Quantitative fluorescence studies of the effects of catecholamines and hydrocortisone on endogenous amine levels in neurones and small intensely fluorescent cells of embryonic chick sympathetic ganglia in vivo and in vitro

1976 ◽  
Vol 175 (3) ◽  
Author(s):  
R.M. Santer ◽  
R. Presley ◽  
J.D. Lever ◽  
K.-S. Lu
Keyword(s):  
Sympathetic Ganglia ◽  
Embryonic Chick ◽  
Fluorescence Studies ◽  
Fluorescent Cells ◽  
Quantitative Fluorescence ◽  
Small Intensely Fluorescent Cells

scholarly journals ATP:Mg2+ shapes condensate properties of rRNA-NPM1 in vitro nucleolus model and its partitioning of ribosomes

2021 ◽  
Author(s):  
N. Amy Yewdall ◽  
Alain A. M. André ◽  
Merlijn H. I. van Haren ◽  
Frank H. T. Nelissen ◽  
Aafke Jonker ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Enzymatic Reaction ◽  
Atp Depletion ◽  
Gel Point ◽  
Dependent Manner ◽  
Biophysical Properties ◽  
Temperature Dependent ◽  
Quantitative Fluorescence

Nucleoli have viscoelastic gel-like condensate dynamics that are not well represented in vitro. Nucleoli models, such as those formed by nucleophosmin 1 (NPM1) and ribosomal RNA (rRNA), exhibit condensate dynamics orders of magnitude faster than in vivo nucleoli. Here we show that an interplay between magnesium ions (Mg2+) and ATP governs rRNA dynamics, and this ultimately shapes the physical state of these condensates. Using quantitative fluorescence microscopy, we demonstrate that increased RNA compaction occurs in the condensates at high Mg2+ concentrations, contributing to the slowed RNA dynamics. At Mg2+ concentrations above 7 mM, rRNA is fully arrested and the condensates are gels. Below the critical gel point, NPM1-rRNA droplets age in a temperature-dependent manner, suggesting that condensates are viscoelastic materials, undergoing maturation driven by weak multivalent interactions. ATP addition reverses the dynamic arrest of rRNA, resulting in liquefaction of these gel-like structures. Surprisingly, ATP and Mg2+ both act to increase partitioning of NPM1-proteins as well as rRNA, which influences the partitioning of small client molecules. By contrast, larger ribosomes form a halo around NPM1-rRNA coacervates when Mg2+ concentrations are higher than ATP concentrations. Within cells, ATP levels fluctuate due to biomolecular reactions, and we demonstrate that a dissipative enzymatic reaction can control the biophysical properties of in vitro condensates through depletion of ATP. This enzymatic ATP depletion also reverses the formation of the ribosome halos. Our results illustrate how cells, by changing local ATP concentrations, may regulate the state and client partitioning of RNA-containing condensates such as the nucleolus.

Small, intensely fluorescent cells of human sympathetic ganglia

1979 ◽  
Vol 12 (1) ◽  
pp. 97-101 ◽  
Author(s):  
Antti Hervonen ◽  
Hannu Alho ◽  
Pauli Helen ◽  
Lasse Kanerva
Keyword(s):  
Sympathetic Ganglia ◽  
Fluorescent Cells ◽  
Small Intensely Fluorescent Cells

scholarly journals THE FINE STRUCTURE OF EMBRYONIC CHICK SKELETAL MUSCLE CELLS DIFFERENTIATED IN VITRO

1967 ◽  
Vol 35 (2) ◽  
pp. 445-453 ◽  
Author(s):  
Y. Shimada ◽  
D. A. Fischman ◽  
A. A. Moscona
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Fine Structure ◽  
Muscle Cells ◽  
Skeletal Muscle Cells ◽  
Chick Embryos ◽  
Embryonic Chick ◽  
Developing Muscle

Dissociated myoblasts from 12-day chick embryos were cultured in monolayer, and the differentiation of skeletal muscle cells was studied by electron microscopy. The results have revealed a striking ultrastructural similarity between the in vivo and the in vitro developing muscle, particularly with respect to the myofibrils and sarcoplasmic reticulum. This study demonstrates that all the characteristic organelles of mature skeletal muscle can develop in vitro in the absence of nerves.

scholarly journals Study on sex-organ development. Oestrogen-receptor translocation in the developing chick Müllerian duct

1976 ◽  
Vol 154 (1) ◽  
pp. 1-9 ◽  
Author(s):  
C S Teng ◽  
C T Teng
Keyword(s):  
Binding Sites ◽  
Developmental Stages ◽  
Dissociation Constants ◽  
Duct Cell ◽  
Mullerian Duct ◽  
Embryonic Chick ◽  
Müllerian Duct ◽  
Initial Content

