Tyrosine Nitration of Flagellins: a Response of Sinorhizobium meliloti to Nitrosative Stress.

Rhizobia are bacteria which can either live as free organisms in the soil or interact with plants of the legume family with, as a result, the formation of root organs called nodules in which differentiated endosymbiotic bacteria fix atmospheric nitrogen to the plant's benefit. In both lifestyles, rhizobia are exposed to nitric oxide (NO) which can be perceived as a signaling or toxic molecule. NO can act at the transcriptional level but can also modify proteins by -nitrosylation of cysteine or nitration of tyrosine residues.

Evaluation of the cost and length of hospital stays related to the management of an intestinal Clostridium difficile infection.

Introduction: Intestinal Clostridium difficile Infection (CDI) treated in hospitals may concern patients whose reason for admission is CDI (primary diagnosis) or who have acquired CDI during their stay (secondary diagnosis).

Objectives: The objective of this study is to evaluate the cost for social security and hospitals and the length of hospital stays related to CDIs as the main reason for admission.

Method: This study was carried out in 2012 in 13 Belgian hospitals.

Sinorhizobium meliloti Controls Nitric Oxide-Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein.

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction.

Control of NO level in rhizobium-legume root nodules: not only a plant globin story.

Nitric oxide (NO ) is a gaseous signaling molecule which plays both regulatory and defense roles in animals and plants. In the symbiosis between legumes and rhizobia, NO has been shown to be involved in bacterial infection and nodule development steps as well as in mature nodule functioning. We recently showed that an increase in NO level inside Medicago truncatula root nodules also could trigger premature nodule senescence.

Temporal assessment of histone H3 phospho-acetylation and casein kinase 2 activation in dentate gyrus from ischemic rats.

Hippocampal dentate gyrus possesses an exceptional capacity of adaptation to ischemic insults. Recently, using a transient global ischemic model in the adult rat, we identified a neuroprotective signalling cascade in the dentate gyrus involving calcium/calmodulin-dependent protein kinase IV (CaMKIV), cyclic AMP response element (CRE)-binding protein (CREB) and brain-derived neurotrophic factor (BDNF), a major regulator of survival. We have shown that intracerebroventricular injections of anti-BDNF and anti-CREB are sufficient to cause substantial tissular damages and apoptotic deaths in late periods (48-72 h) after ischemia.

Identification of a biphasic signaling pathway involved in ischemic resistance of the hippocampal dentate gyrus.

Dentate gyrus is usually assumed to be resistant to ischemia. However, the mechanisms underlying this functional plasticity are not fully understood. Herein, we aimed at identifying a neuroprotective mechanism in the dentate gyrus of the adult rat after global ischemia.

A calcium/calmodulin kinase pathway connects brain-derived neurotrophic factor to the cyclic AMP-responsive transcription factor in the rat hippocampus.

Brain-derived neurotrophic factor (BDNF) plays fundamental roles in synaptic plasticity in rat hippocampus. Recently, using rat hippocampal slices, we found that BDNF induces activation of calcium/calmodulin-dependent protein kinase 2 (CaMKII), a critical mediator of synaptic plasticity. CaMKII in turn activates the p38 subfamily of mitogen-activated protein kinases (MAPK) and its downstream effector, MAPK-activated protein kinase 2 (MAPKAPK-2).

Identification of two persistently activated neurotrophin-regulated pathways in rat hippocampus.

Brain-derived neurotrophic factor contributes profoundly to modulate activity-dependent synaptic plasticity in adult brain areas such as the hippocampus, but the mechanisms underlying this important role still remain unclear. Recently, we have shown that two serine/threonine kinases, calcium/calmodulin-dependent protein kinase-2 and casein kinase-2, are capable of mediating brain-derived neurotrophic factor responses in adult rat hippocampus. In the present study, using hippocampal slices from adult rat, we show that phospholipase C-regulated calcium signals couple the brain-derived neurotrophic factor receptor to two distinct pathways: a pathway in which calcium/calmodulin-dependent protein kinase-2 stimulates a signalling module involving the p38 subfamily of mitogen-activated protein kinases and its downstream target, usually named mitogen-activated protein kinase-activated protein kinase-2; and a pathway in which the extracellular signal-regulated kinase subfamily of mitogen-activated protein kinases activates casein kinase-2.

Casein kinase 2 as a potentially important enzyme in the nervous system.

Protein kinase CK2 is a ubiquitous and pleiotropic seryl/threonyl protein kinase which is highly conserved in evolution indicating a vital cellular role for this kinase. The holoenzyme is generally composed of two catalytic (alpha and/or alpha') and two regulatory (beta) subunits, but the free alpha/alpha' subunits are catalytically active by themselves and can be present in cells under some circumstances. Special attention has been devoted to phosphorylation status and structure of these enzymic molecules, however, their regulation and roles remain intriguing.

Neurotrophin-induced activation of casein kinase 2 in rat hippocampal slices.

