Zsolt Szegletes

Responses in polyamine titer under osmotic and salt stress in Sorghum and maize seedlings

Summary Changes in leaf water potential, K + /Na + ratio and in polyamine titers in maize ( Zea mays L. cv. Pioneer 3950) and sorghum ( Sorghum bicolor (L.) Moench cv. ICSV 112) were investigated as the function of increasing equi-osmotic concentrations of NaCl or polyethylene glycol 6000 applied for 3 days in the nutrient solution. For both treatments, sorghum responded with a more intensive decrease in leaf water potential compared with maize. Plant analysis showed that under salinity, a higher level of Na + was accumulated in the roots of maize than in those of sorghum while shoot Na + concentrations were nearly the same. Internal K + concentration was always higher in sorghum, leading to a K + /Na + ratio 2 to 4-fold higher in the roots of sorghum compared with that in maize. In maize, NaCl and osmotic stresses evoked similar rates of polyamine accumulation, while in sorghum, osmotic stress was more effective in comparison to salt treatment. In maize, spermidine was apparently absent, indicating its fast turnover. In sorghum, the accumulation of di- and polyamines was proportional to the strength of the osmotic stress and the impairment of polyamine synthesis was also observed at high degrees of stress. The present data support the idea that the initiation of polyamine accumulation needs an osmotic signal; however, when a permeable ion is present, salt accumulation can contribute to the osmotic adjustment and thus the onset of polyamine biosynthesis is delayed or does not take place. Some enzymes in the polyamine biosynthetic pathway may be sensitive for high salinity and the biosynthetic processes shift towards oxidative degradation.

Melanoma cell-derived exosomes alter macrophage and dendritic cell functions in vitro.

To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic cells; we quantified NF-κB activation in mature macrophages stimulated with mcd-exosomes, and we compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages. We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-κB activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may play a role in the tumor progression and metastasis formation through supporting tumor immune escape mechanisms.

syrM Is Involved in the Determination of the Amount and Ratio of the Two Forms of the Acidic Exopolysaccharide EPSI in Rhizobium meliloti

For the invasion of root nodule cells of alfalfa by its symbiotic partner Rhizobium meliloti, the bacterial succinoglycan (EPSI) is required. Nitrogen starvation of R. meliloti results in a considerable increase of EPSI production. We have shown that the presence of nitrogen also altered the ratio of high and low molecular weight (HMW and LMW, respectively) forms of EPSI, favoring the production of LMW molecules. The transcription of two exo genes (exoF, exoP) was found to be under the control of combined nitrogen. In contrast, exoH and exoK were expressed independently of the nitrogen level. ntrC and syrM, regulatory genes for the nitrogen control of nod gene expression, were shown to be involved in the regulation of EPSI production. In an syrM mutant both the amount and the ratio of the two main forms of EPSI were altered, compared with the wild-type strain: a reduced level of EPSI and the dominance of the LMW form in nitrogenstarved cultures, while an increased level of EPSI and the accumulation of the HMW fraction in nitrogen-containing samples was observed. An enhanced expression level of the exoK gene and the higher ratio of the LMW form to the total EPSI in nitrogen-free samples of the syrM mutant suggest a regulatory role for syrM in exoK expression and a contribution of exoK to the accumulation of the LMW form of EPSI. During the formation of nitrogen-fixing root nodules, signal molecules are exchanged between the host plant alfalfa (Medicago sativa) and its symbiotic partner Rhizobium meliloti. The signal molecules are required for the control of the highly specific plant-bacterium recognition and for the coordinated interaction of the two organisms. Molecular mechanisms acting at the early steps of nodule formation have been characterized in the R. meliloti‐M. sativa

Effects of excess UV-B irradiation on the antioxidant defence mechanisms in wheat (Triticum aestivum L) seedlings

Summary In the present experiments, changes in the antioxidant defence systems of wheat ( Triticum aestivum L. cv. Tiszataj) as affected by excess UV-B irradiation were investigated. Wheat seedlings were grown hydroponically in phytotrons for 11 days. In one of the phytotrons 2.5 Wm −2 excess UV-B irradiation was applied and samples were taken daily. Catalase, guaiacol peroxidase, superoxide dismutase (SOD) and glutathione reductase (GR) activities were measured and changes in the level of glutathione disulfide (GSSG) were determined by HPLC technique. It was found that catalase, guaiacol peroxidase and SOD activities decreased, while GSSG level and GR activity increased under excess UV-B irradiation. In plant responses, the alarm and adaptation phases were recognised. During the alarm reaction, inhibition of stem and leaf elongation, accumulation of flavonoid pigments, decreased antioxidant enzyme activities, increased GSSG level and triggering of GR activity were observed, while normalisation of growth, disappearance of stem pigmentation, levelling of antioxidant enzyme activities, high GR activity and decreased level of GSSG were found as parts of adaptation to UV-B irradiation. Afier 3 days of UV-B treatment, the activities of Ca 2+ -dependent protein kinases (52 and 65 kDa) increased, suggesting that they may participate in the UV-B signal transduction pathway.

