Hyperosmotic stress induces phosphorylation of cytosolic phospholipase A(2) in HaCaT cells by an epidermal growth factor receptor-mediated process

Cytosolic phospholipase A(2) (cPLA(2)) is an enzyme involved in the formation of proinflammatory mediators by catalyzing the release of arachidonic acid, thereby mediating eicosanoid biosynthesis. Using HaCaT keratinocytes as a model system, we present experimental evidence that in these cells, cPLA(2) is constitutively phosphorylated and that the degree of phosphorylation dramatically increases in cells under hyperosmotic stress induced by sorbitol. In parallel, a rapid release of arachidonic acid followed by prostaglandin E(2) formation was detected. Elucidating the mechanism of cPLA(2) upregulation, we observed that it is mediated via epidermal growth factor receptor (EGFR) activation, since tyrphostin AG1478, a selective inhibitor of EGFR tyrosine kinase, completely inhibited cPLA(2) phosphorylation. Furthermore, addition of PD98059, which is an inhibitor of MEK1 activation, but not of SB203580, which is an inhibitor of p38 stress kinase, inhibited cPLA(2) phosphorylation, indicating that the ras-raf-MEK cascade is the major signalling pathway involved in cPLA(2) phosphorylation. In addition, depletion of the cells from intracellular calcium does not prevent sorbitol-elicited cPLA(2) phosphorylation, suggesting that this process is independent of the presence of calcium. Together, our results demonstrate that hyperosmotic stress phosphorylates cPLA(2) in human keratinocytes by an EGFR-mediated process.     

The tail that wags the dog: p12, the smallest subunit of DNA polymerase δ, is degraded by ubiquitin ligases in response to DNA damage and during cell cycle progression

DNA polymerase δ (Pol δ) is a key enzyme in eukaryotic DNA replication. Human Pol δ is a heterotetramer whose p12 subunit is degraded in response to DNA damage, leading to the in vivo conversion of Pol δ4 to Pol δ3. Two E3 ubiquitin ligases, RNF8 and CRL4^Cdt2, participate in the DNA damage-induced degradation of p12. We discuss how these E3 ligases integrate the formation of Pol δ3 and ubiquitinated PCNA for DNA repair processes. CRL4^Cdt2 partially degrades p12 during normal cell cycle progression, thereby generating Pol δ3 during S phase. This novel finding extends the current view of the role of Pol δ3 in DNA repair and leads to the hypothesis that it participates in DNA replication. The coordinated regulation of licensing factors and Pol δ3 by CRL4^Cdt2 now opens new avenues for control of DNA replication. A parallel study of Pol δ4 and Pol δ3 in Okazaki fragment processing provides evidence for a role of Pol δ3 in DNA replication. We discuss several new perspectives of the role of the 2 forms of Pol δ in DNA replication and repair, as well the significance of the integration of p12 regulation in DNA repair and cell cycle progression.     

Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC,MEK1 and NFκB Activation

Clostridium perfringens phospholipase C (CpPLC), also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC''s cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.     

Influence of Pigment Epithelium-Derived Factor on Outcome after Striatal Cerebral Ischemia in the Mouse

We here suggest that pigment epithelium-derived factor (PEDF) does not have an effect on lesion size, behavioral outcome, cell proliferation, or cell death after striatal ischemia in the mouse. PEDF is a neurotrophic factor with neuroprotective, antiangiogenic, and antipermeability effects. It influences self-renewal of neural stem cells and proliferation of microglia. We investigated whether intraventricular infusion of PEDF reduces infarct size and cell death, ameliorates behavioral outcome, and influences cell proliferation in the one-hour middle cerebral artery occlusion (MCAO) mouse model of focal cerebral ischemia. C57Bl6/N mice were implanted with PEDF or artificial cerebrospinal fluid (control) osmotic pumps and subjected to 60-minute MCAO 48 hours after pump implantation. They received daily BrdU injections for 7 days after MCAO in order to investigate cell proliferation. Infarct volumes were determined 24 hours after reperfusion using magnetic resonance imaging. We removed the pumps on day 5 and performed behavioral testing between day 7 and 21. Immunohistochemical staining was performed to determine the effect of PEDF on cell proliferation and cell death. Our model produced an ischemic injury confined solely to striatal damage. We detected no reduction in infarct sizes and cell death in PEDF- vs. CSF-infused MCAO mice. Behavioral outcome and cell proliferation did not differ between the groups. However, we cannot exclude that PEDF might work under different conditions in stroke. Further studies will elucidate the effect of PEDF treatment on cell proliferation and behavioral outcome in moderate to severe ischemic injury in the brain.     

