Structural Changes of Yeast tRNATyr Caused by the Binding of Divalent Ions in the Presence of Spermine

Abstract

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429–2446], However, our earlier studies of the binding of Mg²⁺ and Mn²⁺ to yeast tRNA^Tyr have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys. Acta, 479, 332–344; Nöthig-Laslo et al. (1981) Eur. J. Biochem. 117, 263–267]. We have now used yeast tRNA^Tyr, spin labeled at the hypermodified purine (i⁶A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA^Tyr at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine-HCl) and at low temperature (7°C) enhances the binding of manganese to tRNA^Tyr. This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA^Tyr affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i⁶A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA^Tyr by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn²⁺ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.     

Molecular modeling and in vitro reactivation study between the oxime BI-6 and acetylcholinesterase inhibited by different nerve agents

Nerve agents are organophosphates acting as potent inhibitors of acetylcholinesterase (AChE), the enzyme responsible for the hydrolysis of acetylcholine and, consequently, the termination of the transmission of nerve impulses. The inhibition of AChE by an organophosphate can be reversed by a nucleophilic agent able to dephosphorylate a serine residue in the active site of AChE. In this sense, the oximes are compounds capable of removing the nerve agent and reactivate the enzyme. Here, we have applied a methodology involving theoretical docking and Quantum Mechanics/Molecular Mechanics, using the softwares Molegro^® and Spartan^®, to evaluate the kinetic constants of reactivation and the interactions of the oxime BI-6 with AChE inhibited by different organophosphorus compounds in comparison to in vitro data. Results confirm that this method is suitable for the prediction of kinetic and thermodynamic parameters of oximes, which may be useful in the design and selection of new and more effective oximes.     

Edible blue-green algae reduce the production of pro-inflammatory cytokines by inhibiting NF-κB pathway in macrophages and splenocytes

Background

Chronic inflammation contributes to the development of pathological disorders including insulin resistance and atherosclerosis. Identification of anti-inflammatory natural products can prevent the inflammatory diseases.

Methods

Anti-inflammatory effects of blue-green algae (BGA), i.e., Nostoc commune var. sphaeroides Kützing (NO) and Spirulina platensis (SP), were compared in RAW 264.7 and mouse bone marrow-derived macrophages (BMM) as well as splenocytes from apolipoprotein E knockout (apoE^−/−) mice fed BGA.

Results

When macrophages pretreated with 100 μg/ml NO lipid extract (NOE) or SP lipid extract (SPE) were activated by lipopolysaccharide (LPS), expression and secretion of pro-inflammatory cytokines, such as tumor necrosis factor α (TNFα), interleukin 1β (IL-1β), and IL-6, were significantly repressed. NOE and SPE also significantly repressed the expression of TNFα and IL-1β in BMM. LPS-induced secretion of IL-6 was lower in splenocytes from apoE^−/− fed an atherogenic diet containing 5% NO or SP for 12 weeks. In RAW 264.7 macrophages, NOE and SPE markedly decreased nuclear translocation of NF-κB. The degree of repression of pro-inflammatory gene expression by algal extracts was much stronger than that of SN50, an inhibitor of NF-κB nuclear translocation. Trichostatin A, a pan histone deacetylase inhibitor, increased basal expression of IL-1β and attenuated the repression of the gene expression by SPE. SPE significantly down-regulated mRNA abundance of 11 HDAC isoforms, consequently increasing acetylated histone 3 levels.

Conclusion

NOE and SPE repress pro-inflammatory cytokine expression and secretion in macrophages and splenocytes via inhibition of NF-κB pathway. Histone acetylation state is likely involved in the inhibition.

General significance

This study underscores natural products can exert anti-inflammatory effects by epigenetic modifications such as histone acetylation.     

In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites

Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described–one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.     

