JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Quantification of lamotrigine in patient plasma utilizing a fast liquid chromatography-tandem mass spectrometry method with backflush technology.
Ther Drug Monit
PUBLISHED: 08-26-2014
Show Abstract
Hide Abstract
Recent concerns have been raised by neurologists and patients with epilepsy regarding the bioequivalence of generic lamotrigine to brand Lamictal. Bioequivalence studies require the quantification of lamotrigine in human plasma, including in the presence of other drugs, for studies that will employ patients with epilepsy rather than healthy volunteers.
Related JoVE Video
Retinoic acid biosynthesis is impaired in human and murine endometriosis.
Biol. Reprod.
PUBLISHED: 08-20-2014
Show Abstract
Hide Abstract
Endometriosis is characterized by the presence of endometrial glands and stroma in extrauterine sites. Our objective was to determine whether endometriotic lesions (ELs) from women with endometriosis have altered retinoid levels compared with their eutopic endometrium, and to test the hypothesis that defects in all-trans retinoic acid (ATRA) biosynthesis in EL is related to reduced expression of cellular retinol-binding protein type 1 (RBP1). Retinoids were evaluated by liquid chromatography-tandem mass spectrometry and high-performance liquid chromatography in eutopic endometrial biopsies (EBs) and ELs from 42 patients with pathologically confirmed endometriosis. The ATRA levels were reduced, whereas the retinol and retinyl ester concentrations were elevated in EL compared with EB tissue. Similar results were found in a mouse model of endometriosis that used green fluorescent protein-positive endometrial tissue injected into the peritoneum of syngeneic hosts to mimic retrograde menses. The ATRA biosynthesis in vitro in retinol-treated primary human endometrial stromal cell (ESC) cultures derived from ELs was reduced compared with that of ESCs derived from patient-matched EBs. Correspondingly, RBP1 expression was reduced in tissue and ESCs derived from EL versus EB. Rbp1(-/-) mice showed reduced endometrial ATRA concentrations compared with wild type, associated with loss of tissue organization and hypercellularity. These findings provide the first quantitative measurements of ATRA in human endometrium and endometriosis, demonstrating reduced ATRA in ectopic tissue and corresponding ESC cultures. Quantitation of retinoids in murine endometriosis and in Rbp1(-/-) mice supports the contention that impaired ATRA synthesis caused by reduced RBP1 promotes an "endometriosis phenotype" that enables cells to implant and grow at ectopic sites.
Related JoVE Video
A Mollusk Retinoic Acid Receptor (RAR) Ortholog Sheds Light on the Evolution of Ligand Binding.
Endocrinology
PUBLISHED: 08-13-2014
Show Abstract
Hide Abstract
Nuclear receptors are transcription factors that regulate networks of target genes in response to small molecules. There is a strong bias in our knowledge of these receptors because they were mainly characterized in classical model organisms, mostly vertebrates. Therefore, the evolutionary origins of specific ligand-receptor couples still remain elusive. Here we present the identification and characterization of a retinoic acid receptor (RAR) from the mollusk Nucella lapillus (NlRAR). We show that this receptor specifically binds to DNA response elements organized in direct repeats as a heterodimer with retinoid X receptor. Surprisingly, we also find that NlRAR does not bind all-trans retinoic acid or any other retinoid we tested. Furthermore, NlRAR is unable to activate the transcription of reporter genes in response to stimulation by retinoids and to recruit coactivators in the presence of these compounds. Three-dimensional modeling of the ligand-binding domain of NlRAR reveals an overall structure that is similar to vertebrate RARs. However, in the ligand-binding pocket (LBP) of the mollusk receptor, the alteration of several residues interacting with the ligand has apparently led to an overall decrease in the strength of the interaction with the ligand. Accordingly, mutations of NlRAR at key positions within the LBP generate receptors that are responsive to retinoids. Altogether our data suggest that, in mollusks, RAR has lost its affinity for all-trans retinoic acid, highlighting the evolutionary plasticity of its LBP. When put in an evolutionary context, our results reveal new structural and functional features of nuclear receptors validated by millions of years of evolution that were impossible to reveal in model organisms.
Related JoVE Video
Development and validation of a LC-MS/MS assay for quantitation of plasma citrulline for application to animal models of the acute radiation syndrome across multiple species.
Anal Bioanal Chem
PUBLISHED: 03-25-2014
Show Abstract
Hide Abstract
The potential risk of a radiological catastrophe highlights the need for identifying and validating potential biomarkers that accurately predict radiation-induced organ damage. A key target organ that is acutely sensitive to the effects of irradiation is the gastrointestinal (GI) tract, referred to as the GI acute radiation syndrome (GI-ARS). Recently, citrulline has been identified as a potential circulating biomarker for radiation-induced GI damage. Prior to biologically validating citrulline as a biomarker for radiation-induced GI injury, there is the important task of developing and validating a quantitation assay for citrulline detection within the radiation animal models used for biomarker validation. Herein, we describe the analytical development and validation of citrulline detection using a liquid chromatography tandem mass spectrometry assay that incorporates stable-label isotope internal standards. Analytical validation for specificity, linearity, lower limit of quantitation, accuracy, intra- and interday precision, extraction recovery, matrix effects, and stability was performed under sample collection and storage conditions according to the Guidance for Industry, Bioanalytical Methods Validation issued by the US Food and Drug Administration. In addition, the method was biologically validated using plasma from well-characterized mouse, minipig, and nonhuman primate GI-ARS models. The results demonstrated that circulating citrulline can be confidently quantified from plasma. Additionally, circulating citrulline displayed a time-dependent response for radiological doses covering GI-ARS across multiple species.
