicate that immunological tolerance to complex antigens such as sheep erythrocytes and bacterial extracts, as well as to serum proteins or chemical haptens, can be induced in neonatal or adult animals^9–14. In this regard, relatively large doses of antigen are reportedly necessary to induce tolerance in adult rodents to a bacterial antigen such as Escherichia coli lipopolysaccharide (LPS). For example, 10–15 mg of the LPS is necessary to induce tolerance, as measured by an indirect haemolytic plaque assay with antigen coated sheep erythrocytes^15,16. Lower doses of LPS reportedly induce only immunity.     

Depressed Cellular Formation of Antibody to Shigella Antigens In Vitro by Spleen Cell Cultures from Unresponsive Mice

Specific antibody plaque-forming cells (PFC) to Shigella-soluble antigen did not appear in spleen cell cultures from Shigella-tolerant mice, as occurred with similar cultures prepared from normal mice immunized with Shigella antigen prior to sacrifice. Cultures from tolerant mice also failed to form serologically detectable amounts of agglutinins in vitro. Exposure of cell cultures from tolerant mice in vitro to additional antigen had little or no effect on appearance of plaque-forming cells to Shigella. Spleen cells from normal control mice formed readily detectable levels of antibody, as well as specific antibody plaque-forming cells, after similar stimulation with antigen either in vivo or in vitro. The absence of antibody-forming cells in cultures prepared from spleens of tolerant mice was specific since such cultures, as well as those from normal control mice, formed numerous antibody plaques to unsensitized sheep erythrocytes in vitro after in vivo challenge of the mice with sheep erythrocytes. Tolerance to Shigella antigen, as assessed by absence of antibody-forming cells in vitro, persisted for several months. Spleen cell cultures from tolerant mice less than 3 to 4 months of age did not form significant numbers of antibody plaques, even after in vitro exposure to specific antigen. However, spleen cultures prepared from neonatally treated mice, approximately 6 to 8 months old, formed essentially normal numbers of specific PFC in vitro, indicating that the animals had "recovered" from tolerance and that their lymphoid cells were capable of responding to Shigella antigen in vitro. Absence of specific PFC in cell cultures from tolerant animals supports the concept that tolerance is due to a central failure of specific immunocompetent cells and not due to an inhibitory effect caused by either "excess" antigen or humoral antibody.     

Gene and Enhancer Trap Transposable Elements Reveal Oxygen Deprivation-regulated Genes and their Complex Patterns of Expression in Arabidopsis

Transposon tagging with modified maize Ds–GUS constructswas used to isolate genes induced by oxygen deprivation in Arabidopsisthaliana. Seedlings of 800 gene-trap (DsG) and 600 enhancer-trap(DsE) lines were grown on vertically positioned plates for 1 week,oxygen deprived for up to 24 h and stained for GUS activity.Oxygen deprivation induced intricate patterns of gene expressionin seedlings of 65 lines. The insertion site and phenotypesof 15 lines were examined. Surprisingly, none of the insertionswere into genes that encode known anaerobic polypeptides. Insertionswere identified within or adjacent to genes encoding proteinsof regulatory, enzymatic, mitochondrial protein import and unknownfunction, as well as adjacent to genes encoding a putative receptor-likekinase and putative sensor-histidine kinase. Four lines hadsignificantly lower ADH activity after 24 h of oxygen deprivationand three of these showed reduced stress tolerance. Two lineswith wild-type levels of ADH were low-oxygen intolerant. Paradoxically,several lines had significantly higher ADH activity after 12 hof oxygen deprivation but reduced stress tolerance. Caffeinetreatment, which increased ADH specific activity in wild-typeseedlings under aerobic conditions, was sufficient to increaseGUS staining in seven of the 15 lines, providing evidence thatthese genes may be regulated by cytosolic calcium levels. Theseresults demonstrate the effectiveness of the Ds–GUS taggingsystem in the identification of genes that are regulated inresponse to oxygen deprivation and a calcium second messenger.     

Neuropilin-1 Expression Is Induced on Tolerant Self-Reactive CD8+ T Cells but Is Dispensable for the Tolerant Phenotype

Establishing peripheral CD8⁺ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8⁺ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8⁺ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4⁺ and CD8⁺ T cells. Despite high expression on tolerant CD8⁺ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.     

