Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.

Transcriptional networks defining stemness in adult neural stem cells (NSCs) are largely unknown. We used the proximal cis-regulatory element (pCRE) of the retina-specific homeobox gene 2 (rx2) to address such a network. Lineage analysis in the fish retina identified rx2 as marker for multipotent NSCs. rx2-positive cells located in the peripheral ciliary marginal zone behave as stem cells for the neuroretina, or the retinal pigmented epithelium. We identified upstream regulators of rx2 interrogating the rx2 pCRE in a trans-regulation screen and focused on four TFs (Sox2, Tlx, Gli3, and Her9) activating or repressing rx2 expression. We demonstrated direct interaction of the rx2 pCRE with the four factors in vitro and in vivo. By conditional mosaic gain- and loss-of-function analyses, we validated the activity of those factors on regulating rx2 transcription and consequently modulating neuroretinal and RPE stem cell features. This becomes obvious by the rx2-mutant phenotypes that together with the data presented above identify rx2 as a transcriptional hub balancing stemness of neuroretinal and RPE stem cells in the adult fish retina.

Membrane currents and changes in the intracellular Ca ] i was observed after the slow superfusion of ATP, ADPb-S and UTP; a,b-meATP was ineffective. These data, in conjunction with results obtained by using the P2 receptor antagonists TNP-ATP, PPADS and MRS2179 indicate that the current response to a,b-meATP is due to P2X 3 receptor activation, while the ATP-induced rise in [Ca 2+ ] i is evoked by P2Y 1 and P2Y 4 receptor activation. TCE depressed the a,b-meATP current in a manner compatible with a non-competitive antagonism. The ATP-induced increase of [Ca 2+ ] i was much less sensitive to the inhibitory effect of TCE than the current response to a,b-meATP. The present study indicates that in HEK293-hP2X 3 cells, TCE, but not ethanol, potently inhibits ligand-gated P2X 3 receptors and, in addition, moderately interferes with G protein-coupled P2Y 1 and P2Y 4 receptors. Such an effect may be relevant for the interruption of pain transmission in dorsal root ganglion neurons following ingestion of chloral hydrate or trichloroethylene.

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The key molecular interactions governing vertebrate limb bud development are a paradigm for studying the mechanisms controlling progenitor cell proliferation and specification during vertebrate organogenesis. However, little is known about the cellular heterogeneity of the mesenchymal progenitors in early limb buds that ultimately contribute to the chondrogenic condensations prefiguring the skeleton. We combined flow cytometric and transcriptome analyses to identify the molecular signatures of several distinct mesenchymal progenitor cell populations present in early mouse forelimb buds. In particular, jagged 1 (JAG1)-positive cells located in the posterior-distal mesenchyme were identified as the most immature limb bud mesenchymal progenitors (LMPs), which crucially depend on SHH and FGF signaling in culture. The analysis of gremlin 1 ( Grem1 )-deficient forelimb buds showed that JAG1-expressing LMPs are protected from apoptosis by GREM1-mediated BMP antagonism. At the same stage, the osteo-chondrogenic progenitors (OCPs) located in the core mesenchyme are already actively responding to BMP signaling. This analysis sheds light on the cellular heterogeneity of the early mouse limb bud mesenchyme and on the distinct response of LMPs and OCPs to morphogen signaling.

Dyeing of cotton fabrics treated with dimethyloldihydroxyethyleneurea and triethanolamine is discussed. Adsorption time curves from finite baths of untreated, mercerized, and crosslinked cotton print cloths have been studied. Mercerization did not change heterogeneous diffusion resistance but increased adsorption equilibrium constants, dyeing affinities, and diffusion coefficients. Maximum dyeing capacities of mercerized cotton were lower. Crosslinking reduced most of the kinetic constants (rate constants, structural diffusion resistance constants, maximum dyeing capacities, activation energies, and diffusion coefficients). Triethanolamine additive enhanced the kinetic constants and the dyeabilities of crosslinked cotton fabrics. The results indicated that in the crosslinked cotton, triethanolamine reacted preferably with the crosslinks and not with the remaining free reactive groups of cotton fabric.Crosslinking by dimethyloldihydroxyethyleneurea (DMDHEU) reduces the equilibria and dyeing rates of cotton or other cellulosic materials [7,9,10]. On the other hand, mercerization increases the dyeing rates. When the cotton fabrics are crosslinked and treated with triethanolamine (TEA), nitrogen content and dyeability increase [ 1 ] . In order to better understand the effect of crosslinking by DMDHEU and TEA on dyeing kinetics from finite baths, we have studied the time curves under different dyeing conditions using the following diffusion kinetic equation [2][3][4][5] :where a is the adsorption of the dye on cotton in time t, a~ is the equilibrium adsorption at t -oo , k is the specific rate constant, Co is the concentration of cotton in a finite bath, and n is the structural diffusion resistance constant. The half-life of dyeing is then From these constants, we have determined other kinetic constants (maximum adsorption capacities ~B, adsorption equilibrium constants Ko, diffusion coe~-cients D, activation energies AE*, enthalpies 011, and exhaustions a ) [ 2 ] . By means of these kinetic parameters, we can predict the structural changes of cotton fabrics and the dyeing kinetic profiles from finite baths under different conditions ( dye and cotton concentrations and temperatures).We determined the constants of Equation 1 using the computer mathematical regressions of the time curves [3]. Graphical determination [2] was less accurate but agreed well in the limits of errors with the computer analysis. The maximum adsorption capacities, the adsorption equilibrium constants, the acti-. vation energies of dyeing, and the apparent enthalpies were determined statistically from two or more time curves with two or more dye and cotton concentrations and two or more temperatures as described previously [ 2 ] . The apparent diffusion coefficients were calculated by comparing Equation 1 with the integrated Fwk's diffusion equations [2, 6, 12] under the assumption that cotton fibers are cylinders:All these kinetic constants obtained from the time curves were a useful tool for elucidating the dyeing mechanism and some structural ...

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SummaryBone-derived mesenchymal stromal cells (MSCs) differentiate into multiple lineages including chondro- and osteogenic fates and function in establishing the hematopoietic compartment of the bone marrow. Here, we analyze the emergence of different MSC types during mouse limb and long bone development. In particular, PDGFRαposSCA-1pos (PαS) cells and mouse skeletal stem cells (mSSCs) are detected within the PDGFRαposCD51pos (PαCD51) mesenchymal progenitors, which are the most abundant progenitors in early limb buds and developing long bones until birth. Long-bone-derived PαS cells and mSSCs are most prevalent in newborn mice, and molecular analysis shows that they constitute distinct progenitor populations from the earliest stages onward. Differential expression of CD90 and CD73 identifies four PαS subpopulations that display distinct chondro- and osteogenic differentiation potentials. Finally, we show that cartilage constructs generated from CD90pos PαS cells are remodeled into bone organoids encompassing functional endothelial and hematopoietic compartments, which makes these cells suited for bone tissue engineering.