Related JoVE Video
BCL-xL/MCL-1 inhibition and RAR? antagonism work cooperatively in human HL60 leukemia cells.
Exp. Cell Res.
PUBLISHED: 01-24-2014
Show Abstract
Hide Abstract
The acute promyelocytic leukemia (APL) subtype of acute myeloid leukemia (AML) is characterized by chromosomal translocations that result in fusion proteins, including the promyelocytic leukemia-retinoic acid receptor, alpha fusion protein (PML-RAR?). All-trans retinoic acid (atRA) treatment is the standard drug treatment for APL yielding cure rates > 80% by activating transcription and proteasomal degradation of retinoic acid receptor, alpha (RAR?). Whereas combination therapy with As2O3 has increased survival further, patients that experience relapse and are refractory to atRA and/or As2O3 is a clinically significant problem. BCL-2 family proteins regulate apoptosis and over-expression of anti-apoptotic B-cell leukemia/lymphoma 2 (BCL-2) family proteins has been associated with chemotherapeutic resistance in APL including impairment of the ability of atRA to induce growth arrest and differentiation. Here we investigated the novel BH3 domain mimetic, JY-1-106, which antagonizes the anti-apoptotic BCL-2 family members B-cell lymphoma-extra large (BCL-xL) and myeloid cell leukemia-1 (MCL-1) alone and in combination with retinoids including atRA, AM580 (RAR? agonist), and SR11253 (RAR? antagonist). JY-1-106 reduced cell viability in HL-60 cells alone and in combination with retinoids. The combination of JY-1-106 and SR11253 had the greatest impact on cell viability by stimulating apoptosis. These studies indicate that dual BCL-xL/MCL-1 inhibitors and retinoids could work cooperatively in leukemia treatment.
Related JoVE Video
Mass spectrometry imaging enriches biomarker discovery approaches with candidate mapping.
Health Phys
PUBLISHED: 01-24-2014
Show Abstract
Hide Abstract
Integral to the characterization of radiation-induced tissue damage is the identification of unique biomarkers. Biomarker discovery is a challenging and complex endeavor requiring both sophisticated experimental design and accessible technology. The resources within the National Institute of Allergy and Infectious Diseases (NIAID)-sponsored Consortium, Medical Countermeasures Against Radiological Threats (MCART), allow for leveraging robust animal models with novel molecular imaging techniques. One such imaging technique, MALDI (matrix-assisted laser desorption ionization) mass spectrometry imaging (MSI), allows for the direct spatial visualization of lipids, proteins, small molecules, and drugs/drug metabolites-or biomarkers-in an unbiased manner. MALDI-MSI acquires mass spectra directly from an intact tissue slice in discrete locations across an x, y grid that are then rendered into a spatial distribution map composed of ion mass and intensity. The unique mass signals can be plotted to generate a spatial map of biomarkers that reflects pathology and molecular events. The crucial unanswered questions that can be addressed with MALDI-MSI include identification of biomarkers for radiation damage that reflect the response to radiation dose over time and the efficacy of therapeutic interventions. Techniques in MALDI-MSI also enable integration of biomarker identification among diverse animal models. Analysis of early, sublethally irradiated tissue injury samples from diverse mouse tissues (lung and ileum) shows membrane phospholipid signatures correlated with histological features of these unique tissues. This paper will discuss the application of MALDI-MSI for use in a larger biomarker discovery pipeline.
Related JoVE Video
Identification and quantitation of biomarkers for radiation-induced injury via mass spectrometry.
Health Phys
PUBLISHED: 01-24-2014
Show Abstract
Hide Abstract
Biomarker identification and validation for radiation exposure is a rapidly expanding field encompassing the need for well defined animal models and advanced analytical techniques. The resources within the consortium, Medical Countermeasures Against Radiological Threats (MCART), provide a unique opportunity for accessing well defined animal models that simulate the key sequelae of the acute radiation syndrome and the delayed effects of acute radiation exposure. Likewise, the use of mass spectrometry-based analytical techniques for biomarker discovery and validation enables a robust analytical platform that is amenable to a variety of sample matrices and considered the benchmark for biomolecular identification and quantitation. Herein, the authors demonstrate the use of two targeted mass spectrometry approaches to link established MCART animal models to identified metabolite biomarkers. Circulating citrulline concentration was correlated to gross histological gastrointestinal tissue damage, and retinoic acid production in lung tissue was established to be reduced at early and late time points post high dose irradiation. Going forward, the use of mass spectrometry-based metabolomics coupled to well defined animal models provides the unique opportunity for comprehensive biomarker discovery.
Related JoVE Video
Effects of diet and strain on mouse serum and tissue retinoid concentrations.
