The Positive Correlations Between the Activity of Superoxide Dismutase and Dehydration Tolerance in Wheat Seedlings

Damage to crops by drought is still a serious problem in large areas of the world. Considerable research has been undertaken to discover the mechanisms of drought injury and drought resistance of plants. However, the critical features of drought injury have not yet been identified. In the past ten years a free radical hypothesis has been suggested to account for subcellular damage caused by severe environments. Superoxide (oxygen radical) is normally produced in hydrated tissues. It is controlled by free radical scavenging reactions. One such scavenger is the enzyme superoxide dismutase (SOD). Under water stress, production of excess free radicals may occur in dehydrated plant tissues and this probably damages the membranes by causing peroxidation of the lipid components. So far few studies have been done to determine if drought injury is correlated with the free radical mechanism. In the present study, the SOD activities in wheat seedlings under water stress have been investigated by measuring the photoreduction of nitro blue tetrazolium using a spectrometric method. Meanwhile, the viabilities of wheat seedlings during drying were followed by tetrazolium test. These results provided information on the relationship between SOD activity and the dehydration tolerance of the plant. Results indicated that SOD activity changed with the time after germination. The activity of SOD of 24 h seedlings was 1.9 times higher than those of 72 h seedlings based on fresh weight. SOD activity in shoot was also higher than in root. These results were consistent with the results obtained from rating of the viabilities of seedlings during drying. The 24h seedlings were more tolerant of dehydration than 72 h seedlings and root were more sensitive of drought than shoot. In addition, shoot and root tips showed the higher SOD activities than non-tip region and they also showed a higher survival ability upon dehydration. In dehydration and subsequent rehydration, SOD activity, different from many other enzymes in plants, increased rather than declined during drying. After rehydration SOD activity returned to nearly the original level. Therefore, the positive correlations were found to exist between SOD activity and dehydration tolerance. It is reasonable to suggest that SOD enzyme may play a protective role against damage caused by free radicals which may be produced excessively during dehydration in wheat seedling.     

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25? effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.     

Estimating dataset size requirements for classifying DNA microarray data.

A statistical methodology for estimating dataset size requirements for classifying microarray data using learning curves is introduced. The goal is to use existing classification results to estimate dataset size requirements for future classification experiments and to evaluate the gain in accuracy and significance of classifiers built with additional data. The method is based on fitting inverse power-law models to construct empirical learning curves. It also includes a permutation test procedure to assess the statistical significance of classification performance for a given dataset size. This procedure is applied to several molecular classification problems representing a broad spectrum of levels of complexity.     

Cell cycle specific changes in the human cyclin B1 gene regulatory region as revealed by response to trichostatin A

The human cyclin Bl gene is cell cycle regulated with maximal activity during G(2)/M. We examined the role of histone deacetylation in cyclin Bl regulation using the histone deacetylase inhibitor trichostatin A (TSA). TSA treatment (100 ng/ml) of NIH3T3 cells containing the luciferase reporter construct pCycB(-287)-LUC caused an increase in promoter activity in G(0) and G(1) but no significant change in G(2). Removal of upstream sequences including an E-box and Sp1 site eliminated the TSA induced increase in G(0) and G(1), and caused a decrease in promoter activity during S and G(2). Promoter activity increased only 2-fold following TSA treatment of G(0) cells containing the construct pCycB(MUT-E-Box)-LUC with an E-box mutation, and a decrease in activity was detected during G(2). We conclude that histone deacetylation contributes to the repression of cyclin B1 expression in G(0) and G(1), and that this mechanism requires, in part, the E-box. TSA reduction of cyclin B1 promoter activity in G(2), however, involves sequences within the first 119 bp. A working model for cyclin B1 regulation is provided.     

