Cognitive training increases platelet PLA2 activity in healthy elderly subjects

Phospholipases A(2) (PLA(2)) are ubiquitous enzymes involved in membrane fatty acid metabolism and intracellular signalling. Recent studies have shown that PLA(2) subtypes are implicated in the modulation of pathways related to memory acquisition and retrieval. We investigated the effects of cognitive training on platelet PLA(2) activity in healthy elderly individuals. Twenty-three cognitively unimpaired older adults were randomly assigned to receive memory training or standard outpatient care only. Both groups were cognitively assessed by the same protocol, and the experimental group (EG) underwent a four-session memory training intervention. Pre- and post-test measures included prose and list recall, WAIS-III digit symbol, strategy use measures and platelet PLA(2) group activity. After cognitive training, patients in the EG group had significant increase in cytosolic, calcium-dependent PLA(2) (cPLA(2)), extracellular (or secreted), calcium-dependent PLA(2) (sPLA(2)), total platelet PLA(2) activity, and significant decrease in platelet calcium-independent PLA(2) (iPLA(2)) activity. Our results suggest that memory training may have a modulating effect in PLA(2)-mediated biological systems associated with cognitive functions and neurodegenerative diseases.     

Role of antioxidants in protecting cellular DNA from damage by oxidative stress

We have previously derived 2 V79 clones resistant to menadione (Md1 cells) and cadmium (Cd1 cells), respectively. They both were shown to be cross-resistant to hydrogen peroxide. There was a modification in the antioxidant repertoire in these cells as compared to the parental cells. Md1 presented an increase in catalase and glutathione peroxidase activities whereas Cd1 cells exhibited an increase in metallothionein and glutathione contents. The susceptibility of the DNA of these cells to the damaging effect of H₂O₂ was tested using the DNA precipitation assay. Both Md1 and Cd1 DNAs were more resistant to the peroxide action. In the case of Md1 cells it seems clear that the extra resistance is provided by the increase in the two H₂O₂ scavenger enzymes, catalase and glutathione peroxidase. In the case of Cd1 cells the activities of these enzymes as well as of superoxide dismutases (Cu/Zn and Mn) are unaltered as compared to the parental cells. The facts that parental cells exposed to 100 μM Zn²⁺ in the medium exhibit an increase in metallothionein but not in glutathione and that these cells become more resistant to the DNA-damaging effect of H₂O₂ suggest that this protein might play a protective role in vivo against the OH radical attack on DNA.     

Role of the exosporium in the stability of the Bacillus sphaericus binary toxin

Abstract The persistence of toxicity of the Bacillus sphaericus 1593 binary toxin was compared when produced in B. sphaericus , inside the exosporium, or in a recombinant B. thuringiensis strain, outside the exosporium. The stability of the toxin crystal was affected by temperature and quality of the water, but not by the location of the production in the bacterial cell.     

applications of electroporation of adherent cellsIn Situ,on a partly conductive slide

One of the most important factors affecting the quality of PCR is the choice of primers. In general, the longer the PCR product the more difficult it is to select efficient primers and set appropriate designing primers, and in general, the more DNA sequence information is available, the better the ch0ance of finding an optimal primer pair. Efficient primers can be designed by avoiding the following flaws: primer-dimer formation, self-complementarity, too lowT _m of the primers, and/or their incorrect internal stability profile. Tips on subcloning PCR products, calculating duplex stability (predicting dimer formation strength), and designing degenerate primers are given.     

PCR amplification of the rDNA internal transcribed spacer region for differentiation of Saccharomyces cultures

Abstract The size of the internal transcribed spacer (ITS) region as measured by gel electrophoresis of PCR products, amplified by primers ITS1 and ITS4, was over 800 bp for all Saccharomyces sensu stricto species, but yeasts belonging to other Saccharomyces species had a shorter ITS region, making this characteristic potentially useful in the identification of Saccharomyces isolates. The ITS product length was homogeneous within the species Saccharomyces cerevisiae .     

Metabolism of Ethionine in Ethionine-Sensitive and Ethionine-Resistant Cells of the Enteric Yeast Candida slooffii

In a defined medium with added ethionine plus low methionine, phenylalanine, tryptophan, tyrosine, adenine, and additional methionine reversed inhibition of the enteric yeast Candida slooffii by ethionine. Isoleucine and 7-methylguanine restored half-maximal growth. Choline but not triethylcholine inhibited C. slooffii. 6-Mercaptopurine reversed ethionine inhibition and also synergistic inhibition by ethionine plus choline. Protection against ethionine by adenine plus aromatics was also evident with log-phase cells in the absence of methionine. Incorporation of ethionine-ethyl-1-(14)C by resting cells was partially inhibited by aromatic amino acids and methionine. Ethionine depressed incorporation of (3)H-phenylalanine but not of (3)H-adenine. Ethionine-resistant mutants were isolated which incorporated ethionine efficiently and degraded it to yet unidentified substances not including 5'-ethylthioadenosine. Ethionine-sensitive cells accumulated more S-adenosylethionine (SAE) than resistant mutants. Adenine was a good precursor of SAE. Radioactivity from ethionine-ethyl-1-(14)C was recovered from cell fractions of ethionine-sensitive cells with the following distribution: cold trichloroacetic acid-soluble > hot trichloroacetic acid-insoluble > lipids > deoxyribonucleic acid > ribonucleic acid. Total radioactivity recovered from ethionine-sensitive cells was twice as much as that from ethionine-resistant mutants.     

