Oxidative stress and cellular immunity in patients with recurrent aphthous ulcers

Recurrent aphthous ulcer (RAU) is an inflammatory condition of the oral mucosa characterized by painful, well-circumscribed, single or multiple round or ovoid ulcerations. The exact etiologic factor(s) of these ulcerations are not yet understood. The objective of this study was to evaluate inflammatory processes and free radical metabolism of 25 patients with RAUs compared to 25 healthy controls. The levels of malondialdehyde (MDA) and glutathione (GSH) were determined by high-performance liquid chromatography. Tumor necrosis factor-alpha (TNF-α), interleukin-2 (IL-2), IL-10, and IL-12 were determined by ELISA. Nitric oxide (NO), myeloperoxidase (MPO), total antioxidant status (TAS), and total oxidant status (TOS) levels were measured spectroscopically in serum. The levels of MDA, GSH, TNF-α, IL-2, IL-12, MPO, and TOS, and oxidative stress index (OSI) were higher, and the levels of NO, IL-10, and TAS were lower in patients with RAU than in controls. Statistical analysis showed that GSH, TNF-α, IL-2, IL-10, and OSI differed significantly in patients with RAU compared to controls. These parameters have important roles in oxidant/antioxidant defense.

Ultrastructural changes of Fusobacterium nucleatum as a defense mechanism against human neutrophil peptide-1 - A Transmission Electron Microscopy (TEM) study

Aims: The aim of this work was to assess the ultrastructural changes, cellular proliferation, and the biofilm formation ability of F. nucleatum as defense mechanisms against the effect of HNP-1. Materials and methods: The type strain of F. nucleatum (ssp. nucleatum ATCC 25586) and two clinical strains (ssp. polymorphum AHN 9910 and ssp. nucleatum AHN 9508) were cultured and incubated with four different test concentrations of recombinant HNP-1 (1, 5, 10 and 20 µg/ml) and one control group (0 µg/ml). Bacterial pellets from each concentration were processed for TEM imaging. Planktonic growth was assessed and colony forming units (CFU) were measured to determine the cellular proliferation. Scrambled HNP-1 was used for confirmation. Results: TEM analyses revealed a decrease in the outer membrane surface corrugations and roughness of the strain AHN 9508 with increasing HNP-1 concentrations. In higher concentrations of HNP-1, the strain AHN 9910 showed thicker outer membranes with a number of associated rough vesicles attached to the outer surface. For ATCC 25586, the treated bacterial cells contained higher numbers of intracellular granules with increasing the peptide concentration. Planktonic growth of the two clinical strains were significantly enhanced (P<0.001) with gradually increased concentrations of HNP-1. None of the planktonic growth results of the 3 strains incubated with the scrambled HNP-1 was statistically significant. HNP-1 decreased the biofilm formation of the two clinical strains, AHN 9910 and 9508, significantly (P<0.01 and P<0.001; respectively). Conclusions: The present in vitro study demonstrates that F. nucleatum has the ability to withstand the lethal effects of HNP-1 even at concentrations simulating the diseased periodontium in vivo. The increase in planktonic growth could act as defense mechanisms of F. nucleatum against HNP-1.

