A Flexible Method to Study Neuronal Differentiation of Mouse Embryonic Stem Cells

Embryonic Stem Cells (ESCs) represent an invaluable tool for the study of early mammalian development, for regenerative medicine and for drug discovery. To fulfill these promises, efficient and easy protocols to differentiate ESCs have to be developed. Most of these protocols results in low efficiency of neural induction and/or requires extended in vitro culture. Here we describe in detail an easy and efficient method to differentiate ESCs into neurons, that can be used to identify molecules required for proper neuronal differentiation. Moreover, we present a modification of this method that allows to clearly evaluate the ability of some molecules to favor neuron formation in vitro. These methods can represent an efficient platform for studying the molecular mechanisms underlying early events of neural induction and differentiation in ESCs, as well as for testing molecule efficacy in the pharmaceutical testing.     

Purinergic receptors mediate two distinct glutamate release pathways in hippocampal astrocytes

The purinergic P2X(7) receptor (P2X(7)R) can mediate glutamate release from cultured astrocytes. Using patch clamp recordings, we investigated whether P2X(7)Rs have the same action in hippocampal astrocytes in situ. We found that 2- and 3-O-(4-benzoylbenzoyl)ATP (BzATP), a potent, although unselective P2X(7)R agonist, triggers two different glutamate-mediated responses in CA1 pyramidal neurons; they are transient inward currents, which have the kinetic and pharmacological properties of previously described slow inward currents (SICs) due to Ca(2+)-dependent glutamate release from astrocytes, and a sustained tonic current. Although SICs were unaffected by P2X(7)Rs antagonists, the tonic current was inhibited, was amplified in low extracellular Ca(2+), and was insensitive to glutamate transporter and hemichannel inhibitors. BzATP triggered in astrocytes a large depolarization that was inhibited by P2X(7)R antagonists and amplified in low Ca(2+). In low Ca(2+) BzATP also induced lucifer yellow uptake into a subpopulation of astrocytes and CA3 neurons. Our results demonstrate that purinergic receptors other than the P2X(7)R mediate glutamate release that evokes SICs, whereas activation of a receptor that has features similar to the P2X(7)R, mediates a sustained glutamate efflux that generates a tonic current in CA1 neurons. This sustained glutamate efflux, which is potentiated under non-physiological conditions, may have important pathological actions in the brain.     

Controlled loading of oligodeoxyribonucleotide monolayers onto unoxidized crystalline silicon; fluorescence-based determination of the surface coverage and of the hybridization efficiency; parallel imaging of the process by Atomic Force Microscopy

Unoxidized crystalline silicon, characterized by high purity, high homogeneity, sturdiness and an atomically flat surface, offers many advantages for the construction of electronic miniaturized biosensor arrays upon attachment of biomolecules (DNA, proteins or small organic compounds). This allows to study the incidence of molecular interactions through the simultaneous analysis, within a single experiment, of a number of samples containing small quantities of potential targets, in the presence of thousands of variables. A simple, accurate and robust methodology was established and is here presented, for the assembling of DNA sensors on the unoxidized, crystalline Si(100) surface, by loading controlled amounts of a monolayer DNA-probe through a two-step procedure. At first a monolayer of a spacer molecule, such as 10-undecynoic acid, was deposited, under optimized conditions, via controlled cathodic electrografting, then a synthetic DNA-probe was anchored to it, through amidation in aqueous solution. The surface coverage of several DNA-probes and the control of their efficiency in recognizing a complementary target-DNA upon hybridization were evaluated by fluorescence measurements. The whole process was also monitored in parallel by Atomic Force Microscopy (AFM).     

Conifers in cold environments synchronize maximum growth rate of tree-ring formation with day length

Intra-annual radial growth rates and durations in trees are reported to differ greatly in relation to species, site and environmental conditions. However, very similar dynamics of cambial activity and wood formation are observed in temperate and boreal zones. Here, we compared weekly xylem cell production and variation in stem circumference in the main northern hemisphere conifer species (genera Picea, Pinus, Abies and Larix) from 1996 to 2003. Dynamics of radial growth were modeled with a Gompertz function, defining the upper asymptote (A), x-axis placement (beta) and rate of change (kappa). A strong linear relationship was found between the constants beta and kappa for both types of analysis. The slope of the linear regression, which corresponds to the time at which maximum growth rate occurred, appeared to converge towards the summer solstice. The maximum growth rate occurred around the time of maximum day length, and not during the warmest period of the year as previously suggested. The achievements of photoperiod could act as a growth constraint or a limit after which the rate of tree-ring formation tends to decrease, thus allowing plants to safely complete secondary cell wall lignification before winter.     

Sterol dependent regulation of human TM7SF2 gene expression: role of the encoded 3beta-hydroxysterol Delta14-reductase in human cholesterol biosynthesis

3Beta-hydroxysterol Delta(14)-reductase operates during the conversion of lanosterol to cholesterol in mammalian cells. Besides the endoplasmic reticulum 3beta-hydroxysterol Delta(14)-reductase (C14SR) encoded by TM7SF2 gene, the lamin B receptor (LBR) of the inner nuclear membrane possesses 3beta-hydroxysterol Delta(14)-reductase activity, based on its ability to complement C14SR-defective yeast strains. LBR was indicated as the primary 3beta-hydroxysterol Delta(14)-reductase in human cholesterol biosynthesis, since mutations in LBR gene were found in Greenberg skeletal dysplasia, characterized by accumulation of Delta(14)-unsaturated sterols. This study addresses the issue of C14SR and LBR role in cholesterol biosynthesis. Both human C14SR and LBR expressed in COS-1 cells exhibit 3beta-hydroxysterol Delta(14)-reductase activity in vitro. TM7SF2 mRNA and C14SR protein expression in HepG2 cells grown in delipidated serum (LPDS) plus lovastatin (sterol starvation) were 4- and 8-fold higher, respectively, than in LPDS plus 25-hydroxycholesterol (sterol feeding), resulting in 4-fold higher 3beta-hydroxysterol Delta(14)-reductase activity. No variations in LBR mRNA and protein levels were detected in the same conditions. The induction of TM7SF2 gene expression is turned-on by promoter activation in response to low cell sterol levels and is mediated by SREBP-2. The results suggest a primary role of C14SR in human cholesterol biosynthesis, whereas LBR role in the pathway remains unclear.     