Beckwith–Wiedemann syndrome prenatal diagnosis by methylation analysis in chorionic villi

Beckwith–Wiedemann syndrome (BWS) is an imprinting disorder that can be prenatally suspected or diagnosed based on established clinical guidelines. Molecular confirmation is commonly performed on amniocytes. The possibility to use fresh (CVF) and cultured (CVC) chorionic villi has never been investigated. To verify whether CVF and CVC are reliable sources of DNA to study fetal methylation, we used pyrosequencing to test the methylation level of a number of differentially methylated regions (DMRs) at several imprinted loci (ICR1, ICR2, H19, PWS/AS-ICR, GNASXL, GNAS1A, ZAC/PLAGL1, and MEST) and at non-imprinted MGMT and RASSF1A promoters. We analyzed these regions in 19 healthy pregnancies and highlighted stable methylation levels between CVF and CVC at ICR1, ICR2, GNASXL, PWS/AS-ICR, and MEST. Conversely, the methylation levels at H19 promoter, GNAS1A and ZAC/PLAGL1 were different in CVC compared to fresh CV. We also investigated ICR1 and ICR2 methylation level of CVF/CVC of 2 BWS-suspected fetuses (P1 and P2). P1 showed ICR2 hypomethylation, P2 showed normal methylation at both ICR1 and ICR2. Our findings, although limited to one case of BWS fetus with an imprinting defect, can suggest that ICR1 and ICR2, but not H19, could be reliable targets for prenatal BWS diagnosis by methylation test in CVF and CVC. In addition, PWS/AS-ICR, GNASXL, and MEST, but not GNAS1A and ZAC/PLAGL1, are steadily hemimethylated in CV from healthy pregnancies, independently from culture. Thus, prenatal investigation of genomic imprinting in CV needs to be validated in a locus-specific manner.     

Transient TLR activation restores inflammatory response and ability to control pulmonary bacterial infection in germfree mice

Mammals are colonized by an astronomical number of commensal microorganisms on their environmental exposed surfaces. These symbiotic species build up a complex community that aids their hosts in several physiological activities. We have shown that lack of intestinal microbiota is accompanied by a state of active IL-10-mediated inflammatory hyporesponsiveness. The present study investigated whether the germfree state and its hyporesponsive phenotype alter host resistance to an infectious bacterial insult. Experiments performed in germfree mice infected with Klebsiella pneumoniae showed that these animals are drastically susceptible to bacterial infection in an IL-10-dependent manner. In germfree mice, IL-10 restrains proinflammatory mediator production and neutrophil recruitment and favors pathogen growth and dissemination. Germfree mice were resistant to LPS treatment. However, priming of these animals with several TLR agonists recovered their inflammatory responsiveness to sterile injury. LPS pretreatment also rendered germfree mice resistant to pulmonary K. pneumoniae infection, abrogated IL-10 production, and restored TNF-α and CXCL1 production and neutrophil mobilization into lungs of infected germfree mice. This effective inflammatory response mounted by LPS-treated germfree mice resulted in bacterial clearance and enhanced survival upon infection. Therefore, host colonization by indigenous microbiota alters the way the host reacts to environmental infectious stimuli, probably through activation of TLR-dependent pathways. Symbiotic gut colonization enables proper inflammatory response to harmful insults to the host, and increases resilience of the entire mammal-microbiota consortium to environmental pressures.     

A leucine-rich diet and exercise affect the biomechanical characteristics of the digital flexor tendon in rats after nutritional recovery

An increase in the capacity of athletic performance depends on adequate nutrition, which ensures optimal function of the musculoskeletal system, including tendon stability. However, little is known about the status of tendons and extracellular matrix modifications during malnutrition and nutritional recovery when leucine is used in response to exercise conditioning. The purpose of this study was to evaluate the collagen content and biomechanical aspects of the deep digital flexor tendon (DDFT) in malnourished rats submitted to nutritional recovery (control diet or leucine-rich diet) and aerobic physical activity. After 60 days of undernourishment (6% protein diet), the malnourished rats were subsequently nutritionally recovered with a control diet or leucine-rich diet and trained or not (swimming, without overload) for 5 weeks. The biomechanical analysis and quantification of hydroxyproline were assessed in the DDFT in all experimental groups. The leucine-rich diet increased hydroxyproline content in the tension region, independently of the training. In the compression region, hydroxyproline content was higher in the malnourished and leucine-trained groups. Biomechanical analysis showed a lower load in the malnourished and all-trained groups. The lowest stress was observed with control-trained animals. The nutritional-recovered groups showed higher strain values corresponding to control group, while the lowest values were observed in malnourished and trained groups. The results suggest that a leucine-rich diet stimulates collagen synthesis of the DDFT, especially when in combination with physical exercise, and seems to determine the increase of resistance and the biomechanical characteristics of tendons.     

The synthetic purine reversine selectively induces cell death of cancer cells

The synthetic purine reversine has been shown to possess a dual activity as it promotes the de‐differentiation of adult cells, including fibroblasts, into stem‐cell‐like progenitors, but it also induces cell growth arrest and ultimately cell death of cancer cells, suggesting its possible application as an anti‐cancer agent. Aim of this study was to investigate the mechanism underneath reversine selectivity in inducing cell death of cancer cells by a comparative analysis of its effects on several tumor cells and normal dermal fibroblasts. We found that reversine is lethal for all cancer cells studied as it induces cell endoreplication, a process that malignant cells cannot effectively oppose due to aberrations in cell cycle checkpoints. On the other hand, normal cells, like dermal fibroblasts, can control reversine activity by blocking the cell cycle, entering a reversible quiescent state. However, they can be induced to become sensitive to the molecule when key cell cycle proteins, e.g., p53, are silenced. J. Cell. Biochem. 113: 3207–3217, 2012. © 2012 Wiley Periodicals, Inc.