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Carl Denef - One of the best experts on this subject based on the ideXlab platform.
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Melanocortin peptides stimulate prolactin gene expression and prolactin accumulation in rat pituitary Aggregate Cell cultures.
Journal of Neuroendocrinology, 2004Co-Authors: Lies Langouche, Nicole Hersmus, Anna Papageorgiou, Hugo Vankelecom, Carl DenefAbstract:Treatment for 40 h of reAggregate pituitary Cell cultures from 14-day-old female rats with nanomolar concentrations of gamma3-melanocyte-stimulating hormone (MSH) increased prolactin mRNA but not growth hormone (GH) mRNA expression levels as measured by quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). During the 40 h incubation, gamma3-MSH stimulated prolactin accumulation in the culture medium. alpha-MSH, a potent agonist of the rat melanocortin-3 receptor (MC3R) and Ala(8)-gamma2-MSH, a very weak agonist of the MC3R, increased prolactin mRNA expression at a similar concentration range as gamma3-MSH. The effect of gamma3-MSH on prolactin mRNA expression was abolished when Aggregates were cultured in the presence of thyroid or glucocorticoid hormones, but not of oestradiol. By contrast, oestradiol abolished the stimulatory effect of Ala(8)-gamma2-MSH on prolactin mRNA expression. In GH3 Cells stably transfected with the enhanced green fluorescent protein (eGFP) gene under control of a 3-kb prolactin promoter fragment, a dose as low as 1 nMgamma3-MSH, added for 24 h, significantly increased eGFP fluorescence. Agouti-related protein (AgRP(83-132)), a known endogenous MC3R and MC4R antagonist, did not reduce the stimulation of prolactin mRNA expression by gamma3-MSH or Ala(8)-gamma2-MSH. On its own, AgRP(83-132) significantly increased prolactin mRNA expression level and prolactin accumulation. Both gamma2-MSH and Ala(8)-gamma2-MSH increased [S(35)]GTPgammaS binding in membrane preparations of 14-day-old rat pituitaries and of GH3 Cells. Whereas MC3R and MC5R mRNA were detectable by RT-PCR in normal pituitary, these receptor mRNAs were undetectable in GH3 Cells using various oligonucleotide primer sets. The present findings indicate that melanocortin peptides stimulate prolactin gene expression and production and that, at least in part, a receptor different from the classic MCR is involved. AgRP appears to have other actions than its known antagonistic activity on the MC3R and MC4R.
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Effect of POMC(1-76), its C-terminal fragment gamma3-MSH and anti-POMC(1-76) antibodies on DNA replication in lactotrophs in Aggregate Cell cultures of immature rat pituitary.
Journal of Neuroendocrinology, 2003Co-Authors: Diane Tilemans, Dirk Ramaekers, Maria Andries, Carl DenefAbstract:Treatment of Aggregate Cell cultures of 14-day-old rat pituitary for 40 h with purified human (h) POMC(1-76) dose-dependently augmented the number of DNA replicating lactotrophs as estimated by autoradiography of [3H]-thymidine (3H-T) incorporation in Cells immunostained for prolactin (PRL). No such effect was seen on the total number of 3H-T labelled Cells (the majority of which did not contain any pituitary hormone in a detectable amount) or on the total number of lactotrophs. The effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs was blocked by concomitant treatment with anti-hPOMC(1-76) monoclonal and polyclonal antibodies cross-reactive with rat POMC(1-74). The latter anti-hPOMC(1-76) antibodies also decreased the number of 3H-T incorporating lactotrophs in the absence of hPOMC(1-76). Gamma3-MSH, which is the C-terminal domain of hPOMC(1-76), mimicked the effect of hPOMC(1-76) on 3H-T incorporation in lactotrophs but its potency was lower than that of hPOMC(1-76). Other melanocortin (MC) peptides such as alpha- and beta-MSH were also effective but were less potent than gamma3-MSH. The difference in potency was not due to partial degradation of the peptides. hPOMC(1-76) did not affect 3H-T incorporation in other pituitary Cell types. In contrast gamma3-MSH also augmented the number of 3H-T labelled somatotrophs and thyrotrophs. In the embryonic kidney 293 Cell line stably transfected with the MC-3 receptor, gamma3-MSH (10 nM) augmented cAMP formation up to 30 times. In contrast, hPOMC(1-76) (100 nM) was inactive in this test system, indicating this peptide is not an agonist at the MC-3 receptor. The present investigation further supports the role of rat POMC(1-74) as a paracrine growth factor in the development of lactotrophs. The active core of POMC(1-76) does not seem to be restricted to its C-terminal domain gamma3-MSH as the latter peptide displays a growth promoting effect that is different from that of POMC(1-76): it is less potent, it is not specific for lactotrophs and whereas the effect of gamma3-MSH may be mediated by the MC-3 receptor that of POMC(1-76) is not.