Casein kinase 2 is present in the brain, including the hippocampus. It is associated with long-term potentiation and is known to be involved in phosphorylation of proteins potentially important for neuroplasticity, but regulation of its activity in neuronal cells is not yet known. In the present work, it was found that brain-derived neurotrophic factor and neurotrophin-4 control the activity of casein kinase 2 in hippocampal slices of adult rat.

Brain-derived neurotrophic factor increases Ca2+/calmodulin-dependent protein kinase 2 activity in hippocampus.

Here we show that brain-derived neurotrophic factor (BDNF) stimulates both the phosphorylation of the Ca2+/calmodulin-dependent protein kinase 2 (CaMK2) and its kinase activity in rat hippocampal slices. In addition, we find that: (i) the time course of BDNF action is not accompanied by a change in the spectrum of either alpha- and beta-subunits of CaMK2 detected by immunoblotting; (ii) both treatment of solubilized CaMK2 with alkaline phosphatase and treatment of immunoprecipitated CaMK2 with protein phosphatase 1 reverse phosphorylation and activation of the kinase; (iii) phospholipase C inhibitor D609 and intracellular Ca2+ chelation by 1,2-bis-(o-aminophenoxy)ethane-N,N,N",N',-tetracetic acid tetra(acetoxymethyl)ester or 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate but not omission of Ca2+ or Ca2+ chelation by EGTA, abolish the stimulatory effect of BDNF on phosphorylation and activation of CaMK2. These results strongly suggest that the conversion of CaMK2 into its active, autophosphorylated form, but not its concentration, is increased by BDNF via stimulation of phospholipase C and subsequent intracellular Ca2+ mobilization.

Signal-regulated proteins and fibroblast growth factor receptors: comparative immunolocalization in rat retina.

We compared the immunolocalization of fibroblast growth factor (FGF)-R1 and FGF-R2 with that of several intracellular signalling proteins in rat neural retina. Only the serine/threonine extracellular signal-related kinases (ERK) and lipocortin/annexin 6, a major calcium-binding protein, appeared to be co-localized with FGF-R1 and FGF-R2 in all subfields of the neural retina. In particular, ERK appeared to be present in perikarya of ganglion cells and synaptic layers as did these receptors.

Activation of phosphotyrosine phosphatase activity is associated with decreased differentiation in adult bovine lens.

The postnatal vertebrate eye lens provides an opportunity to study possible involvement of reversible protein phosphorylation in the differentiation process of epithelial cells. Epithelial cells at the lens equator, indeed, differentiate continuously into fiber cells throughout life but this capacity progressively decreases with age. Here we describe the characterization of a phosphotyrosine-protein phosphatase(s) (PTPase(s)) in the equatorial epithelium of bovine lens which exhibits a high level of specific activity.

A high phosphotyrosine phosphatase activity is present in differentiating bovine lens cells.

Activity of phosphotyrosine-protein phosphatases (PTPases) has been investigated in the different cellular regions of bovine eye lens. PTPases were tested in cellular detergent extracts using phospholabelled synthetic peptides and p-nitrophenyl phosphate. We show that a high PTPase activity is only present in cells which undergo differentiation, namely the equatorial epithelium and cortex fiber cells.

Immunochemical evidence for a fibroblast growth factor receptor in adult retinal optic fiber and synaptic layers.

Evidence for fibroblast growth factor receptors in the central nervous system has only been obtained using autoradiographic localization of fibroblast growth factor binding sites and messenger RNA. To clarify those neuronal functions that are regulated by fibroblast growth factor receptors, we have localized immunocytochemically the fibroblast growth factor receptor protein in bovine retina, a neural tissue of well-defined structure and function. The extracellular domain of the gene product referred to as fibroblast growth factor receptor 2 was expressed genetically in bacteria to obtain a polyclonal antibody.

Immunohistochemical analysis of fibroblast growth factor receptor in bovine retina.

The distribution of fibroblast growth factor (FGF) receptor in bovine retina was established using a polyclonal antibody against the extracellular domain of this receptor. Different conditions of tissue fixation and development of the secondary antibody were tested. The ability of the antiserum to map precisely the receptor was obtained on fresh-frozen sections which had been treated with paraformaldehyde prior to incubation with this antiserum.

Purification of an active receptor for acidic and basic fibroblast growth factor from bovine retina.

Acidic and basic fibroblast growth factors (FGFs) influence cell division and differentiation in retina cells. Their effects are thought to be mainly mediated through stimulation of a specific membrane receptor and subsequent generation of an intracellular signal pathway. In this study, we purified a FGF receptor of 130 kDa from bovine neural retina using wheat germ agglutinin affinity chromatography followed by FGF-affinity chromatography.

Possible involvement of arachidonic acid metabolites in the synergistic action of endothelial mitogenesis by basic fibroblast growth factor and phorbol ester.