A comparative study of ultraviolet radiation tolerance in different populations of Diaptomus minutus

A series of experiments was conducted to test the hypothesis that populations of Diaptomus minutus routinely experiencing high levels of ultraviolet radiation (UVR) are more tolerant of UVR than are those that routinely experience low levels of UVR. The relative degree of UVR tolerance was determined by monitoring mortality induced by either lamp or solar UVR. Diaptomus minutus from the low-dissolved organic carbon (DOC), high-UVR Lake Giles were consistently more tolerant of lamp and solar UVR than were those from the moderate-DOC, low-UVR Lake Lacawac. This difference in UVR tolerance was apparent throughout the year in freshly collected animals, but it did not persist in cultured animals. The lamp UVR tolerances of cultured D.minutus were similar, except for those initiated from September collections. The September culture of Lake Giles animals was significantly more tolerant than the Lake Giles cultures initiated in December, May and June. The September culture of Lake Lacawac animals was significantly more tolerant than the June culture. The lamp UVR tolerance of freshly collected Lake Giles animals was greatest from late June through October, but the Lake Lacawac population was least tolerant in July and August. No differences in lamp UVR tolerances were detected between animals collected from the surface and those collected from deeper water of either lake. It is concluded that the Lake Giles population was more tolerant than the Lake Lacawac population and that the period of greatest tolerance occurred several months after the time period with the highest ambient levels of solar UVR. Collectively, the experiments with both field and laboratory-cultured animals suggest that acclimation time plays a large role in UVR tolerance.      

Sulphydryl and Disulphide Levels in Protein Fractions from Hydrated and Dry Leaves of Resurrection Plants

Significant changes in sulphydryl (‘SH’) and disulphide(‘SS’) levels during air-drying in leaves of ‘resurrection’plants (whose protoplasm survives dehydration) stemmed mainlyfrom protein turnover effects. No significant changes were foundin the SH, SS levels in leaves of the desiccation sensitivespecies Sporobolus pyramidalis following air-drying. The three tolerant species studied differed in the directionof change. Some data were consistent with Levitt's SH, SS hypothesis:increases in protein-SS levels in Sporobolus stapfianus (desiccationtolerant) were consistent with a stabilization of new proteinby SS bonds; lower reactivity of protein-SH in the tolerantspecies Talbotia elegans (which on the other hand has decreasedprotein-SS) is consistent with a second mechanism of decreasingprotein denaturation proposed in Levitt's hypothesis. Evidence of some conversion of SH to SS in the soluble proteinsof Xerophyta viscosa (a tolerant species) would on Levitt'shypothesis indicate an injurious process. Some degree of proteindenaturation might be indicated by partial inactivation of thesoluble enzyme ribulose bisphosphate carboxylase in this species,and loss of some soluble isoenzymes (peroxidase and alkalinephosphatase). An apparent lack of SH conversion to SS in thesensitive species Sporobolus pyramidalis was not consistentwith the SH, SS hypothesis. Resurrection plants, Sporobolus pyramidalis, Sporobolus stapfianus, Talbotia elegans, Xerophyta viscosa, drought resistance, desiccation tolerance, protein turnover, sulphydryl groups     

Salt Tolerance in the Triticeae: Solute Accumulation and Distribution in an Amphidiploid derived from Triticum aestivum cv. Chinese Spring and Thinopyrum bessarabicum

An amphidiploid derived by colchicine treatment of a hybridbetween Triticum aestivum cv Chinese Spring and Thinopyrum bessarabicumwas found to be more salt tolerant than the wheat cultivarsChinese Spring, Kharchia and Ciano 79 in terms of survival andgrain yield at 250 mol m^–3 NaCl. Tolerance was relatedto the ability of the amphidiploid to exclude Na and Cl fromthe shoots, and particularly from the young leaves, developinginflorescence and grain. There was no relationship between thesalt tolerance of the different species and varieties testedand changes in the concentrations of other solutes. The amphidiploiddid not inherit the high glycinebetaine concentrations characteristicof the wheatgrass parent. Amphidiploids produced from crossesbetween Thinopyrum species and wheat may be useful as stress-resistantnew crops. Key words: Salt stress, solute accumulation, Thinopyrum, Triticum     

