Stereospecific effects of opiate antagonists on superficial and deep nociception and on motor activity suggest involvement of endorphins on different opioid receptors.

It is known that various opiate antagonists enhance stereospecifically reactions to superficial nociceptive stimuli (e.g. in the hot plate test) suggesting the involvement of endogenous ligands in these reactions. In mice and rats the writhing responses to deep nociceptive stimuli (intraperitoneal test) were also enhanced stereospecifically by (-) naloxone, Mr 2266 and GPA 2163 but some other antagonists (naltrexone, levallorphan, diprenorphine) were inactive probably as a consequence of interfering agonist (antinociceptive) properties. An another antagonist, (-) Win 44441 suggested to bind principally with κ receptors did not enhance either superficial or deep nociception indicating that the former antagonists are probably interfering with endorphins at the level of μ receptors. The motor reaction of mice to a novel environment was stereospecifically depressed by opioid antagonists including (-) Win 44441 suggesting an involvement of endorphins at the level of κ receptors ; Mr 2266 and GPA 2163 were ineffective in this test and hyperalgesic in the two antinociceptive tests ; they might be relatively pure μ antagonists.     

Characterization of membrane association of Rinderpest virus matrix protein

Paramyxovirus matrix protein is believed to play a crucial role in the assembly and maturation of the virus particle by bringing the major viral components together at the budding site in the host cell. The membrane association capability of many enveloped virus matrix proteins has been characterized to be their intrinsic property. In this work, we have characterized the membrane association of Rinderpest virus matrix (M) protein. The M protein of Rinderpest virus when expressed in the absence of other viral proteins is present both in the cytoplasm and plasma membrane. When expressed as GFP fusion protein, the M protein gets localized into plasma membrane protrusions. High salt and alkaline conditions resulted in partial dissociation of M protein from cell membrane. Thus, M protein behaves like an integral membrane protein although its primary structure suggests it to be a peripheral membrane protein.     

Effects of N-methylnaloxone and N-methylnaltrexone on nociception and precipitated abstinence in mice

Subcutaneous administrations of naloxone and naltrexone have already been shown to enhance nociceptive reactions in mice. The present study was undertaken to examine the effects of N-methyl-naloxone and N-methylnaltrexone on nociception using the hot plate test (dose range: 0.3 to 30 mg kg-1s.c.). The latter compounds were selected to differentiate the central and peripheral components of hyperalgesia. Unlike naloxone, N-methyl-naloxone did not produce hyperalgesia. Similarly low doses of N-methylnaltrexone did not enhance the jumping response. However, a high dose of N-methylnaltrexone (30 mg kg-1 s.c.) significantly reduced the jumping latencies 2 h after its administration. This phenomenon indicated that it might be converted to an active metabolite. Further, N-methylnaloxone and N-methylnaltrexone were very weak in precipitating the signs of abstinence in mice rendered acutely dependent on morphine. Two factors, poorer penetration into the CNS and steric hindrance, might render the N-methylated antagonists weak. Hence, both these factors should be considered when interpreting the effects after quaternary derivatives of opioid antagonists.     

Processing of the intermediate form of the iron-sulfur protein of the BC1 complex to the mature form after import into yeast mitochondria.

The Rieske iron-sulfur protein of the cytochrome bc1 complex is synthesized in the cytosol as a precursor with an additional 30 amino acids at the amino terminus. After import into the mitochondrial matrix, the precursor is processed to the mature form by two distinct proteolytic cleavages. Addition of 2.5 mM EDTA and 0.5 mM o-phenanthroline to the incubation mixture during import of the iron-sulfur protein precursor in vitro resulted in the selective inhibition of the second processing step with the concomitant accumulation of the intermediate form. The intermediate form was chased to the mature form in the presence of antimycin and oligomycin (to block the formation of a membrane potential) provided that 0.5 nM ATP and a metal ion such as Ca2+, Mn2+, or Mg2+ were added. Ca2+ ion was the most effective and at a concentration of 2.5 mM resulted in the complete cleavage of the intermediate to the mature form. Addition of Zn2+, Co2+, Mo2+, and Fe2+ was not effective in restoring the second cleavage. The pH optimum for the processing of the intermediate form of the iron-sulfur protein to the mature form was between 6.8-8.0. Processing of the intermediate form of the iron-sulfur protein to the mature form was observed at temperatures ranging from 12 to 27 degrees C in a temperature-dependent manner. The time course during the chase indicated that the second processing step was completed within 2 min after addition of Ca2+ ions. Attempts to isolate the second processing enzyme by sonication of mitochondria or by solubilization with detergents such as digitonin, Triton X-100, dodecyl-maltoside, or octyl-glucoside were unsuccessful as only the first cleavage was observed. Hence, the second processing enzyme may be present in the inner membrane or matrix in a conformation disrupted by detergents or alternatively the enzyme may be very labile.     

