Distribution of leucine-enkephalin in bone and joint tissues

Wu, Qinyang; Lindgren, J. U.; Elhassan, Adlan M.; Hultenby, Kjell

doi:10.1016/s0143-4179(02)00046-x

Cited by 4 publications

(2 citation statements)

References 24 publications

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“…The cells were incubated with primary antibodies against p70S6K (T421/S424), ERK1/2 (T202/ Y204) and S6 (S235/236) at 1 : 200 overnight at 4°C, and then followed by incubation with biotinylated anti-rabbit IgG at a dilution of 1 : 200 for 2 h and visualized with the avidin-biotin-peroxidase complex kit (Vector, Burlingame, CA, USA) with 3-3¢-diaminobenzidine-4 HCl/H 2 0 2 (DAB, Sigma) as a substrate. For immunoelectronic microscopy, after zinc treatment cells were fixed in a mixture of 3% paraformaldehyde plus 0.1% glutaraldehyde in 0.1 M phosphate buffer (PB) pH 7.4, and ultra-thin sections of cell pellets were prepared following previously developed protocols (Qinyang et al 2002). Non-specific bindings were blocked by 10% BSA for 2 h. Subsequently, the sections were incubated with the primary antibody against p70S6K (T421/S424) 1 : 200 or PHF-1 (1 : 100) overnight in a humidified chamber at 21°C and bound antibodies were detected with protein A coated with 10 nm gold.…”

Section: Western Blottingmentioning

confidence: 99%

Mechanism of zinc‐induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH‐SY5Y neuroblastoma cells

Björkdahl

Liu

et al. 2005

Journal of Neurochemistry

102

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Section: Western Blottingmentioning

confidence: 99%

Mechanism of zinc‐induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH‐SY5Y neuroblastoma cells

Björkdahl

Liu

et al. 2005

Journal of Neurochemistry

102

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“…These compounds exert a profound influence on many physiological and pathological states such as pain transmission, addiction, and dementia [2][3][4]. Opioid peptide levels are typically measured by radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), or radioreceptorassay (RRA) [5,6]. However, these modes of quantitation require a large sample volume and are reported to be less sensitive than chromatographic methods coupled with electrochemical detection [7].…”

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confidence: 99%

Reaction of naphthalene-2,3-dicarboxaldehyde with enkephalins for LC-fluorescence and lc-ms analysis: Conformational studies by molecular modeling and H/D Exchange mass spectrometry

Lacroix

Garrigues

Couderc

2007

J. Am. Soc. Mass Spectrom.

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A new labeling method compatible with both laser-induced fluorescence (LIF) and MS detection for enkephalins, which uses naphthalene-2,3-dicarboxaldehyde (NDA) and a new nucleophilic agent (N,N-dimethylaminoethanethiol) is described. When the derivative is separated via reverse phase HPLC and detected via MS, two different peaks with similar exact mass but different fluorescence and fragmentation properties are obtained. To interpret these results, molecular modeling and H/D exchange mass spectrometry studies were investigated to test the hypothesis that the peak obtained by LC/LIF/MS analysis depends on the site of protonation of the labeled enkephalins. The peptides labeled with NDA and N,Ndimethylaminoethanethiol were separated on a reverse phase C18 column with a gradient of aqueous 0.1% formic acid and acetonitrile. In mass spectrometry, two peaks are observed with the same exact mass for each molecule while only one peak is detected using fluorescence. Tandem mass spectrometry experiments of ion m/z 809.5 were performed on each chromatographic peak; the first peak (which is not observed by LIF detection) gives a fragment corresponding to the loss of the aminothiol side chain while no fragmentation is observed on the second peak, which was detected by fluorescence. The hypothesis is that each peak represents the labeled enkephalin with different sites of protonation. According to this hypothesis, three fundamental conformations that were closed to the unlabeled leucine-enkephalin were obtained by molecular modeling: a ␤-turn like conformation with two hydrogen bonds, a 3 10 -helix with an H bond, and finally, the extended form without any intramolecular interactions. H/D exchange mass spectrometry experiments with D 2 O and d 2-formic acid as eluent was used to determine which conformation is involved in each peak. Hughes et al. in 1975 [1] with analgesic properties similar to that of morphine. The two enkephalins, leucine enkephalin (Leu-Enk) and methionine-enkephalin (Met-Enk) are pentapeptides, which differ at the C-terminal function (Tyr-Gly-Gly-Phe-Leu or Met) and act as neurotransmitters and neuromodulators [2]. These compounds exert a profound influence on many physiological and pathological states such as pain transmission, addiction, and dementia [2][3][4]. Opioid peptide levels are typically measured by radioimmunoassay (RIA), enzyme-linked immunoassay (ELISA), or radioreceptorassay (RRA) [5,6]. However, these modes of quantitation require a large sample volume and are reported to be less sensitive than chromatographic methods coupled with electrochemical detection [7]. The two most common methods for quantitation are capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) [8] coupled to electrochemical detectors [7], UV detectors [9], laser-induced fluorescence (LIF) detectors [10], or mass spectrometry detection [11,12].Stobaugh et al. have developed chromatographic methods to monitor leucine-enkephalin in biological samples using fluorescence detection. These workers ...

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Tissue levels of leu-enkephalin in rats with adjuvant arthritis

Hultenby

Lindgren

2005

Journal of Neuroimmunology

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Distribution of leucine-enkephalin in bone and joint tissues

Cited by 4 publications

References 24 publications

Mechanism of zinc‐induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH‐SY5Y neuroblastoma cells

Mechanism of zinc‐induced phosphorylation of p70 S6 kinase and glycogen synthase kinase 3β in SH‐SY5Y neuroblastoma cells

Reaction of naphthalene-2,3-dicarboxaldehyde with enkephalins for LC-fluorescence and lc-ms analysis: Conformational studies by molecular modeling and H/D Exchange mass spectrometry

Tissue levels of leu-enkephalin in rats with adjuvant arthritis

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