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  • 1
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 56 (1991), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: The rabbit cerebellum has been shown to contain significant quantities of opioid receptors consisting of both γ- and k-subtypes. To determine the nature of the endogenous opioid ligands in this tissue, extracts from rabbit cerebellum were separated by various chromatography techniques and fractions were assayed initially for opioid peptides with a radioimmunoassay capable of detecting all peptides with an amino-terminal Tyr-Gly-Gly-Phe sequence. This sequence is common to all mammalian opioid peptides and is critical for recognition by all known opioid receptors. Each of the three immunoreactive opioid peptide peaks detected was purified to homogeneity and subjected to amino acid composition and sequence analysis. One peak was analyzed further by mass spectrometry. This identified the major opioid peptides in the cerebellum as [Met5]enkephalin, [Leu5]enkephalin, and heptapeptide [Met5]enkephalyl-Arg6-Phe7. The comprehensiveness of this initial detection scheme in identifying biologically active opioid peptides was substantiated through subsequent analysis. Using specific radioimmunoassays for representative opioid peptides of the three opioid systems currently known, no other peptides of either the proenkephalin, proopiomelanocortin, or prodynorphin series were detected in any appreciable amounts. Collectively, these results are consistent with the position that rabbit cerebellar opioids are derived from proenkephalin. However, given that no appreciable quantities of either [Met5]enkephalyl-Arg6-Arg7-Val8-NH2 (metorphamide) or [Met5]enkephalyl-Arg6-Gly7-Leu8 were detected suggests that rabbit proenkephalin may have a slightly altered sequence and/or is differentially processed relative to other mammalian species studied.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 62 (1994), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 57 (1991), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Physiological release of endogenous opioids in guinea pig hippocampal slices was detected in an in vitro competition binding assay using [3H]U69,593, a K1-selective radioligand. Veratridine-induced opioid release caused a decrease in [3H]U69,593 binding that was blocked by either tetrodotoxin addition or the removal of calcium from the incubation buffer. Focal electrical stimulation of opioid pep-tide-containing afferent pathways resulted in a decrease in [3H]U69,593 binding, whereas stimulation of a major afferent lacking endogenous opioid immunoreactivity had no effect. The addition of 6-cyano-7-nitroquinoxaline-2,3-dione blocked the reduction in [3H]U69,593 binding caused by perforant path stimulation, but not the reduction caused by mossy fiber stimulation, suggesting that the primary source of endogenous K ligands was likely to be the dentate granule cells. Antisera against dynorphin A(1–8) or dynorphin B peptides inhibited the effects of mossy fiber stimulation in the [3H]U69,593 displacement assay. Antisera against other prodynorphin-and proenkephalin-derived opioid peptides had no effect. As shown by receptor autoradiography, the distribution of K1 binding sites was limited to the molecular layer of the dentate gyrus and the presubiculum region of temporal hippocampal slices. These results indicate that pro-dynorphin-derived opioids released under physiological conditions from the mossy fibers act at K1 receptors in the guinea pig dentate gyrus.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 56 (1991), S. 0 
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Microdialysis combined with a solid-phase radioimmunoassay was used to monitor changes in extracellular opioid peptide levels in the rat globus pallidus/ventral pallidum as a result of terminal brain ischemia. Ischemia was induced by anesthetic overdose or by severance of blood vessels supplying the brain. In control animals the recovered immunoreactivity increased an average of 13-fold in the 30-min sample following anesthetic overdose. Perfusion of a calcium-free, 10 mM EGTA-containing medium through the dialysis probe significantly attenuated the amplitude of this response, with the average increase being only threefold. Shorter sampling intervals (5 min) indicated that release of opioid peptide material into the extracellular environment occurs within the first 5 min of ischemia resulting from severance of the blood supply to the brain. HPLC analysis identified the majority of the postmortem-induced immunoreactive material as Met- and Leu-enkephalin.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Antibodies specific for α-N-acetyl-β-endorphins have been prepared by injecting into rabbits either α-N-acetyl-β-endorphin(1-31) or [α-N-acetyl, ε-acetyl-Lys9]-β-endorphin(1-9) linked by carbodiimide to bovine thyroglobulin. Both antisera were used to develop specific radioimmunoassays for α-N-acetyl-β-endorphins. The radioimmunoassays were used to measure α-N-acetylated β-endorphins in extracts of pituitary regions from different species. By comparison of the amounts of total β-endorphin and α-N-acetyl-β-endorphin immunoreactivity, a relative ratio of β-endorphin acetylation was obtained. The relative acetylation of β-endorphin was highest in rat posterior-intermediate lobe extracts (〉90%). Beef and monkey intermediate lobes had a lower degree of acetylation (53 and 31%, respectively). Anterior lobe extracts from all three species contained low amounts of acetylated β-endorphin. Human pituitary extracts did not contain acetylated β-endorphins. By the use of cation exchange and high performance liquid chromatography, six different acetylated derivatives and fragments of β-endorphin were resolved in extracts of rat posterior-intermediate pituitaries. Two of these peptides corresponded to α-N-acetyl-β-endorphin(1-31) and -(1-27). One acetylated