Oral bioavailability of triamcinolone tablets and a triamcinolone diacetate suspension.
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Administration, Oral↗
Biomedical subjects
G Hochhaus
Publications and source records attributed to G Hochhaus.
Neuronal cell differentiation of human neuroblastoma cells by retinoic acid plus herbimycin A.
We investigated the effect of retinoic acid (RA) and herbimycin A (herb-A) on cell growth, cell differentiation, and colony formation of human neuroblastoma cell lines. The neuroblastoma line SK-N-SH expressed both neuroblast and nonneuronal phenotypes, whereas its subclone SH-SY5Y and the Kelly cell line were predominantly neuroblastic. Both herb-A and RA, given alone, moderately reduced cell growth and colony formation of the neuroblastic cell lines. Growth curve analyses with SK-N-SH suggested that herb-A greatly reduced the number of initially growing cells, whereas RA slightly enhanced initial cell growth. Morphological changes were determined with the use of rhodaminephalloidin staining of actin. Retinoic acid caused an increase in the fraction of neuroblast cell in SK-N-SH, and conversely of nonneuronal cells in SH-SY5Y and Kelly cell lines. Both drugs also caused partial differentiation towards a neuronal phenotype, and herb-A induced selective lysis of nonneuronal cells of SK-N-SH. Because of their discrepant effects, RA (10 microM) and herb-A (236 nM) were tested in combination at a concentration that had only moderate effects when given alone. The combination further reduced cell growth and colony formation and dramatically enhanced differentiation towards a neuronal morphology. The Kelly cell line with amplified N-myc genome, which correlates with clinical progression of neuroblastoma, was also sensitive to RA plus herb-A. These results recommend the combination of RA and herb-A for differentiation therapy of neuroblastoma.
Anti-Bacterial Agents↗
Biotinylated human beta-endorphins as probes for the opioid receptor.
The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human beta-endorphin to cross-link the mu and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.
Animals↗
[Biocytin13]dynorphin A 1-13 amide: a potential probe for the kappa-opioid receptor.
A dynorphin A 1-13 amide (DYN) derivative biotinylated in position lysine 13 (B-DYN) has been prepared by automated solid-phase peptide synthesis. The derivative retained its ability to bind to avidin, and B-DYN-avidin complex showed a dissociated half-life of 10 hr at 37 degrees C. Opioid receptor binding was measured in membrane preparations of rat brain (mu), NG-108-15 neuroblastoma-glioma hybrid cells (delta), and guinea pig cerebellum (kappa). Biotinyl substitution of DYN either did not effect receptor binding (delta) or slightly reduced binding affinity (mu and kappa). Binding of B-DYN to the kappa receptor was very tight, with an IC50 value in the low picomolar range, while binding to mu and delta sites was over two orders of magnitude lower. Preassociation of B-DYN with avidin resulted in a reduction of the affinities to the investigated opioid receptors by 100- to 1000-fold. However, the apparent affinity of B-DYN-avidin for the kappa-opioid receptor is sufficient to suggest that B-DYN may be a useful tool for kappa-opioid receptor assay, localization, and purification.
Animals↗
A biotin-avidin-based enzyme immunoassay for beta h-endorphin.
An avidin-biotin enzyme-linked immunosorbent assay (ELISA) is described for beta h-endorphin (beta h-EP). Microtiter plates coated with commercially available antibodies were used together with beta h-EP tracer derivatives that were biotinylated in positions 24, 28, and 29 via a C6 spacer arm. Nonspecific binding of biotinylated derivatives to the microtiter plates was blocked with a mixture of 1% casein and 10% ethanolamine in 0.1 M NaHCO3. A sequential saturation procedure using a high-affinity antiserum in combination with an avidin-alkaline phosphatase complex matched the sensitivity of reported radioimmunoassays (RIAs), with a detection limit of 0.5 fmol/assay. The intra- and interassay coefficients of variation were 5 and 12%, respectively. Results obtained by ELISA and RIA showed good correlations (r = 0.95). The beta-EP concentration in extracted rat plasma after high-performance liquid chromatographic (HPLC) fractionation was determined by this method to be 1600 fmol/mol.
