Disulfide reduction abolishes tissue factor cofactor function

Background

Tissue factor (TF), an in vivo initiator of blood coagulation, is a transmembrane protein and has two disulfides in the extracellular domain. The integrity of one cysteine pair, Cys186–Cys209, has been hypothesized to be essential for an allosteric “decryption” phenomenon, presumably regulating TF procoagulant function, which has been the subject of a lengthy debate. The conclusions of published studies on this subject are based on indirect evidences obtained by the use of reagents with potentially oxidizing/reducing properties.

Methods

The status of disulfides in recombinant TF_1–263 and natural placental TF in their non-reduced native and reduced forms was determined by mass-spectrometry. Functional assays were performed to assess TF cofactor function.

Results

In native proteins, all four cysteines of the extracellular domain of TF are oxidized. Reduced TF retains factor VIIa binding capacity but completely loses the cofactor function.

Conclusion

The reduction of TF disulfides (with or without alkylation) eliminates TF regulation of factor VIIa catalytic function in both membrane dependent FX activation and membrane independent synthetic substrate hydrolysis.

General significance

Results of this study advance our knowledge on TF structure/function relationships.     

Neuronal Autophagy: Self-eating or Self-cannibalism in Alzheimer’s Disease

Autophagy is a major intracellular degeneration pathway involved in the elimination and recycling of damaged organelles and long-lived proteins by lysosomes. Many of the pathological factors, which trigger neurodegenerative diseases, can perturb the autophagy activity, which is associated with misfolded protein aggregates accumulation in these disorders. Alzheimer’s disease, the first neurodegenerative disorder between dementias, is characterized by two aggregating proteins, β-amyloid peptide (plaques) and τ-protein (tangles). In Alzheimer’s disease autophagosomes dynamically form along neurites within neuronal cells and in synapses but effective clearance of these structures needs retrograde transportation towards the neuronal soma where there is a major concentration of lysosomes. Maturation of autophago-lysosomes and their retrograde trafficking are perturbed in Alzheimer’s disease, which causes a massive concentration of autophagy elements along degenerating neurites. Transportation system is disturbed along defected microtubules in Alzheimer’s disease brains. τ-protein has been found to control the stability of microtubules, however, phosphorylation of τ-protein or an increase in the total level of τ-protein can cause dysfunction of neuronal cells microtubules. Current evidence has shown that autophagy is developing in Alzheimer’s disease brains because of ineffective degradation of autophagosomes, which hold amyloid precursor protein-rich organelles and secretases important for β-amyloid peptides generation from amyloid precursor. The combination of raised autophagy induction and abnormal clearance of β-amyloid peptide-generating autophagic vacuoles creates circumstances helpful for β-amyloid peptide aggregation and accumulation in Alzheimer’s disease. However, the key role of autophagy in Alzheimer’s disease development is still under consideration today. One point of view suggests that abnormal autophagy induction causes a concentration of autophagic vacuoles rich in amyloid precursor protein, β-amyloid peptide and the elements crucial for its formation, whereas other hypothesis points to marred autophagic clearance or even decrease in autophagic effectiveness playing a role in maturation of Alzheimer’s disease. In this review we present the recent evidence linking autophagy to Alzheimer’s disease and the role of autophagic regulation in the development of full-blown Alzheimer’s disease.     

Nutlin-3a, an MDM2 antagonist and p53 activator, helps to preserve the replicative potential of cancer cells treated with a genotoxic dose of resveratrol

Resveratrol is a natural compound that has been intensely studied due to its role in cancer prevention and potential as an anti-cancer therapy. Its effects include induction of apoptosis and senescence-like growth inhibition. Here, we report that two cancer cell lines (U-2 OS and A549) differ significantly in their molecular responses to resveratrol. Specifically, in U-2 OS cells, the activation of the p53 pathway is attenuated when compared to the activation in A549 cells. This attenuation is accompanied by a point mutation (458: CGA→TGA) in the PPM1D gene and overexpression of the encoded protein, which is a negative regulator of p53. Experimentally induced knockdown of PPM1D in U-2 OS cells resulted in slightly increased activation of the p53 pathway, most clearly visible as stronger phosphorylation of p53 Ser³⁷. When treated with nutlin-3a, a non-genotoxic activator of p53, U-2 OS and A549 cells both responded with substantial activation of the p53 pathway. Nutlin-3a improved the clonogenic survival of both cell lines treated with resveratrol. This improvement was associated with lower activation of DNA-damage signaling (phosphorylation of ATM, CHK2, and histone H2AX) and higher accumulation of cells in the G1 phase of the cell cycle. Thus, the hyperactivation of p53 by nutlin-3a helps to preserve the replicative potential of cells exposed to resveratrol.     

