Effects of intramuscular administration of Platelet-Rich Plasma on denervated muscle after peripheral nerve injury.

Purpose: After peripheral nerve injury (PNI), prolonged denervation of the target muscle prevents adequate reinnervation even if the nerve is repaired. The aim of this work is to analyze the effect of intramuscular Platelet-Rich Plasma (PRP) in a denervated muscle due to PNI.Materials and.

PLCβ1 by-passes early growth response -1 to induce the differentiation of neuronal cells.

The Gα/phospholipase C-β (PLCβ) signaling system mediates calcium responses to a variety of hormones and neurotransmitters. Recent studies suggest that PLCβ1 expression plays a role in the differentiation of two types of cultured neuronal cells (PC12 and SK-N-SH) through a mechanism independent of Gα. Here, we show that, similar to that observed in PC12 and SK-N-SH cells, PLCβ1 expression increases when human NT2 cells are induced to differentiate either through cytosine-β-D-arabinofuranoside or retinoic acid.

Endocannabinoid 2-Arachidonoylglycerol Synthesis and Metabolism at Neuronal Nuclear Matrix Fractions Derived from Adult Rat Brain Cortex.

In this report, we describe the kinetics characteristics of the diacylglycerol lipase-α (DGLα) located at the nuclear matrix of nuclei derived from adult cortical neurons. Thus, using high-resolution fluorescence microscopy, classical biochemical subcellular fractionation, and Western blot techniques, we demonstrate that the DGLα enzyme is located in the matrix of neuronal nuclei. Furthermore, by quantifying the 2-arachidonoylglycerol (2-AG) level by liquid chromatography and mass spectrometry when 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG) was exogenously added as substrate, we describe the presence of a mechanism for 2-AG production through DGLα dependent biosynthesis with an apparent () of 180 µM and a of 1.

Up-regulation of CB cannabinoid receptors located at glutamatergic terminals in the medial prefrontal cortex of the obese Zucker rat.

The present study describes a detailed neuroanatomical distribution map of the cannabinoid type 1 (CB) receptor, along with the biochemical characterization of the expression and functional coupling to their cognate G proteins in the medial prefrontal cortex (mPCx) of the obese Zucker rats. The CB receptor density was higher in the prelimbic (PL) and infralimbic (IL) subregions of the mPCx of obese Zucker rats relative to their lean littermates which was associated with a higher percentage of CB receptor immunopositive excitatory presynaptic terminals in PL and IL. Also, a higher expression of CB receptors and WIN55,212-2-stimulated [S]GTPγS binding was observed in the mPCx but not in the neocortex (NCx) and hippocampus of obese rats.

Isolation of Platelet-Derived Exosomes from Human Platelet-Rich Plasma: Biochemical and Morphological Characterization.

Platelet-Rich Plasma (PRP) is enriched in molecular messengers with restorative effects on altered tissue environments. Upon activation, platelets release a plethora of growth factors and cytokines, either in free form or encapsulated in exosomes, which have been proven to promote tissue repair and regeneration. Translational research on the potential of exosomes as a safe nanosystem for therapeutic cargo delivery requires standardizing exosome isolation methods along with their molecular and morphological characterization.

Fit-for-purpose based testing and validation of antibodies to amino- and carboxy-terminal domains of cannabinoid receptor 1.

Specific and selective anti-CB antibodies are among the most powerful research tools to unravel the complex biological processes mediated by the CB receptor in both physiological and pathological conditions. However, low performance of antibodies remains a major source of inconsistency between results from different laboratories. Using a variety of techniques, including some of the most commonly accepted ones for antibody specificity testing, we identified three of five commercial antibodies against different regions of CB receptor as the best choice for specific end-use purposes.

Effects of Platelet-Rich Plasma on Cellular Populations of the Central Nervous System: The Influence of Donor Age.

Platelet-rich plasma (PRP) is a biologic therapy that promotes healing responses across multiple medical fields, including the central nervous system (CNS). The efficacy of this therapy depends on several factors such as the donor's health status and age. This work aims to prove the effect of PRP on cellular models of the CNS, considering the differences between PRP from young and elderly donors.

Data for the morphometric characterization of NT2-derived postmitotic neurons.