Animals↗
Differentiation of human neuroblastoma cells: marked potentiation of prostaglandin E-stimulated accumulation of cyclic AMP by retinoic acid.
Neuroblastoma cells in culture contain low levels of cyclic AMP, a second messenger which plays a major role in neuronal maturation. In this study, human neuroblastoma cells, SK-N-SH-SY5Y, were induced to differentiate by treatment with either nerve growth factor (50 ng/ml), retinoic acid (10 microM), dibutyryl cyclic AMP (1 mM), or 12-O-tetradecanoylphorbol-13-acetate (0.1 microM), and the ability of several neurotransmitters or hormones to stimulate adenylyl cyclase was tested. Although all four differentiation factors caused morphological changes towards a neuronal phenotype, only retinoic acid dramatically enhanced cyclic AMP accumulation, specifically upon stimulation with prostaglandin E1 (PGE1). PGE2 was also active, but less potent, than PGE1, whereas the other cyclic AMP-stimulating agents tested were largely unaffected. Further, the rapid desensitization of the PGE1-cyclic AMP response observed in control cells after 20 min of PGE1 exposure did not occur in retinoic acid-treated cells, and the EC50 values for PGE1 were reduced from approximately 240 to 14 nM after retinoic acid treatment. The increased sensitivity to PGE was associated with an increase of high-affinity PGE1 binding sites, whereas the Gs coupling proteins and adenylyl cyclase were not measurably affected. A similar enhancement of the PGE1-cyclic AMP response by retinoic acid was also observed in two additional human neuroblastoma cell lines tested, Kelly and IMR-32, suggesting that up-regulation of the prostaglandin response by retinoic acid is common among neuroblastoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenylyl Cyclases↗
Effects of opioid peptides on human neuroblastoma cells.
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Adenylyl Cyclases↗
Delta opioid receptors in human neuroblastoma cell lines.
Opioid receptor sites were detectable in 4 out of 9 human neuroblastoma cell lines tested, in the human retinoblastoma line Y79 NHT C10 and in the mouse neuroblastoma line Neuro 2A. All of these cell lines expressed delta sites, while only one coexpressed mu sites (SK-N-SH). Together with delta sites previously found in rodent neuroblastoma lines, these results suggest that the expression of delta sites is under less stringent control than that of mu and chi sites. A large number of delta sites (greater than 10,000 sites per cell) is expressed in IMR-32 and NMB neuroblastoma lines. Agonist binding was sensitive to Na+ and guanine nucleotides. The delta sites in IMR-32 and NMB cells were further characterized with delta selective ligands and [3H]DADL tracer. Their delta binding affinities were identical to those of the mu and delta cell line SK-N-SH; therefore the presence of mu sites does not appear to affect the binding behavior of the delta sites by any potential interaction among the binding proteins. Further, close correlations were found when comparing ligand binding in the human neuroblastoma cell lines with those of mouse neuroblastoma cells and rodent brain, an indication that the delta receptor is highly preserved among different species.
Binding, Competitive↗
Affinities of glucocorticoids for glucocorticoid receptors in the human lung.
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Binding, Competitive↗
HPLC determination of glucocorticoid alcohols, their phosphates and hydrocortisone in aqueous solutions and biological fluids.
High-performance liquid chromatographic (HPLC) assays are described for the determination of dexamethasone phosphate, dexamethasone and hydrocortisone and for the determination of triamcinolone, triamcinolone acetonide, triamcinolone acetonide phosphate and hydrocortisone in aqueous solutions and biological fluids. These assays allow quantification of the glucocorticoids in plasma down to a concentration of 100 ng ml(-1) (dexamethasone phosphate 300 ng ml(-1), hydrocortisone 40 ng ml(-1)). During the development of optimum extraction procedures the pK(a) values of the esters were determined by extractions performed at different pH values. The stability in vitro of the phosphate esters in ampoules, plasma and blood was studied. The esters are stable in their formulated ampoules after long-term storage, whereas the half-life in vitro of dexamethasone phosphate in plasma at 37 degrees C is 5 h and that of triamcinolone acetonide phosphate is 3.5 h. Determination by HPLC of endogenous hydrocortisone in samples from patients who received either dexamethasone phosphate or triamcinolone acetonide phosphate gave results identical with those obtained by radio-immunoassay (RIA).
