Stem cell factor and H2O2 induce GLUT1 translocation in M07e cells

This work aims to elucidate the mechanisms involved in the early activation of glucose transport in hematopoietic M07e cells by stem cell factor (SCF) and a reactive oxygen species (ROS) as H2O2. SCF and H2O2 increase Vmax for glucose transport; this enhancement is due to a higher content in GLUT1 in plasma membranes, possibly through a translocation from intracellular stores. Inhibitors of tyrosine kinases or phospholipase C (PLC) remove glucose transport enhancement and prevent translocation. The inhibitory effect of STI-571 suggests a role for c-kit tyrosine kinase on glucose transport activation not only by SCF, but also by H2O2. On the other hand, neither protein kinase C nor phosphoinositide-3-kinase appear to be involved in the acute activation of glucose transport. Our data suggest that i) in M07e cells, SCF and exogenous H2O2 elicit a short-term activation of glucose transport through a translocation of GLUT1 from intracellular stores to plasma membranes; ii) both stimuli could share at least some signaling pathways leading to glucose uptake activation, involving protein tyrosine kinases and PLC iii) H2O2 could act increasing the level of tyrosine phosphorylation through the inhibition of tyrosine phosphatases and mimicking the regulation role of endogenous ROS.     

The capacity of Enterobacteriaceae species to produce biogenic amines in cheese

The amino acid decarboxylating activity and production of biogenic amines by 104 cheese-associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated. All strains could decarboxylate at least two amino acids in M?ller's broth and in Niven's medium, and the decarboxylase activity was strain specific. In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains. Tyramine and histamine were produced in the lowest concentrations. A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content. This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.     

Signal processes and ROS production in glucose transport regulation by thrombopoietin and granulocyte macrophage-colony stimulation factor in a human leukaemic cell line

In M07e cells, a human megakaryocytic leukaemia line, reactive oxygen species (ROS) are generated in response to cytokines acting as intracellular messengers to modulate glucose transport. The aim of this work was to study the signal cascade involved in the acute glucose transport activation in cells exposed to growth factors, such as granulocyte macrophage-colony stimulation factor (GM-CSF) and thrombopoietin (TPO), to better understand some aspects of the aberrant proliferation in leukaemia. Results confirm ROS involvement in modulation of glucose transport in this cell line. Furthermore, GM-CSF and TPO produced changes in Glut1 phosphorylation and specific inhibitors employed to identify protein kinases involved in Glut activation by these cytokines proved that Akt, PLCγ, Syk and the Src family take part in signal transduction leading to Glut1 activation.     

Chirality as a tool in nucleic acid recognition: principles and relevance in biotechnology and in medicinal chemistry

The understanding of the interaction of chiral species with DNA or RNA is very important for the development of new tools in biology and of new drugs. Several cases in which chirality is a crucial point in determining the DNA binding mode are reviewed and discussed, with the aim of illustrating how chirality can be considered as a tool for improving the understanding of mechanisms and the effectiveness of nucleic acid recognition. The review is divided into two parts: the former describes examples of chiral species interacting with DNA: intercalators, metal complexes, and groove binders; the latter part is dedicated to chirality in DNA analogs, with discussion of phosphate stereochemistry and chirality of ribose substitutes, in particular of peptide nucleic acids (PNAs) for which a number of works have been published recently dealing with the effect of chirality in DNA recognition. The discussion is intended to show how enantiomeric recognition originates at the molecular level, by exploiting the enormous progresses recently achieved in the field of structural characterization of complexes formed by nucleic acid with their ligands by crystallographic and spectroscopic methods. Examples of application of the DNA binding molecules described and the role of chirality in DNA recognition relevant for biotechnology or medicinal chemistry are reported.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

Possible role of RKIP in cytotrophoblast migration: immunohistochemical and in vitro studies

Raf kinase inhibitor protein (RKIP) regulates growth and differentiation signaling of mitogen-activated protein kinases (MAPK), GRK2 and NF-kappaB pathways each of which regulates cytotrophoblast differentiation and normal placental development. We show here that RKIP is expressed in human normal and preeclampic placentas as detected by immunostaining. RKIP was detected in villous cytotrophoblast in normal placenta and switched to syncytiotrophoblast in pre-eclampsia (PE)-complicated pregnancies. RKIP was also localized in extravillous cytotrophoblast of cell islands and cell columns both in normal and in PE placentas, although staining was less uniform in the latter specimens. In order to test RKIP involvement in cytotrophoblast function, we performed in vitro studies on HTR-8/SVneo cells, a first trimester cytotrophoblast cell line. We show that the RKIP inhibitor locostatin reduces ERK phosphorylation and impairs HTR-8/SV neo cells motility in wound closure experiments. We also document the presence of GRK2 mRNA, the reduction of phosphorylated RKIP expression by locostatin and the induction of PAI mRNA expression in HTR-8/SV neo cells, suggesting the involvement of GRK2 and NF-kappaB pathways in these cells. In conclusion, our work provides evidence that RKIP is a novel factor expressed in cytotrophoblast cells where it likely regulates cell migration.