After oestradiol administration in vivo, 87-95% of the initial concentration of oestradiol receptor in the cytoplasm of the embryonic-chick Müllerian-duct cell was translocated into the nucleus. The process of translocation depends on the amount of oestardiol administered in vivo. At 6 h after oestradiol administration in vivo, about 30% replenishment of the initial content of the cytosol receptor was observed in the cytoplasm. The Müllerian-duct nuclei, after exposure to non-radioactive oestradiol, exhibit saturable exchange with [3H]oestradiol in vitro. The exchange of oestradiol is temperature- and time-dependent. The optimal temperature and time for exchange are 37-41 degrees C and 2h respectively. The [3H]oestradiol-receptor complex extracted from the exchanged nuclei is present in 5-6S form, and its isoelectric point is 6.8. The number of nuclear oestradiol-binding sites of the developing Müllerian duct are 1.66, 2.22, 2.63, and 2.50 pmol/mg of DNA respectively for embryos of 10, 12, 15 and 18 days. The dissociation constants of the nuclear oestradiol receptor of the four observed developmental stages range from 3.0 to 3.1 nM.

scholarly journals Cerebrovascular Responses to Insulin in Rats

2009 ◽  
Vol 29 (12) ◽  
pp. 1955-1967 ◽  
Author(s):  
Prasad Venkateswera Gurunath Katakam ◽  
Ferenc Domoki ◽  
Laura Lenti ◽  
Tamás Gáspár ◽  
Adam Institoris ◽  
...  
Keyword(s):  
Insulin Treatment ◽  
Cerebral Arteries ◽  
Receptor Expression ◽  
Type B ◽  
Fluorescence Studies ◽  
Akt Phosphorylation ◽  
Endothelial Denudation ◽  
Insulin Receptor Expression

Effects of insulin on cerebral arteries have never been examined. Therefore, we determined cerebrovascular actions of insulin in rats. Both PCR and immunoblot studies identified insulin receptor expression in cerebral arteries and in cultured cerebral microvascular endothelial cells (CMVECs). Diameter measurements (% change) of isolated rat cerebral arteries showed a biphasic dose response to insulin with an initial vasoconstriction at 0.1 ng/mL (−9.7%±1.6%), followed by vasodilation at 1 to 100 ng/mL (31.9%±1.4%). Insulin also increased cortical blood flow in vivo (30%±8% at 120 ng/mL) when applied topically. Removal of reactive oxygen species (ROS) abolished the vasoconstriction to insulin. Endothelial denudation, inhibition of K+ channels, and nitric oxide (NO) synthase, all diminished insulin-induced vasodilation. Inhibition of cytochrome P450 enhanced vasodilation in endothelium-intact arteries, but promoted vasoconstriction after endothelial denudation. Inhibition of cyclooxygenase abolished vasoconstriction and enhanced vasodilation to insulin in all arteries. Inhibition of endothelin type A receptors enhanced vasodilation, whereas endothelin type B receptor blockade diminished vasodilation. Insulin treatment in vitro increased Akt phosphorylation in cerebral arteries and CMVECs. Fluorescence studies of CMVECs showed that insulin increased intracellular calcium and enhanced the generation of NO and ROS. Thus, cerebrovascular responses to insulin were mediated by complex mechanisms originating in both the endothelium and smooth muscle.

Multiple mechanisms regulate sympathetic neuronal phenotype

Development ◽  
1995 ◽  
Vol 121 (8) ◽  
pp. 2361-2371
Author(s):  
A.K. Hall ◽  
S.E. MacPhedran
Keyword(s):  
Sympathetic Neurons ◽  
Intrinsic Property ◽  
Sympathetic Ganglia ◽  
Environmental Cues ◽  
Peripheral Target ◽  
Neuronal Phenotype ◽  
Adult Rat ◽  
Cervical Ganglion

Adult rat sympathetic neurons can possess specific neuropeptides utilized as cotransmitters along with norepinephrine, but the factors that regulate their expression remain unknown. 60% of adult rat superior cervical ganglion (SCG) neurons express neuropeptide Y (NPY) in vivo. To determine whether the restricted expression was an intrinsic property of sympathetic ganglia, we examined if embryonic sympathetic precursors gave rise to NPY immunoreactive (-IR) neurons in vitro. After one week in culture, 60% of neurons derived from the E14.5 rat SCG were NPY-IR. Thus, ganglia isolated before peripheral target contact or preganglionic innervation were capable of regulating NPY expression both in the number of neurons with NPY and in the developmental timing of NPY expression. To determine if the restricted expression of NPY was a reflection of neuroblasts committed to an NPY fate, SCG precursors were labeled with a replication incompetent retrovirus carrying lacZ, and NPY expression in lacZ-labeled clones examined after one week. Two thirds of neuronal clones obtained were uniformly NPY-IR; that is, all neurons in a clone either possessed or lacked NPY. One-third of the neuronal clones were mixed and contained both neurons with and without NPY. We provide a novel demonstration that both lineage and environmental cues contribute to neuropeptide phenotype.

