PAR1 activation induces the release by Schwann cells of factors promoting cell survival and neuritogenesis

Protease-activated receptor 1 (PAR1) is a member of a family of four G-protein-coupled receptors which are activated by proteolytic cleavage of their N-terminal extracellular domain. The expression and the role of PAR1 in peripheral nervous system (PNS) is still poorly investigated, although high PAR1 mRNA expression was found in the dorsal root ganglia and in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier. Schwann cells (SCs) are the principal population of glial cells of the PNS which myelinate axons and play a key role in axonal regeneration and remyelination. Aim of the present study was to determine if the activation of PAR1 affects the neurotrophic properties of SCs. By double immunofluorescence we observed a specific staining for PAR1 in S100ȕ-positive cells of rat sciatic nerve and sciatic teased fibers. Moreover, PAR1 was highly expressed in SC cultures obtained from both neonatal and adult rat sciatic nerves. When PAR1 specific agonists were added to these cultures an increased proliferation rate was observed. Moreover, the conditioned medium obtained from primary SCs treated with PAR1 agonists increased cell survival and neurite outgrowth on PC12 cells respect to controls. By proteomics, western blot and RT-PCR analyses we identified five proteins which are released by SCs following PAR1 stimulation: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). Conversely, a significant decrease in the level of three proteins was observed: Complement C1r subcomponent (C1r) and Complement component 1 Q subcomponent-bindingprotein (C1qbp). When PAR1 expression was silenced by siRNA the observed pro-survival and neurotrophic properties of SCs appear to be reduced respect to controls. References PAR1 activation affects the neurotrophic properties of Schwann cells. Pompili E1, Fabrizi C2, Somma F2, Correani V3, Maras B3, Schininà ME3, Ciraci V2, Artico M4, Fornai F5, Fumagalli L2. 2017 Jan 4;79:23-33. doi: 10.1016/j.mcn.2017.01.001.Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest

Degeneration and regeneration of peripheral nerves: role of thrombin and its receptor PAR-1

The peripheral nervous system has a striking regeneration potential and after damage extensive changes in the differentiation state both of the injured neurons and of the Schwann cells are observed. Schwann cells, in particular, undergo a large scale change in gene expression becoming able to support axonal regeneration. Nerve injury is generally associated to inflammation and activation of the coagulation cascade. Thrombin acts as a polyfunctional signalling molecule exerting its physiological function through soluble target proteins and G-protein-coupled receptors, the protease-activated receptors (PARs) [1]. Recently, we have demonstrated that the activation of the main thrombin receptor, PAR-1, in Schwann cells favours their regenerative potential determining the release of factors which promote axonal regrowth [2]. The pro-regenerative potential of thrombin seems to be exerted in a narrow range of concentrations (pM-nM range). In fact, our preliminary data indicate that high levels of thrombin in the micromolar range slow down Schwann cell proliferation and induce cell death. On the contrary, PAR-1 activating peptides mimic the pro-survival but not the pro-apoptotic effects of thrombin. Controlling thrombin concentration may preserve neuronal health during nerve injury and represent a novel target for pharmacologic therapies

Metacognitive monitoring and control of eyewitness memory reports in autism

Providing eyewitness testimony involves monitoring one's memory to provide a detailed and accurate account: reporting details likely to be accurate and withholding potentially inaccurate details. Autistic individuals reportedly experience difficulties in both retrieving episodic memories and monitoring their accuracy, which has important implications for eyewitness testimony. Thirty autistic and 33 IQ‐matched typically developing (TD) participants viewed a video of a mock bank robbery followed by three phases of questions (with judgments of confidence). In Phase 1, participants freely generated the granularity of their responses (i.e., fine‐ or coarse‐grained). In Phase 2, participants answered the same questions but provided both a fine‐ and a coarse‐grained answer. In Phase 3, participants were instructed to maximize accuracy over informativeness by selecting one of their Phase 2 answers as their final answer. They either received the questions socially (from the experimenter) or answered them online. There were no group differences in accuracy or metacognitive monitoring, with both autistic and TD witnesses demonstrating: (a) a strong preference for reporting fine‐grained details at the expense of accuracy; (b) improved though still suboptimal grain size reporting when instructed to maximize accuracy over informativeness; (c) effective accuracy monitoring; and (d) higher overall accuracy when questions were delivered socially. There was, however, a subtle difference in metacognitive control, with autistic witnesses performing more poorly than TD witnesses when questions were delivered socially, but not when they were delivered online. These findings contrast with evidence suggesting that autism is marked by impairments in episodic memory and metacognitive monitoring and control

Primary structure and tetrahydropteroylglutamate binding site of rabbit liver cytosolic 5,10-methenyltetrahydrofolate synthetase.