Probable loss of tolerance and simulation of autoimmunization in induced chimeras in doves

Chimeras were induced in doves (Streptopelia) by making parabionts of embryonating eggs that carried genes for erythrocyte antigens, which were readily identified. The parabiotic pairs were chosen so that new antigenic specificities would appear if somatic cell mating took place. However, no evidence of somatic cell mating was noted. Erythrocytic chimerism was no longer. detectable in some birds after varying periods of time. In a few others tolerance was presumably lost, since their plasma contained antibodies against cellular antigens that either were present, or had been present, in the bird's circulation.     

Cellular events in tolerance IX: Maintenance of immunological tolerance in the presence of normal B-cell precursors and in the absence of demonstrable suppression

The cellular events involved in immunological tolerance to fluoresceinated sheep gammaglobulin (FL-SGG) were analyzed at the level of hapten-specific B cells. One single iv injection of FL-SGG induced tolerance as measured by challenge with thymus-dependent (FL-KLH) or thymus-independent (FL-Ficoll) antigens in vivo or thymus-independent (FL-LPS) antigen in vitro. As noted earlier, unresponsiveness was maintained until 6–8 weeks after tolerance induction. Limiting-dilution precursor analysis demonstrated a reduction in B-cell precursors on Day 7 after tolerogen treatment; precursor frequencies returned to control levels by 3–4 weeks. This recovery of precursors in the presence of stable tolerance was not due to suppressor activity. Rather, results show that tolerant hapten-specific B cells are clonally anergic and display a reduced burst size in response to antigen. Hence, unresponsiveness is maintained in the presence of apparently normal precursor levels by an intrinsic defect in antigen-specific B cells.     

Suppression of the High Affinity Phosphate Uptake System: A Mechanism of Arsenate Tolerance in Holcus lanatus L.

In Holcus lanatus L. phosphate and arsenate are taken up bythe same transport system. Short-term uptake kinetics of thehigh affinity arsenate transport system were determined in excisedroots of arsenate-tolerant and non-tolerant genotypes. In tolerantplants the V_max of ion uptake in plants grown in phosphate-freemedia was decreased compared to non-tolerant plants, and theaffinity of the uptake system was lower than in the non-tolerantplants. Both the reduction in V_max and the increase in K_m ledto reduced arsenate influx into tolerant roots. When the twogenotypes were grown in nutrient solution containing high levelsof phosphate, there was little change in the uptake kineticsin tolerant plants. In non-tolerant plants, however, there wasa marked decrease in the V_max to the level of the tolerant plantsbut with little change in the K_m. This suggests that the lowrate of arsenate uptake over a wide range of differing rootphosphate status is due to loss of induction of the synthesisof the arsenate (phosphate) carrier. Key words: Arsenate, Holcus lanatus L., phosphate uptake, tolerance mechanisms, uptake mechanisms     

Hapten-specific tolerance induced by hapten conjugates of D-glutamic acid, D-lysine (D-Gl) or isologous gamma-globulin: evidence for central B cell tolerance in the presence of carrier-primed helper T cells.

A comparison has been made of the well known hapten-specific tolerance systems induced, respectively, by hapten-D-GL or hapten-isologous gamma-globulin conjugates. The principal question addressed in this study concerned the comparative maintenance of B cell tolerance, induced by one or the other method, after adoptive transfer into carrier-primed, irradiated recipient animals and, in addition, what role, if any, might be played by T lymphocytes in the tolerant donor cell population in maintaining such tolerance. The results clearly show that insofar as the hapten-specific B cell is concerned, no obvious difference exists in the capacity to maintain tolerance adoptive transfer between the hapten-D-GL and hapten-isologous gamma-globulin systems; such cells remained tolerant even in the presence of excess helper T cell activity. Moreover, under the conditions employed, depletion of T lymphocytes from the tolerant donor cell population did not affect the maintenance of hapten-specific B cell tolerance after adoptive transfer to irradiated recipients.