The impact of humic and fulvic acids on the dynamic properties of liposome membranes: the ESR method

This paper presents the results of research on the influence of two fractions of humic substances (HS): fulvic acids (FA) and humic acids (HA), as a function of concentration, on the liposome membranes formed from egg yolk lecithin (EYL). The concentration of HS in relation to EYL changed from 0% to 10% by weight. The influence of HS on various areas of membranes: interphase water-lipid, in the lipid layer just below the polar part of the membrane and in the middle of the lipid bilayer, was investigated by different spin labels (TEMPO, DOXYL 5, DOXYL 16). The study showed that HA slightly decreased the fluidity of the analyzed membranes on the surface layer, while FA significantly liquidated the center of the lipid bilayer. The strong effect of both fractions of HS on the concentration of free radicals as a function of time was also described.     

Distinctive structural motifs of RNA G-quadruplexes composed of AGG,CGG and UGG trinucleotide repeats

Trinucleotide repeats are microsatellite sequences that are polymorphic in length. Their expansion in specific genes underlies a number of neurodegenerative disorders. Using ultraviolet-visible, circular dichroism, nuclear magnetic resonance (NMR) spectroscopies and electrospray ionization mass spectrometry, the structural preferences of RNA molecules composed of two and four repeats of AGG, CGG and UGG in the presence of K⁺, Na⁺ and NH₄⁺ were analysed. (AGG)₂A, (AGG)₄A, p(UGG)₂U and p(UGG)₄U strongly prefer folding into G-quadruplexes, whereas CGG-containing sequences can adopt different types of structure depending on the cation and on the number of repeats. In particular, the two-repeat CGG sequence folds into a G-quadruplex in potassium buffer. We also found that each G-quadruplex fold is different: A:(G:G:G:G)A hexads were found for (AGG)₂A, whereas mixed G:C:G:C tetrads and U-tetrads were observed in the NMR spectra of G(CGG)₂C and p(UGG)₂U, respectively. Finally, our NMR study highlights the influence of the strand sequence on the structure formed, and the influence of the intracellular environment on the folding. Importantly, we highlight that although potassium ions are prevalent in cells, the structures observed in the HeLa cell extract are not always the same as those prevailing in biophysical studies in the presence of K⁺ ions.     

Activity of AMP-Regulated Protein Kinase and AMP-Deaminase in the Heart of Mice Fed High-Fat Diet

AMP-regulated protein kinase (AMPK) is involved in numerous regulatory processes and its role in control of cardiac energy metabolism is particularly important. This activity could be affected by AMP-deaminase (AMPD) since substrate of AMPD is AMPK activator. Hearts of male mouse, fed for six weeks with normal or high-fat diet, were fractionated to enrich AMPK activity. Purified fraction was incubated with AMARA peptide for up to 5 minutes and then conversion of AMARA to pAMARA was determined by liquid chromatography—mass spectrometry (LC/MS) using mass detector. Activity of AMPK in heart was 0.038 ± 0.012 pmol/min/mg protein for mice fed high-fat diet and that was not different to control (0.032 ± 0.01 pmol/min/mg protein). We observed change in AMPD activity. It was 5.39 ± 1.5 nmol/mg tissue/min in heart of mice fed high-fat diet while in heart of mice fed low-fat diet it was 2.29 ± 0.32 nmol/mg tissue/min. Data we present indicate that while total AMPK activity is not changed decrease in AMPD activity may affect AMPK signaling in diabetic heart.     

Genome-scale patterns in the loss of heterozygosity incidence in Saccharomyces cerevisiae

Former studies have established that loss of heterozygosity can be a key driver of sequence evolution in unicellular eukaryotes and tissues of metazoans. However, little is known about whether the distribution of loss of heterozygosity events is largely random or forms discernible patterns across genomes. To initiate our experiments, we introduced selectable markers to both arms of all chromosomes of the budding yeast. Subsequent extensive assays, repeated over several genetic backgrounds and environments, provided a wealth of information on the genetic and environmental determinants of loss of heterozygosity. Three findings stand out. First, the number of loss of heterozygosity events per unit time was more than 25 times higher for growing than starving cells. Second, loss of heterozygosity was most frequent when regions of homology around a recombination site were identical, about a half-% sequence divergence was sufficient to reduce its incidence. Finally, the density of loss of heterozygosity events was highly dependent on the genome’s physical architecture. It was several-fold higher on short chromosomal arms than on long ones. Comparably large differences were seen within a single arm where regions close to a centromere were visibly less affected than regions close, though usually not strictly adjacent, to a telomere. We suggest that the observed uneven distribution of loss of heterozygosity events could have been caused not only by an uneven density of initial DNA damages. Location-depended differences in the mode of DNA repair, or its effect on fitness, were likely to operate as well.     

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.     

Multimodal analysis of metals in copper–zinc superoxide dismutase isoforms separated on electrophoresis gels

It has been suggested that copper–zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.     

Vascular extracellular adenosine metabolism in mice correlates with susceptibility to atherosclerosis

Abstract

Animal models are widely used in atherosclerosis research. The most useful, economic and valid is mouse genetic model of this pathology. Purinergic signaling is an important mechanism regulating processes involved in the vascular inflammation and atherosclerosis. The aim of this study was to measure vascular activities of nucleotide and adenosine-degrading ecto-enzymes in different strains of mice and to compare them to atherosclerotic susceptibility.

The vascular extracellular nucleotide catabolism pathway was analyzed in 6-month-old male genetically unmodified mouse strains: FVB/NJ, DBA/2J, BALB/c, C57Bl/6J and mouse knock-outs on C57Bl/6J background for LDLR (LDLR-/-) and for ApoE and LDLR (ApoE-/-LDLR-/-). LDLR-/- mice were a model of moderate hypercholesterolemia, while ApoE-/-LDLR-/- mice, a model of severe hypercholesterolemia with advanced atherosclerosis.

FVB/NJ, DBA/2J and BALB/c mice showed high rates of vascular extracellular AMP hydrolysis and low activity of adenosine deamination. In turn, all mice with the C57Bl/6J background expressed diminished activity of vascular AMP hydrolysis. Mice with genetically-induced hyperlipidemia and atherosclerosis on the C57Bl/6J background revealed increased ecto-adenosine deaminase activity.

Mouse strains that were resistant to atherosclerosis (FVB/NJ, DBA/2J, BALB/c) exhibited a protective extracellular vascular ecto-enzyme pattern directed toward the production of anti-inflammatory and anti-atherosclerotic adenosine. In turn, mice with genetically induced hypercholesterolemia and atherosclerosis expressed disturbed activities of ecto-5’nucleotidase and ecto-adenosine deaminase related to decreased production and increased degradation of extracellular adenosine.