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In vitro immunoneutralization of a cleaved prolactin variant: evidence for a local paracrine action of cleaved prolactin in the development of gonadotrophs and thyrotrophs in rat pituitary.
Journal of Neuroendocrinology, 1996Co-Authors: Maria Andries, Diane Tilemans, Greta F. M. Jacobs, Carl DenefAbstract:We have previously isolated a cleaved prolactin variant, secreted by rat pituitary Cells in culture, that stimulated [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs when added to pituitary Aggregate Cell cultures of 14-day-old female rats. Using synthetic peptides homologous to the new C- and N-termini of the cleavage site, we made antisera recognizing this cleaved variant without significant cross reaction with native prolactin. Addition of these antisera to pituitary Aggregate Cell cultures decreased [3H]thymidine incorporation into DNA in gonadotrophs and thyrotrophs but not in the other pituitary Cell types. These data are further evidence that this prolactin variant, cleaved between Tyr-145 and Leu-146, may have an important role as growth regulator of the gonadotrophs and thyrotrophs in the rat pituitary.
Rainer Krull - One of the best experts on this subject based on the ideXlab platform.
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Viability characterization of Taxus chinensis plant Cell suspension cultures by rapid colorimetric- and image analysis-based techniques
Bioprocess and Biosystems Engineering, 2014Co-Authors: Thomas Wucherpfennig, Annika Schulz, Jaime Arturo Pimentel, Gabriel Corkidi, Dominik Sieblitz, Matthias Pump, Gilbert Gorr, Kai Schütte, Christoph Wittmann, Rainer KrullAbstract:For the commercially established process of paclitaxel production with Taxus chinensis plant Cell culture, the size of plant Cell Aggregates and phenotypic changes in coloration during cultivation have long been acknowledged as intangible parameters. So far, the variability of Aggregates and coloration of Cells are challenging parameters for any viability assay. The aim of this study was to investigate simple and non-toxic methods for viability determination of Taxus cultures in order to provide a practicable, rapid, robust and reproducible way to sample large amounts of material. A further goal was to examine whether Taxus Aggregate Cell coloration is related to general Cell viability and might be exploited by microscopy and image analysis to gain easy access to general Cell viability. The Alamar Blue assay was found to be exceptionally eligible for viability estimation. Moreover, Aggregate coloration, as a morphologic attribute, was quantified by image analysis and found to be a good and traceable indicator of T. chinensis viability.
Sergei Zuyev - One of the best experts on this subject based on the ideXlab platform.
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Aggregate and fractal tessellations
Probability Theory and Related Fields, 2001Co-Authors: Konstantin Tchoumatchenko, Sergei ZuyevAbstract:Consider a sequence of stationary tessellations {‹n}, n=0,1,..., of  d consisting of Cells {Cn(xin)}with the nuclei {xin}. An Aggregate Cell of level one, C01(xi0), is the result of merging the Cells of ‹1 whose nuclei lie in C0(xi0). An Aggregate tessellation ‹0n consists of the Aggregate Cells of level n, C0n(xi0), defined recursively by merging those Cells of ‹n whose nuclei lie in Cnm1(xi0). We find an expression for the probability for a point to belong to atypical Aggregate Cell, and obtain bounds for the rate of itsexpansion. We give necessary conditions for the limittessellation to exist as nMX and provide upperbounds for the Hausdorff dimension of its fractal boundary and forthe spherical contact distribution function in the case ofPoisson-Voronoi tessellations {‹n}.
