Temperature downshift induces antioxidant response in fungi isolated from Antarctica

Although investigators have been studying the cold-shock response in a variety of organisms for the last two decades or more, comparatively little is known about the difference between antioxidant cell response to cold stress in Antarctic and temperate microorganisms. The change of environmental temperature, which is one of the most common stresses, could be crucial for their use in the biotechnological industry and in ecological research. We compared the effect of short-term temperature downshift on antioxidant cell response in Antarctic and temperate fungi belonging to the genus Penicillium. Our study showed that downshift from an optimal temperature to 15° or 6°C led to a cell response typical of oxidative stress: significant reduction of biomass production; increase in the levels of oxidative damaged proteins and accumulation of storage carbohydrates (glycogen and trehalose) in comparison to growth at optimal temperature. Cell response against cold stress includes also increase in the activities of SOD and CAT, which are key enzymes for directly scavenging reactive oxygen species. This response is more species-dependent than dependent on the degree of cold-shock. Antarctic psychrotolerant strain Penicillium olsonii p14 that is adapted to life in extremely cold conditions demonstrated enhanced tolerance to temperature downshift in comparison with both mesophilic strains (Antarctic Penicillium waksmanii m12 and temperate Penicillium sp. t35).     

Nitrogen Fixation of Faba Bean (Vicia faba L.) Interacting with a Non-legume in Two Contrasting Intercropping Systems

A field experiment was carried out to quantify biological nitrogen fixation (BNF) using the ¹⁵N isotope natural abundance method in maize (Zea mays L.)/faba bean (Vicia faba L.) and wheat (Triticum aestivum L.)/faba bean intercropping systems. Faba bean was yielding more in the maize/faba bean intercropping, but not in the wheat/faba bean intercropping. Biomass, grain yield and N acquisition of faba bean were significantly increased when intercropped with maize, and decreased significantly with wheat, irrespective of N-fertilizer application, indicating that the legume could gain or lose productivity in an intercropping situation. There was yield advantage of maize/faba bean intercropping, but no in wheat/faba bean intercropping. The grain yield of the faba bean intercropped with maize was greater than that of faba bean monoculture due to increases of the stems per plant and the pods per stem of faba bean. N fertilization inhibited N fixation of faba bean in maize/faba bean and wheat/faba bean intercropping and faba bean monoculture. The responses of different cropping systems to N-fertilizer application, however, were not identical, with competitive intercropping (wheat/faba bean) being more sensitive than facilitative intercropping (maize/faba bean). Intercropping increased the percentage of N derived from air (%Ndfa) of the wheat/faba bean system, but not that of the maize/faba bean system when no N fertilizer was applied. When receiving 120 kg N/ha, however, intercropping did not significantly increase %Ndfa either in the wheat/faba bean system or in the maize/faba bean system in comparison with faba bean in monoculture. The amount of shoot N derived from air (Ndfa), however, increased significantly when intercropped with maize, irrespective of N-fertilizer application. Ndfa decreased when intercropped with wheat, albeit not significantly at 120 kg N/ha. Ndfa was correlated more closely with dry matter yield, grain yield and competitive ratio, than with %Ndfa. This indicates that that total dry matter yield (sink strength), not %Ndfa, was more critical for the legume to increase Ndfa. The results suggested that N fixation could be improved by yield maximization in an intercropping system.     

N-Glycans differentially regulate eosinophil and neutrophil recruitment during allergic airway inflammation

Allergic airway inflammation, including asthma, is usually characterized by the predominant recruitment of eosinophils. However, neutrophilia is also prominent during severe exacerbations. Cell surface-expressed glycans play a role in leukocyte trafficking and recruitment during inflammation. Here, the involvement of UDP-N-acetylglucosamine:α-6-D-mannoside β1,6-N-acetylglucosaminyltransferase V (MGAT5)-modified N-glycans in eosinophil and neutrophil recruitment during allergic airway inflammation was investigated. Allergen-challenged Mgat5-deficient (Mgat5(-/-)) mice exhibited significantly attenuated airway eosinophilia and inflammation (decreased Th2 cytokines, mucus production) compared with WT counterparts, attributable to decreased rolling, adhesion, and survival of Mgat5(-/-) eosinophils. Interestingly, allergen-challenged Mgat5(-/-) mice developed airway neutrophilia and increased airway reactivity with persistent elevated levels of proinflammatory cytokines (IL-17A, TNFα, IFNγ)). This increased neutrophil recruitment was also observed in LPS- and thioglycollate (TG)-induced inflammation in Mgat5(-/-) mice. Furthermore, there was significantly increased recruitment of infused Mgat5(-/-) neutrophils compared with WT neutrophils in the peritoneal cavity of TG-exposed WT mice. Mgat5(-/-) neutrophils demonstrated enhanced adhesion to P-selectin as well as increased migration toward keratinocyte-derived chemokine compared with WT neutrophils in vitro along with increased calcium mobilization upon activation and expression of elevated levels of CXCR2, which may contribute to the increased neutrophil recruitment. These data indicate an important role for MGAT5-modified N-glycans in differential regulation of eosinophil and neutrophil recruitment during allergic airway inflammation.     