Immunology of nerve growth factor (NGF). The effect of NGF-antiserum on sensory ganglia in vitro

Development ◽  
1970 ◽  
Vol 23 (2) ◽  
pp. 273-287
Author(s):  
H. Hoffman
Keyword(s):  
Molecular Weight ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Sympathetic Ganglion ◽  
Sympathetic Ganglia ◽  
Sensory Ganglia ◽  
Nerve Growth ◽  
Adult Organism

The properties of the ‘Nerve Growth Factor’ (NGF) have been described extensively (Levi-Montalcini & Booker, 1960; Levi-Montalcini, 1965) and reviewed recently (Levi-Montalcini, 1966). This factor is a protein of molecular weight about 130000 in its aggregated form (Varon, Nomura & Shooter, 1967, 1968) but may be active in lower molecular weight forms (Cohen, 1959, 1960; Banks et al. 1968). It is widely distributed in the adult organism (Bueker, Schenkein & Bane, 1960) and exerts a controlling influence on the differentiation of sensory and sympathetic ganglia in developing chick embryos. In newborn mammals its administration influences sympathetic ganglion growth only. A possible role in the adult nervous system is suggested by Scott, Gutmann & Horsky (1966), who showed that injected NGF will increase protein synthesis in regenerating sensory neurons in vivo. Active proteins in complex biological systems may be removed in a highly selective fashion by specific antibodies which thus provide a valuable means of studying their action.

In Vitro and In Vivo Mesenchymal Stem Cells Neurotrophin Production but Limited Neuronal Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1687-1687
Author(s):  
Patrizia Bossolasco ◽  
Davide Soligo ◽  
Yvan Torrente ◽  
Federica Pisati ◽  
Mirella Meregalli ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Differentiation ◽  
Human Mscs ◽  
Post Injection ◽  
Rt Pcr ◽  
Differentiation Potential ◽  
Fluorescent Cells ◽  
The Brain

Abstract The aim of the present work was to explore mesenchymal stem cells (MSCs) differentiation potential towards neural phenotype. MSCs are self-renewable multipotent cells shown to be able to support hematopoiesis and to improve functional outcomes in animal models of neurological disorders. MSCs were obtained by plastic adherence from iliac crest bone marrow of healthy donors for allogeneic transplantation and, for the in vitro studies, were cultured on laminin-coated dishes in a B27 Neurobasal medium with 3% to 10% FBS for 3 weeks. Few cell (11%) showing bipolar morphologies, expressed β-tubulin III and GFAP. Furthermore, only GAP43 expression was detected by RT-PCR. Addition of exogenous neurotrophins to cultures did not improve neural differentiation. To investigate the brain microenvironment effect on MSCs, cells were cultured on brain sections and supernatant of the cultures analyzed by ELISA. In this condition, MSCs were shown to release soluble human NT3/NT4 and NGF and to express p75 and TrkC receptors by immunocytochemistry. In order to improve these observations, we analyzed the human neurotrophin and receptor gene expression profile by GEArray technology. The expression patterns of human trkC, NT3, NT4, and NGF mRNA were consistent with the results of immunostaining. To evaluate the in vivo MSCs differentiation potential, 50.000 cells labeled with a fluorescent dye (PKH26) were injected into the right parietal cortex of newborn Balb/C and nude mice (4 and 7 days old). Seven and 45 days later, immunocytochemistry and RT-PCR were performed on brain sections using the following neural specific markers: neurofilament-M, NSE, GFAP, b-tubulin III, MAP-2ab, nestin, Gal-C, and anti TrkC, TrkA and p75NFGR. FISH analysis was also performed using both Cy-3 labeled human Pan Centromeric and FITC labeled mouse Pan Centromeric probes. In 7 out of 52 Balb/C mice analyzed, fluorescent cells were detected 30 days post-injection but only one mouse showed NF and MAP-2ab expression by immunocytochemistry on FISH positive cells thirty days after transplantation. These data were confirmed by RT-PCR for the presence of human GAPDH. In nude mice, fluorescent cells were also detected away from the site of injection indicating cells migration throughout the brain. Moreover, 7 and 45 days post-injection, a high percentage of cells was shown to express the TrkC and p75 receptors. Isolation of the single human MSCs transplanted cells from brain sections was performed by laser microdissection analysis. ELISA analysis from these dissected areas showed the expression of human NT3/NT4 and NGF neurotrophin’s. Finally, several transplanted human MSCs expressing the Ve-cadherin were found close to blood vessels after 45 days of transplantation, whereas these cells were negative for human KDR and CD45. In addition, we determined the capability of conditioned MSCs media to regulate the angiogenesis in a tube formation assay. In conclusion, our data show an in vitro and in vivo capacity of MSCs to express neurotrophins under epigenetic stimuli rather than a real neural differentiation potential.

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