Expression, purification, and characterization of a new heterotetramer structure of leucyl-tRNA synthetase from Aquifex aeolicus in Escherichia coli

Aminoacyl-tRNA synthetases are key players in the interpretation of the genetic code. They constitute a textbook example of multi-domain proteins including insertion and terminal functional modules appended to one of the two class-specific active site domains. The non-catalytic domains usually have distinct roles in the aminoacylation reaction. Aquifex aeolicus leucyl-tRNA synthetase (LeuRS) is composed of a separated catalytic site and tRNA anticodon-binding site, which would represent one of the closest relics of the primordial aminoacyl-tRNA synthetase. Moreover, the essential catalytic site residues are split into the two different subunits. In all other class-I aminoacyl-tRNA synthetases, those two functional polypeptides are nowadays fused into a single protein chain. In this work, we report the isolation and the characterization, in Escherichia coli, of a novel oligomeric form (alphabeta)2 for A. aeolicus LeuRS, which is present in addition to the alphabeta heterodimer. A. aeolicus (alphabeta)2 LeuRS has been characterized by biochemical and biophysical methods. Native gel electrophoresis, mass spectrometry, analytical ultracentrifugation, and kinetic analysis confirmed that the (alphabeta)2 enzyme was a stable and active entity. By mass spectrometry we confirmed that the heterodimer alphabeta can bind one tRNALeu molecule whereas the heterotetramer (alphabeta)2 can bind two tRNALeu molecules. Active site titration and aminoacylation assays showed that two functional active sites are found per heterotetramer, suggesting that this molecular species might exist and be active in vivo. All those data suggest that the existence of the heterotetramer is certainly not an artifact of overexpression in E. coli.     

Cytokine-stimulated phosphorylation of GSK-3 is primarily dependent upon PKCs, not PKB.

The regulation of glycogen synthase kinase-3 (GSK-3) by phosphorylation at inhibitory sites has been well documented. In many, but not all, cases, the phosphatidylinositol 3-kinase pathway, and particularly the downstream kinase protein kinase B (PKB)/akt, have been shown to be responsible for GSK-3 phosphorylation. Given that no studies have ever reported cytokine-mediated phosphorylation of GSK-3, we investigated the phosphorylation of this kinase in several hemopoietic cell types in response to either interleukin (IL)-3, IL-4 or granulocyte-macrophage colony stimulating factor (GM-CSF). Each of the cytokines was able to stimulate phosphorylation of the isoforms GSK-3alpha and GSK-3beta. However, only in the case of IL-4 stimulation was there any dependence on PKB for this phosphorylation. We were clearly able to show that PKB was capable of phosphorylating GSK-3 in these cells, but studies using inhibitors of the protein kinase C (PKC) family of kinases have shown that these enzymes are more likely to play a key role in GSK-3 phosphorylation. Cytokine-mediated generation of diacylglycerol was demonstrated, supporting the possible activation of PKC family members. Thus, cytokine-dependent GSK-3 phosphorylation in hemopoietic cells proceeds primarily through PKB independent pathways.     

Molecular characterization of cytotoxic and resistance mechanisms induced by NCX 4040, a novel NO-NSAID, in pancreatic cancer cell lines*

Although non steroidal antiinflammatory drugs (NSAIDs) have been shown to be effective as chemopreventive agents, important side-effects limit their clinical use. A promising novel class of drugs, nitric oxide-donating NSAIDs (NO-NSAIDs), has been found to be more active than classical NSAIDs. This study explored the effect of the NO-donating aspirin derivative, NCX 4040, on three human pancreatic adenocarcinoma cell lines (Capan-2, MIA PaCa-2 and T3M4). NCX 4040 activity was compared with that of NCX 4016 (an NO₂-positional isomer of NCX 4040), SNAP (a standard NO-releasing molecule), NCX 4042 (denitrated analog of NCX 4040), and aspirin. NCX 4040 showed a striking cytocidal activity in all cell lines, already inducing significant percentages of apoptotic cells at 10 μM in Capan-2 cell lines. This study focused on the biological mechanisms of sensitivity and resistance to NCX 4040, highlighting that the cytotoxic action of this drug may be due to the hyperexpression of Bax, its translocation to the mitochondria, the release of Cytochrome C, and the activation of caspases-9 and -3, overall in a p53-independent manner. Moreover, the use of a specific COX-2 inhibitor (NS 398) in the experimental models showed that COX-2 hyperexpression could partially explain the resistance mechanisms to NCX 4040. This study was supported by Istituto Oncologico Romagnolo, Forlì, and by the Italian, Ministry of Health, 2004.     