The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

Systemic Acquired Resistance (SAR) is a type of plant systemic resistance occurring against a broad spectrum of pathogens. It can be activated in response to pathogen infection in the model plant Arabidopsis thaliana and many agriculturally important crops. Upon SAR activation, the infected plant undergoes transcriptional reprogramming, marked by the induction of a battery of defense genes, including Pathogenesis-related (PR) genes. Activation of the PR-1 gene serves as a molecular marker for the deployment of SAR. The accumulation of a defense hormone, salicylic acid (SA) is crucial for the infected plant to mount SAR. Increased cellular levels of SA lead to the downstream activation of the PR-1 gene, triggered by the combined action of the Non-expressor of Pathogenesis-related Gene 1 (NPR1) protein and the TGA II-clade transcription factor (namely TGA2). Despite the importance of SA, its receptor has remained elusive for decades. In this study, we demonstrated that in Arabidopsis the NPR1 protein is a receptor for SA. SA physically binds to the C-terminal transactivation domain of NPR1. The two cysteines (Cys521 and Cys529), which are important for NPR1’s coactivator function, within this transactivation domain are critical for the binding of SA to NPR1. The interaction between SA and NPR1 requires a transition metal, copper, as a cofactor. Our results also suggested a conformational change in NPR1 upon SA binding, releasing the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. These results advance our understanding of the plant immune function, specifically related to the molecular mechanisms underlying SAR. The discovery of NPR1 as a SA receptor enables future chemical screening for small molecules that activate plant immune responses through their interaction with NPR1 or NPR1-like proteins in commercially important plants. This will help in identifying the next generation of non-biocidal pesticides.

Fur controls iron homeostasis and oxidative stress defense in the oligotrophic alpha-proteobacterium Caulobacter crescentus

In most bacteria, the ferric uptake regulator (Fur) is a global regulator that controls iron homeostasis and other cellular processes, such as oxidative stress defense. In this work, we apply a combination of bioinformatics, in vitro and in vivo assays to identify the Caulobacter crescentus Fur regulon. A C. crescentus fur deletion mutant showed a slow growth phenotype, and was hypersensitive to H(2)O(2) and organic peroxide. Using a position weight matrix approach, several predicted Fur-binding sites were detected in the genome of C. crescentus, located in regulatory regions of genes not only involved in iron uptake and usage but also in other functions. Selected Fur-binding sites were validated using electrophoretic mobility shift assay and DNAse I footprinting analysis. Gene expression assays revealed that genes involved in iron uptake were repressed by iron-Fur and induced under conditions of iron limitation, whereas genes encoding iron-using proteins were activated by Fur under conditions of iron sufficiency. Furthermore, several genes that are regulated via small RNAs in other bacteria were found to be directly regulated by Fur in C. crescentus. In conclusion, Fur functions as an activator and as a repressor, integrating iron metabolism and oxidative stress response in C. crescentus.

14-3-3: Defense and regulatory proteins coding by Eucalyptus genome

The data mining of Eucalyptus ESTs genome finds four clusters (EGCEST2257E11.g, EGBGRT3213F11.g, and EGCCFB1223H11.g) from highly conservative 14-3-3 protein family which modulates a wide variety of cellular processes. Multiple alignments were built from twenty four sequences of 14-3-3 proteins searched into the GenBank databases and into the four pools of Eucalyptus genome programs. The alignment has shown two regions highly conservative on the sequences corresponding to the motifs of protein phosphorylation and nine highly conservative regions on the sequence corresponding to the linkage regions of alpha helices structure based on three dimensional of dimer functional structure. The differences of amino acid into the structural and functional domains of 14-3-3 plant protein were identified and can explain the functional diversity of different isoforms. The phylogenic protein trees were built by the maximum parsimony and neighborjoining procedures of Clustal X alignments and PAUP software for phylogenic analysis.

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. on the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction. (c) 2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.

Cancer intelligence acquired (CIA): tumor glycosylation and sialylation codes dismantling antitumor defense

Aberrant glycosylation is a key feature of malignant transformation and reflects epigenetic and genetic anomalies among the multitude of molecules involved in glycan biosynthesis. Although glycan biosynthesis is not template bound, altered tumor glycosylation is not random, but associated with common glycosylation patterns. Evidence suggests that acquisition of distinct glycosylation patterns evolves from a ‘microevolutionary’ process conferring advantages in terms of tumor growth, tumor dissemination, and immune escape. Such glycosylation modifications also involve xeno- and hypersialylation. Xeno-autoantigens such as Neu5Gc-gangliosides provide potential targets for immunotherapy. Hypersialylation may display ‘enhanced self’ to escape immunosurveillance and involves several not mutually exclusive inhibitory pathways that all rely on protein–glycan interactions. A better understanding of tumor ‘glycan codes’ as deciphered by lectins, such as siglecs, selectins, C-type lectins and galectins, may lead to novel treatment strategies, not only in cancer, but also in autoimmune disease or transplantation.