PLoS ONE
PUBLISHED: 01-01-2014
Show Abstract
Hide Abstract
The relationship between dietary vitamin A and all-trans-retinoic acid levels in serum and tissues had not been quantified. We determined the impact of dietary vitamin A on retinoid levels in serum, liver, kidney, testis, and epididymal white adipose of five mouse strains: AKR/J; BALB/cByJ; C3H/HeJ; C57BL/6J; 129S1/SvImJ. Retinoids were quantified in mice fed copious vitamin A (lab chow, ?20 IU/g) followed by one month feeding a vitamin A-sufficient diet (4 IU/g), or after three generations of feeding a vitamin A-sufficient diet. Retinol and retinyl esters were measured by high-performance liquid chromatography with ultraviolet absorbance detection. All-trans-retinoic acid was quantified by liquid chromatography tandem mass spectrometry. The amounts of dietary vitamin A had long-term strain-specific effects on tissue retinyl ester, retinol and all-trans-retinoic acid concentrations. Three generations of feeding a vitamin A-sufficient diet decreased all-trans-retinoic acid in most tissues of most strains, in some cases more than 60%, compared to a diet with copious vitamin A. With both diets, all-trans-retinoic acid concentrations maintained an order of liver ? testis > kidney > white adipose tissue ? serum. Neither retinol nor all-trans-retinoic acid in serum reflected all-trans-retinoic acid concentrations in tissues. Strain and tissue-specific differences in retinol and all-trans-retinoic acid altered by different amounts of dietary vitamin A could have profound effects on retinoid action. This would be the case especially with the increased all-trans-retinoic acid values associated with the amounts of vitamin A and its precursors (carotenoids) in chow diets.
Related JoVE Video
The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development.
FASEB J.
PUBLISHED: 09-04-2013
Show Abstract
Hide Abstract
Oxidation of retinol via retinaldehyde results in the formation of the essential morphogen all-trans-retinoic acid (ATRA). Previous studies have identified critical roles in the regulation of embryonic ATRA levels for retinol, retinaldehyde, and ATRA-oxidizing enzymes; however, the contribution of retinaldehyde reductases to ATRA metabolism is not completely understood. Herein, we investigate the role of the retinaldehyde reductase Dhrs3 in embryonic retinoid metabolism using a Dhrs3-deficient mouse. Lack of DHRS3 leads to a 40% increase in the levels of ATRA and a 60% and 55% decrease in the levels of retinol and retinyl esters, respectively, in Dhrs3(-/-) embryos compared to wild-type littermates. Furthermore, accumulation of excess ATRA is accompanied by a compensatory 30-50% reduction in the expression of ATRA synthetic genes and a 120% increase in the expression of the ATRA catabolic enzyme Cyp26a1 in Dhrs3(-/-) embryos vs. controls. Excess ATRA also leads to alterations (40-80%) in the expression of several developmentally important ATRA target genes. Consequently, Dhrs3(-/-) embryos die late in gestation and display defects in cardiac outflow tract formation, atrial and ventricular septation, skeletal development, and palatogenesis. These data demonstrate that the reduction of retinaldehyde by DHRS3 is critical for preventing formation of excess ATRA during embryonic development.-Billings, S. E., Pierzchalski, K., Butler Tjaden, N. E., Pang, X.-Y., Trainor, P. A., Kane, M. A., Moise, A. R. The retinaldehyde reductase DHRS3 is essential for preventing the formation of excess retinoic acid during embryonic development.
Related JoVE Video
Disruption of Myc-Max heterodimerization with improved cell-penetrating analogs of the small molecule 10074-G5.
Oncotarget
PUBLISHED: 06-27-2013
Show Abstract
Hide Abstract
The c-Myc (Myc) oncoprotein is a high-value therapeutic target given that it is deregulated in multiple types of cancer. However, potent small molecule inhibitors of Myc have been difficult to identify, particularly those whose mechanism relies on blocking the association between Myc and its obligate heterodimerization partner, Max. We have recently reported a structure-activity relationship study of one such small molecule, 10074-G5, and generated an analog, JY-3-094, with significantly improved ability to prevent or disrupt the association between recombinant Myc and Max proteins. However, JY-3094 penetrates cells poorly. Here, we show that esterification of a critical para-carboxylic acid function of JY-3-094 by various blocking groups significantly improves cellular uptake although it impairs the ability to disrupt Myc-Max association in vitro. These pro-drugs are highly concentrated within cells where JY-3-094 is then generated by the action of esterases. However, the pro-drugs are also variably susceptible to extracellular esterases, which can deplete extracellular reservoirs. Furthermore, while JY-3-094 is retained by cells for long periods of time, much of it is compartmentalized within the cytoplasm in a form that appears to be less available to interact with Myc. Our results suggest that persistently high extracellular levels of pro-drug, without excessive susceptibility to extracellular esterases, are critical to establishing and maintaining intracellular levels of JY-3-094 that are sufficient to provide for long-term inhibition of Myc-Max association. Analogs of JY-3-094 appear to represent promising small molecule Myc inhibitors that warrant further optimization.
Related JoVE Video
Synthesis, modeling, and pharmacological evaluation of UMB 425, a mixed ? agonist/? antagonist opioid analgesic with reduced tolerance liabilities.
ACS Chem Neurosci
PUBLISHED: 06-11-2013
Show Abstract
Hide Abstract
Opioid narcotics are used for the treatment of moderate-to-severe pain and primarily exert their analgesic effects through ? receptors. Although traditional ? agonists can cause undesired side effects, including tolerance, addition of ? antagonists can attenuate said side effects. Herein, we report 4a,9-dihydroxy-7a-(hydroxymethyl)-3-methyl-2,3,4,4a,5,6-hexahydro-1H-4,12-methanobenzofuro[3,2-e]isoquinolin-7(7aH)-one (UMB 425) a 5,14-bridged morphinan-based orvinol precursor synthesized from thebaine. Although UMB 425 lacks ?-specific motifs, conformationally sampled pharmacophore models for ? and ? receptors predict it to have efficacy similar to morphine at ? receptors and similar to naltrexone at ? receptors, due to the compound sampling conformations in which the hydroxyl moiety interacts with the receptors similar to orvinols. As predicted, UMB 425 exhibits a mixed ? agonist/? antagonist profile as determined in receptor binding and [(35)S]GTP?S functional assays in CHO cells. In vivo studies in mice show that UMB 425 displays potent antinociception in the hot plate and tail-flick assays. The antinociceptive effects of UMB 425 are blocked by naloxone, but not by the ?-selective antagonist norbinaltorphimine. During a 6-day tolerance paradigm, UMB 425 maintains significantly greater antinociception compared to morphine. These studies thus indicate that, even in the absence of ?-specific motifs fused to the C-ring, UMB 425 has mixed ? agonist/? antagonist properties in vitro that translate to reduced tolerance liabilities in vivo.