Assembly of Deletion Mutants of the Rieske Iron–Sulfur Protein into the Cytochrome bc 1 Complex of Yeast Mitochondria

The assembly of two deletion mutants of the Rieske iron-sulfur protein into the cytochrome bc ₁ complex was investigated after import in vitro into mitochondria isolated from a strain of yeast, JPJ1, from which the iron-sulfur protein gene (RIP) had been deleted. The assembly process was investigated by immunoprecipitation of the labeled iron-sulfur protein or the two deletion mutants from detergent-solubilized mitochondria with specific antisera against either the iron-sulfur protein or the bc ₁ complex (complex III) [Fu and Beattie (1991). J. Biol. Chem. 266, 16212–16218]. The deletion mutants lacking amino acid residues 55–66 or residues 161–180 were imported into mitochondria in vitro and processed to the mature form via an intermediate form. After import in vitro, the protein lacking residues 161–180 was not assembled into the complex, suggesting that the region of the iron-sulfur protein containing these residues may be involved in the assembly of the protein into the bc ₁ complex; however, the protein lacking residues 55–66 was assembled in vitro into the bc ₁ complex as effectively as the wild type iron-sulfur protein. Moreover, this mutant protein was present in the mitochondrial membrane fraction obtained from JPJ1 cells transformed with a single-copy plasmid containing the gene for this protein lacking residues 55–66. This deletion mutant protein was also assembled into the bc ₁ complex in vivo, suggesting that the hydrophobic stretch of amino acids, residues 55–66, is not required for assembly of the iron-sulfur protein into the bc ₁ complex; however, this association did not lead to enzymatic activity of the bc ₁ complex, as the Rieske FeS cluster was not epr detectable in these mitochondria.     

Mutation@A Glance: An Integrative Web Application for Analysing Mutations from Human Genetic Diseases

Although mutation analysis serves as a key part in making a definitive diagnosis about a genetic disease, it still remains a time-consuming step to interpret their biological implications through integration of various lines of archived information about genes in question. To expedite this evaluation step of disease-causing genetic variations, here we developed Mutation@A Glance (http://rapid.rcai.riken.jp/mutation/), a highly integrated web-based analysis tool for analysing human disease mutations; it implements a user-friendly graphical interface to visualize about 40 000 known disease-associated mutations and genetic polymorphisms from more than 2600 protein-coding human disease-causing genes. Mutation@A Glance locates already known genetic variation data individually on the nucleotide and the amino acid sequences and makes it possible to cross-reference them with tertiary and/or quaternary protein structures and various functional features associated with specific amino acid residues in the proteins. We showed that the disease-associated missense mutations had a stronger tendency to reside in positions relevant to the structure/function of proteins than neutral genetic variations. From a practical viewpoint, Mutation@A Glance could certainly function as a ‘one-stop’ analysis platform for newly determined DNA sequences, which enables us to readily identify and evaluate new genetic variations by integrating multiple lines of information about the disease-causing candidate genes.     

Synthesis and biological evaluation of the major metabolite of atomoxetine: elucidation of a partial kappa-opioid agonist effect

The major human metabolite of atomoxetine (4-hydroxyatomoxetine) was tested against a panel of receptors and enzymes, and was found to interact with the mu, delta, and kappa-opioid receptors based upon studies involving both binding and functional assays. 4-hydroxyatomoxetine was determined to be a partial agonist of the kappa-opioid receptor.