Examination of the requirement for an amphiphilic helical structure in beta-endorphin through the design, synthesis, and study of model peptides

In our approach to beta-endorphin modeling, we have proposed that the biological properties of the natural peptide are determined by the combination of three basic structural units: a highly specific opiate recognition sequence at the NH2 terminus (residues 1-5) connected via a hydrophilic peptide link (residues 6-12) to a potential amphiphilic helix in the COOH-terminal residues 13-31. In the alpha-helical conformation the hydrophobic domain twists around the length of the helix and covers almost one-half of its surface. The other distinctive features of the helix include its basicity and the two aromatic residues Phe18 and Tyr27. In contrast to previous models we have studied, peptide 4 is a "negative" model in the sense that it was designed and examined in order to determine how the lack of a well defined amphiphilic structure affects the biological properties of beta-endorphin. For this purpose, peptide 4 retains the three structural units previously postulated for beta-endorphin, but the amino acids of the 13-31 region are arranged in such a way that no definite continuous hydrophobic zone could be formed in an alpha- or pi-helical conformation of this region. In aqueous buffered solutions, peptide 4 showed almost the same amount of alpha-helical structure as beta-endorphin, with a slight tendency toward less helicity in 50% aqueous 2,2,2-trifluoroethanol. In rat brain homogenate, peptide 4 was degraded slightly slower than beta-endorphin, in contrast to the apparently much higher stability of previous models under the same conditions. With regard to opiate receptor binding, peptide 4 was twice as potent as beta-endorphin in mu-receptor assays but half as potent in delta-receptor assays. The opiate potency of peptide 4 on the guinea pig ileum was higher than that of beta-endorphin. In contrast, in the rat vas deferens assay, which is very specific for beta-endorphin, the potency of peptide 4 was very low and could be shown not to be mediated by the same opiate mechanism or by the same opiate receptor. A comparison of these results with those of previous model peptides provides further evidence for the importance of an amphiphilic helical structure in beta-endorphin residues 13-31, which determines the resistance to proteolysis of the natural molecule and contributes to the delta- and mu-opiate receptor interaction. The amphiphilicity of this helical structure must also be essential for high opiate activity on the rat vas deferens (epsilon-receptors), whereas no such structural requirement appears to be necessary for interaction with the opiate receptors on the guinea pig ileum.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism VI. The Influence of the Epiphytic Bacteria on the Content of Extractable Auxin in the Plant

Sterile plants of maize, pea, and cucumber contain less auxin (extracted with methanol or ether) than nonsterile ones. The auxin content is restored within one day by reinfecting sterile plants (or only the shoots, with roots and culture medium remaining sterile) with epiphytic bacteria strains able to produce IAA or with soaking water of nonsterile seeds. Reinfection with bacteria, strains unable to produce IAA is ineffective. — The possibility of a bacterial auxin production during methanol extraction was excluded.     

Studies on the control of mitotic activity

Summary A tetraploid cell population was produced in the primary root meristem of Pisum sativum by one-half hour treatments with various concentrations of colchicine. The tetraploid population so produced was found to be reasonably synchronous in its passage through successive mitotic cycles with the degree of synchrony being more or less proportional to the concentration of colchicine used. The average time between mitoses appears to be of the order of 12 hours which agrees well with previous estimates. Treatments of roots containing tetraploid populations with 2.52 × 10^–5 M. actidione for 15 minutes were used to demonstrate the possibility of using the system for studies on the differential susceptibility of cells at different stages of the mitotic cycle.This work was carried out under contract number RG-4835 of the National Institutes of Health, United States Public Health Service, and an Institutional Grant from the American Cancer Society.Contribution number 59-26 of the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Michigan.Predoctoral Fellow (CF-9871) of the National Cancer Institute, United States Public Health Service     

dsg, a gene required for cell-cell interaction early in Myxococcus development.

dsg mutants of Myxococcus xanthus are conditionally defective in fruiting body development, including sporulation. Unable to develop on their own, these mutants can assemble fruiting bodies with spores if they are mixed with wild-type cells. To elucidate the developmental defect in dsg mutants by close comparison with wild type, such mutants have been backcrossed by transduction, using a closely linked insertion of transposon Tn5 for selection. Backcrossed dsg mutants form aggregates that are larger, less compact, and less symmetrical than dsg+ fruiting bodies. Also, the starvation-induced sporulation in dsg aggregates is delayed and reduced. However, dsg mutants can be induced by glycerol or dimethyl sulfoxide to sporulate at levels approaching those of wild type. dsg mutants may thus have a primary defect early in development which diminishes their capacity to aggregate and which indirectly decreases the number of fruiting body spores. The linked insertion of Tn5 also facilitated cloning the dsg gene. The cloned dsg+ allele was shown to be dominant to both the dsg-429 and dsg-439 alleles, and both mutant alleles were shown to belong to the same genetic complementation group. Subcloning of restriction fragments, deletions, and insertions of transposon Tn5 agree in locating the dsg gene to an 850-base-pair segment of the cloned region.     

Separation and characterization of isoforms of DT-diaphorase from rat liver cytosol.

Rats were treated with 3-methylcholanthrene (MC) and DT-diaphorase from liver was partially purified on an azodicoumarol-Sepharose 6B column and applied to an FPLC-chromatofocusing column in order to resolve isoforms. Six peaks showing significant DT-diaphorase activity were eluted from this column with a pH gradient between 7.30 to 4.80. The amino acid compositions of the two major peaks (II and VIb) were found to be nearly identical, suggesting existence of isoforms rather than isozymes of DT-diaphorase. The isoforms of DT-diaphorase showed broad substrate specificities towards four different quinones (menadione, vitamin K-1, benzo(a)pyrene 3,6-quinone and cyclized-dopamine ortho-quinone), although quantitative differences in the specific activities were also found. All isoforms are glycoproteins but contain different carbohydrates. Thus isoform II reacts with biotinylated lectins which are specific for N-acetylgalactosamine, mannose, fucose and galactosyl(beta-1,3)N-acetylgalactosamine, while isoform VIb reacts only with biotinylated lectins specific for mannose and N-acetylgalactosamine. Separation of DT-diaphorase isoforms from control rat liver cytosol using FPLC-chromatofocusing revealed that the induction of the isoforms is not uniform, since isform II was not found and the major isoform was composed of three peaks, whereas the major isoform of DT-diaphorase from liver cytosol of rats treated with 3-methylcholanthrene was composed of only two peaks.