Related JoVE Video
Analysis of follicular fluid retinoids in women undergoing in vitro fertilization: retinoic acid influences embryo quality and is reduced in women with endometriosis.
Reprod Sci
PUBLISHED: 02-20-2013
Show Abstract
Hide Abstract
Retinol (ROL) and its biologically active metabolite, all-trans retinoic acid (ATRA), are essential for a number of reproductive processes. However, there is a paucity of information regarding their roles in ovarian folliculogenesis, oocyte maturation, and early embryogenesis. The objectives of this study were to quantify and compare peripheral plasma (PP) and follicular fluid (FF) retinoid levels, including ATRA in women undergoing in vitro fertilization (IVF) and to investigate the relationship between retinoid levels and embryo quality. Retinoid levels were evaluated in PP and FF from 79 women undergoing IVF at the time of oocyte retrieval and corresponding embryo quality assessed on a daily basis after retrieval for 3 days until uterine transfer. Analysis compared the retinoid levels with day 3 embryo grades and between endometriosis versus control patients. Results demonstrated distinctive levels of retinoid metabolites and isomers in FF versus PP. There was a significantly larger percentage of high-quality grade I embryos derived from the largest versus smallest follicles. An increase in follicle size also correlated with a >50% increase in FF ROL and ATRA concentrations. Independent of follicle size, FF yielding grade I versus nongrade I embryos showed higher mean levels of ATRA but not ROL. In a nested case-control analysis, control participants had 50% higher mean levels of ATRA in their FF and PP than women with endometriosis. These findings strongly support the proposition that ATRA plays a fundamental role in oocyte development and quality, and that reduced ATRA synthesis may contribute to decreased fecundity of participants with endometriosis.
Related JoVE Video
CrbpI regulates mammary retinoic acid homeostasis and the mammary microenvironment.
FASEB J.
PUBLISHED: 01-29-2013
Show Abstract
Hide Abstract
Cellular retinol-binding protein, type I (CrbpI), encoded by retinol-binding protein, type 1 (Rbp1), is a chaperone of vitamin A (retinol) that is epigenetically silenced in ~25% of human breast cancers. CrbpI delivers vitamin A to enzymes for metabolism into an active metabolite, all-trans retinoic acid (atRA), where atRA is essential to cell proliferation, apoptosis, differentiation, and migration. Here, we show the effect of CrbpI loss on mammary atRA homeostasis using the Rbp1(-/-) mouse model. Rbp1(-/-) mouse mammary tissue has disrupted retinoid homeostasis that results in 40% depleted endogenous atRA. CrbpI loss and atRA depletion precede defects in atRA biosynthesis enzyme expression. Compensation by CrbpIII as a retinoid chaperone does not functionally replace CrbpI. Mammary subcellular fractions isolated from Rbp1(-/-) mice have altered retinol dehydrogenase/reductase (Rdh) enzyme activity that results in 24-42% less atRA production. Rbp1(-/-) mammary tissue has epithelial hyperplasia, stromal hypercellularity, increased collagen, and increased oxidative stress characteristic of atRA deficiency and early tissue dysfunction that precedes tumor formation. Consistent with the findings from the Rbp1(-/-) mouse, tumorigenic epithelial cells lacking CrbpI expression produce 51% less atRA. Together, these data show that CrbpI loss disrupts atRA homeostasis in mammary tissue, resulting in microenvironmental defects similar to those observed at the early stages of tumorigenesis.
Related JoVE Video
Analysis, occurrence, and function of 9-cis-retinoic acid.
Biochim. Biophys. Acta
PUBLISHED: 06-15-2011
Show Abstract
Hide Abstract
Metabolic conversion of vitamin A (retinol) into retinoic acid (RA) controls numerous physiological processes. 9-cis-retinoic acid (9cRA), an active metabolite of vitamin A, is a high affinity ligand for retinoid X receptor (RXR) and also activates retinoic acid receptor (RAR). Despite the identification of candidate enzymes that produce 9cRA and the importance of RXRs as established by knockout experiments, in vivo detection of 9cRA in tissue was elusive until recently when 9cRA was identified as an endogenous pancreas retinoid by validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) methodology. This review will discuss the current status of the analysis, occurrence, and function of 9cRA. Understanding both the nuclear receptor-mediated and non-genomic mechanisms of 9cRA will aid in the elucidation of disease physiology and possibly lead to the development of new retinoid-based therapeutics. This article is part of a Special Issue entitled Retinoid and Lipid Metabolism.
Related JoVE Video
CrbpI modulates glucose homeostasis and pancreas 9-cis-retinoic acid concentrations.
Mol. Cell. Biol.