Significant relationships among frost tolerance and net photosynthetic rate,water use efficiency and dehydrin accumulation in cold-treated winter oilseed rapes

Five winter oilseed rape cultivars (Benefit, Californium, Cortes, Ladoga, Navajo) were subjected to 30 days of cold treatment (4 °C) to examine the effect of cold on acquired frost tolerance (FT), dehydrin (DHN) content, and photosynthesis-related parameters. The main aim of this study was to determine whether there are relationships between FT (expressed as LT₅₀ values) and the other parameters measured in the cultivars. While the cultivar Benefit accumulated two types of DHNs (D45 and D35), the other cultivars accumulated three additional DHNs (D97, D47, and D37). The similar-sized DHNs (D45 and D47) were the most abundant; the others exhibited significantly lower accumulations. The highest correlations were detected between LT₅₀ and DHN accumulation (r = −0.815), intrinsic water use efficiency (WUEi; r = −0.643), net photosynthetic rate (r = −0.628), stomatal conductance (r = 0.511), and intracellular/intercellular CO₂ concentration (r = 0.505). Those cultivars that exhibited higher Pn rate in cold (and further a significant increase in WUEi) had higher levels of DHNs and also higher FT. No significant correlation was observed between LT₅₀ and E, PRI, or NDVI. Overall, we have shown the selected physiological parameters to be able to distinguish different FT cultivars of winter oilseed rape.     

Low genetic polymorphism in the re-introduced Eurasian beaver (Castor fiber) population in Finland: implications for conservation

Mammal Research - Reduction of genetic diversity can lead to reduced fitness of species, such as the loss of adaptability to changing environments. The native Eurasian beaver (Castor fiber) was...     

Photophobic and phototactic responses of Amoeba proteus in KCN and SHAM solutions

The effects of the metabolic inhibitors KCN and SHAM on phototaxis and photophobic response in Amoeba proteus have been studied. Both drugs neither change amoebae photophobic response nor the phototactic reaction. The results indicate clearly that the negative phototactic orientation is not impaired by impediment of respiration thus it is not directly coupled to the differences in energy production in different parts of A. proteus body.     

Structural changes of yeast tRNA(Tyr) caused by the binding of divalent ions in the presence of spermine

The existence of specific sites in tRNA for the binding of divalent cations has been seriously questioned by electrostatic considerations [Leroy & Guéron (1979) Biopolymers, 16, 2429-2446]. However, our earlier studies of the binding of Mg2+ and Mn2+ to yeast tRNA(Tyr) have indicated that spermine creates new binding sites for divalent cations [Weygand-Durasevi? et al. (1977) Biochim. Biophys, Acta, 479, 332-344; N?thig-Laslo et al. (1981) Eur. J. Biochem. 117, 263-267]. We have now used yeast tRNA(Tyr), spin labeled at the hypermodified purine (i6A-37) in the anticodon loop, to study the effect of spermine on the binding of manganese ions. The presence of eight spermine molecules per tRNA(Tyr) at high ionic strength (0.2 M NaCl, 0.05 M triethanolamine.HCl) and at low temperature (7 degrees C) enhances the binding of manganese to tRNA(Tyr). This effect could not be explained by electrostatic binding. The initial binding of manganese to tRNA(Tyr) affects the motional properties of the spin label indicating a change of the conformation of the anticodon loop. From the absence of the paramagnetic effect of manganese on the ESR spectra of the spin label one can conclude that the first binding site for manganese is at a distance from i6A-37, influencing the spin label motion through a long-range effect. The enhancement of the binding of manganese to tRNA(Tyr) by spermine is lost upon destruction of its specific macromolecular structure and it does not occur in single stranded or in double-stranded polynucleotides. The observed effect can be explained by the binding of Mn2+ to new sites, created by the binding of spermine, which are specific for the macromolecular structure of tRNA.     

Effects ofN,N-bis(3-aminopropyl)dodecylamine on antioxidant enzyme activities, mitochondrial morphology and metabolism inAspergillus niger

The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSP) as well as of succinate dehydrogenase (SDG), NADH dehydrogenase (NDG) and fumarate hydratase (FHT) were examined in relation to mitochondrial ultrastructure changes in Aspergillus niger exposed to N,N-bis(3-aminopropyl)dodecylamine (Apd) that was shown to exhibit fungicidal activity. There was a progressive increase in SOD, CAT and GSP activities 1 and 4 h after 0.05 and 0.1 % Apd application. However, this was followed by a pronounced activity decrease when 0.05 % Apd treatment was prolonged by 1 d. The destructive effect on fungal morphology was observed when this fungicidal agent was applied at the concentration of 0.1 % for 1 d. In the treated hyphae mitochondria degenerated after all organelles. The morphological malformations of mitochondria had an impact on their metabolic state; however, the activities of SDG, NDG and FHT were affected to a different extent. In A. niger the fungicidal effect of Apd could be mediated by oxidative stress impairing the vital mitochondria-related cellular functions.