Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-γ1 catalytic activity

Buckley, Colin T

doi:10.1016/s0003-2697(03)00394-4

Cited by 9 publications

(5 citation statements)

References 12 publications

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“…Assessment of protein levels was performed using previously established immunoblotting protocols ( Tyler et al, 2015a ). Frontal cortex tissue lysates from male and female offspring exposed to arsenic (or control) and treated with valproate (or saline) were prepared from tissue collected within 15–17 h of the afternoon session following obtaining reversal criteria; the nuclear fraction was isolated for protein analysis ( Buckley and Caldwell, 2003 ; Caldwell et al, 2015 ). Antibody concentrations and total protein quantity (15 μg) were optimized to fit the linear range of signal detection.…”

Section: Methodsmentioning

confidence: 99%

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure

et al. 2018

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Several studies have demonstrated that exposure to arsenic in drinking water adversely affects brain development and cognitive function in adulthood. While the mechanism by which arsenic induces adverse neurological outcomes remains elusive, studies suggest a link between reduced levels of histone acetylation and impaired performance on a variety of behavioral tasks following arsenic exposure. Using our developmental arsenic exposure (DAE) paradigm, we have previously reported reduced histone acetylation and associated histone acetyltransferase enzyme expression in the frontal cortex of C57BL/6J adult male mice, with no changes observed in the female frontal cortex. In the present study, we sought to determine if DAE produced sex-dependent deficits in frontal cortical executive function using the Y-maze acquisition and reversal learning tasks, which are specific for assessing cognitive flexibility. Further, we tested whether the administration of valproic acid, a class I–IIa histone deacetylase inhibitor, was able to mitigate behavioral and biochemical changes resulting from DAE. As anticipated, DAE inhibited acquisition and reversal learning performance in adult male, but not female, mice. Valproate treatment for 2 weeks restored reversal performance in the male arsenic-exposed offspring, while not affecting female performance. Protein levels of HDACs 1, 2, and 5 were elevated following behavioral assessment but only in DAE male mice; restoration of appropriate HDAC levels occurred after valproate treatment and was concurrent with improved behavioral performance, particularly during reversal learning. Female frontal cortical levels of HDAC enzymes were not impacted by DAE or valproate treatment. Finally, mRNA expression levels of brain-derived neurotrophic factor, Bdnf, which has been implicated in the control of frontal cortical flexibility and is regulated by HDAC5, were elevated in DAE male mice and restored to normal levels following HDACi treatment. Levels of mRNA encoding glutamate receptor ionotropic NMDA type subunits, which have been linked to cognitive flexibility, were not related to the reversal learning deficit in the DAE mice and were not altered by HDACi treatments. These findings demonstrate that DAE alters frontal cortical HDAC levels and Bdnf expression in males, but not females, and that these molecular changes are associated with sex-dependent differences in cognitive flexibility in a reversal-learning task.

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Section: Methodsmentioning

confidence: 99%

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure

et al. 2018

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“…Fetal telencephalons were homogenized according to previously established protocols [7]. Individual telencephalons were homogenized in a Kontes RNase-Free Pellet Pestle Grinder (cat: KT749520-0090, VWR) in 100 μL ice cold homogenization buffer (HB) (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 320 mM sucrose, 1:1000 protease inhibitor cocktail (Sigma P8340), 200 μM sodium orthovanadate).…”

Section: Methodsmentioning

confidence: 99%

Arsenic exposure during embryonic development alters the expression of the long noncoding RNA growth arrest specific-5 (Gas5) in a sex-dependent manner