PUBLISHED: 06-13-2011
Show Abstract
Hide Abstract
Cellular retinol-binding protein type I (CrbpI), encoded by Rpb1, serves as a chaperone of retinol homeostasis, but its physiological effects remain incompletely understood. We show here that the Rbp1(-/-) mouse has disrupted retinoid homeostasis in multiple tissues, with abnormally high 9-cis-retinoic acid (9cRA), a pancreas autacoid that attenuates glucose-stimulated insulin secretion. The Rbp1(-/-) pancreas has increased retinol and intense ectopic expression of Rpb2 mRNA, which encodes CrbpII: both would contribute to increased ?-cell 9cRA biosynthesis. 9cRA in Rbp1(-/-) pancreas resists postprandial and glucose-induced decreases. Rbp1(-/-) mice have defective islet expression of genes involved in glucose sensing and insulin secretion, as well as islet ?-cell infiltration, which contribute to reduced glucose-stimulated insulin secretion, high glucagon secretion, an abnormally high rate of gluconeogenesis, and hyperglycemia. A diet rich in vitamin A (as in a standard chow diet) increases pancreas 9cRA and impairs glucose tolerance. Crbp1 attenuates the negative impact of vitamin A (retinol) on glucose tolerance, regardless of the dietary retinol content. Rbp1(-/-) mice have an increased rate of fatty acid oxidation and resist obesity when fed a high-fat diet. Thus, glucose homeostasis and energy metabolism rely on Rbp1 expression and its moderation of pancreas retinol and of the autacoid 9cRA.
Related JoVE Video
MyD88 and retinoic acid signaling pathways interact to modulate gastrointestinal activities of dendritic cells.
Gastroenterology
PUBLISHED: 04-01-2011
Show Abstract
Hide Abstract
Gut-associated dendritic cells (DC) metabolize vitamin A into all-trans retinoic acid (RA), which is required to induce lymphocytes to localize to the gastrointestinal tract and promotes the differentiation of Foxp3+ regulatory T cells and IgA antibody-secreting cells. We investigated whether RA functions in a positive-feedback loop in DC to induce its own synthesis.
Related JoVE Video
In vivo magnetic resonance imaging to detect biliary excretion of 19F-labeled drug in mice.
Drug Metab. Dispos.
PUBLISHED: 01-26-2011
Show Abstract
Hide Abstract
Isoflurane is an inhaled halogenated hydrocarbon anesthetic commonly used for animal research. In our quest to develop a method for measuring bile acid transport in live animals using (19)F magnetic resonance (MR) imaging, it occurred to us that isoflurane, which contains five fluorines per molecule and is probably widely distributed, would provide an excellent test drug to evaluate the merits of this approach. Experiments in 20- to 28-g male C57BL/6 mice were performed using a horizontal bore scanner with a 30-mm (19)F/(1)H dual-tuned surface coil used to transmit and receive radiofrequency signals at 300.28 MHz for (1)H and 282.55 MHz for (19)F nuclei. Proton MR imaging was used to identify the mouse gallbladder in vivo, which was verified by anatomical dissection. Subsequent experiments in mice inhaling 1.5% isoflurane for 1 to 2 h revealed robust (19)F signals from the gallbladder, verified by overlying (1)H and (19)F signals. No (19)F signal was detected in mice anesthetized with nonhalogenated anesthetics. The presence of isoflurane in gallbladder bile of isoflurane-treated mice was verified using liquid chromatography-mass spectrometry. Gallbladder bile isoflurane content ranged from 3.2 to 4.7 ?g. The data presented here provide proof of concept that this novel approach can be used for in vivo measurement of biliary excretion of both existing and novel (19)F-labeled drugs.
Related JoVE Video
Binding affinities of CRBPI and CRBPII for 9-cis-retinoids.
Biochim. Biophys. Acta
PUBLISHED: 01-05-2011
Show Abstract
Hide Abstract
Cellular retinol binding-protein I (CRBPI) and cellular retinol binding-protein II (CRBPII) serve as intracellular retinoid chaperones that bind retinol and retinal with high affinity and facilitate substrate delivery to select enzymes that catalyze retinoic acid (RA) and retinyl ester biosynthesis. Recently, 9-cis-RA has been identified in vivo in the pancreas, where it contributes to regulating glucose-stimulated insulin secretion. In vitro, 9-cis-RA activates RXR (retinoid × receptors), which serve as therapeutic targets for treating cancer and metabolic diseases. Binding affinities and structure-function relationships have been well characterized for CRBPI and CRBPII with all-trans-retinoids, but not for 9-cis-retinoids. This study extended current knowledge by establishing binding affinities for CRBPI and CRBPII with 9-cis-retinoids.
Related JoVE Video
Multiple retinol and retinal dehydrogenases catalyze all-trans-retinoic acid biosynthesis in astrocytes.
J. Biol. Chem.
PUBLISHED: 12-07-2010
Show Abstract
Hide Abstract
All-trans-retinoic acid (atRA) stimulates neurogenesis, dendritic growth of hippocampal neurons, and higher cognitive functions, such as spatial learning and memory formation. Although astrocyte-derived atRA has been considered a key factor in neurogenesis, little direct evidence identifies hippocampus cell types and the enzymes that biosynthesize atRA. Here we show that primary rat astrocytes, but not neurons, biosynthesize atRA using multiple retinol dehydrogenases (Rdh) of the short chain dehydrogenase/reductase gene family and retinaldehyde dehydrogenases (Raldh). Astrocytes secrete atRA into their medium; neurons sequester atRA. The first step, conversion of retinol into retinal, is rate-limiting. Neurons and astrocytes both synthesize retinyl esters and reduce retinal into retinol. siRNA knockdown indicates that Rdh10, Rdh2 (mRdh1), and Raldh1, -2, and -3 contribute to atRA production. Knockdown of the Rdh Dhrs9 increased atRA synthesis ?40% by increasing Raldh1 expression. Immunocytochemistry revealed cytosolic and nuclear expression of Raldh1 and cytosol and perinuclear expression of Raldh2. atRA autoregulated its concentrations by inducing retinyl ester synthesis via lecithin:retinol acyltransferase and stimulating its catabolism via inducing Cyp26B1. These data show that adult hippocampus astrocytes rely on multiple Rdh and Raldh to provide a paracrine source of atRA to neurons, and atRA regulates its own biosynthesis in astrocytes by directing flux of retinol. Observation of cross-talk between Dhrs9 and Raldh1 provides a novel mechanism of regulating atRA biosynthesis.