Formation of oxidised (OX) proteins in Entamoeba histolytica exposed to auranofin and consequences on the parasite virulence

Metronidazole (MNZ), the first line drug for amoebiasis and auranofin (AF), an emerging antiprotozoan drug, are both inhibiting Entamoeba histolytica thioredoxin reductase. The nature of oxidised proteins (OXs) formed in AF‐ or MNZ‐treated E. histolytica trophozoites is unknown. In order to fill this knowledge gap, we performed a large‐scale identification and quantification of the OXs formed in AF‐ or MNZ‐treated E. histolytica trophozoites using resin‐assisted capture coupled to mass spectrometry (MS). We detected 661 OXs in MNZ‐treated trophozoites and 583 OXs in AF‐treated trophozoites. More than 50% of these OXs were shared, and their functions include hydrolases, enzyme modulators, transferases, nucleic acid binding proteins, oxidoreductases, cytoskeletal proteins, chaperones, and ligases. Here, we report that the formation of actin filaments (F‐actin) is impaired in AF‐treated trophozoites. Consequently, their erythrophagocytosis, cytopathic activity, and their motility are impaired. We also observed that less than 15% of OXs present in H₂O₂‐treated trophozoites are also present in AF‐ or MNZ‐treated trophozoites. These results strongly suggest that the formation of OXs in AF‐ or MNZ‐treated trophozoites and in H₂O₂‐treated trophozoites occurred by two different mechanisms.     

Mitochondrial H<Subscript>2</Subscript>O<Subscript>2</Subscript> as an enable signal for triggering autophosphorylation of insulin receptor in neurons

Background

Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H₂O₂ production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H₂O₂ signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date.

Results

Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H₂O₂ signal precedes receptor autophosphorylation and represents a single spike with a peak at 5–10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H₂O₂ signal. The mechanism by which insulin triggers the H₂O₂ signal involves modulation of succinate dehydrogenase activity. Insulin dose–response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, n_H, of 1.1±0.1; R²=0.99). N-acetylcysteine (NAC), a scavenger of H₂O₂, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, n_H, of 8.0±2.3; R²=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H₂O₂ signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H₂O₂ signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H₂O₂ generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H₂O₂-generating system in neurons during the autophosphorylation process.

Conclusions

In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H₂O₂ signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H₂O₂ signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H₂O₂ signaling may lead to the development of cerebral insulin resistance.

     

Computer simulation based selection of optimal monomer for imprinting of tri-O-acetiladenosine in polymer matrix: vacuum calculations

Molecularly imprinted polymers can be anticipated as synthetic imitation of natural antibodies, receptors and enzymes. In case of successful imprinting the selectivity and affinity of the imprint for substrate molecules are comparable with those of natural counterparts. The selection of the optimal functional monomer, monomer/template ratio as well as choosing of polymerization solvent is crucial determinants of the successful imprinting. In the present study the simulation approach to the development of molecular imprinting polymers for the extraction of new protein kinase ATP-competitive inhibitors is presented. By imprinting tri-O-acetyladenosine into polymer matrix the synthetic reproduction of adenosine triphosphate binding site to protein kinases can be fabricated and further used for adenosine triphosphate analogs screening in different sources. The optimized geometrical structure and energy of the pre-polymerization complexes of tri-O-acetyladenosine (template) with three different monomers—methacrylic acid, 3-vinyl benzoic acid and acrylamide in vacuum were calculated using hybrid quantum mechanical/molecular mechanical (QM/MM) approach. These calculations demonstrate that methacrylic acid forms the most stable complex with template, the next is 3-vinyl benzoic acid complex and the third—acrylamide one. The bond energies of the complexes are shown to increase monotonically as more monomers are linked to the template. The same conclusions are made from purely quantum self-consistent field calculations of pre-polymerization complex energy and structure. Hybrid calculation is shown to be effective and can substantially accelerate the development of the imprinting technology.

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Figure Pre-polymerization complex of MIP with tri-O-acetiladenosine template with 5 metacrylic acid monomers