The maize lipoxygenase, ZmLOX10 , mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack

Fatty acid derivatives are of central importance for plant immunity against insect herbivores; however, majorregulatory genes and the signals that modulate these defense metabolites are vastly understudied, especiallyin important agro-economic monocot species. Here we show that products and signals derived from a singleZea mays (maize) lipoxygenase (LOX), ZmLOX10, are critical for both direct and indirect defenses to herbiv-ory. We provide genetic evidence that two 13-LOXs, ZmLOX10 and ZmLOX8, specialize in providing substratefor the green leaf volatile (GLV) and jasmonate (JA) biosynthesis pathways, respectively. Supporting the spe-cialization of these LOX isoforms, LOX8 and LOX10 are localized to two distinct cellular compartments, indi-cating that the JA and GLV biosynthesis pathways are physically separated in maize. Reduced expression ofJA biosynthesis genes and diminished levels of JA in lox10 mutants indicate that LOX10-derived signaling isrequired for LOX8-mediated JA. The possible role of GLVs in JA signaling is supported by their ability to par-tially restore wound-induced JA levels in lox10 mutants. The impaired ability of lox10 mutants to produceGLVs and JA led to dramatic reductions in herbivore-induced plant volatiles (HIPVs) and attractiveness toparasitoid wasps. Because LOX10 is under circadian rhythm regulation, this study provides a mechanistic linkto the diurnal regulation of GLVs and HIPVs. GLV-, JA- and HIPV-deficient lox10 mutants display compro-mised resistance to insect feeding, both under laboratory and field conditions, which is strong evidence thatLOX10-dependent metabolites confer immunity against insect attack. Hence, this comprehensive gene toagro-ecosystem study reveals the broad implications of a single LOX isoform in herbivore defense.

Reduced stress defense in heme oxygenase 1-deficient cells

Stressed mammalian cells up-regulate heme oxygenase 1 (Hmox1; EC 1.14.99.3), which catabolizes heme to biliverdin, carbon monoxide, and free iron. To assess the potential role of Hmox1 in cellular antioxidant defense, we analyzed the responses of cells from mice lacking functional Hmox1 to oxidative challenges. Cultured Hmox1−/− embryonic fibroblasts demonstrated high oxygen free radical production when exposed to hemin, hydrogen peroxide, paraquat, or cadmium chloride, and they were hypersensitive to cytotoxicity caused by hemin and hydrogen peroxide. Furthermore, young adult Hmox1−/− mice were vulnerable to mortality and hepatic necrosis when challenged with endotoxin. Our in vitro and in vivo results provide genetic evidence that up-regulation of Hmox1 serves as an adaptive mechanism to protect cells from oxidative damage during stress.

An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense

Plants contain RNA-dependent RNA polymerase (RdRP) activities that synthesize short cRNAs by using cellular or viral RNAs as templates. During studies of salicylic acid (SA)-induced resistance to viral pathogens, we recently found that the activity of a tobacco RdRP was increased in virus-infected or SA-treated plants. Biologically active SA analogs capable of activating plant defense response also induced the RdRP activity, whereas biologically inactive analogs did not. A tobacco RdRP gene, NtRDRP1, was isolated and found to be induced both by virus infection and by treatment with SA or its biologically active analogs. Tobacco lines deficient in the inducible RDRP activity were obtained by expressing antisense RNA for the NtRDRP1 gene in transgenic plants. When infected by tobacco mosaic virus, these transgenic plants accumulated significantly higher levels of viral RNA and developed more severe disease symptoms than wild-type plants. After infection by a strain of potato virus X that does not spread in wild-type tobacco plants, the transgenic NtRDRP1 antisense plants accumulated virus and developed symptoms not only locally in inoculated leaves but also systemically in upper uninoculated leaves. These results strongly suggest that inducible RdRP activity plays an important role in plant antiviral defense.