Related JoVE Video
Identification of 9-cis-retinoic acid as a pancreas-specific autacoid that attenuates glucose-stimulated insulin secretion.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 11-29-2010
Show Abstract
Hide Abstract
The all-trans-retinoic acid (atRA) isomer, 9-cis-retinoic acid (9cRA), activates retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in vitro. RARs control multiple genes, whereas RXRs serve as partners for RARs and other nuclear receptors that regulate metabolism. Physiological function has not been determined for 9cRA, because it has not been detected in serum or multiple tissues with analytically validated assays. Here, we identify 9cRA in mouse pancreas by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and show that 9cRA decreases with feeding and after glucose dosing and varies inversely with serum insulin. 9cRA reduces glucose-stimulated insulin secretion (GSIS) in mouse islets and in the rat ?-cell line 832/13 within 15 min by reducing glucose transporter type 2 (Glut2) and glucokinase (GK) activities. 9cRA also reduces Pdx-1 and HNF4? mRNA expression, ?8- and 80-fold, respectively: defects in Pdx-1 or HNF4? cause maturity onset diabetes of the young (MODY4 and 1, respectively), as does a defective GK gene (MODY2). Pancreas ?-cells generate 9cRA, and mouse models of reduced ?-cell number, heterozygous Akita mice, and streptozotocin-treated mice have reduced 9cRA. 9cRA is abnormally high in glucose-intolerant mice, which have ?-cell hypertropy, including mice with diet-induced obesity (DIO) and ob/ob and db/db mice. These data establish 9cRA as a pancreas-specific autacoid with multiple mechanisms of action and provide unique insight into GSIS.
Related JoVE Video
Endogenous retinoids in mammalian growth plate cartilage: analysis and roles in matrix homeostasis and turnover.
J. Biol. Chem.
PUBLISHED: 09-14-2010
Show Abstract
Hide Abstract
The growth plate contains resting and proliferating chondrocytes in its upper zones (UGP) and maturing and hypertrophic chondrocytes in its lower zones (LGP), but the mechanisms by which it operates to sustain skeletal growth are not fully clear. Retinoid signaling was previously found to be nearly absent in UGP, but to be much stronger in LGP coincident with hypertrophy, extracellular matrix turnover and endochondral bone formation. To determine whether such distinct signaling levels and phenotypic events reflect different endogenous retinoid levels, the upper two-thirds and lower one-third of rabbit rib growth plates were microsurgically isolated and processed for ultrasensitive retinoid LC-tandem MS quantification. Indeed, the UGP samples contained only about a 0.6 nm concentration of all-trans-retinoic acid (atRA) that is the most active natural retinoid in tissues, whereas LGP samples contained nearly 3-fold higher atRA levels (about 1.8 nM). Perichondrium was quite rich in atRA (about 4.9 nM). Interestingly, the levels of retinol, the major but inactive atRA precursor, were similar in all tissues (1.1-1.6 ?M), suggesting that the distinct atRA levels in UGP and LGP reflect different retinoid anabolic capacity. Indeed, RALDH2 and CRABP1 transcript levels were much higher in LGP than UGP samples. To determine the minimum effective atRA concentration, chondrogenic cells transfected with a retinoic acid response element (RARE)-luc reporter plasmid were treated with different concentrations of exogenous atRA (0-100 nM). About 3 nm atRA was needed to elicit appreciable RARE-luc reporter activity and to decrease proteoglycan synthesis and activity of an aggrecan enhancer reporter plasmid. In sum, the data indicate that (i) the endogenous levels of atRA are significantly higher in hypertrophic than upper zones of growth plate; (ii) such difference likely reflects distinct retinoid anabolic capacity; and (iii) importantly, atRA levels in hypertrophic portion are within effective ranges to elicit retinoid signaling and action, but those in upper zones are not.
Related JoVE Video
Quantification of endogenous retinoids.
Methods Mol. Biol.
PUBLISHED: 06-17-2010
Show Abstract
Hide Abstract
Numerous physiological processes require retinoids, including development, nervous system function, immune responsiveness, proliferation, differentiation, and all aspects of reproduction. Reliable retinoid quantification requires suitable handling and, in some cases, resolution of geometric isomers that have different biological activities. Here we describe procedures for reliable and accurate quantification of retinoids, including detailed descriptions for handling retinoids, preparing standard solutions, collecting samples and harvesting tissues, extracting samples, resolving isomers, and detecting with high sensitivity. Sample-specific strategies are provided for optimizing quantification. Approaches to evaluate assay performance also are provided. Retinoid assays described here for mice also are applicable to other organisms including zebrafish, rat, rabbit, and human and for cells in culture. Retinoid quantification, especially that of retinoic acid, should provide insight into many diseases, including Alzheimers disease, type 2 diabetes, obesity, and cancer.
Related JoVE Video
Altered retinoic acid metabolism in diabetic mouse kidney identified by O isotopic labeling and 2D mass spectrometry.