NTERA2/D1 human teratocarcinoma progenitors induced to differentiate into postmitotic neurons by either long-term treatment with retinoic acid or short-term treatment with the nucleoside analog cytosine β-D-arabinofuranoside were subjected to morphometric analysis and compared. Our data provide a methodological and conceptual framework for future investigations aiming at distinguishing neuronal phenotypes on the basis of morphometric analysis. Data presented here are related to research concurrently published in "Highly Efficient Generation of Glutamatergic/Cholinergic NT2-Derived Postmitotic Human Neurons by Short-Term treatment with the Nucleoside Analogue Cytosine β-D-Arabinofuranoside" [1].

Highly efficient generation of glutamatergic/cholinergic NT2-derived postmitotic human neurons by short-term treatment with the nucleoside analogue cytosine β-D-arabinofuranoside.

The human NTERA2/D1 (NT2) cells generate postmitotic neurons (NT2N cells) upon retinoic acid (RA) treatment and are functionally integrated in the host tissue following grafting into the rodent and human brain, thus representing a promising source for neuronal replacement therapy. Yet the major limitations of this model are the lengthy differentiation procedure and its low efficiency, although recent studies suggest that the differentiation process can be shortened to less than 1 week using nucleoside analogues. To explore whether short-term exposure of NT2 cells to the nucleoside analogue cytosine β-d-arabinofuranoside (AraC) could be a suitable method to efficiently generate mature neurons, we conducted a neurochemical and morphometric characterization of AraC-differentiated NT2N (AraC/NT2N) neurons and improved the differentiation efficiency by modifying the cell culture schedule.

New Insights into Gene Delivery to Human Neuronal Precursor NT2 Cells: A Comparative Study between Lipoplexes, Nioplexes, and Polyplexes.

The transfection of human NTera2/D1 teratocarcinoma-derived cell line (or NT2 cells) represents a promising strategy for the delivery of exogenous proteins or biological agents into the central nervous system (CNS). The development of suitable nonviral vectors with high transfection efficiencies requires a profound knowledge of the whole transfection process. In this work, we elaborated and characterized in terms of size and zeta potential three different nonviral vectors: lipoplexes (144 nm; -29.

Nuclear diacylglycerol lipase-α in rat brain cortical neurons: evidence of 2-arachidonoylglycerol production in concert with phospholipase C-β activity.

In this report, we describe the localization of diacylglycerol lipase-α (DAGLα) in nuclei from adult cortical neurons, as assessed by double-immunofluorescence staining of rat brain cortical sections and purified intact nuclei and by western blot analysis of subnuclear fractions. Double-labeling assays using the anti-DAGLα antibody and NeuN combined with Hoechst staining showed that only nuclei of neuronal origin were DAGLα positive. At high resolution, DAGLα-signal displayed a punctate pattern in nuclear subdomains poor in Hoechst's chromatin and lamin B1 staining.

Nuclear phospholipase C-β1 and diacylglycerol LIPASE-α in brain cortical neurons.

Phosphoinositide (PtdIns) signaling involves the generation of lipid second messengers in response to stimuli in a receptor-mediated manner at the plasma membrane. In neuronal cells of adult brain, the standard model proposes that activation of metabotropic receptors coupled to Phospholipase C-β1 (PLC-β1) is linked to endocannabinoid signaling through the production of diacylglycerol (DAG), which could be systematically metabolized by 1,2-diacylglycerol Lipases (DAGL) to produce an increase of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. However, the existence of a nuclear PtdIns metabolism independent from that occurring elsewhere in the cell is now widely accepted, suggesting that the nucleus constitutes both a functional and a distinct compartment for PtdIns metabolism.

Levels of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclase isoforms (1, 5/6, 8) in post-mortem human brain caudate and cortical membranes: comparison with rat brain membranes and potential stoichiometric relationships.

The levels of expression of Gsα(short and long), Gα(olf) and Gβ(common) subunits, and calcium-sensitive adenylyl cyclases isoforms (AC1, 5/6, and 8) in human brain cortical and caudate membranes were quantified by western blot analysis in order to establish their contribution to the patterns of AC functioning. Both areas expressed Gsα(long) (52 kDa) with values ranging from about 1400 ng/mg of membrane protein in cerebral cortex to close to 600 ng/mg of membrane protein in caudate nucleus. In contrast, Gsα(short) and Gsα(olf) were expressed separately, Gsα(short) in cortical membranes with values around 500 ng/mg of membrane protein and Gα(olf) in caudate membranes with values around 1300 ng/mg of membrane protein.

Opposite changes in cannabinoid CB1 and CB2 receptor expression in human gliomas.