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Aggregate and Fractal Tessellations
1999Co-Authors: Konstantin Tchoumatchenko, Sergei ZuyevAbstract:Consider a sequence of stationary tessellations {Theta^n, n=0,1,...} of R^d consisting of Cells {C^n(x_i^n)} with the nuclei {x_i^n}. An Aggregate Cell of level one, C_0^1(x_i^0), is the result of merging the Cells of Theta^1 whose nuclei lie in C^0(x_i^0). An Aggregate tessellation Theta_0^n consists of the Aggregate Cells of level n, C_0^n(x_i^0), defined recursively by merging those Cells of Theta^n whose nuclei lie in C_0^n-1(x_i^0). We find an expression for the probability for a point to belong to a typical Aggregate Cell and obtain bounds for the probability of Cell's expansion and extinction. We give necessary conditions for the limit tessellation to exist as n to infinity and provide upper bounds for the Hausdorff dimension of its fractal boundary and for the spherical contact distribution function in the case of Poisson-Voronoi tessellations {Theta^n}.
Shastri V. Prasad - One of the best experts on this subject based on the ideXlab platform.
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Cell number in mesenchymal stem Cell Aggregates dictates Cell stiffness and chondrogenesis
BioMed Central, 2019Co-Authors: Sarem Melika, Otto Oliver, Tanaka Simon, Shastri V. PrasadAbstract:Background Although mesenchymal stem/stromal Cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the Cell agglomerate, we inquired if the initial number of Cells forming the Aggregate (Aggregate Cell number (ACN)) can impact chondrogenesis. Methods Aggregates of varying ACN were formed using well-established centrifugation approach. Progression of chondrogenic differentiation in the Aggregates was assessed over 3 weeks in presence and absence of transforming growth factor-beta 1 (TGF-β1). Mechanical properties of the Cells were characterized using high-throughput real-time deformability cytometry (RT-DC), and gene expression was analyzed using Affymetrix gene array. Expression of molecular markers linked to chondrogenesis was assessed using western blot and immunofluorescence. Results Reducing ACN from 500 k to 70 k lead to activation and acceleration of the chondrogenic differentiation, independent of soluble chondro-inductive factors, which involves changes to β-catenin-dependent TCF/LEF transcriptional activity and expression of anti-apoptotic protein survivin. RT-DC analysis revealed that stiffness and size of Cells within Aggregates are modulated by ACN. A direct correlation between progression of chondrogenesis and emergence of stiffer Cell phenotype was found. Affymetrix gene array analysis revealed a downregulation of genes associated with lipid synthesis and regulation, which could account for observed changes in Cell stiffness. Immunofluorescence and western blot analysis revealed that increasing ACN upregulates the expression of lipid raft protein caveolin-1, a β-catenin binding partner, and downregulates the expression of N-cadherin. As a demonstration of the relevance of these findings in MSC-based strategies for skeletal repair, it is shown that implanting Aggregates within collagenous matrix not only decreases the necessity for high Cell numbers but also leads to marked improvement in the quality of the deposited tissue. Conclusions This study presents a simple and donor-independent strategy to enhance the efficiency of MSC chondrogenic differentiation and identifies changes in Cell mechanics coincident with MSC chondrogenesis with potential translational applications
V. Prasad Shastri - One of the best experts on this subject based on the ideXlab platform.
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Cell number in mesenchymal stem Cell Aggregates dictates Cell stiffness and chondrogenesis
Stem Cell Research & Therapy, 2019Co-Authors: Melika Sarem, Oliver Otto, Simon Tanaka, V. Prasad ShastriAbstract:Background Although mesenchymal stem/stromal Cell (MSC) chondrogenic differentiation has been thoroughly investigated, the rudiments for enhancing chondrogenesis have remained largely dependent on external cues. Focus to date has been on extrinsic variables such as soluble signals, culture conditions (bioreactors), and mechanical stimulation. However, the role of intrinsic mechanisms of MSC programming-based mechanobiology remains to be explored. Since aggregation of MSCs, a prerequisite for chondrogenesis, generates tension within the Cell agglomerate, we inquired if the initial number of Cells forming the Aggregate (Aggregate Cell number (ACN)) can impact chondrogenesis.