PLoS ONE
PUBLISHED: 02-02-2010
Show Abstract
Hide Abstract
Numerous metabolic pathways have been implicated in diabetes-induced renal injury, yet few studies have utilized unbiased systems biology approaches for mapping the interconnectivity of diabetes-dysregulated proteins that are involved. We utilized a global, quantitative, differential proteomic approach to identify a novel retinoic acid hub in renal cortical protein networks dysregulated by type 2 diabetes.
Related JoVE Video
Vitamin A facilitates enteric nervous system precursor migration by reducing Pten accumulation.
Development
PUBLISHED: 01-30-2010
Show Abstract
Hide Abstract
Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration, cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinol-binding-protein-deficient (Rbp4(-/-)) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis induced by vitamin A deficiency in Rbp4(-/-) animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.
Related JoVE Video
Retinoic acid is a cofactor for translational regulation of vascular endothelial growth factor in human endometrial stromal cells.
Mol. Endocrinol.
PUBLISHED: 11-12-2009
Show Abstract
Hide Abstract
Vascular endothelial growth factor (VEGF) and endometrial angiogenesis play a critical role in successful embryonic implantation. Despite many studies of the effects of estrogen and progesterone on VEGF expression, its focal regulation at the site of implantation is unknown. Retinoic acid (RA) has been reported to regulate VEGF in a variety of cell types. Because localized RA synthesis occurs within the periimplantation endometrium, we tested the possibility that RA regulates VEGF production in endometrial stromal cells. Using primary and telomerase-immortalized human endometrial stromal cells, we determined that RA alone did not alter constitutive levels of VEGF production, but markedly amplified secretion when the cells were cotreated with activators of VEGF gene transcription (12-O-tetradecanoyl phorbol-13-acetate, TPA; TGF-beta; and IL-1beta). Whereas TPA or TGF-beta alone stimulated VEGF promoter activity and up-regulated mRNA levels, significant protein secretion was detected only after RA was added to the culture systems. Analysis of retinoids in secretory phase endometrial biopsies indicated that endogenous RA accumulated at concentrations sufficient to induce VEGF secretion. Polyribosome profile analysis showed that the addition of RA to transcriptional activators of VEGF shifted the translational suppressed VEGF mRNA transcripts into larger polyribosome complexes engaged in active translation. Although the precise mechanism(s) of the RA effect remains to be defined, it appears to be mediated by reactive oxygen species; the antioxidant N-acetylcysteine inhibited RA+TPA-stimulated secretion of VEGF by more than 80%. Together, our results demonstrate that in human endometrial stromal cells, RA can combine with transcriptional activators of VEGF to augment VEGF secretion through a translational mechanism of action mediated by reactive oxygen species. These findings suggest a link between the spatiotemporal changes of retinoid synthesis in the periimplantation stroma and the capacity to quickly up-regulate focal VEGF secretion needed to induce early angiogenic events of pregnancy.
Related JoVE Video
Ethanol elevates physiological all-trans-retinoic acid levels in select loci through altering retinoid metabolism in multiple loci: a potential mechanism of ethanol toxicity.
FASEB J.
PUBLISHED: 11-04-2009
Show Abstract
Hide Abstract
All-trans-retinoic acid (atRA) supports embryonic development, central nervous system function, and the immune response. atRA initiates neurogenesis and dendritic growth in the hippocampus and is required for spatial memory; superphysiological atRA inhibits neurogenesis, causes teratology and/or embryo toxicity, and alters cognitive function and behavior. Because abnormal atRA shares pathological conditions with alcoholism, inhibition of retinol (vitamin A) activation into atRA has been credited widely as a mechanism of ethanol toxicity. Here, we analyze the effects of ethanol on retinoid concentrations in vivo during normal vitamin A nutriture, using sensitive and analytically robust assays. Ethanol either increased or had no effect on atRA, regardless of changes in retinol and retinyl esters. Acute ethanol (3.5 g/kg) increased atRA in adult hippocampus (1.6-fold), liver (2.4-fold), and testis (1.5-fold). Feeding dams a liquid diet with 6.5% ethanol from embryonic day 13 (e13) to e19 increased atRA in fetal hippocampus (up to 20-fold) and cortex (up to 50-fold), depending on blood alcohol content. One-month feeding of the 6.5% ethanol diet increased atRA in adult hippocampus (20-fold), cortex (2-fold), testis (2-fold), and serum (10-fold). Tissue-specific increases in retinoid dehydrogenase mRNAs and activities, extrahepatic retinol concentrations, and atRA catabolism combined to produce site-specific effects. Because a sustained increase in atRA has deleterious effects on the central nervous system and embryo development, these data suggest that superphysiological atRA contributes to ethanol pathological conditions, including cognitive dysfunction and fetal alcohol syndrome.-Kane, M. A., Folias, A. E., Wang, C., Napoli, J. L. Ethanol elevates physiological all-trans-retinoic acid levels in select loci through altering retinoid metabolism in multiple loci: a potential mechanism of ethanol toxicity.
Related JoVE Video
Retinoic acid from the meninges regulates cortical neuron generation.