β-endorphin fragment had the same size as α-N-acetyl-β-endorphin(1-27) but was eluted earlier from the cation exchange column. This peptide had full cross-reactivity with antibodies directed against the middle and amino-terminal parts of β-endorphin. Compared with α-N-acetyl-β-endorphin(1-27), it had much less cross-reactivity with antibodies directed against the COOH-terminal part of β-endorphin, suggesting that it was a COOH-terminally modified derivative of β-endorphin(1-27). The remaining N-acetylated β-endorphin derivatives were eluted even earlier from the cation exchange column. The majority of these fragments were slightly larger in size than y-endorphin, i.e., β-endorphin(1-17), but smaller than β-endorphin(1-27). They had full cross-reactivity in an amino-terminally directed β-endorphin radioimmunoassay and a greatly diminished cross-reactivity with antibodies to the middle region of β-endorphin.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A sensitive assay, which cross-reacts with and is specific for diverse opioid peptides, is described. This is based on the prior acetylation of samples and subsequent radioimmunoassay with an antiserum highly specific for the acetylated NH2 terminus of opioid peptides. The result is a procedure that can be used to investigate multiple forms of opioid peptides in extracts of biological material. The sensitivity of the assay is ˜15 fmol of β-endorphin per incubation tube, i.e., ˜ 100-fold greater sensitivity than the radioreceptor assay used in our laboratory. The peptide concentration required for 50% displacement of trace ranged from 0.65 nM (β-endorphin) to 1.6 nM (Met-enkephalin). The assay apparently shows an absolute requirement for a free (or acetylated) NH2 terminus corresponding to either a Leu- or Met-enkephalin sequence. Use of the assay with and without prior acetylation of sample provides a method for estimation of the ratio of acetylated:nonacetylated opioid peptides in crude or fractionated extracts. The procedure is used to investigate the forms of opioid peptide found in rat brain and pituitary.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    [s.l.] : Nature Publishing Group
    Nature 299 (1982), S. 77-79 
    ISSN: 1476-4687
    Source: Nature Archives 1869 - 2009
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
    Notes: [Auszug] The various peptides were detected in acid acetone extracts of rat brain regions with three different highly specific RIAs using antibodies raised against synthetic dynorphini_i7, dynor-phini_8 and a-neo-endorphin. All the RIAs were highly specific for their respective antigen. There was no ...
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular evolution 43 (1996), S. 179-184 
    ISSN: 1432-1432
    Keywords: Key words: Mu opioid receptor gene — Vertebrates — Molecular evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORL1) (59%). The hagfish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular evolution 43 (1996), S. 179-184 
    ISSN: 1432-1432
    Keywords: Mu opioid receptor gene ; Vertebrates ; Molecular evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The sequence of the mu opioid receptor is highly conserved among human, rat, and mouse. In order to gain insights into the evolution of the mu opioid receptor, polymerase chain reaction (PCR) was used to screen genomic DNA from a number of different species using degenerate oligonucleotides which recognize a highly conserved region. DNA was assayed from representative species of both the protostome and deuterostome branches of the metazoan phylogenetic tree. Mu opioid receptor-like sequences were found in all vertebrate species that were analyzed. These species included bovine, chicken, bullfrog, striped bass, thresher shark, and Pacific hagfish. However, no mu opioid receptor-like sequences were detected from protostomes or from any invertebrates. The PCR results demonstrate that the region of the mu opioid receptor gene between the first intracellular loop and the third transmembrane domain (TM3) has been highly conserved during evolution and that mu opioid receptor-like sequences are present in the earliest stages of vertebrate evolution. Additional opioid receptor-like sequence was obtained from mRNA isolated from Pacific hagfish brain using rapid amplification of cDNA ends (RACE). The sequence of the Pacific hagfish was most homologous with the human mu opioid receptor (72% at the amino acid level between intracellular loop 1 and transmembrane domain 6) although over the same region high homology was also observed with the delta opioid receptor (69%), the kappa receptor (63%), and opioid receptor-like (ORLI) (59%). The hag-fish sequence showed low conservation with the mammalian opioid receptors in the first and second extracellular loops but high conservation in the transmembrane and intracellular domains.
    Type of Medium: Electronic Resource
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  • 10
    ISSN: 1573-3297
    Keywords: β-endorphin ; enkephalins ; opiate receptors ; opioid peptides ; learned help-lessness ; social behavior ; depression ; schizophrenia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Psychology
    Notes: Abstract The opioid peptides have been related to behavior in both animal and human studies. Further investigation can be anticipated which could lead to the elucidation of genetic controls over enzymes which process these peptides and the receptors upon which the peptides act. The enzymes, both synthetic and degradative, can lead to the formation of different forms of the opiate peptides. Differential control of these enzymes or of the multiple forms of opiate receptors could lead to discrete changes in opiate status and subsequent behavioral changes. Conversely, genetically regulated behavioral modification could also lead secondarily to opiate changes.
    Type of Medium: Electronic Resource
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