Accumulation of overoxidized Peroxiredoxin III in aged rat liver mitochondria

Overoxidation and subsequent inactivation of Peroxiredoxin III (PrxIII), a mitochondrial H₂O₂ scavenging enzyme, have been reported in oxidative stress conditions. No data are available in the literature about the presence of overoxidized forms of PrxIII in aged tissues. Liver mitochondria from 12-month-old rats and 28-month-old rats were here analyzed by two-dimensional gel electrophoresis. A spot corresponding to the native form of PrxIII was present in adult and old rats with the same volume, whereas an additional, more acidic spot, of the same molecular weight of the native form, accumulated only in old rats. The acidic spot was identified, by MALDI-MS analysis, as a form of PrxIII bearing the cysteine of the catalytic site overoxidized to sulphonic acid. This modified PrxIII form corresponds to the irreversibly inactivated enzyme, here reported, for the first time, in aging. Three groups of 28-month-old rats treated with acetyl-l-carnitine were also examined. Reduced accumulation of the overoxidized PrxIII form was found in all ALCAR-treated groups.     

Exon 1 polymorphisms in the equine CSN3 gene: SNPs distribution analysis in Murgese horse breed

The aim of this study was to assess genetic polymorphism at two loci in the exon 1 of the CSN3 gene in Murgese horse breed by PCR-RFLP analysis. The overall frequencies of alleles A and G at c.-66A?>?G locus were 0.80 and 0.20, respectively, and no GG animals were found in the population. At the c.-36C?>?A locus allelic frequencies were 0.74 and 0.26 for allele C and A, respectively, and no AA animals were detected. Population genetic indexes, namely gene heterozygosity, gene homozygosity, effective allele numbers, fixation index, and polymorphism information index were calculated. Combined genotypic frequencies and possible haplotypes frequencies were also reported. Only three out of nine possible genotypic combinations were found in the studied population. The most frequent genotype was AACC (0.49) while the frequency of AGCA was 0.40. Only five animals were genotyped as AACA (11%). Consequently, the most frequent haplotype in the population was AC (0.744), followed by GA (0.200) and AA (0.056).     

Microbial dioxygenase gene population shifts during polycyclic aromatic hydrocarbon biodegradation

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.     

SrnR from Streptomyces griseus is a nickel-binding transcriptional activator

JBIC Journal of Biological Inorganic Chemistry - Nickel ions are crucial components for the catalysis of biological reactions in prokaryotic organisms. As an uncontrolled nickel trafficking is...     

LUVs Recovered with Chitosan: A New Preparation for Vaccine Delivery

Chitosan, α-(1–4)-amino-2-deoxy-β-D-glucan, is a deacetylated form of chitin, an abundant natural polysaccharide present in crustacean shells. Its unique characteristics such as positive charge, biodegradability, biocompatibility, nontoxicity, and rigid linear molecular structure make this macromolecule ideal as drug carrier. The association between chitosan and liposomes was carefully described, where REVs (reverse phase evaporation vesicles) were sandwiched by chitosan. The usage of these particles in vaccine formulation is here proposed for the first time in the literature. The Chitosan-REVs now stabilized by polyvinilic alcohol were the vehicle for Diphtheria toxoid (Dtxd). Round chitosan-sandwiched REVs (REVs-Chi) particles of 373 ± 17 nm containing 65% Dtxd were obtained. After 200 min of incubation in a simulated gastric fluid, 70% of the Dtxd was liberated from REVs-Chi in comparison to 100% of Dtxd liberated from pure REVs. In PBS, the Dtxd liberation from REVS-Chi was about 60%. Mice were immunized with Dtxd encapsulated within REVs-Chi and with other REVs/Dtxd formulations adsorbed onto Freund adjuvant or alumen [AIF and Al(OH)₃]. The response patterns and the immune maturity were measured by IgG₁ and IgG_2a titrations. REVs-Chi containing Dtxd elicited both antibodies production giving the animals higher immune response and selectivity. It was interesting that the memory of those mice immunized with REVs-Chi containing Dtxd enhanced, after booster, antibody production by 47% in contrast with 17 and 7% in mice immunized with the antigen vehiculated in REVs-AIF or REVs-Al(OH)₃, respectively.     