Defense Responses to Tetrapyrrole-Induced Oxidative Stress in Transgenic Plants with Reduced Uroporphyrinogen Decarboxylase or Coproporphyrinogen Oxidase Activity1

We analyzed the antioxidative defense responses of transgenic tobacco (Nicotiana tabacum) plants expressing antisense RNA for uroporphyrinogen decarboxylase or coproporphyrinogen oxidase. These plants are characterized by necrotic leaf lesions resulting from the accumulation of potentially photosensitizing tetrapyrroles. Compared with control plants, the transformants had increased levels of antioxidant mRNAs, particularly those encoding superoxide dismutase (SOD), catalase, and glutathione peroxidase. These elevated transcript levels correlated with increased activities of cytosolic Cu/Zn-SOD and mitochondrial Mn-SOD. Total catalase activity decreased in the older leaves of the transformants to levels lower than in the wild-type plants, reflecting an enhanced turnover of this photosensitive enzyme. Most of the enzymes of the Halliwell-Asada pathway displayed increased activities in transgenic plants. Despite the elevated enzyme activities, the limited capacity of the antioxidative system was apparent from decreased levels of ascorbate and glutathione, as well as from necrotic leaf lesions and growth retardation. Our data demonstrate the induction of the enzymatic detoxifying defense system in several compartments, suggesting a photosensitization of the entire cell. It is proposed that the tetrapyrroles that initially accumulate in the plastids leak out into other cellular compartments, thereby necessitating the local detoxification of reactive oxygen species.

Inhibition of ascorbate peroxidase by salicylic acid and 2,6-dichloroisonicotinic acid, two inducers of plant defense responses.

In recent years, it has become apparent that salicylic acid (SA) plays an important role in plant defense responses to pathogen attack. Previous studies have suggested that one of SA's mechanisms of action is the inhibition of catalase, resulting in elevated levels of H2O2, which activate defense-related genes. Here we demonstrate that SA also inhibits ascorbate peroxoidase (APX), the other key enzyme for scavenging H2O2. The synthetic inducer of defense responses, 2,6-dichloroisonicotinic acid (INA), was also found to be an effective inhibitor of APX. In the presence of 750 microM ascorbic acid (AsA), substrate-dependent IC50 values of 78 microM and 95 microM were obtained for SA and INA, respectively. Furthermore, the ability of SA analogues to block APX activity correlated with their ability to induce defense-related genes in tobacco and enhance resistance to tobacco mosaic virus. Inhibition of APX by SA appears to be reversible, thus differing from the time-dependent, irreversible inactivation by suicide substrates such as p-aminophenol. In contrast to APX, the guaiacol-utilizing peroxidases, which participate in the synthesis and crosslinking of cell wall components as part of the defense response, are not inhibited by SA or INA. The inhibition of both catalase and APX, but not guaiacol peroxidases, supports the hypothesis that SA-induced defense responses are mediated, in part, through elevated H2O2 levels or coupled perturbations of the cellular redox state.

Induction, modification, and transduction of the salicylic acid signal in plant defense responses.