Environmental stress and the biological clock in plants: Changes of rhythmic behavior of carbohydrates, antioxidant enzymes and stomatal resistance by salinity

Summary In this report, the influence of environmental stress as effector on rhythmic phenomena in plants is discussed based on data from literature and from own studies. In our experiments, the effects of NaCl salinity (0, 50, 100 and 150 mM) on the levels of total soluble carbohydrates and on the activities of the antioxidant enzymes, superoxide dismutase and glutathione reductase, were followed as the function of time in young winter wheat ( Triticum aestivum L.) plants grown under controlled conditions. Salinity effects on stomatal movements of the halophytic Aster tripolium L. were also investigated. Periodicity was calculated by Fourier analysis. The results indicate that the fluctuations in the above functions were modulated by the stressor suggesting the connection between environmental stress and biological rhythmicity. On the basis of these observations and literature data we hypothesize that the stressor may modify and desynchronize the biochemical and physiological oscillations by acting either on the input (from receptor till oscillator) or on the output (from the oscillator till the oscillating component) pathways. In these terms, adaptation means re-setting of the new circadian and/or ultradian rhythms of biochemical/physiological processes and the restoration of their concerted action results in maximal resistance for the organism under the new environmental conditions.

Effect of Antimicrobial Peptide-Amide: Indolicidin on Biological Membranes

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity is intensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here an in situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membrane of dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependent interaction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purple membrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the border of membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool in the study of indolicidin-membrane interaction.

Antimicrobial nodule-specific cysteine-rich peptides disturb the integrity of bacterial outer and inner membranes and cause loss of membrane potential

BackgroundCertain legume plants produce a plethora of AMP-like peptides in their symbiotic cells. The cationic subgroup of the nodule-specific cysteine-rich (NCR) peptides has potent antimicrobial activity against gram-negative and gram-positive bacteria as well as unicellular and filamentous fungi.FindingsIt was shown by scanning and atomic force microscopies that the cationic peptides NCR335, NCR247 and Polymyxin B (PMB) affect differentially on the surfaces of Sinorhizobium meliloti bacteria. Similarly to PMB, both NCR peptides caused damages of the outer and inner membranes but at different extent and resulted in the loss of membrane potential that could be the primary reason of their antimicrobial activity.ConclusionsThe primary reason for bacterial cell death upon treatment with cationic NCR peptides is the loss of membrane potential.

Differences in photorespiration, glutamine synthetase and polyamines between fragmented and closed stands of Phragmites australis

Abstract Physiological processes related to C and N metabolism were investigated in closed healthy, and fragmented die-back stands of Phragmites australis (Cav.) Trin. ex Steudel along the shores of Lake Balaton, Hungary. In the leaves, similar concentrations of total N and P, K+, Na+, Ca2+ and Mg2+ were found. However, higher concentrations of soluble proteins in the fragmented stand indicated alterations in N metabolism. In both types of stands, nitrate reductase (NR) activity was detectable only in the period of vegetative growth and it was higher in the fragmented than in the closed stands. Glutamine synthetase (GS) activity showed three-fold higher activities in the leaves from the fragmented stands compared to those in closed stands, indicating high substrate (NH3/NH4+) availability. Polyamine concentrations were 4–10-fold higher in the leaves of the fragmented stands than in those of closed stands. Photosynthetic activity was nearly equal in both stands, however, photorespiration was about two-fold higher in the fragmented than in the closed stands. A linear correlation between photorespiration and GS activity indicated a causal relationship (R2=0.86). Stomatal conductance data suggest that the higher photorespiration in the fragmented stands could be the consequence of disturbed stomatal regulation. It is concluded that fragmented stands of Phragmites possess an altered C/N metabolism, due to high photorespiration and intensive N metabolism. The primary reason of the cascade of events is still not clear but apparently, these metabolic malfunctions accompany an accelerated die-back of Phragmites around Lake Balaton.

Spatial and temporal dependence of the cerebral endothelial cells elasticity

The reliable determination of the mechanical properties of a living cell is one of the most important challenges of the atomic force microscopic measurements. In the present study the spatial and temporal dependency of the force measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two different sequences of force measurements were applied: Acquisition of force curves in short time at several points across the cell surface investigating spatial dependence of the elasticity. Acquisition of force curves for long time at a previously determined place, over the cell nucleus, which provides the temporal stability/variation of the measured forces/values.

Interaction of cysteine-rich cationic antimicrobial peptides with intact bacteria and model membranes.

Antimicrobial peptides are small proteins that exhibit a broad spectrum of antimicrobial activity. Their chemical structure allows them to interact (attach and insert) with membranes. The fine details about this interaction and their mode of action are not fully clarified yet. In order to better understand this mechanism, we have performed in situ atomic force microscopy studies using two types of nodule specific cysteine-rich NCR peptides on Escherichia coli bacteria and on natural purple membrane. On intact bacteria, both NCR247 and NCR335 caused increase in the surface roughness, indicating the damage of the bacterial cell envelope. In case of the tightly packed purple membrane, it is clear that the peptides prefer to disrupt the border of the disks indicating a strong lipid preference of the interaction. These results verify the concept that the first target of NCR peptides is probably the bacterial cell envelope, especially the lipid matrix.