Effect of salt stress on photosynthesis and chlorophyll fluorescence in Medicago truncatula

In the present study, photosynthetic parameters including gas exchanges, pigment contents, and chlorophyll fluorescence, were compared in two contrasting local Medicago truncatula lines TN6.18 and TN8.20, in response to salt added to the nutrient solution. Plants were cultivated under symbiotic nitrogen fixation (SNF) after inoculation with a reference strain Sinorhizobium meliloti 2011, a very tolerant strain to salinity (700 mM NaCl), and grown in a controlled glasshouse. On one month old plants (with active SNF), salt treatment (75 mM NaCl) was gradually applied. Photosynthesis, assimilating pigments and chlorophyll fluorescence were monitored throughout the experiment during both short and long terms, compared to control (non-saline) conditions. A genotypic variation in salt tolerance was found; TN6.18 was the more sensitive to salinity. The relative tolerance of TN8.20 was concomitant with the highest photochemical quenching coefficient (q_P) affecting the maximum quantum yield of PSII (Y); the real quantum yield (?_exc) was the most affected in the sensitive line. Moreover, stomatal and PSII reaction centers activities differed clearly between the studied lines. We found that the effect of salinity on photosynthesis of M. truncatula was related to PSII activity reduction rather than to stomatal conductance limitation. Photosynthesis was reduced by the inhibition of CO₂ assimilation caused by PSII damage. This was clearly estimated by the Y, ?_exc and especially by the quantum yield of electron transport of PSII (Φ_PSII). Thus, on the basis of our results on the two local M. truncatula lines, we recommend the use of chlorophyll fluorescence as non-destructive screening method to discriminate susceptible and resistant legumes to salt stress.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

Preparation and Evaluation of Microemulsion Systems Containing Salicylic Acid

Microemulsions (MEs) are clear, thermodynamically stable systems. They were used to solubilize drugs and to improve topical drug availability. Salicylic acid (SA) is a keratolytic agent used in topical products with antimicrobial actions. The objective of this work was to prepare and evaluate SA ME systems. Different concentrations of SA were incorporated in an ME base composed of isopropyl myristate, water, and Tween 80: propylene glycol in the ratio of 15:1. Three ME systems were prepared: S_2%, S_5%, and S_10% which contain 2%, 5%, and 10% of SA, respectively. Evaluation by examination under cross-polarizing microscope, measuring of percent transmittance, pH measurement, determination of the specific gravity, assessment of rheological properties, and accelerated stability study were carried out. The data showed that the addition of SA markedly affected the physical properties of the base. All systems were not affected by accelerated stability tests. Stability study for 6 months under ambient conditions was carried out for S_10%. No remarkable changes were recorded except a decrease in the viscosity value after 1 month. The results suggested that ME could be a suitable vehicle for topical application of different concentrations of SA.     

Axisymmetric Drop Shape Analysis for Estimating the Surface Tension of Cell Aggregates by Centrifugation

Biological tissues behave in certain respects like liquids. Consequently, the surface tension concept can be used to explain aspects of the in vitro and in vivo behavior of multicellular aggregates. Unfortunately, conventional methods of surface tension measurement cannot be readily applied to small cell aggregates. This difficulty can be overcome by an experimentally straightforward method consisting of centrifugation followed by axisymmetric drop shape analysis (ADSA). Since the aggregates typically show roughness, standard ADSA cannot be applied and we introduce a novel numerical method called ADSA-IP (ADSA for imperfect profile) for this purpose. To examine the new methodology, embryonic tissues from the gastrula of the frog, Xenopus laevis, deformed in the centrifuge are used. It is confirmed that surface tension measurements are independent of centrifugal force and aggregate size. Surface tension is measured for ectodermal cells in four sample batches, and varies between 1.1 and 7.7 mJ/m². Surface tension is also measured for aggregates of cells expressing cytoplasmically truncated EP/C-cadherin, and is approximately half as large. In parallel, such aggregates show a reduction in convergent extension-driven elongation after activin treatment, reflecting diminished intercellular cohesion.     

Two dimensional electrophoresis of the exo-proteome produced from community acquired methicillin resistant Staphylococcus aureus belonging to clonal complex 80

Two-dimensional electrophoresis (2DE) combined with mass spectrometry was used to characterize the exo-proteome secreted by two strains (ER13 and ER21) representing community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) belonging to clonal complex 80 (CC80). Common spots were detected between the 2 gels using the Progenesis SameSpots software. Two hundred and fifty-one and 312 spots from the exo-proteome of ER13 and ER21 were resolved, respectively. 2DE overlap comparison showed that 59 spots were shared. LC–MS/MS analysis identified 57 proteins from these spots comprising about 21% extracellular, 48% cytoplasmic, 2% cytoplasmic membrane, 2% cell wall, and 26% with unknown localization. The identified proteins were classified with respect to their Gene Ontology (GO) annotation as ～24% virulence determinants and toxins, ～17% involved in carbohydrate metabolism, ～14% involved in environmental stress, and ～12% associated with cell division. The identification of the enterotoxin B from the exo-products of both strains used in our study, as belonging to CC80 was interesting.     