Structure determination of feline panleukopenia virus empty particles

Various crystal forms of the single-stranded DNA, feline panleukopenia virus (FPV), a parvovirus, have been grown of both full virions and empty particles. The structure of empty particles crystallized in an orthorhombic space group P2₁2₁2₁, with unit cell dimensions a = 380.1 Å, b = 379.3 Å, and c = 350.9 Å, has been determined to 3.3 Å resolution. The data were collected using oscillation photography with synchrotron radiation. The orientations of the empty capsids in the unit cell were determined using a self-rotation function and their positions were obtained with an R-factor search using canine parvovirus (CPV) as a model. Phases were then calculated, based on the CPV model, to 6.0 Å resolution and gradually extended to 3.3 Å resolution by molecular replacement electron density averaging. The resultant electron density was readily interpreted in terms of the known amino acid sequence. The structure is contrasted to that of CPV in terms of host range, neutralization by antibodies, hemagglutination properties, and binding of genomic DNA. © Wiley-Liss, Inc.     

Inhibition of ozone-induced SP-A oxidation by plant polyphenols

Surfactant protein-A (SP-A) is the best studied and most abundant of the protein components of lung surfactant and plays an important role in host defense of the lung. It has been shown that ozone-induced oxidation of SP-A protein changes its functional and biochemical properties. In the present study, eight plant polyphenols (three flavonoids, three hydroxycinnamic acids, and two hydroxybenzoic acids) known as strong antioxidants, were tested for their ability to inhibit ozone-induced SP-A oxidation as a mechanism for chemoprevention against lung damage. SP-A isolated from alveolar proteinosis patients was exposed to ozone (1 ppm) for 4 h. The flavonoids protected SP-A from oxidation in a dose dependent manner. ( - )-Epicatechin was the most potent flavonoid and exhibited inhibition of ozone-induced formation of carbonyls by 35% at a concentration as low as 5 μM. Hydroxybenzoic acids inhibited SP-A oxidation in a dose-dependent manner although they were less potent than flavonoids. On the other hand, hydroxycinnamic acids exhibited a different inhibitory pattern. Inhibition was observed only at medium concentrations. The results indicate that inhibition of SP-A oxidation by plant polyphenols may be a mechanism accounting for the protective activity of natural antioxidants against the effects of ozone exposure on lungs.     

Phosphodiesterase inhibition attenuates alterations to the tight junction proteins occludin and ZO-1 in immunostimulated Caco-2 intestinal monolayers

AimsUnder normal conditions, the intestinal mucosa acts as a local barrier to prevent the influx of luminal contents. The intestinal epithelial tight junction is comprised of several membrane associated proteins, including zonula occludens-1 (ZO-1) and occludin. Disruption of this barrier can lead to the production of pro-inflammatory mediators and ultimately multiple organ failure. We have previously shown that Pentoxifylline (PTX) decreases histologic gut injury and pro-inflammatory mediator synthesis. We hypothesize that PTX prevents the breakdown of ZO-1 and occludin in an in vitro model of immunostimulated intestinal cell monolayers.Main methodsCaco-2 human enterocytes were grown as confluent monolayers and incubated under control conditions, or with PTX (2 mM), Cytomix (TNF-α, IFN-γ, IL-1), or Cytomix + PTX for 24 h. Occludin and ZO-1 protein levels were analyzed by Western blot. Confocal microscopy was used to assess the cytoplasmic localization of ZO-1 and occludin.Key findingsCytomix stimulation of Caco-2 cells resulted in a 50% decrease in both occludin and ZO-1 protein. Treatment with Cytomix + PTX restored both occludin and ZO-1 protein to control levels. Confocal microscopy images show that Cytomix caused an irregular, undulating appearance of ZO-1 and occludin at the cell junctions. Treatment with PTX prevented the Cytomix-induced changes in ZO-1 and occludin localization.SignificanceTreatment with PTX decreases the pro-inflammatory cytokine induced changes in the intestinal tight junction proteins occludin and ZO-1. Pentoxifylline may be a useful adjunct in the treatment of sepsis and shock by attenuating intestinal barrier breakdown.     

Role of claudin interactions in airway tight junctional permeability

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.