Gliomas are the most important group of malignant primary brain tumors and one of the most aggressive forms of cancer. During the last years, several studies have demonstrated that cannabinoids induce apoptosis of glioma cells and inhibit angiogenesis of gliomas in vivo. As the effects of cannabinoids rely on CB(1) and CB(2) receptors activation, the aim of the present study was to investigate both receptors protein expression in cellular membrane homogenates of human glial tumors using specific antibodies raised against these proteins.

B cell receptor-induced growth arrest and apoptosis in WEHI-231 immature B lymphoma cells involve cyclic AMP and Epac proteins.

Signaling by the B cell antigen receptor (BCR) is essential for B lymphocyte homeostasis and immune function. In immature B cells, ligation of the BCR promotes growth arrest and apoptosis, and BCR-driven balancing between pro-apoptotic extracellular signal-regulated kinase 1 and 2 (ERK1/2) and anti-apoptotic phosphoinositide 3-kinase-dependent Akt seems to define the final cellular apoptotic response. Dysfunction of these late BCR signaling events can lead to the development of immunological diseases.

Direct stimulation of receptor-controlled phospholipase D1 by phospho-cofilin.

The activity state of cofilin, which controls actin dynamics, is driven by a phosphorylation-dephosphorylation cycle. Phosphorylation of cofilin by LIM-kinases results in its inactivation, a process supported by 14-3-3zeta and reversed by dephosphorylation by slingshot phosphatases. Here we report on a novel cellular function for the phosphorylation-dephosphorylation cycle of cofilin.

Phospholipase D signaling: orchestration by PIP2 and small GTPases.

Hydrolysis of phosphatidylcholine by phospholipase D (PLD) leads to the generation of the versatile lipid second messenger, phosphatidic acid (PA), which is involved in fundamental cellular processes, including membrane trafficking, actin cytoskeleton remodeling, cell proliferation and cell survival. PLD activity can be dramatically stimulated by a large number of cell surface receptors and is elaborately regulated by intracellular factors, including protein kinase C isoforms, small GTPases of the ARF, Rho and Ras families and, particularly, by the phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP(2)). PIP(2) is well known as substrate for the generation of second messengers by phospholipase C, but is now also understood to recruit and/or activate a variety of actin regulatory proteins, ion channels and other signaling proteins, including PLD, by direct interaction.

Cyclic AMP-dependent and Epac-mediated activation of R-Ras by G protein-coupled receptors leads to phospholipase D stimulation.

The activation of the Ras-related GTPase R-Ras, which has been implicated in the regulation of various cellular functions, by G protein-coupled receptors (GPCRs) was studied in HEK-293 cells stably expressing the M3 muscarinic acetylcholine receptor (mAChR), which can couple to several types of heterotrimeric G proteins. Activation of the receptor induced a very rapid and transient activation of R-Ras. Studies with inhibitors and activators of various signaling pathways indicated that R-Ras activation by the M3 mAChR is dependent on cyclic AMP formation but is independent of protein kinase A.

Levels of G-protein alpha q/11 subunits and of phospholipase C-beta(1-4), -gamma, and -delta1 isoforms in postmortem human brain caudate and cortical membranes: potential functional implications.

The levels of expression of G-protein alpha(q/11) (Galpha(q/11)) subunits and PLC-beta(1-4), -gamma, and -delta(1) isoforms were quantified by Western blot analysis in order to establish their contribution to the patterns of PLC functioning reported here. Quantitative measurements of the levels of Galpha(q/11) subunits in each region were obtained by comparison with known amounts of Escherichia coli expressed recombinant Galpha(q) subunits. Quantitative analysis indicated that Galpha(q/11) subunits are abundant polypeptides in human brain, with values ranging from about 1200 ng/mg in cerebral cortex to close to 900 ng/mg of membrane protein in caudate.

Differential postmortem delay effect on agonist-mediated phospholipase Cbeta activity in human cortical crude and synaptosomal brain membranes.

The phosphoinositide signal transduction system, and particularly, phospholipase Cbeta isozymes, are relevant in the etiopathogeny of human neuropsychiatric pathologies such as depression. Stimulation of phospholipase Cbeta activity by muscarinic receptors and G proteins was determined in crude and synaptosomal membrane preparations from nine postmortem human frontal cortices (postmortem delay range 8 to 50 h). Thus, the phospholipase Cbeta activity was determined by measuring the hydrolysis of exogenous [3H]-phosphatidylinositol 4,5-bisphosphate.