Journal Article↗
Development of a biotin-avidin probe for detecting opioid receptors.
Biotinylated derivatives of beta h-endorphin (beta h-EP) with C6 spacer arm, inserted between biotin and beta h-EP, were synthesized and isolated by HPLC. Liquid secondary ion mass spectrometry (LSIMS) indicated the presence of 1 to 4 biotin substituents per beta h-EP molecule, and in combination with the analysis of tryptic peptide fragments, specified the location of the biotinylated lysine residue. Affinities to mu receptors decreased with increasing biotinylation number. Association of the biotinylated ligands with avidin retained or even enhanced IC50 values at the mu site, thus, matching the relative binding affinity of underivatized beta h-EP with the monobiotinylated derivatives. Hence, monobiotinylated beta h-EP represents a versatile opioid receptor probe.
Avidin↗
Simultaneous determination of glucocorticoid alcohols, their succinates and hydrocortisone in plasma.
A reversed-phase high-performance liquid chromatographic (HPLC) assay is described for the simultaneous determination of methylprednisolone, methylprednisolone-21-hemisuccinate and endogenous hydrocortisone in biological fluids. This assay is also applicable to the determination of prednisolone-21-hemisuccinate in the presence of prednisolone and hydrocortisone in biological fluids. Prednisolone and hydrocortisone are determined by normal-phase HPLC. The stability of hemisuccinate esters in ampoules, in saline and in plasma has been studied. Whereas the esters were stable in saline at 37 degrees C, they were considerably hydrolysed in plasma at the same temperature. Comparison of hydrocortisone levels obtained by HPLC and radioimmunoassay (RIA) showed the large influence of the cross-reactivity of methylprednisolone and hydrocortisone in the presence of high serum concentrations of methylprednisolone.
Journal Article↗
Identification of glucocorticoid receptors in normal and neoplastic adult human lung.
A dexamethasone-binding protein was detected in cytosol of five normal and three neoplastic lung tissue samples. The apparent dissociation constant of the dexamethasone protein complex ranged from 5.8 to 21 nM (11.4 +/- 5.7 nM for normal tissue cytosol, 12.1 +/- 7.7 nM for the neoplastic tissue cytosol). The binding protein sedimented at 8 S in sucrose density gradient at low ionic strength and showed a high specificity for glucocorticoids. It was concluded that the dexamethasone-binding protein had the characteristics of a glucocorticoid receptor. The glucocorticoid receptor content was determined to be 12-77 fmol/mg protein (37.1 +/- 18.9 fmol/mg protein for the normal tissue cytosol, 51.9 +/- 23.9 fmol/mg protein for the neoplastic tissue cytosol).
Aged↗
A selective HPLC/RIA for dexamethasone and its prodrug dexamethasone-21-sulphobenzoate sodium in biological fluids.
A combined high performance liquid chromatography/radioimmunoassay procedure is described for the simultaneous determination of dexamethasone (DEX) and its prodrug dexamethasone-21-sulphobenzoate sodium (DSS) in plasma. After precipitation of the plasma proteins by acetonitrile, the protein-free supernatant was injected onto a C18 reversed phase liquid chromatographic system and DSS- and DEX-containing fractions were collected. Hydrolysis of DSS by 0.01 N NaOH, followed by fractions extraction of both hydrolysed DSS and DEX fractions with ethyl acetate allowed the use of a dexamethasone-specific radioimmunoassay for the selective determination of both compounds. The method is accurate and reproducible (intraday variability for DSS and DEX < 6%, interday variability for DEX 14%), allowing quantification of DEX and DSS as low as 0.3 ng/mL and 0.7 ng/mL, respectively.
Chromatography, High Pressure Liquid↗