Evidence for the Critical Role of Sucrose Synthase for Anoxic Tolerance of Maize Roots using a Double Mutant

The induction of the sucrose synthase (SuSy) gene (SuSy) by low O2, low temperature, and limiting carbohydrate supply suggested a role in carbohydrate metabolism under stress conditions. The isolation of a maize (Zea mays L.) line mutant for the two known SuSy genes but functionally normal showed that SuSy activity might not be required for aerobic growth and allowed the possibility of investigating its importance during anaerobic stress. As assessed by root elongation after return to air, hypoxic pretreatment improved anoxic tolerance, in correlation with the number of SuSy genes and the level of SuSy expression. Furthermore, root death in double-mutant seedlings during anoxic incubation could be attributed to the impaired utilization of sucrose (Suc). Collectively, these data provide unequivocal evidence that Suc is the principal C source and that SuSy is the main enzyme active in Suc breakdown in roots of maize seedlings deprived of O2. In this situation, SuSy plays a critical role in anoxic tolerance.

Fourier Transform Infrared Microspectroscopy Detects Changes in Protein Secondary Structure Associated with Desiccation Tolerance in Developing Maize Embryos1

Isolated immature maize (Zea mays L.) embryos have been shown to acquire tolerance to rapid drying between 22 and 25 d after pollination (DAP) and to slow drying from 18 DAP onward. To investigate adaptations in protein profile in association with the acquisition of desiccation tolerance in isolated, immature maize embryos, we applied in situ Fourier transform infrared microspectroscopy. In fresh, viable, 20- and 25-DAP embryo axes, the shapes of the different amide-I bands were identical, and this was maintained after flash drying. On rapid drying, the 20-DAP axes had a reduced relative proportion of α-helical protein structure and lost viability. Rapidly dried 25-DAP embryos germinated (74%) and had a protein profile similar to the fresh control axes. On slow drying, the α-helical contribution in both the 20- and 25-DAP embryo axes increased compared with that in the fresh control axes, and survival of desiccation was high. The protein profile in dry, mature axes resembled that after slow drying of the immature axes. Rapid drying resulted in an almost complete loss of membrane integrity in the 20-DAP embryo axes and much less so in the 25-DAP axes. After slow drying, low plasma membrane permeability ensued in both the 20- and 25-DAP axes. We conclude that slow drying of excised, immature embryos leads to an increased proportion of α-helical protein structures in their axes, which coincides with additional tolerance of desiccation stress.

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth1

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family.

Molecular and Physiological Responses to Water Deficit in Drought-Tolerant and Drought-Sensitive Lines of Sunflower1 : Accumulation of Dehydrin Transcripts Correlates with Tolerance

To investigate correlations between phenotypic adaptation to water limitation and drought-induced gene expression, we have studied a model system consisting of a drought-tolerant line (R1) and a drought-sensitive line (S1) of sunflowers (Helianthus annuus L.) subjected to progressive drought. R1 tolerance is characterized by the maintenance of shoot cellular turgor. Drought-induced genes (HaElip1, HaDhn1, and HaDhn2) were previously identified in the tolerant line. The accumulation of the corresponding transcripts was compared as a function of soil and leaf water status in R1 and S1 plants during progressive drought. In leaves of R1 plants the accumulation of HaDhn1 and HaDhn2 transcripts, but not HaElip1 transcripts, was correlated with the drought-adaptive response. Drought-induced abscisic acid (ABA) concentration was not associated with the varietal difference in drought tolerance. Stomata of both lines displayed similar sensitivity to ABA. ABA-induced accumulation of HaDhn2 transcripts was higher in the tolerant than in the sensitive genotype. HaDhn1 transcripts were similarly accumulated in the tolerant and in the sensitive plants in response to ABA, suggesting that additional factors involved in drought regulation of HaDhn1 expression might exist in tolerant plants.

Unifying theory of hypoxia tolerance: molecular/metabolic defense and rescue mechanisms for surviving oxygen lack.

We develop a unifying theory of hypoxia tolerance based on information from two cell level models (brain cortical cells and isolated hepatocytes) from the highly anoxia tolerant aquatic turtle and from other more hypoxia sensitive systems. We propose that the response of hypoxia tolerant systems to oxygen lack occurs in two phases (defense and rescue). The first lines of defense against hypoxia include a balanced suppression of ATP-demand and ATP-supply pathways; this regulation stabilizes (adenylates) at new steady-state levels even while ATP turnover rates greatly decline. The ATP demands of ion pumping are down-regulated by generalized "channel" arrest in hepatocytes and by "spike" arrest in neurons. Hypoxic ATP demands of protein synthesis are down-regulated probably by translational arrest. In hypoxia sensitive cells this translational arrest seems irreversible, but hypoxia-tolerant systems activate "rescue" mechanisms if the period of oxygen lack is extended by preferentially regulating the expression of several proteins. In these cells, a cascade of processes underpinning hypoxia rescue and defense begins with an oxygen sensor (a heme protein) and a signal-transduction pathway, which leads to significant gene-based metabolic reprogramming-the rescue process-with maintained down-regulation of energy-demand and energy-supply pathways in metabolism throughout the hypoxic period. This recent work begins to clarify how normoxic maintenance ATP turnover rates can be drastically (10-fold) down-regulated to a new hypometabolic steady state, which is prerequisite for surviving prolonged hypoxia or anoxia. The implications of these developments are extensive in biology and medicine.

