ROS production during symbiotic infection suppresses pathogenesis-related gene expression

Leguminous plants have exclusive ability to form symbiotic relationship with soil bacteria of the genus Rhizobium. Symbiosis is a complex process that involves multiple molecular signaling activities, such as calcium fluxes, production of reactive oxygen species (ROS) and synthesis of nodulation genes. We analyzed the role of ROS in defense gene expression in Medicago truncatula during symbiosis and pathogenesis. Studies in Arabidopsis thaliana showed that the induction of pathogenesis-related (PR) genes during systemic acquired resistance (SAR) is regulated by NPR1 protein, which resides in the cytoplasm as an oligomer. After oxidative burst and return of reducing conditions, the NPR1 undergoes monomerization and becomes translocated to the nucleus, where it functions in PR genes induction. We show that ROS production is both stronger and longer during symbiotic interactions than during interactions with pathogenic, nonhost or common nonpathogenic soil bacteria. Moreover, root cells inoculated with Sinorhizobium meliloti accumulated ROS in the cytosol but not in vacuoles, as opposed to Pseudomonas putida inoculation or salt stress treatment. Furthermore, increased ROS accumulation by addition of H₂O₂ reduced the PR gene expression, while catalase had an opposite effect, establishing that the PR gene expression is opposite to the level of cytoplasmic ROS. In addition, we show that salicylic acid pretreatment significantly reduced ROS production in root cells during symbiotic interaction.     

Effects of mechanical stimulation induced by compression and medium perfusion on cardiac tissue engineering

Cardiac tissue engineering presents a challenge due to the complexity of the muscle tissue and the need for multiple signals to induce tissue regeneration in vitro. We investigated the effects of compression (1 Hz, 15% strain) combined with fluid shear stress (10^?2–10^?1 dynes/cm²) provided by medium perfusion on the outcome of cardiac tissue engineering. Neonatal rat cardiac cells were seeded in Arginine‐Glycine‐Aspartate (RGD)‐attached alginate scaffolds, and the constructs were cultivated in a compression bioreactor. A daily, short‐term (30 min) compression (i.e., “intermittent compression”) for 4 days induced the formation of cardiac tissue with typical striation, while in the continuously compressed constructs (i.e., “continuous compression”), the cells remained spherical. By Western blot, on day 4 the expression of the gap junction protein connexin 43 was significantly greater in the “intermittent compression” constructs and the cardiomyocyte markers (α‐actinin and N‐cadherin) showed a trend of better preservation compared to the noncompressed constructs. This regime of compression had no effect on the proliferation of nonmyocyte cells, which maintained low expression level of proliferating cell nuclear antigen. Elevated secretion levels of basic fibroblast growth factor and transforming growth factor‐β in the daily, intermittently compressed constructs likely attributed to tissue formation. Our study thus establishes the formation of an improved cardiac tissue in vitro, when induced by combined mechanical signals of compression and fluid shear stress provided by perfusion. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

The LysM Receptor-Like Kinase LysM RLK1 Is Required to Activate Defense and Abiotic-Stress Responses Induced by Overexpression of Fungal Chitinases in Arabidopsis Plants

Application of crab shell chitin or pentamer chitin oligosaccharide to Arabidopsis seedlings increased tolerance to salinity in wild-type but not in knockout mutants of the LysM Receptor-Like Kinase1 (CERK1/LysM RLK1) gene, known to play a critical role in signaling defense responses induced by exogenous chitin. Arabidopsis plants overexpressing the endochitinase chit36 and hexoaminidase excy1 genes from the fungus Trichoderma asperelleoides T203 showed increased tolerance to salinity, heavy-metal stresses, and Botrytis cinerea infection. Resistant lines, overexpressing fungal chitinases at different levels, were outcrossed to lysm rlk1 mutants. Independent homozygous hybrids lost resistance to biotic and abiotic stresses, despite enhanced chitinase activity. Expression analysis of 270 stress-related genes, including those induced by reactive oxygen species (ROS) and chitin, revealed constant up-regulation (at least twofold) of 10 genes in the chitinase-overexpressing line and an additional 76 salt-induced genes whose expression was not elevated in the lysm rlk1 knockout mutant or the hybrids harboring the mutation. These findings elucidate that chitin-induced signaling mediated by LysM RLK1 receptor is not limited to biotic stress response but also encompasses abiotic-stress signaling and can be conveyed by ectopic expression of chitinases in plants.     

