Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture

Mates, G.; Daniel, M.R.; Walker, Carol

doi:10.1016/0309-1651(86)90141-4

Cited by 18 publications

(6 citation statements)

References 11 publications

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“…DNA was determined by using a modified (37) fluorimetric procedure (38). Briefly, methanol was evaporated under N 2 , and the dried material was resuspended in 200 μl of assay buffer (10 mM EDTA, 10 mM Tris pH 7.4, 10 mM NaCl) by sonication.…”

Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

et al. 2002

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Glucosylceramide (GlcCer) accumulates in the inherited metabolic disorder, Gaucher disease, because of the defective activity of lysosomal glucocerebrosidase. We previously demonstrated that upon GlcCer accumulation, cultured hippocampal neurons exhibit modified growth patterns, altered endoplasmic reticulum density, and altered calcium release from intracellular stores. We here examined the relationship between GlcCer accumulation and phospholipid synthesis. After treatment of neurons with an active site-directed inhibitor of glucocerebrosidase, or in neurons obtained from a mouse model of Gaucher disease, [14C]methyl choline incorporation into [14C]phosphatidylcholine ([14C]PC) and [14C]sphingomyelin was elevated, as were [14C]CDP-choline levels, suggesting that CTP:phosphocholine cytidylyltransferase (CCT) is activated. Indeed, CCT activity was elevated in neurons that had accumulated GlcCer. GlcCer, but not galactosylceramide (GalCer), stimulated CCT activity in rat brain homogenates, and significantly higher levels of CCT were membrane associated in cortical homogenates from a mouse model of Gaucher disease compared with wild-type mice. Because CCT mRNA and protein levels were unaltered in either neurons or brain tissue that had accumulated GlcCer, it appeared likely that GlcCer activates CCT by a post-translational mechanism. This was verified by examination of the effect of GlcCer on CCT purified about 1200-fold from rat brain. GlcCer stimulated CCT activity, with stimulation observed at levels as low as 2.5 mol% and with maximal activation reached at 10 mol%. In contrast, GalCer had no effect. Together, these data demonstrate that GlcCer directly activates CCT, which results in elevated PC synthesis, which may account for some of the changes in growth rates observed upon neuronal GlcCer accumulation.

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Section: Methodsmentioning

confidence: 99%

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

et al. 2002

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“…DNA quantification was performed using a modified version of the method of Mates et al [22], as previously described [7].…”

Section: Dna Quantificationmentioning

confidence: 99%

Interaction of Prostaglandin F<sub>2α</sub> and Prostaglandin-E<sub>2</sub> on Progesterone Production in Human Granulosa-Luteal Cells

Väänänen¹,

Tong²,

Väänänen³

et al. 2001

Neurosignals

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This study examined the effects of prostaglandin-F_2α (PGF_2α), prostaglandin-E₂ (PGE₂) and their interactions on progesterone production in human granulosa-luteal cells (GLCs). Human GLCs collected from in vitro fertilization patients were cultured for 1 (D₁) or 8 days (D₈), followed by a 24-hour treatment period, after which media were collected and radioimmunoassayed for progesterone. Seven-point PGF_2α and PGE₂ concentration-response curves were crossed into a matrix of 49 separate treatments. Responses were plotted in three dimensions and as two-dimensional ‘slices’. In D₁ cultured human GLCs neither PGF_2α nor PGE₂ alone had any effect on progesterone production, however two different combinations of these hormones led to at least a 3-fold increase in progesterone production. This stimulation was seen when cells were treated with 10^–6M PGF_2α plus 10^–9M PGE₂, and when they were treated with 10^–10M PGF_2α plus 10^–9M PGE₂. In D₈ GLCs, PGF_2α stimulated progesterone production maximally at 10^–9M, while the lowest (10^–11 M) and highest concentrations (10^–6 M) tested were ineffectual. On the contrary, in the presence of high concentrations of PGE₂ (10^–6 to 10^–7 M), PGF_2α-mediated stimulation of progesterone production was attenuated. In a similar fashion to PGF_2α, PGE₂ also acted in a luteotrophic manner, although the maximal stimulation of progesterone production was seen at a higher concentration (10^–8 to 10^–7 M). Likewise, PGE₂-mediated progesterone production was attenuated by the presence of high concentrations of PGF_2α (10^–6 to 10^–7 M). In conclusion, in D₁ human GLCs neither PGF_2α nor PGE₂ alone were luteotrophic, although specific combinations of these hormones were. Conversely, in D₈ GLCs both PGF_2α and PGE₂ stimulated progesterone production in a biphasic manner, while the presence of a high concentration of either of these prostaglandins attenuated the luteotrophic effects of the other. Therefore, PGF_2α and PGE₂ interacted in a concentration-dependent manner, resulting in a multimodal progesterone response, which was easily visualized using three-dimensional plots.

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“…DNA was quantified using a modified version of the method of Mates et al [38]. Briefly, after the treatment period, media were removed and replaced with DNase-free trypsin (50 jig/ml; Sigma), in a final volume of 500 il in PBS.…”

Section: Dna Quantificationmentioning

confidence: 99%

Interaction of prostaglandin F2alpha and gonadotropin-releasing hormone on progesterone and estradiol production in human granulosa-luteal cells

Väänänen

Tong

Väänänen

et al. 1997

Biology of Reproduction

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This study examined the effects of prostaglandin-F2alpha (PGF2alpha), GnRH, and their interactions on steroidogenesis in human granulosa-luteal cells (GLCs). Human GLCs collected from in vitro fertilization patients were cultured for one or eight days, followed by a 24-h treatment period, after which media were collected and radioimmunoassayed for progesterone and estradiol. In the first experiment, GLCs were treated with vehicle, PGF2alpha (10(-9) M), GnRH (10(-6) M), or PGF2alpha plus GnRH, with or without hCG (1 IU/ml). Neither PGF2alpha nor GnRH alone had a significant effect; however, the combination of PGF2alpha plus GnRH significantly stimulated steroidogenesis. Similarly, co-application enhanced the luteolytic effects of PGF2alpha. In a second experiment, PGF2alpha and GnRH concentration-response curves were crossed into a matrix of 49 separate treatments. Responses were plotted in three-dimensions and as two-dimensional "slices" that were analyzed statistically. In the presence of high concentrations of GnRH (10(-6) M), PGF2alpha stimulated progesterone production in a biphasic manner, as middle concentrations significantly stimulated (10(-9) M) whereas low and high concentrations did not. In the presence of middle concentrations of PGF2alpha (10(-9) M), GnRH significantly stimulated progesterone production in a linear concentration-dependent manner. Similar complexities were seen with respect to estradiol response. Thus, in the human GLC, GnRH potentiates the luteolytic effects of PGF2alpha, while it acts as a permissive factor for the luteotropic effects. Furthermore, we have revealed the complex interaction of these hormones using a three-dimensional experimental design.

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Factors affecting the reproducibility of a spectrofluorimetric assay for the enumeration of human venous endothelium in culture

Cited by 18 publications

References 11 publications

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

Phosphatidylcholine synthesis is elevated in neuronal models of Gaucher disease due to direct activation of CTP:phosphocholine cytidylyltransferase by glucosylceramide

Interaction of Prostaglandin F<sub>2α</sub> and Prostaglandin-E<sub>2</sub> on Progesterone Production in Human Granulosa-Luteal Cells

Interaction of prostaglandin F2alpha and gonadotropin-releasing hormone on progesterone and estradiol production in human granulosa-luteal cells

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