Characterization of the osmotic response element of the human aldose reductase gene promoter.

Aldose reductase (EC 1.1.1.21) catalyzes the NADPH-mediated conversion of glucose to sorbitol. The hyperglycemia of diabetes increases sorbitol production primarily through substrate availability and is thought to contribute to the pathogenesis of many diabetic complications. Increased sorbitol production can also occur at normoglycemic levels via rapid increases in aldose reductase transcription and expression, which have been shown to occur upon exposure of many cell types to hyperosmotic conditions. The induction of aldose reductase transcription and the accumulation of sorbitol, an organic osmolyte, have been shown to be part of the physiological osmoregulatory mechanism whereby renal tubular cells adjust to the intraluminal hyperosmolality during urinary concentration. Previously, to explore the mechanism regulating aldose reductase levels, we partially characterized the human aldose reductase gene promoter present in a 4.2-kb fragment upstream of the transcription initiation start site. A fragment (-192 to +31 bp) was shown to contain several elements that control the basal expression of the enzyme. In this study, we examined the entire 4.2-kb human AR gene promoter fragment by deletion mutagenesis and transfection studies for the presence of osmotic response enhancer elements. An 11-bp nucleotide sequence (TGGAAAATTAC) was located 3.7 kb upstream of the transcription initiation site that mediates hypertonicity-responsive enhancer activity. This osmotic response element (ORE) increased the expression of the chloramphenicol acetyltransferase reporter gene product 2-fold in transfected HepG2 cells exposed to hypertonic NaCl media as compared with isoosmotic media. A more distal homologous sequence is also described; however, this sequence has no osmotic enhancer activity in transfected cells. Specific ORE mutant constructs, gel shift, and DNA fragment competition studies confirm the nature of the element and identify specific nucleotides essential for enhancer activity. A plasmid construct containing three repeat OREs and a heterologous promoter increased expression 8-fold in isoosmotic media and an additional 4-fold when the transfected cells are subjected to hyperosmotic stress (total approximately 30-fold). These findings will permit future studies to identify the transcription factors involved in the normal regulatory response mechanism to hypertonicity and to identify whether and how this response is altered in a variety of pathologic states, including diabetes.

Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator.

In many plants, osmotic stress induces a rapid accumulation of proline through de novo synthesis from glutamate. This response is thought to play a pivotal role in osmotic stress tolerance [Kishor, P. B. K., Hong, Z., Miao, G.-H., Hu, C.-A. A. and Verma, D. P. S. (1995) Plant Physiol. 108, 1387-1394]. During recovery from osmotic stress, accumulated proline is rapidly oxidized to glutamate and the first step of this process is catalyzed by proline oxidase. We have isolated a full-length cDNA from Arabidopsis thaliana, At-POX, which maps to a single locus on chromosome 3 and that encodes a predicted polypeptide of 499 amino acids showing significant similarity with proline oxidase sequences from Drosophila and Saccharomyces cerevisiae (55.5% and 45.1%, respectively). The predicted location of the encoded polypeptide is the inner mitochondrial membrane. RNA gel blot analysis revealed that At-POX mRNA levels declined rapidly upon osmotic stress and this decline preceded proline accumulation. On the other hand, At-POX mRNA levels rapidly increased during recovery. Free proline, exogenously added to plants, was found to be an effective inducer of At-POX expression; indeed, At-POX was highly expressed in flowers and mature seeds where the proline level is higher relative to other organs of Arabidopsis. Our results indicate that stress- and developmentally derived signals interact to determine proline homeostasis in Arabidopsis.

Peptide vaccination can lead to enhanced tumor growth through specific T-cell tolerance induction.