Cell
PUBLISHED: 07-10-2009
Show Abstract
Hide Abstract
Extrinsic signals controlling generation of neocortical neurons during embryonic life have been difficult to identify. In this study we demonstrate that the dorsal forebrain meninges communicate with the adjacent radial glial endfeet and influence cortical development. We took advantage of Foxc1 mutant mice with defects in forebrain meningeal formation. Foxc1 dosage and loss of meninges correlated with a dramatic reduction in both neuron and intermediate progenitor production and elongation of the neuroepithelium. Several types of experiments demonstrate that retinoic acid (RA) is the key component of this secreted activity. In addition, Rdh10- and Raldh2-expressing cells in the dorsal meninges were either reduced or absent in the Foxc1 mutants, and Rdh10 mutants had a cortical phenotype similar to the Foxc1 null mutants. Lastly, in utero RA treatment rescued the cortical phenotype in Foxc1 mutants. These results establish RA as a potent, meningeal-derived cue required for successful corticogenesis.
Related JoVE Video
Rdh12 activity and effects on retinoid processing in the murine retina.
J. Biol. Chem.
PUBLISHED: 06-08-2009
Show Abstract
Hide Abstract
RDH12 mutations are responsible for early-onset autosomal recessive retinal dystrophy, which results in profound retinal pathology and severe visual handicap in patients. To investigate the function of RDH12 within the network of retinoid dehydrogenases/reductases (RDHs) present in retina, we studied the retinal phenotype of Rdh12-deficient mice. In vivo rates of all-trans-retinal reduction and 11-cis-retinal formation during recovery from bleaching were similar in Rdh12-deficient and wild-type mice matched for an Rpe65 polymorphism that impacts visual cycle efficiency. However, retinal homogenates from Rdh12-deficient mice exhibited markedly decreased capacity to reduce exogenous retinaldehydes in vitro. Furthermore, in vivo levels of the bisretinoid compound diretinoid-pyridinium-ethanolamine (A2E) were increased in Rdh12-deficient mice of various genetic backgrounds. Conversely, in vivo levels of retinoic acid and total retinol were significantly decreased. Rdh12 transcript levels in wild-type mice homozygous for the Rpe65-Leu(450) polymorphism were greater than in Rpe65-Met(450) mice and increased during postnatal development in wild-type mice and Nrl-deficient mice having an all-cone retina. Rdh12-deficient mice did not exhibit increased retinal degeneration relative to wild-type mice at advanced ages, when bred on the light-sensitive BALB/c background, or when heterozygous for a null allele of superoxide dismutase 2 (Sod2(+/-)). Our findings suggest that a critical function of RDH12 is the reduction of all-trans-retinal that exceeds the reductive capacity of the photoreceptor outer segments.
Related JoVE Video
Retinol Esterification by DGAT1 Is Essential for Retinoid Homeostasis in Murine Skin.
J. Biol. Chem.
PUBLISHED: 04-01-2009
Show Abstract
Hide Abstract
Retinoic acid (RA) is a potent signaling molecule that is essential for many biological processes, and its levels are tightly regulated by mechanisms that are only partially understood. The synthesis of RA from its precursor retinol (vitamin A) is an important regulatory mechanism. Therefore, the esterification of retinol with fatty acyl moieties to generate retinyl esters, the main storage form of retinol, may also regulate RA levels. Here we show that the neutral lipid synthesis enzyme acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) functions as the major acyl-CoA:retinol acyltransferase (ARAT) in murine skin. When dietary retinol is abundant, DGAT1 deficiency results in elevated levels of RA in skin and cyclical hair loss; both are prevented by dietary retinol deprivation. Further, DGAT1-deficient skin exhibits enhanced sensitivity to topically administered retinol. Deletion of the enzyme specifically in the epidermis causes alopecia, indicating that the regulation of RA homeostasis by DGAT1 is autonomous in the epidermis. These findings show that DGAT1 functions as an ARAT in the skin, where it acts to maintain retinoid homeostasis and prevent retinoid toxicity. Our findings may have implications for human skin or hair disorders treated with agents that modulate RA signaling.
Related JoVE Video
Retinoic acid receptors are required for skeletal growth, matrix homeostasis and growth plate function in postnatal mouse.
Dev. Biol.
PUBLISHED: 01-20-2009
Show Abstract
Hide Abstract
The retinoic acid receptors alpha, beta and gamma (RARalpha, RARbeta and RARgamma) are nuclear hormone receptors that regulate fundamental processes during embryogenesis, but their roles in skeletal development and growth remain unclear. To study skeletal-specific RAR function, we created conditional mouse mutants deficient in RAR expression in cartilage. We find that mice deficient in RARalpha and RARgamma (or RARbeta and RARgamma) exhibit severe growth retardation obvious by about 3 weeks postnatally. Their growth plates are defective and, importantly, display a major drop in aggrecan expression and content. Mice deficient in RARalpha and RARbeta, however, are virtually normal, suggesting that RARgamma is essential. In good correlation, we find that RARgamma is the most strongly expressed RAR in mouse growth plate and its expression characterizes the proliferative and pre-hypertrophic zones where aggrecan is strongly expressed also. By being avascular, those zones lack endogenous retinoids as indicated by previous RARE reporter mice and our direct biochemical measurements and thus, RARgamma is likely to exert ligand-less repressor function. Indeed, our data indicate that: aggrecan production is enhanced by RARgamma over-expression in chondrocytes under retinoid-free culture conditions; production is further boosted by co-repressor Zac1 or pharmacologic agents that enhance RAR repressor function; and RAR/Zac1 function on aggrecan expression may involve Sox proteins. In sum, our data reveal that RARs, and RARgamma in particular, exert previously unappreciated roles in growth plate function and skeletal growth and regulate aggrecan expression and content. Since aggrecan is critical for growth plate function, its deficiency in RAR-mutant mice is likely to have contributed directly to their growth retardation.
Related JoVE Video

What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

How does it work?

We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

Video X seems to be unrelated to Abstract Y...

In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.