The primary sequence of 5,10-methenyltetrahydrofolate synthetase from rabbit liver was determined by amino acid sequencing of the purified enzyme. The enzyme contains 201 amino acid residues with a predicted mass of 22,779 Da. The enzyme is located in the cytosolic fraction of liver homogenates. Carbodiimide-activated 5-formyltetrahydropteroylmonoglutamate and the pentaglutamate form of the substrate both irreversibly inactivate the enzyme by forming a covalent bond to Lys-18. Non-activated 5-formyltetrahydropteroylpentaglutamate protected against this inactivation. Substrate specificity studies showed that increasing the number of glutamate residues from zero to five on 5-formyltetrahydropteroate results in a 2 order of magnitude increase in the affinity of the substrate for the enzyme but only a 3-fold increase in the value of Vmax

PAR1 activation induces the release by Schwann cells of factors promoting cell survival and neuritogenesis

Protease-activated receptor 1 (PAR1) is a member of a family of four G-protein-coupled receptors which are activated by proteolytic cleavage of their N-terminal extracellular domain. The expression and the role of PAR1 in peripheral nervous system (PNS) is still poorly investigated, although high PAR1 mRNA expression was found in the dorsal root ganglia and in the non-compacted Schwann cell myelin microvilli at the nodes of Ranvier. Schwann cells (SCs) are the principal population of glial cells of the PNS which myelinate axons and play a key role in axonal regeneration and remyelination. Aim of the present study was to determine if the activation of PAR1 affects the neurotrophic properties of SCs. By double immunofluorescence we observed a specific staining for PAR1 in S100ȕ-positive cells of rat sciatic nerve and sciatic teased fibers. Moreover, PAR1 was highly expressed in SC cultures obtained from both neonatal and adult rat sciatic nerves. When PAR1 specific agonists were added to these cultures an increased proliferation rate was observed. Moreover, the conditioned medium obtained from primary SCs treated with PAR1 agonists increased cell survival and neurite outgrowth on PC12 cells respect to controls. By proteomics, western blot and RT-PCR analyses we identified five proteins which are released by SCs following PAR1 stimulation: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). Conversely, a significant decrease in the level of three proteins was observed: Complement C1r subcomponent (C1r) and Complement component 1 Q subcomponent-bindingprotein (C1qbp). When PAR1 expression was silenced by siRNA the observed pro-survival and neurotrophic properties of SCs appear to be reduced respect to controls. References PAR1 activation affects the neurotrophic properties of Schwann cells. Pompili E1, Fabrizi C2, Somma F2, Correani V3, Maras B3, Schininà ME3, Ciraci V2, Artico M4, Fornai F5, Fumagalli L2. 2017 Jan 4;79:23-33. doi: 10.1016/j.mcn.2017.01.001.Schwann cells (SCs) regulate a wide variety of axonal functions in the peripheral nervous system, providing a supportive growth environment following nerve injury (1). Here we show that rat SCs express the protease-activated receptor-1 (PAR1) both in vivo and in vitro. PAR1 is a G-protein coupled receptor eliciting cellular responses to thrombin and other proteases (2). To investigate if PAR1 activation affects the neurotrophic properties of SCs, this receptor was activated by a specific agonist peptide (TFLLR) and the conditioned medium was transferred to PC12 pheocromocytoma cells for assessing cell survival and neurite outgrowth. Culture medium from SCs treated with 10 µM TFLLR reduced significantly the release of LDH and increased the viability of PC12 cells with respect to the medium of the untreated SCs. Furthermore, conditioned medium from TFLLR-treated SCs increased neurite outgrowth on PC12 cells respect to control medium from untreated cells. To identify putative neurotrophic candidates we performed proteomic analysis on SC secretoma and real time PCR experiments after PAR1 activation. Stimulation of SCs with TFLLR increased specifically the release of a subset of five proteins: Macrophage migration inhibitory factor (Mif), Aldose reductase (Akr1b1), Matrix metalloproteinase-2 (Mmp2), Syndecan-4 (Sdc) and Decorin (Dcn). At the same time there was a significant decrease in the level of three proteins: Complement C1r subcomponent (C1r), Complement component 1 Q subcomponent-binding protein (C1qbp) and Angiogenic factor with G patch and FHA domains 1 (Aggf1). These data indicate that PAR1 stimulation does induce the release by SCs of factors promoting cell survival and neuritogenesis. Among these proteins, Mif, Sdc, Dcn and Mmp2 are of particular interest

The primary structure of rabbit liver mitochondrial serine hydroxymethyltransferase.

The complete amino acid sequence of mitochondrial serine hydroxymethyltransferase from rabbit liver was determined. The sequence was obtained from analysis of peptides isolated from chymotryptic, cyanogen bromide, and limited acid cleavages of the protein. The enzyme consists of four identical subunits, each of 475 residues, i.e. 8 residues shorter than the subunit of the corresponding cytosolic isoenzyme. The sequences of the two rabbit proteins are easily aligned, provided a gap of 5 residues near the amino terminus and a gap of 3 residues near the carboxyl terminus are included in the mitochondrial sequence. The overall degree of identity between the two isoenzymes is 61.9%, whereas the structural identity of each eukaryotic isoenzyme with the corresponding Escherichia coli enzyme is about 40%. The rabbit isoenzymes are about 70 residues longer than the E. coli enzyme, with one-half of these residues accounted for by insertions in both isoenzymes near their carboxyl terminus. Predictions of secondary structure and calculations of hydropathy profiles are also presented, suggesting an even more extensive degree of identity in the three-dimensional folding of the three proteins, in accord with the known similarity of their catalytic properties. Evidence was obtained for the existence of additional molecular forms of the mitochondrial protein, differing in the absence of some amino acid residues at the amino terminus of the polypeptide chain