Vaccination with synthetic peptides representing cytotoxic T lymphocyte (CTL) epitopes can lead to a protective CTL-mediated immunity against tumors or viruses. We now report that vaccination with a CTL epitope derived from the human adenovirus type 5 E1A-region (Ad5E1A234-243), which can serve as a target for tumor-eradicating CTL, enhances rather than inhibits the growth of Ad5E1A-expressing tumors. This adverse effect of peptide vaccination was rapidly evoked, required low doses of peptide (10 micrograms), and was achieved by a mode of peptide delivery that induces protective T-cell-mediated immunity in other models. Ad5E1A-specific CTL activity could no longer be isolated from mice after injection of Ad5E1A-peptide, indicating that tolerization of Ad5E1A-specific CTL activity causes the enhanced tumor outgrowth. In contrast to peptide vaccination, immunization with adenovirus, expressing Ad5E1A, induced Ad5E1A-specific immunity and prevented the outgrowth of Ad5E1A-expressing tumors. These results show that immunization with synthetic peptides can lead to the elimination of anti-tumor CTL responses. These findings are important for the design of safe peptide-based vaccines against tumors, allogeneic organ transplants, and T-cell-mediated autoimmune diseases.

Circumventing tolerance to generate autologous monoclonal antibodies to the prion protein.

Prion diseases are disorders of protein conformation and do not provoke an immune response. Raising antibodies to the prion protein (PrP) has been difficult due to conservation of the PrP sequence and to inhibitory activity of alpha-PrP antibodies toward lymphocytes. To circumvent these problems, we immunized mice in which the PrP gene was ablated (Prnp 0/0) and retrieved specific monoclonal antibodies (mAbs) through phage display libraries. This approach yielded alpha-PrP mAbs that recognize mouse PrP. Studies with these mAbs suggest that cellular PrP adopts an unusually open structure consistent with the conformational plasticity of this protein.

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

Epitope-specific tolerance induction with an engineered immunoglobulin.

Isologous and heterologous immunoglobulins have been shown to be extremely effective as tolerogenic carriers for nearly 30 years. The efficacy of these proteins is due in part to their long half-life in vivo, as well as their ability to crosslink surface IgM with Fc receptors. The concept of using IgG as a carrier molecule to induce unresponsiveness in the adult immune system has been exploited for simple haptens, such as nucleosides, as well as for peptides. To further evaluate the in vivo potential of these molecules for inducing tolerance to a defined epitope, we have engineered a fusion protein of mouse IgG1 with the immunodominant epitope 12-26 from bacteriophage lambda cI repressor protein. This 15-mer, which contains both a B-cell and T-cell epitope, has been fused in-frame to the N terminus of a mouse heavy chain IgG1 construct, thus creating a "genetic hapten-carrier" system. We describe a novel in vitro and in vivo experimental system for studying the feasibility of engineered tolerogens, consisting of a recombinant flagellin challenge antigen and a murine IgG1 tolerogen, both expressing the lambda repressor epitope 12-26. Herein, we show that peptide-grafted IgG molecules injected i.v., or expressed by transfected, autologous B cells, can efficiently modulate the cellular and humoral immune responses to immunodominant epitopes. This model displays the feasibility of "tailor-designing" immune responses to whole antigens by selecting epitopes for either tolerance or immunity.

Human V delta 2+ gamma delta T-cell tolerance to foreign antigens of Toxoplasma gondii.

Little is known about the mechanisms involved in human gammadelta T-cell tolerance to self or to foreign antigens. Patients with congenital toxoplasmosis offer a unique opportunity to examine Vdelta2+ gammadelta T-cell tolerance. Analysis of gammadelta T cells in patients with congenital toxoplasmosis revealed evidence for anergy of these cells with or without clonal Vdelta2+ gammadelta T-cell expansion in the acute phase of the Toxoplasma infection. T cells in general were unresponsive and did not proliferate upon exposure to mitogens or to Toxoplasma lysate antigens or in response to live Toxoplasma-infected cells when the congenitally infected infants were 1 month of age, and they exhibited selective anergy to Toxoplasma lysate antigens and live Toxoplasma-infected cells when the infants were aged 5 months. During the chronic phase of congenital toxoplasmosis in the patients who were more than I year of age, the repertoires of the gammadelta T-cell receptors were found to be within normal ranges. In addition, in the chronic phase, the gammadelta T cells proliferated and secreted gamma-interferon in response to exposure to live Toxoplasmia-infected cells. By contrast, alphabeta T cells remained anergic. Vdelta2+ gammadelta T cells have been considered to undergo extrathymic maturation and thus to be subject to development of peripheral tolerance. Our findings indicate that Vdelta2+ gammadelta T-cell tolerance was lost in these infected infants earlier than alphabeta T-cell tolerance. These findings suggest that gammadelta T cells play a role in protection against Toxoplasma gondii in the chronic phase when congenitally infected children are more than 1 year of age, especially in those in whom alphabeta T cells continue to exhibit deficits in specific immune responses to Toxoplasma antigens.

Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.