A 17S multiprotein form of murine cell DNA polymerase mediates polyomavirus DNA replication in vitro

We have identified and purified a multiprotein form of DNA polymerase from the murine mammary carcinoma cell line (FM3A) using a series of centrifugation, polyethylene glycol precipitation, and ion-exchange chromatography steps. Proteins and enzymatic activities associated with this mouse cell multiprotein form of DNA polymerase include the DNA polymerases α and δ, DNA primase, proliferating cell nuclear antigen (PCNA), DNA ligase I, DNA helicase, and DNA topoisomerases I and II. The sedimentation coefficient of the multiprotein form of DNA polymerase is 17S, as determined by sucrose density gradient analysis. The integrity of the murine cell multiprotein form of DNA polymerase is maintained after treatment with detergents, salt, RNase, DNase, and after chromatography on DE52-cellulose, suggesting that the association of the proteins with one another is independent of nonspecific interaction with other cellular macromolecular components. Most importantly, we have demonstrated that this complex of proteins is fully competent to replicate polyomavirus DNA in vitro. This result implies that all of the cellular activities required for large T-antigen dependent in vitro polyomavirus DNA synthesis are present within the isolated 17S multiprotein form of the mouse cell DNA replication activities. A model is proposed to represent the mammalian Multiprotein DNA Replication Complex (MRC) based on the fractionation and chromatographic profiles of the individual proteins found to co-purify with the complex.     

Unisite Hydrolysis of [γ32P]ATP by Soluble Mitochondrial F1ATPase and Its Release by Excess ADP and ATP. Effect of Trifluoperazine

Some of the characteristics of unisite hydrolysis of [³²P]ATP as well as the changes that occur on the transition to multisite catalysis were further studied. It was found that a fraction of [³²P]ATP bound at the catalytic sites of F₁ under unisite conditions undergoes both hydrolysis and release induced by medium nucleotides upon addition of millimolar concentrations of ADP or ATP. The fraction of [³²P]ATP that undergoes release is similar to the fraction that undergoes hydrolytic cleavage, indicating that the rates of the release and hydrolytic reactions of bound [³²P]ATP are in the same range. As part of studies on the mechanisms through which trifluoperazine inhibits ATP hydrolysis, its effect on unisite hydrolysis of [³²P]ATP was also studied. Trifluoperazine diminishes the rate of unisite hydrolysis by 30–40%. The inhibition is accompanied by a nearly tenfold increase in the ratio of [³²P]ATP/³²Pi bound at the catalytic site and a 50% diminution in the rate of ³²Pi release from the enzyme into the media. Trifluoperazine also induces heterogeneity of the three catalytic sites of F₁ in the sense that in a fraction of F₁ molecules, the high-affinity catalytic site has a turnover rate lower than the other two. Trifluoperazine does not modify the release of previously bound [³²P]ATP induced by medium nucleotides. The latter indicates that hindrances in the release of Pi do not necesarily accompany alterations in the release of ATP even though both species lie in the same site.     

Monilinia species in Hungary: morphology, culture characteristics, and molecular analysis

Monilinia is a well-known pathogen of fruit trees affecting fruit production all over the world. Three species of the Monilinia genus are particularly important with regard to fruit trees and ornamentals, causing serious blossom and twig blight and brown rot in fruits: Monilinia fructicola, Monilinia fructigena, and Monilinia laxa. In this study, Monilinia isolates were compared and identified using classical and molecular methods. Morphological and culture characteristics were determined and pathogenicity testing performed. In addition, internal transcribed spacer regions and a genomic sequence with unknown function were analyzed and compared with sequence data from other Monilinia species in an international database. Four Monilinia/Monilia species were identified: M. fructicola, Monilia polystroma, M. fructigena, and M. laxa. M. fructicola was isolated from imported peach fruits. M. polystroma was first reported from Hungary and Europe on apple shoots and fruits. M. fructigena was identified on tea-rose hybrid pseudofruits, which is the first occurrence of this pathogen on this host. M. laxa causes brown rot of grapes, which has only been reported in New Zealand. Substitutions and insertions were detected when comparing M. laxa, M. fructigena, and M. polystroma sequences. In the genomic sequence with unknown function, three repetitive sequence motifs were identified in different numbers, depending on species and isolate. On the phylogram produced in this analysis, the Hungarian M. polystroma isolate (UFT) and M. polystroma reference isolates localized at a different branch than the closely related M. fructigena isolates and other Monilinia species.     

Cyclic AMP regulates the expression and nuclear translocation of RFC40 in MCF7 cells

We have previously shown that the regulatory subunit of PKA, RIalpha, functions as a nuclear transport protein for the second subunit of the replication factor C complex, RFC40, and that this transport appears to be crucial for cell cycle progression from G1 to S phase. In this study, we found that N(6)-monobutyryl cAMP significantly up-regulates the expression of RFC40 mRNA by 1.8-fold and its endogenous protein by 2.3-fold with a subsequent increase in the RIalpha-RFC40 complex formation by 3.2-fold. Additionally, the nuclear to cytoplasmic ratio of RFC40 increased by 26% followed by a parallel increase in the percentage of S phase cells by 33%. However, there was reduction in the percentage of G1 cells by 16% and G2/M cells by 43% with a concurrent accumulation of cells in S phase. Interestingly, the higher percentage of S phase cells did not correlate with a parallel increase in DNA replication. Moreover, although cAMP did not affect the expression of the other RFC subunits, there was a significant decrease in the RFC40-37 complex formation by 81.3%, substantiating the decrease in DNA replication rate. Taken together, these findings suggest that cAMP functions as an upstream modulator that regulates the expression and nuclear translocation of RFC40.