Hafez¹,

Solomon²,

Cunningham³

et al. 2018

Neurotoxicology and Teratology

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Our previous studies suggest that prenatal arsenic exposure (50ppb) modifies epigenetic control of the programming of the glucocorticoid receptor (GR) signaling system in the developing mouse brain. These deficits may lead to long-lasting consequences, including deficits in learning and memory, increased depressive-like behaviors, and an altered set-point of GR feedback throughout life. To understand the arsenic-induced changes within the GR system, we assessed the impact of in utero arsenic exposure on the levels of the GR and growth arrest-specific-5 (Gas5), a noncoding RNA, across a key gestational period for GR programming (gestational days, GD 14-18) in mice. Gas5 contains a glucocorticoid response element (GRE)-like sequence that binds the GR, thereby decreasing GR-GRE-dependent gene transcription and potentially altering GR programming. Prenatal arsenic exposure resulted in sex-dependent and age-dependent shifts in the levels of GR and Gas5 expression in fetal telencephalon. Nuclear GR levels were reduced in males, but unchanged in females, at all gestational time points tested. Total cellular Gas5 levels were lower in arsenic-exposed males with no changes seen in arsenic-exposed females at GD16 and 18. An increase in total cellular Gas-5 along with increased nuclear levels in GD14 arsenic-exposed females, suggests a differential regulation of cellular compartmentalization of Gas5. RIP assays revealed reduced Gas5 associated with the GR on GD14 in the nuclear fraction prepared from arsenic-exposed males and females. This decrease in levels of GR-Gas5 binding continued only in the females at GD18. Thus, nuclear GR signaling potential is decreased in prenatal arsenic-exposed males, while it is increased or maintained at levels approaching normal in prenatal arsenic-exposed females. These findings suggest that females, but not males, exposed to arsenic are able to regulate the levels of nuclear free GR by altering Gas5 levels, thereby keeping GR nuclear signaling closer to control (unexposed) levels.

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“…Frozen (–80 °C) mouse fetal brains were homogenized following established protocols (Buckley and Caldwell, 2003) with the following modifications. Brains were homogenized in 100 μL of ice cold homogenization buffer (HB) (20 mM Tris–HCl, pH = 7.4, 1 mM EDTA, 320 mM sucrose, 1:1000 protease inhibitor cocktail (Sigma P8340), 200 μM sodium orthovanadate).…”

Section: Methodsmentioning

confidence: 99%

Prenatal arsenic exposure alters the programming of the glucocorticoid signaling system during embryonic development

Caldwell

Labrecque

Solomon

et al. 2015

Neurotoxicology and Teratology

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The glucocorticoid system, which plays a critical role in a host of cellular functions including mood disorders and learning and memory, has been reported to be disrupted by arsenic. In previous work we have developed and characterized a prenatal moderate arsenic exposure (50 ppb) model and identified several deficits in learning and memory and mood disorders, as well as alterations within the glucocorticoid receptor signaling system in the adolescent mouse. In these present studies we assessed the effects of arsenic on the glucocorticoid receptor (GR) pathway in both the placenta and the fetal brain in response at two critical periods, embryonic days 14 and 18. The focus of these studies was on the 11β-hydroxysteroid dehydrogenase enzymes (11β-HSD1 and 11β-HSD2) which play a key role in glucorticoid synthesis, as well as the expression and set point of the GR negative feedback regulation. Negative feedback regulation is established early in development. At E14 we found arsenic exposure significantly decreased expression of both protein and message in brain of GR and the 11β-HSD1, while 11β-HSD2 enzyme protein levels were increased but mRNA levels were decreased in the brain. These changes in brain protein continued into the E18 time point, but mRNA levels were no longer significantly altered. Placental HSD11B2 mRNA was not altered by arsenic treatment but protein levels were elevated at E14. GR placental protein levels were decreased at E18 in the arsenic exposed condition. This suggests that arsenic exposure may alter GR expression levels as a consequence of a prolonged developmental imbalance between 11β-HSD1 and 11β-HSD2 protein expression despite decreased 11HSDB2 mRNA. The suppression of GR and the failure to turn down 11β-HSD2 protein expression during fetal development may lead to an altered set point for GR signaling throughout adulthood. To our knowledge, these studies are the first to demonstrate that gestational exposure to moderate levels of arsenic results in altered fetal programming of the glucocorticoid system.

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Two-layer antibody capture of enzymes on the surface of microtiter plates: application to the study of the regulation of phospholipase C-γ1 catalytic activity

Cited by 9 publications

References 12 publications

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure

Sex-Dependent Effects of the Histone Deacetylase Inhibitor, Sodium Valproate, on Reversal Learning After Developmental Arsenic Exposure

Arsenic exposure during embryonic development alters the expression of the long noncoding RNA growth arrest specific-5 (Gas5) in a sex-dependent manner

Prenatal arsenic exposure alters the programming of the glucocorticoid signaling system during embryonic development

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