Effects of stress and beta-funal trexamine pretreatment on morphine analgesia and opioid binding in rats

This study was essentially an in vivo protection experiment designed to test further the hypothesis that stress induces release of endogenous opioids which then act at opioid receptors. Rats that were either subjected to restraint stress for 1 hr or unstressed were injected ICV with either saline or 2.5 micrograms of beta-funaltrexamine (beta-FNA), an irreversible opioid antagonist that alkylates the mu-opioid receptor. Twenty-four hours later, subjects were tested unstressed for morphine analgesia (tail-flick assay) or were sacrificed and opioid binding in brain was determined. [3H]D-Ala2NMePhe4-Gly5(ol)enkephalin (DAGO) served as a specific ligand for mu- opioid receptors, and [3H]-bremazocine as a general ligand for all opioid receptors. Rats injected with saline while stressed were significantly less sensitive to the analgesic action of morphine 24 hr later than were their unstressed counterparts. Beta-FNA pretreatment attenuated morphine analgesia in an insurmountable manner. Animals pretreated with beta-FNA while stressed were significantly more sensitive to the analgesic effect of morphine than were animals that received beta-FNA while unstressed, consistent with the hypothesis that stress induces release of endogenous opioids that would protect opioid receptors from alkylation by beta-FNA. beta-FNA caused small and similar decreases in [3H]-DAGO binding in brain of both stressed and unstressed animals. Stressed rats injected with saline tended to have increased levels of [3H]DAGO and [3H]-bremazocine binding compared to the other groups. This outcome may be relevant to the tolerance to morphine analgesia caused by stress.     

Characterization of the gene for fructose-1,6-bisphosphatase from Saccharomyces cerevisiae and Schizosaccharomyces pombe. Sequence, protein homology, and expression during growth on glucose

We have determined the nucleotide sequence of the gene for fructose-1,6-bisphosphatase from both Saccharomyces cerevisiae and Schizosaccharomyces pombe. The predicted protein sequence for fructose-1,6-bisphosphatase from S. cerevisiae contains 347 amino acids and has a molecular weight of 38,100; that from S. pombe, contains 346 amino acids and has a molecular weight of 38,380. Comparison of these amino acid sequences with each other and that of pig kidney fructose-1,6-bisphosphatase shows several regions of strong homology separated by regions of divergence. These homologous regions are likely candidates for functional domains. A gene cassette was constructed for fructose-1,6-bisphosphatase from S. cerevisiae and the gene cassette expressed from the regulated PHO5 and GAL1 promoters of yeast. Yeast cells expressing fructose-1,6-bisphosphatase, while growing on glucose, accumulated large amounts of enzyme intracellularly, suggesting that glucose-regulated proteolytic inactivation does not operate efficiently under these conditions. Growth on glucose was not inhibited by the expression of fructose 1,6-bisphosphatase.     

Purification of an active opioid-binding protein from bovine striatum

We report the purification to apparent homogeneity of an active opioid-binding protein solubilized from bovine striatal membranes. The purification was accomplished in two steps: affinity chromatography on beta-naltrexylethylenediamine (NED)-CH-Sepharose 4B followed by lectin affinity chromatography on wheat germ agglutinin-agarose. The ligand affinity-purified fraction exhibits stereospecific and saturable binding of opiates and is heat-sensitive. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the NED-purified material gave 6-8 bands by silver staining or autoradiography of radioiodinated material. Under nondenaturing conditions, the NED-purified material elutes in a molecular mass range between 300 and 350 kDa from gel exclusion chromatography on Ultrogel AcA-34. The specific activity of the affinity-purified fraction (800-1500 pmol/mg protein) is enriched 4000 to 7000-fold over that of the membrane-bound or unpurified soluble receptor. Further purification (10-20-fold) is achieved by chromatography of the NED eluate on wheat germ agglutinin-agarose. The eluted fraction shows a single protein (65 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified material is an acidic glycoprotein with a pI of 6.0-6.3 and binds opiates with a specific activity (12,000-15,000 pmol/mg) that is 65,000 to 75,000-fold greater (theoretical, 77,000-fold) than that of the membrane-bound or crude soluble receptors.     

Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Licht- und elektronenmikroskopische untersuchungen zur haftborstenentwicklung bei Tarentola m. mauritanica L. (Reptilia,Gekkonidae)

Zusammenfassung Kurz nach einer Hdutung wird bei Tarentola m. m. bereits die übemächste Epidermisgeneration — und somit auch die der Haftborsten —angelegt. Das geschieht vornehmlich in der Oz- (Oberhäutchenzellen-) und in der Hs-Schicht (clear layer). Zunächst entstehen die Aufspaltungen der Haftborstenenden, indem Keratinfilamentbündel nach einem bestimmten System von den Oz-Zellen aus in die Hs-Zellen einwachsen. Auf these Weise fungieren die Zellen der Hs-Schicht als Matrix der Haftborsten. Nach Abschluß dieses Prozesses werden die eigentlichen Haftborsten gebildet unter gleichzeitigem Auseinanderrücken der Hs- und Oz-Schichten. Die Hs-Schicht behdlt ihre Matrizen-Funktion bis zur anschließenden Häutung bei.

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Light and electron microscope studies of developing setae of Tarentola m. mauritanica (Rept., Gekkonidae)

Summary In Tarentola m. mauritanica the next epidermis generation but one and therefore the adhesive setae of the generation after this begin to develop shortly after a skin has been shed. This development takes place principally in the horny layer (Oz) and the clear layer. First bundles of keratin filaments radiate from the horny layer into the clear layer, thus giving rise to the split distal parts of the adhesive bristles. Thus the cells in the clear layer act as a matrix for the setae. When this stage is complete the formation of the setae proper begins, while the horny layer and the clear layer become separated from each other. The clear layer retains its function as matrix for the setae until the next time a skin is shed.

     

Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation

The mobility of purified mu opioid binding protein in SDS-polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration-dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, mu greater than delta much greater than kappa. For mu receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.     

Cell retention-chemostat studies of hybridoma cells-analysis of hybridoma growth and metabolism in continuous suspension culture in serum-free medium

The steady-state metabolic parameters for a hybridoma cell line have been determined in continuous suspension-perfusion culture over a wide range of perfusion rates and cell bleed rates. Significant increases in viable cell concentrations and volumetric productivities were achieved at high perfusion rates and low cell bleed rates. At the low growth rates examined in this study, cellular metabolism shifted to become more oxidative, and as a result, the fraction of consumed substrate converted to inhibitory metabolic by-products was reduced. Specific antibody productivity was found to be non-growth associated. (c) 1993 John Wiley & Sons, Inc.     

Vph6 Mutants of Saccharomyces Cerevisiae Require Calcineurin for Growth and Are Defective in Vacuolar H(+)-Atpase Assembly

We have characterized a Saccharomyces cerevisiae mutant strain that is hypersensitive to cyclosporin A (CsA) and FK506, immunosuppressants that inhibit calcineurin, a serine-threonine-specific phosphatase (PP2B). A single nuclear mutation, designated cev1 for calcineurin essential for viability, is responsible for the CsA-FK506-sensitive phenotype. The peptidyl-prolyl cis-trans isomerases cyclophilin A and FKBP12, respectively, mediate CsA and FK506 toxicity in the cev1 mutant strain. We demonstrate that cev1 is an allele of the VPH6 gene and that vph6 mutant strains fail to assemble the vacuolar H(+)-ATPase (V-ATPase). The VPH6 gene was mapped on chromosome VIII and is predicted to encode a 181-amino acid (21 kD) protein with no identity to other known proteins. We find that calcineurin is essential for viability in many mutant strains with defects in V-ATPase function or vacuolar acidification. In addition, we find that calcineurin modulates extracellular acidification in response to glucose, which we propose occurs via calcineurin regulation of the plasma membrane H(+)-ATPase PMA1. Taken together, our findings suggest calcineurin plays a general role in the regulation of cation transport and homeostasis.     

Analysis of the HLA-ABC linkage disequilibrium: Decreasing strength of gametic association with increasing map distance

Summary 1242 HLA-ABC haplotypes of the North German population (Hamburg) as deduced by family analyses are described. They are in perfect agreement with recently published data by Mayr (1977) from Austria (Vienna) in all parameters tested: frequency of the single HLA-alleles, haplotype distribution and linkage disequilibrium values. Gametic association studies revealed that 69.4% of the B and C genes (map distance 0.2 cM) 36.9% of the A and C genes (0.6 cM), but only 23.2% of the A and B genes (0.8 cM) were significantly more often combined than expected due to their frequencies. From these findings it seems likely that the linkage disequilibrium within the MHC is rather due to a short evolutionary period than to selective forces. Some observations as to the most common European haplotype A1,B8 are discussed.