Citrus chlorophyllase dynamics at ethylene-induced fruit color-break: a study of chlorophyllase expression, posttranslational processing kinetics, and in situ intracellular localization

Fruit color-break is the visual manifestation of the developmentally regulated transition of chloroplasts to chromoplasts during fruit ripening and often involves biosynthesis of copious amounts of carotenoids concomitant with massive breakdown of chlorophyll. Regulation of chlorophyll breakdown at different physiological and developmental stages of the plant life cycle, particularly at fruit color-break, is still not well understood. Here, we present the dynamics of native chlorophyllase (Chlase) and chlorophyll breakdown in lemon (Citrus limon) fruit during ethylene-induced color-break. We show, using in situ immunofluorescence on ethylene-treated fruit peel (flavedo) tissue, that citrus Chlase is located in the plastid, in contrast to recent reports suggesting cytoplasmic localization of Arabidopsis (Arabidopsis thaliana) Chlases. At the intra-organellar level, Chlase signal was found to overlap mostly with chlorophyll fluorescence, suggesting association of most of the Chlase protein with the photosynthetic membranes. Confocal microscopy analysis showed that the kinetics of chlorophyll breakdown was not uniform in the flavedo cells. Chlorophyll quantity at the cellular level was negatively correlated with plastid Chlase accumulation; plastids with reduced chlorophyll content were found by in situ immunofluorescence to contain significant levels of Chlase, while plastids containing still-intact chlorophyll lacked any Chlase signal. Immunoblot and protein-mass spectrometry analyses were used to demonstrate that citrus Chlase initially accumulates as an approximately 35-kD precursor, which is subsequently N-terminally processed to approximately 33-kD mature forms by cleavage at either of three consecutive amino acid positions. Chlase plastid localization, expression kinetics, and the negative correlation with chlorophyll levels support the central role of the enzyme in chlorophyll breakdown during citrus fruit color-break.     

Biodegradation Kinetics of Hydrocarbons in Soil during Land Treatment of Oily Sludge

This study focuses on the processes influencing hydrocarbon residue persistence in soil, following land treatment of refinery oily sludge. Treating sludge applied to soil resulted in 70% to 90% degradation of total petroleum hydrocarbon (TPH) during 2 months, regardless of their initial concentrations (9 to 60 g/kg soil). Kinetic analyses performed on TPH degradation, in laboratory and field systems, revealed a degradation pattern characterized by two consecutive first-order kinetics reactions in all experimental settings. The first stage lasted about 3 weeks and was characterized by a temperature dependent rate constant of 0.047 day^-1 at 24°C. That value was comparable to the rate constant obtained when combining the individual rate constants of the saturated, aromatic, asphaltene and polar fractions. The subsequent slower stage rate constant was 0.012 day^-1, insensitive to temperature and to hydrocarbon composition. The transition between the two stages (about 21 days) was independent of the experimental temperature and the biodegradation extent during the first stage. It was concluded that the extent of residual accumulation in the soil was determined by the biodegradation efficiency during the first three weeks of treatment when biological processes dominated. During the following period, abiotic processes leading to reduced bioavailability of the TPH were limiting the degradation rate. Practically, as the first few weeks of treatment determine its efficiency, efforts to enhance the biological activity should be directed to that period.     

Effects of estrone and 17 β-estradiol on vegetative growth of Medicago sativa

Alfalfa ( Medicago sativa L.) plants were grown in the absence or presence of the steroidal estrogens, estrone and 17β-estradiol, under varying conditions. Plants were analysed for the following parameters: plant weight, estrogen content, phytoestrogen content, degree of nodulation and nitrogen fixation activity. It was found that under controlled greenhouse conditions: (1) Treatment with estrogens in the range of 0.005 to 0.5 μg 1⁻¹ increases both shoot and root dry weitht. In contrast, estrogen in concentrations of 50 to 500 μg 1⁻¹ decreases plant growth. (2) The effect of estrogen of growth is most marked in the absence of nitrogen. (3) Estrone is more effective in increasing growth than 17 β-estradiol. (4) In the plants where estrogen induced growth there was no significant increase in nitrogen fixation activity and nodule number. (5) Endogenous estrogen content of the plant did not increase at concentrations (0.005-0.5 μg 1⁻¹) which increased vegetative growth. (6) Endogenous estrogen content of the plant did increase at concentrations of estrogen (50-500 μg 1⁻¹ which inhibited vegetative growth and nodule weight. It can be concluded that estrogen in concentrations found in sewage water (0.3 μg estrogen 1⁻¹) can affect the vegetative growth of alfalfa plants.     

Three-dimensional porous alginate scaffolds provide a conducive environment for generation of well-vascularized embryoid bodies from human embryonic stem cells

Differentiation of human embryonic stem cells (hESCs) can be instigated through the formation of embryo-like aggregates in suspension, termed human embryoid bodies (hEBs). Controlling cell aggregation and agglomeration during hEBs formation has a profound effect on the extent of cell proliferation and differentiation. In a previous work, we showed that control over hEBs formation and differentiation can be achieved via cultivation of hESC suspensions in a rotating bioreactor system. We now report that hEBs can be generated directly from hESC suspensions within three-dimensional (3D) porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled efficient formation of hEBs with a relatively high degree of cell proliferation and differentiation; encouraged round, small-sized hEBs; and induced vasculogenesis in the forming hEBs to a greater extent than in static or rotating cultures. We therefore conclude that differentiation of hEBs can be induced and directed by physical constraints in addition to chemical cues.     

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.     

Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of 4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.