The influence of age on tooth root colour changes

The purpose of this study was to examine the relationship between tooth root colour and age, and its possible application in age assessment. In this research altogether 100 tooth roots have been analysed. All teeth, that is their roots, were digitally recorded and the colorimetric treatment was made using Adobe Photoshop 7.0 computer program. Studies have shown no significant difference between RGB values analysed on the whole root surface or only on its central part, with certainty p >0.99. It is also established that there is no statistically significant difference in colouration on four anatomical surfaces (buccal, mesial, lingual, distal) of tooth roots with certaintyp>0.99 for red, p>0.99 for green and p>0.50 for blue colouration component. Statistical data interpretation showed that there is a linear correlation between obtained RGB values and age, with r=-0.994, p>0.99 for red component, r=-0.972, p>0.99 for green and r=-0.982, p>0.95 for blue colouration component. From the obtained results it is possible to conclude that analysing the above mentioned parameter we can easily establish dental age and this technique can be the basis of practical application in establishing chronological age of man.     

Identification of Single Nucleotide Polymorphisms in the Nicastrese Goat and Sardinia Sheep Mannose-Binding Lectin

This study was undertaken to detect polymorphisms in the goat and sheep mannose-binding lectin encoding gene (MBL2) and to explore allelic variability of this gene in these two species. The analysis and comparison of the sequences obtained from sheep showed 13 polymorphic sites, six in the promoter and seven in exon 1, four of which were of the missense type. In the goats, 12 polymorphic sites were detected, five intronic, five in the promoter, and one exonic. The exon site was responsible for an amino acid change. Mutations detected at the MBL2 locus in the sheep are of particular interest, being potentially responsible for the alterations of gene expression. A population survey involved 102 ewes of the Sardinian breed and 218 goats of the Nicastrese breed, all reared in southern Italy.     

Increased asymmetric and multi-daughter cell division in mechanically confined microenvironments

As the microenvironment of a cell changes, associated mechanical cues may lead to changes in biochemical signaling and inherently mechanical processes such as mitosis. Here we explore the effects of confined mechanical environments on cellular responses during mitosis. Previously, effects of mechanical confinement have been difficult to optically observe in three-dimensional and in vivo systems. To address this challenge, we present a novel microfluidic perfusion culture system that allows controllable variation in the level of confinement in a single axis allowing observation of cell growth and division at the single-cell level. The device is capable of creating precise confinement conditions in the vertical direction varying from high (3 μm) to low (7 μm) confinement while also varying the substrate stiffness (E = 130 kPa and 1 MPa). The Human cervical carcinoma (HeLa) model with a known 3N+ karyotype was used for this study. For this cell line, we observe that mechanically confined cell cycles resulted in stressed cell divisions: (i) delayed mitosis, (ii) multi- daughter mitosis events (from 3 up to 5 daughter cells), (iii) unevenly sized daughter cells, and (iv) induction of cell death. In the highest confined conditions, the frequency of divisions producing more than two progeny was increased an astounding 50-fold from unconfined environments, representing about one half of all successful mitotic events. Notably, the majority of daughter cells resulting from multipolar divisions were viable after cytokinesis and, perhaps suggesting another regulatory checkpoint in the cell cycle, were in some cases observed to re-fuse with neighboring cells post-cytokinesis. The higher instances of abnormal mitosis that we report in confined mechanically stiff spaces, may lead to increased rates of abnormal, viable, cells in the population. This work provides support to a hypothesis that environmental mechanical cues influences structural mechanisms of mitosis such as geometric orientation of the mitotic plane or planes.     

Developmental programming of cardiovascular dysfunction by prenatal hypoxia and oxidative stress

Fetal hypoxia is a common complication of pregnancy. It has been shown to programme cardiac and endothelial dysfunction in the offspring in adult life. However, the mechanisms via which this occurs remain elusive, precluding the identification of potential therapy. Using an integrative approach at the isolated organ, cellular and molecular levels, we tested the hypothesis that oxidative stress in the fetal heart and vasculature underlies the molecular basis via which prenatal hypoxia programmes cardiovascular dysfunction in later life. In a longitudinal study, the effects of maternal treatment of hypoxic (13% O(2)) pregnancy with an antioxidant on the cardiovascular system of the offspring at the end of gestation and at adulthood were studied. On day 6 of pregnancy, rats (n = 20 per group) were exposed to normoxia or hypoxia ± vitamin C. At gestational day 20, tissues were collected from 1 male fetus per litter per group (n = 10). The remaining 10 litters per group were allowed to deliver. At 4 months, tissues from 1 male adult offspring per litter per group were either perfusion fixed, frozen, or dissected for isolated organ preparations. In the fetus, hypoxic pregnancy promoted aortic thickening with enhanced nitrotyrosine staining and an increase in cardiac HSP70 expression. By adulthood, offspring of hypoxic pregnancy had markedly impaired NO-dependent relaxation in femoral resistance arteries, and increased myocardial contractility with sympathetic dominance. Maternal vitamin C prevented these effects in fetal and adult offspring of hypoxic pregnancy. The data offer insight to mechanism and thereby possible targets for intervention against developmental origins of cardiac and peripheral vascular dysfunction in offspring of risky pregnancy.