Design,synthesis and biological evaluation of coumarin alkylamines as potent and selective dual binding site inhibitors of acetylcholinesterase

Acetylcholinesterase inhibitors (AChEIs) are currently the drugs of choice, although only symptomatic and palliative, for the treatment of Alzheimer’s disease (AD). Donepezil is one of most used AChEIs in AD therapy, acting as a dual binding site, reversible inhibitor of AChE with high selectivity over butyrylcholinesterase (BChE). Through a combined target- and ligand-based approach, a series of coumarin alkylamines matching the structural determinants of donepezil were designed and prepared. 6,7-Dimethoxycoumarin derivatives carrying a protonatable benzylamino group, linked to position 3 by suitable linkers, exhibited fairly good AChE inhibitory activity and a high selectivity over BChE. The inhibitory potency was strongly influenced by the length and shape of the spacer and by the methoxy substituents on the coumarin scaffold. The inhibition mechanism, assessed for the most active compound 13 (IC₅₀ 7.6 nM) resulted in a mixed-type, thus confirming its binding at both the catalytic and peripheral binding sites of AChE.     

Ligand migration in the apolar tunnel of Cerebratulus lacteus mini-hemoglobin

The large apolar tunnel traversing the mini-hemoglobin from Cerebratulus lacteus (CerHb) has been examined by x-ray crystallography, ligand binding kinetics, and molecular dynamic simulations. The addition of 10 atm of xenon causes loss of diffraction in wild-type (wt) CerHbO(2) crystals, but Leu-86(G12)Ala CerHbO(2), which has an increased tunnel volume, stably accommodates two discrete xenon atoms: one adjacent to Leu-86(G12) and another near Ala-55(E18). Molecular dynamics simulations of ligand migration in wt CerHb show a low energy pathway through the apolar tunnel when Leu or Ala, but not Phe or Trp, is present at the 86(G12) position. The addition of 10-15 atm of xenon to solutions of wt CerHbCO and L86A CerHbCO causes 2-3-fold increases in the fraction of geminate ligand recombination, indicating that the bound xenon blocks CO escape. This idea was confirmed by L86F and L86W mutations, which cause even larger increases in the fraction of geminate CO rebinding, 2-5-fold decreases in the bimolecular rate constants for ligand entry, and large increases in the computed energy barriers for ligand movement through the apolar tunnel. Both the addition of xenon to the L86A mutant and oxidation of wt CerHb heme iron cause the appearance of an out Gln-44(E7) conformer, in which the amide side chain points out toward the solvent and appears to lower the barrier for ligand escape through the E7 gate. However, the observed kinetics suggest little entry and escape (≤ 25%) through the E7 pathway, presumably because the in Gln-44(E7) conformer is thermodynamically favored.     

Biological role of interleukin-1beta in defensive-aggressive behaviour

During the past decade, a great deal of data has accumulated supporting the notion that cytokines interact to regulate several aspects of social and emotional behaviour. There are reports of a positive correlation between cytokine levels and aggressive behaviour in healthy populations, and clinical reports describe an increase of aggressive traits in patients who receive cytokine immunotherapy. Interleukin-1beta released during an immune response acts as messenger that helps to modulate behaviour by influencing relevant neurotransmitter systems, and in some cases, by directly acting within the brain. In this site, IL-1beta exerts its actions by acting through 5-HT2 and IL-1 Type I receptors in hypothalamus or by potentially indirect routes, including activation of sensory afferents, and stimulation of cytokine release by brain endothelial cells. This review reports research investigating the relationship between IL-1beta, and the immune and central nervous systems involving or potentially involving defensive aggressive behaviour.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.