Our object was to obtain information about the regulatory mechanism which modulates the effect of basic fibroblast growth factor (bFGF) on commitment to growth in human umbilical vein endothelial (HUVE) cells. Firstly, phorbol ester PMA, a known activator of protein kinase C (PKC), was found to be able to act synergistically with bFGF to stimulate 3H thymidine incorporation in HUVE cells. Secondly, bFGF and PMA induced a stimulated phospholipase A2 (PLA2)-catalyzed release of 14C arachidonate.

Fibroblast growth factor-stimulated phosphorylation of a lipocortin I-like protein is S-phase cell cycle specific in human vascular endothelial cells.

We examined whether the phosphorylation of a 34 kDa lipocortin I-like protein may be associated with internalization process of fibroblast growth factor (FGF) in human umbilical vein endothelial (HUVE) cells. We show that: 1) exposure of synchronized HUVE cells to basic FGF for an appreciable time lag (> or = 30 min) at 37 degrees C and subsequent phosphorylation at 37 degrees C are required to obtain an increased 32P-labelling of a 34 kDa substrate; 2) this FGF-stimulated phosphorylation occurs in S phase but not G1 phase of the growth cycle; 3) the 34 kDa substrate appears to be phosphorylated on tyrosine residues; 4) a major fraction of the 34 kDa 32P-labelled substrate is immunoprecipitated with an antibody that has been raised against human lipocortin/annexin of type I. It is suggested that internalized FGF-receptor/kinase complexes might be primarily responsible for the phosphorylation of the 34 kDa lipocortin I-related protein in S phase HUVE cells.

[Response of human vascular endothelium to angiogenic fibroblast growth factor: role of 2 lipocortins/annexins].

We are interested in whether lipocortins/annexins are involved in the response of human vascular endothelial cells (HUVEC) to angiogenic bFGF. Previously, a lipocortin/annexin of type I (p34) and a lipocortin/annexin of type VI were found to be associated with plasma membranes of HUVEC. Here we show that: i) phorbol ester PMA, a known activator of protein kinase C, possesses the property of acting synergistically with bFGF to stimulate DNA-primary initiation activity; ii) p69 is only detectable in membrane preparations from G1 phase HUVEC, whereas p34 is found to be present in membranes of G1 and S phase HUVEC; iii) the combination of bFGF and PMA induces an increased phosphorylation of p69 in late G1 phase.

Possible involvement of a lipocortin in the initiation of DNA synthesis by human endothelial cells.

This work focused on three themes. First, evidence was obtained for the presence of proteins of 34, 35, 32, and 69 kDa immunologically related to lipocortins I, II, V, and VI, respectively, in human umbilical vein endothelial (HUVE) cells. The 69-kDa protein (p69), but not proteins related to lipocortins I, II, and V, exhibited an increased phosphorylation after exposure of cells to basic fibroblast growth factor (bFGF) and phorbol ester PMA.

Related fibroblast growth factor receptor genes exist in the human genome.

We have isolated, from a human tumor cDNA library, a gene encoding a putative receptor-like protein-tyrosine kinase that we call TK14. The amino acid sequence of the TK14 protein is closely related to the available partial sequence of the mouse protein bek, and more distantly related to the sequences of a chicken basic fibroblast growth factor receptor (73% sequence homology) and the apparent human equivalent of this receptor, the FLG protein (encoded by the fms-like tyrosine kinase gene). Overexpression of the TK14 protein by transfection of COS-1 cells with the corresponding cDNA in a simian virus 40-based expression vector leads to the appearance of new cell-surface binding sites for both acidic and basic fibroblast growth factors.

Phosphorylation and lipocortin-like activity of a 34-kD surface protein in lens epithelial cells: relation to mitogenesis induced by basic fibroblast growth factor.

In order to elucidate the molecular mechanism whereby basic fibroblast growth factor (bFGF) exerts its action on cell proliferation, we investigated the possible relationship between the mitogenic response to bFGF of bovine lens epithelial cells and the phosphorylation and phospholipase A2 (PLA2) activity of a 34-kD protein (p34) present in the basal plasma membrane of these cells. When p34 was obtained from the peripheral epithelial cells which were not capable of being stimulated with bFGF, pretreatment with bFGF led to decreased phosphorylation and PLA2 activity of p34. In contrast, both an increased phosphorylation of p34 and a strong activation of PLA2 occurred when p34 was derived from the central epithelium which was induced to proliferate in the presence of bFGF.

Identification and isolation from bovine epithelial lens cells of two basic fibroblast growth factor receptors that possess bFGF-enhanced phosphorylation activities.

Cross-linking of bound 125I-basic fibroblast growth factor (bFGF) to bovine epithelial lens cells identified two labelled species whose apparent molecular weights were identical with those of two phosphorylated proteins. The bFGF-stimulated phosphorylation of these proteins was shown to be rapid, suggesting an autophosphorylation process. To demonstrate that the phosphorylated proteins were indeed the bFGF-binding molecules, the two components were purified to homogeneity and their bFGF-binding activity was examined.