A comparative study of bipolar disorder and attention deficit hyperactivity disorder through the measurement of regional cerebral blood flow

Attention Deficit Hyperactivity Disorder (ADHD) and Bipolar Disorder (BPD) are two common neuropsychological disorders which are often present in a comorbid state. I used the results of cerebral blood flow studies made with Single Photon Emission Computer Tomography (SPECT), Positron Emission Tomography (PET) and Functional Magnetic Resonance Imaging (fMRI), to investigate a possible relationship between ADHD and BPD. The common areas of the brain involved in both BPD and ADHD appears to be the prefrontal cortex in its various components, the basal ganglia and possibly the cerebellum which, especially in the past, has been little studied by researchers in relation to ADHD and BPD. Among the differences the blood flow lateralization, present in BPD in states of altered mood, is evident with left hypoperfusion and right hyperperfusion during depression, the opposite in the case of manic state; in ADHD, the lateralization is less constant and of questionable interpretation. In BPD the involvement of a greater number of brain areas, especially the temporal lobe, is common. I advance the hypothesis that BPD progresses from ADHD secondary to expansion of perturbation in cerebral blood flow.     

Loss of the preconditioning effect of rosuvastatin during sustained therapy: a human in vivo study

Studies have demonstrated that the acute administration of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors has protective effects in the setting of ischemia-reperfusion (IR). Previously, we demonstrated that a single dose of rosuvastatin prevented IR-induced endothelial dysfunction in humans through a cyclooxygenase-2-dependent mechanism. Whether the chronic administration of HMG-CoA reductase inhibitors provides similar protection remains controversial and is unknown in humans. Eighteen male volunteers were randomized to receive a single dose of rosuvastatin (20 mg) or placebo. Twenty-four hours later, endothelium-dependent, radial artery flow-mediated dilation (FMD) was measured before and after IR (15 min of upper arm ischemia followed by 15 min of reperfusion). In a separate protocol, 30 healthy volunteers were randomized in a double-blind fashion to receive oral rosuvastatin (20 mg/day) and placebo, rosuvastatin, and celecoxib (100 mg bid) or placebo alone, all for 21 days. Twenty-four hours after the final administration of study medication, FMD was measured before and after IR. Pre-IR FMD was similar between groups in both protocols. In the acute administration protocol, rosuvastatin significantly prevented the blunting of FMD associated with IR (FMD pre-IR: 8.4 ± 1.3%; post-IR: 6.2 ± 1.3%; P = 0.01 ANOVA, treatment group interaction). In the daily administration protocol, IR significantly blunted FMD in the placebo group (FMD pre-IR: 7.5 ± 0.9%; post-IR: 3.3 ± 0.7%; P < 0.001). Chronic treatment with rosuvastatin did not modify this ischemic injury (FMD pre-IR: 6.9 ± 0.4%; post-IR: 1.6 ± 1.0%; P < 0.001; P = NS ANOVA, treatment group interaction). Similarly, FMD responses post-IR in volunteers receiving rosuvastatin and celecoxib did not significantly differ from placebo (FMD pre-IR: 8.3 ± 0.9%; post-IR: 2.1 ± 0.8%; P < 0.001; P = NS ANOVA, treatment group interaction). In contrast to acute administration, chronic rosuvastatin does not prevent the development of IR-induced endothelial dysfunction in normal humans.     

Phosphodiesterase type-2 and NO-dependent S-nitrosylation mediate the cardioinhibition of the antihypertensive catestatin

The chromogranin A (CHGA)-derived peptide catestatin (CST: hCHGA(352-372)) is a noncompetitive catecholamine-release inhibitor that exerts vasodilator, antihypertensive, and cardiosuppressive actions. We have shown that CST directly influences the basal performance of the vertebrate heart where CST dose dependently induced a nitric oxide-cGMP-dependent cardiosuppression and counteracted the effects of adrenergic stimulation through a noncompetitive antagonism. Here, we sought to determine the specific intracardiac signaling activated by CST in the rat heart. Physiological analyses performed on isolated, Langendorff-perfused cardiac preparations revealed that CST-induced negative inotropism and lusitropism involve β(2)/β(3)-adrenergic receptors (β(2)/β(3)-AR), showing a higher affinity for β(2)-AR. Interaction with β(2)-AR activated phosphatidylinositol 3-kinase/endothelial nitric oxide synthase (eNOS), increased cGMP levels, and induced activation of phosphodiesterases type 2 (PDE2), which was found to be involved in the antiadrenergic action of CST as evidenced by the decreased cAMP levels. CST-dependent negative cardiomodulation was abolished by functional denudation of the endothelium with Triton. CST also increased the eNOS expression in cardiac tissue and human umbilical vein endothelial cells. cells, confirming the involvement of the vascular endothelium. In ventricular extracts, CST increased S-nitrosylation of both phospholamban and β-arrestin, suggesting an additional mechanism for intracellular calcium modulation and β-adrenergic responsiveness. We conclude that PDE2 and S-nitrosylation play crucial roles in the CST regulation of cardiac function. Our results are of importance in relation to the putative application of CST as a cardioprotective agent against stress, including excessive sympathochromaffin overactivation.     

Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.