The Force-Biased Algorithm for the Irregular Close Packing of Equal Hard Spheres

Abstract

We present a “force-biased” algorithm for generating the irregular close packing of hard spheres. The algorithm is partly based on Jodrey and Tory's ideas [9] and incorporates methods from Molecular Dynamics. Packings generated by means of the two algorithms are consistent up to final packing fraction of 0.65, which seems to be the limit density of Jodrey and Tory's method. Significantly higher densities (up to 0.71) can be achieved for small numbers of spheres by the force-biased algorithm. However the shape of the radial and angle distribution functions implies that a partial short-range ordering occurs in packings of those densities.     

The Influence of Rhamnolipids on Aliphatic Fractions of Diesel Oil Biodegradation by Microorganism Combinations

Twelve different bacteria–yeast combinations were tested for determination of their ability to biodegrade diesel oil. The cell surface properties of the bacterial and yeast strains were correlated with the type of carbon source used in the experiments. The highest biodegradation of diesel oil after 7 days was obtained for the following combinations: Aeromonas hydrophila MR4–Yarrowia lipolytica EH 56 (87 %) and Xantomonas maltophila MRP7–Candida maltosa EH15 (90 %). Degradation performances of 10 of 12 combinations were enhanced by the presence of rhamnolipids. The highest increases were observed for A. hydrophila MR4–C. maltosa EH15 (from 34 to 67 %), A. hydrophila MR4–C. maltosa EH60 (from 47 to 76 %) and for Pseudomonas stutzeri MR7–C. maltosa EH60 (from 29 to 79 %). The addition of rhamnolipids to the system reduces the removal time of diesel oil from the contaminated water and changes the microbial adhesion to hydrocarbons. Modification of the cell surface of the tested strain during biodegradation is a very important factor determining the removal of hydrophobic compounds.     

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

We cloned two genes coding F107-C and K88-1NT fimbrial subunits from strains E. coli C and 1NT isolated from Thua Thien Hue province, Vietnam. The mature peptide of faeG gene from strain E. coli 1NT (called faeG-1NT) is 100 % similarity with faeG gene, while the CDS of fedA gene from strain C (called fedA-C) has a similarity of 97 % with the fedA gene. Expression of the faeG-1NT and fedA-C genes in E. coli BL21 Star™ (DE3) produced proteins of ~31 and 22 kDa, respectively. The effect of IPTG concentration on the K88-1NT and F107-C fimbriae production was investigated. The results showed that 0.5 mM IPTG is suitable for higher expression of K88-1NT subunit, while 0.75 mM IPTG strongly stimulated expression of F107-C subunit. The optimal induction time for expression was also examined. Generally, highest expression of K88-1NT subunit occurred after 6 h of induction, while that of F107-C subunit is after 14 h.     

Production of phenolics and the emission of volatile organic compounds by perennial ryegrass (Lolium perenne L.)/Neotyphodium lolii association as a response to infection by Fusarium poae