Studies in our laboratory as well as others strongly suggest that salicylic acid (SA) plays an important signaling role in plant defense against pathogens. We have found that increases in endogenous SA levels correlates with both resistance of tobacco to infection with tobacco mosaic virus and induction of defense-related genes such as that encoding pathogenesis-related protein 1 (PR-1). Some of this newly synthesized SA was conjugated to glucose to form SA beta-glucoside. A cell wall-associated beta-glucosidase activity that releases SA from this glucoside has been identified, suggesting that SA beta-glucoside serves as an inactive storage form of SA. By purifying a soluble SA-binding protein and isolating its encoding cDNA from tobacco, we have been able to further characterize the mechanism of SA signaling. This protein is a catalase, and binding of SA and its biologically active analogues inhibited catalase's ability to convert H2O2 to O2 and H2O. The resulting elevated levels of cellular H2O2 appeared to induce PR-1 gene expression, perhaps by acting as a second messenger. Additionally, transgenic tobacco expressing an antisense copy of the catalase gene and exhibiting depressed levels of catalase also showed constitutive expression of PR-1 genes. To further dissect the SA signaling pathway, we have tested several abiotic inducers of PR gene expression and disease resistance for their ability to stimulate SA production. Levels of SA and its glucoside rose following application of all of the inducers except 2,6-dichloroisonicotinic acid. 2,6-Dichloroisonicotinic acid was found to bind catalase directly and inhibit its enzymatic activity. Thus, it appears that many compounds that induce PR gene expression and disease resistance in plants inactivate catalases directly or indirectly.

Involvement of small GTP-binding proteins in defense signal-transduction pathways of higher plants.

Small GTP-binding proteins play a critical role in the regulation of a range of cellular processes--including growth, differentiation, and intracellular transportation. Previously, we isolated a gene, rgp1, encoding a small GTP-binding protein, by differential screening of a rice cDNA library with probe DNAs from rice tissues treated with or without 5-azacytidine, a powerful inhibitor of DNA methylation. To determine the physiological role of rgp1, the coding region was introduced into tobacco plants. Transformants, with rgp1 in either sense or antisense orientations, showed distinct phenotypic changes with reduced apical dominance, dwarfism, and abnormal flower development. These abnormal phenotypes appeared to be associated with the higher levels of endogenous cytokinins that were 6-fold those of wild-type plants. In addition, the transgenic plants produced salicylic acid and salicylic acid-beta-glucoside in an unusual response to wounding, thus conferring increased resistance to tobacco mosaic virus infection. In normal plants, the wound- and pathogen-induced signal-transduction pathways are considered to function independently. However, the wound induction of salicylic acid in the transgenic plants suggests that expression of rgp1 somehow interfered with the normal signaling pathways and resulted in cross-signaling between these distinct transduction systems. The results imply that the defense signal-transduction system consists of a complicated and finely tuned network of several regulatory factors, including cytokinins, salicylic acid, and small GTP-binding proteins.

Molecular mechanisms of defense by rhizobacteria against root disease.

Genetic resistance in plants to root diseases is rare, and agriculture depends instead on practices such as crop rotation and soil fumigation to control these diseases. "Induced suppression" is a natural phenomenon whereby a soil due to microbiological changes converts from conducive to suppressive to a soilborne pathogen during prolonged monoculture of the susceptible host. Our studies have focused on the wheat root disease "take-all," caused by the fungus Gaeumannomyces graminis var. tritici, and the role of bacteria in the wheat rhizosphere (rhizobacteria) in a well-documented induced suppression (take-all decline) that occurs in response to the disease and continued monoculture of wheat. The results summarized herein show that antibiotic production plays a significant role in both plant defense by and ecological competence of rhizobacteria. Production of phenazine and phloroglucinol antibiotics, as examples, account for most of the natural defense provided by fluorescent Pseudomonas strains isolated from among the diversity of rhizobacteria associated with take-all decline. There appear to be at least three levels of regulation of genes for antibiotic biosynthesis: environmental sensing, global regulation that ties antibiotic production to cellular metabolism, and regulatory loci linked to genes for pathway enzymes. Plant defense by rhizobacteria producing antibiotics on roots and as cohabitants with pathogens in infected tissues is analogous to defense by the plant's production of phytoalexins, even to the extent that an enzyme of the same chalcone/stilbene synthase family used to produce phytoalexins is used to produce 2,4-diacetylphloroglucinol. The defense strategy favored by selection pressure imposed on plants by soilborne pathogens may well be the ability of plants to support and respond to rhizosphere microorganisms antagonistic to these pathogens.