MESENCHIMAL STEM CELLS IN INFLAMMATORY BOWEL DISEASES: CLINICAL EVIDENCES AND POTENTIAL INSIGHTS FOR THE CLINICIANS

Mesenchimal stem cells (MSCs) have been used experimentally and clinically in the treatment of a wide variety of pathologies MSCs can be safely transplanted in autologous and allogenic ways as they are non-immunogenic, and consequently represent a therapeutic option for refractory connective tissue diseases, fibrosing diseases like scleroderma and fistulizing colitis like in Crohn's disease (CD). The immunomodulatory properties of MSCs have already shown promise when used as therapy for otherwise medically refractory CD. Accumulating evidence suggests that the properties may also be exploited of several other conditions. The currently available experimental and clinical data indicate that, similar to previously obtained data in the setting of HSCT, MSC treatment for IBD is feasible and safe. aim of this review is to analyze the pathophysiological insights for the use of MSCs in inflammatory bowel diseases, and to summarize the clinical evidences about the efficacy and safety of stem cell therapy in such disorders

The Effect of the Pairing Interaction on the Energies of Isobar Analog Resonances in 112−124^{112-124}Sb and Isospin Admixture in 100−124^{100-124}Sn Isotopes

In the present study, the effect of the pairing interaction and the isovector correlation between nucleons on the properties of the isobar analog resonances (IAR) in $^{112-124}$Sb isotopes and the isospin admixture in $^{100-124}$Sn isotopes is investigated within the framework of the quasiparticle random phase approximation (QRPA). The form of the interaction strength parameter is related to the shell model potential by restoring the isotopic invariance of the nuclear part of the total Hamiltonian. In this respect, the isospin admixtures in the $^{100-124}$Sn isotopes are calculated, and the dependence of the differential cross section and the volume integral $J_{F}$ for the Sn($^{3}$He,t)Sb reactions at E($^{3}$He)$=200$ MeV occurring by the excitation of IAR on mass number A is examined. Our results show that the calculated value for the isospin mixing in the $^{100}$Sn isotope is in good agreement with Colo et al.'s estimates $(4-5$, and the obtained values for the volume integral change within the error range of the value reported by Fujiwara et al. (53$\pm$5 MeV fm$^{3}$). Moreover, it is concluded that although the differential cross section of the isobar analog resonance for the ($^{3}$He,t) reactions is not sensitive to pairing correlations between nucleons, a considerable effect on the isospin admixtures in $N\approx Z$ isotopes can be seen with the presence of these correlations.Comment: 16 pages, 5 EPS figures and 2 tables, Late

The effects of early leaf removal and cluster thinning treatments on berry growth and grape composition in cultivars Vranac and Cabernet Sauvignon

Background: The aim of the present work was to investigate the effect of early leaf removal and cluster thinning treatments in the Mediterranean climate on berry growth and how these two techniques affect phenolic profile (especially proathocyanidins) and color characteristics for later wine production. The study was conducted in 2011 in Podgorica, Montenegro. Two grapevine cultivars were selected to compare different ability in flavonoid accumulation: Vranac, with moderate accumulation and Cabernet Sauvignon, usually showing very good accumulation of polyphenols. Four treatments were compared: only leaf removal, only cluster thinning, leaf removal combined with cluster thinning, and no treatment that was used for control (control set). Results: Early defoliation reduced the yield in both varieties. In Cabernet Sauvignon, defoliation initially delayed berry growth, but at the end, defoliation slightly affected almost all yield parameters (cluster weight, berry weight, and number of berries per cluster), while in cultivar Vranac, defoliation did not modify the berry growth and berry weight. In both varieties, cluster thinning did not affect the berry weight. In the treatments where both defoliation and cluster thinning was applied, a reduction of the cluster weight, berry weight, and berry numbers per cluster was observed. Cabernet Sauvignon showed a greater reactivity to the applied techniques, while Vranac was less reactive. At harvest, no damaged bunches (caused by sunburn) were found in defoliated treatment. Conclusions: It can be concluded that for both varieties, early defoliation and cluster thinning lead to better soluble solids accumulation than in the control set. The treatments lead to raised concentration of anthocyanins and proanthocyanidins in both varieties. It is confirmed that the highest content of anthocyanins and proanthocyanidins was in the skin extracts of the grapes where both treatments were applied. This is followed by the treatment where only defoliation was applied. The enhanced contents of these compounds per berry in grape variety Vranac are the result of increased synthesis, while in Cabernet sauvignon variety, increased content was due to the less berry weight. The best wine characteristics (alcohol, color intensity, color hue, total anthocyanins, total polyphenols) were found in products, where defoliation was applied