PTPN22 gene polymorphism in Egyptian alopecia areata patients and its impact on response to diphencyprone immunotherapy

PTPN22 1858C>T gene polymorphism has been associated with several autoimmune disorders including alopecia areata. The aim of the current study was to investigate the effect of the inherited genetic polymorphism 1858C>T of PTPN22 gene on the predisposition to severe forms of alopecia areata and its effect on the response to DPC treatment. To achieve our aim, PTPN22 1858C>T genotyping was performed by PCR-based restricted fragment length polymorphism (PCR-RFLP) analysis. The study included 103 Egyptian patients with extensive alopecia areata treated by DPC. Hundred healthy age and sex matched blood donors were included in the current study as a control group. Results of genotyping showed that PTPN22 CT and TT mutant genotypes were significantly higher in AA patients compared to controls and conferred increase risk of AA (OR = 2.601, 95% CI = 1.081–6.255). Statistical comparison between AA patients with wild and mutant genotypes revealed that the duration of the illness was significantly longer in those harboring the mutant genotypes. Moreover, the association of other autoimmune diseases as atopy and diabetes mellitus was higher in patients with mutant genotypes. Furthermore, PTPN22 1858C>T genetic polymorphism did not affect the patients' response to DPC immunotherapy.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

Dependence of pathogen molecule-induced Toll-like receptor activation and cell function on Neu1 sialidase

The signaling pathways of mammalian Toll-like receptors (TLR) are well characterized, but the initial molecular mechanisms activated following ligand interactions with the receptors remain poorly defined. Here, we show a membrane controlling mechanism that is initiated by ligand binding to TLR-2, -3 and-4 to induce Neu1 sialidase activity within minutes in live primary bone marrow (BM) macrophage cells and macrophage and dendritic cell lines. Central to this process is that Neu1 and not Neu2,-3 and-4 forms a complex with TLR-2,-3 and-4 on the cell surface of naïve macrophage cells. Neuraminidase inhibitors BCX1827, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA), zanamivir and oseltamivir carboxylate have a limited significant inhibition of the LPS-induced sialidase activity in live BMC-2 macrophage cells but Tamiflu (oseltamivir phosphate) completely blocks this activity. Tamiflu inhibits LPS-induced sialidase activity in live BMC-2 cells with an IC₅₀ of 1.2?μM compared to an IC₅₀ of 1015?μM for its hydrolytic metabolite oseltamivir carboxylate. Tamiflu blockage of LPS-induced Neu1 sialidase activity is not affected in BMC-2 cells pretreated with anticarboxylesterase agent clopidogrel. Endotoxin LPS binding to TLR4 induces Neu1 with subsequent activation of NFκB and the production of nitric oxide and pro-inflammatory IL-6 and TNFα cytokines in primary and macrophage cell lines. Hypomorphic cathepsin A mice with a secondary Neu1 deficiency respond poorly to LPS-induced pro-inflammatory cytokines compared to the wild-type or hypomorphic cathepsin A with normal Neu1 mice. Our findings establish an unprecedented mechanism for pathogen molecule-induced TLR activation and cell function, which is critically dependent on Neu1 sialidase activity associated with TLR ligand treated live primary macrophage cells and macrophage and dendritic cell lines.     

How the N-terminal extremity of Saccharomyces cerevisiae IF1 interacts with ATP synthase: a kinetic approach

The N-terminal part of the inhibitory peptide IF1 interacts with the central γ subunit of mitochondrial isolated extrinsic part of ATP synthase in the inhibited complex (J.R. Gledhill, M.G. Montgomery, G.W. Leslie, J.E. Walker, 2007). To explore its role in the different steps of IF1 binding, kinetics of inhibition of the isolated and membrane-bound enzymes were investigated using Saccharomyces cerevisiae IF1 derivatives modified in N-terminal extremity. First, we studied peptides truncated in Nter up to the amino acid immediately preceding Phe17, a well-conserved residue thought to play a key role. These deletions did not affect or even improve the access of IF1 to its target. They decreased the stability of the inhibited complex but much less than previously proposed. We also mutated IF1-Phe17 and found this amino acid not mandatory for the inhibitory effect. The most striking finding came from experiments in which PsaE, a 8 kDa globular-like protein, was attached in Nter of IF1. Unexpectedly, such a modification did not appreciably affect the rate of IF1 binding. Taken together, these data show that IF1-Nter plays no role in the recognition step but contributes to stabilize the inhibited complex. Moreover, the data obtained using chimeric PsaE-IF1 suggest that before binding IF1 presents to the enzyme with its middle part facing a catalytic interface and its Nter extremity folded in the opposite direction.