Grasses very often form symbiotic associations with Neotyphodium/Epichloë endophytic fungi. These endophytes often allow the host grass to be protected from different pathogens. However, there is little known about the mechanisms of such endophyte influence on the host. Thus, the purpose of this research was to examine the effect of the N. lolii endophyte on the total production of phenolic compounds, VOCs emission and the resistance of three perennial ryegrass genotypes infected by pathogenic Fusarium poae. Analyses of total phenolics content were performed in control (not inoculated) and inoculated plants after 1, 2, 3, 4, 5, and 6 days (DAI) and for VOCs after 0, 3, 6 and 12 DAI. The presence of endophytes significantly reduced the disease index in two of the three genotypes relative to that in E−. Plants infected by N. lolii exhibited higher production of phenolics relative to the E− plants. The highest amounts of phenolics were observed on the second and sixth DAI. Genotype Nl22 showed the strongest effect of the endophyte on the production of phenolics, which increased by over 61%. Both the endophyte infected and non-infected plants emitted most abundantly two GLVs ((Z)-3-hexenal, (Z)-3-hexen-1-yl acetate), three terpenes (linalool, (Z)-ocimene, β-caryophyllene) and three shikimic acid pathway derivatives (benzyl acetate, indole, and methyl salicylate). The endophyte presence and the intervals of VOCs detection were a highly significant source of variation for all emitted volatiles (P < 0.001). The genotype of the perennial ryegrass significantly affected only the emission of methyl salicylate (P < 0.05) and β-caryophyllene (P < 0.05). Most of the VOCs ((Z)-3-hexen-1-yl acetate, (Z)-3-hexenal, linalool and methyl salicylate) reached their highest levels of emission on the sixth DAI, when averaged over genotypes and endophyte status. The results highlight the role of Neotyphodium spp. in the mediation of quadro-trophic interactions among plants, symbiotic endophytes, invertebrate herbivores and plant pathogenic fungi. Our results also confirm the fact that symbiotic plants can activate a defense reaction faster than non-symbiotic plants after a pathogen attack. Thus, N. lolii can be involved in the defense of perennial ryegrass against pathogens and potentially could be central to the host plants’ protection.     

Climate change impact on development rates of the codling moth (Cydia pomonella L.) in the Wielkopolska region, Poland

The main goal of this paper is to estimate how the observed and predicted climate changes may affect the development rates and emergence of the codling moth in the southern part of the Wielkopolska region in Poland. In order to simulate the future climate conditions one of the most frequently used A1B SRES scenarios and two different IPCC climate models (HadCM3 and GISS modelE) are considered. A daily weather generator (WGENK) was used to generate temperature values for present and future climate conditions (time horizons 2020–2040 and 2040–2060). Based on the generated data set, the degree-days values were then calculated and the emergence dates of the codling moth at key stages were estimated basing on the defined thresholds. Our analyses showed that the average air surface temperature in the Wielkopolska region may increase from 2.8°C (according to GISS modelE) even up to 3.3°C (HadCM3) in the period of 2040–2060. With the warming climate conditions the cumulated degree-days values may increase at a rate of about 142 DD per decade when the low temperature threshold (T _low ) of 0°C is considered and 91 DD per decade when T _low ?=?10°C. The key developmental stages of the codling moth may occur much earlier in the future climate conditions than currently, at a rate of about 3.8–6.8 days per decade, depending on the considered GCM model and the pest developmental stage. The fastest changes may be observed in the emergence dates of 95% of larvae of the second codling moth generation. This could increase the emergence probability of the pest third generation that has not currently occurred in Poland.     

Interpretation of the invertebrate-based BMWP-PL index in a gravel-bed river: insight from the Polish Carpathians

Like its British prototype (Biological Monitoring Working Party score system), the Polish benthic invertebrate-based BMWP-PL index is commonly regarded as an indicator of river water quality. This interpretation of the index has been verified in a study of the gravel-bed Bia?a River. Benthic macroinvertebrates were sampled at 10 sites and compared in one channelized and one unmanaged cross-section per site. The resulting taxa richness and BMWP-PL index scores were compared with water quality and physical habitat characteristics in the cross-sections. Channelized and unmanaged cross-sections clearly differed in their physical habitat conditions, and water quality characteristics mostly varied in the downstream direction. Particular cross-sections hosted between 3 and 26 invertebrate taxa, with the respective BMWP-PL scores indicating the water in the surveyed cross-sections varied between high and poor quality. However, the BMWP-PL scores were unrelated to physicochemical characteristics of the river water, which consistently pointed to high water quality. Instead, the scores were significantly related to several physical habitat variables, with the number of low-flow channels in a cross-section explaining the largest proportion of the variance in the index values. The relationship of the scores with the complexity of flow pattern in the river and a lack of their dependence on physicochemical water characteristics show that the BMWP-PL index should not be regarded as an indicator of water quality but rather as an indicator of the ecological status of rivers, dependent both on their hydromorphological and water-quality characteristics.