Mono and two-dimensional 500-MHz characterization of synthetic bombesin and related peptides in DMSO and DMSO-water

The proton nmr characterization of bombesin (BBS) and of two peptide fragments corresponding to the (1-6) and (6-14) sequences has been carried out at 500 MHz in dimethyl sulfoxide (DMSO-d6) using two-dimensional (2D) homo and 1H-13C heterocorrelated techniques. All resonances in the nmr spectra have been assigned and several coupling constants have been measured. The backbone J alpha CH-NH coupling constants are quite similar and around 7.8-8.2 Hz, pointing to an unfolded structure in DMSO-d6. The possibility of secondary structures in highly viscous mixtures of DMSO-d6-water was investigated. The existence of sequential nuclear Overhauser enhancement (NOE) effects in the C-terminal nonapeptide section may indicate a preferential site for secondary structuring.     

Genomic distribution of copia-like transposable elements in somatic tissues and during development of Drosophila melanogaster

The genomic distribution of elements of the copia, 412, B 104, mdg 1, mdg 4 and 1731 transposon families was compared by the Southern technique in DNA preparations extracted from brains, salivary glands and adult flies of two related Drosophila lines. The copia, 412 and mdg 1 sequences were also probed in DNA from sperm, embryos, and 1st and 2nd instar larvae. The homogeneity of the patterns observed shows that somatic transposition is unlikely to occur frequently. A correlation between mobility and the euchromatic or heterochromatic location of transposable elements is discussed. In addition, an explanation of the variable band intensities of transposable elements in Southern autoradiographs is proposed.     

N-terminal region of Proteus mirabilis glutathione transferase is not homologous to mammalian and plant glutathione transferases

The N-terminal amino acid sequence of glutathione transferase, Pm-GST-6.0, purified from Proteus mirabilis [(1988) Biochem. J. 255, 971-975] up to residue 38 and a comparative peptide fingerprint are reported. No obvious homology with the sequences of alpha, pi and mu classes of mammalian glutathione transferases as well as with those of plant glutathione transferases has been noted. These results suggest that the classification so far adopted for glutathione transferases cannot be extended to the bacterial enzyme.     

Assay of L-3-hydroxyacyl-coenzyme A dehydrogenase with substrates of different chain lengths

A method for assaying L-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) which permits rate measurements with L-3-hydroxyacyl-CoA substrates of various chain lengths at physiological pH is described. The method is based on a coupled assay system in which 3-ketoacyl-CoA compounds formed by the dehydrogenase are cleaved by 3-ketoacyl-CoA thiolase (EC 2.3.1.16) in the presence of CoASH. The advantages of this assay method are its irreversibility and elimination of product inhibition. The assay procedure was used to determine the kinetic parameters (Km, Vmax) of pig heart L-3-hydroxyacyl-CoA dehydrogenase with several substrates of various chain lengths. The data obtained show the enzyme to be most active with medium-chain substrates whereas Km values for medium-chain and long-chain substrates are almost equal but much lower than those previously reported.     

Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria

The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.     

Tryptophan environments in glutathione transferase of human placenta from temperature-dependent phosphorescence studies

An investigation of the tryptophan emission properties of glutathione transferase from human placenta was conducted in order to characterize the environments of the two aromatic residues. The low-temperature phosphorescence spectra and temperature dependence of the phosphorescence quantum yield of the tryptophan residues revealed a difference in the chemical nature and dynamical structure of the surrounding protein matrix. Thus, one tryptophan residue seems to be deeply embedded within the polypeptide in a rigid weakly polar environment, characteristic of a beta-type secondary structure. The other is located in a more polar site, probably near the surface, in a rather flexible region of the macromolecule. At high temperature, the heterogeneity in the triplet lifetime of the internal residue attests to the presence of multiple conformers which are not in rapid equilibrium in the phosphorescence time scale. The anisotropy of the phosphorescence emission of glutathione transferase indicates that no energy transfer occurs between the two residues, and measurement of the rotational correlation time yields an hydrodynamic volume which is in good agreement with the molecular weight reported in the literature for the dimer.     

Influence of lactate on isoproterenol-induced lipolysis and beta-adrenoceptors distribution in human fat cells

The influence of lactate on human adipocytes lipolysis and the possible relationship between lactate-induced metabolic effects and beta-adrenoceptor binding sites were investigated. beta-sites were identified in membranes with (125I)-cyanopindolol and in intact cells with (125I)-cyanopindolol and (3H)-CGP 12177. Lactate reduced isoproterenol-induced lipolysis in a dose-response fashion and such inhibition became significant only at 16 mmol/l lactate. Exposure of human fat cells to 16 mmol/l lactate significantly reduced beta-adrenoceptors density on crude membranes. When the binding assay was performed on intact cells using (125I)-cyanopindolol at 37 degrees C, the radioligand identified the same number of receptors, regardless of the presence of lactate in the preincubation medium. When (3H)-CGP 12177 was used, it bound to about 35% less receptors in lactate pre-treated cells than in control. Seemingly, at 37 degrees C, because of its lipophilicity, (125I)-cyanopindolol can cross the plasma membrane and bind to intracellular sites whereas, (3H)-CGP 1277, due to its hydrophilicity, identifies surface receptors only. Thus, the present in vitro study provides evidence that high levels of lactate, similar to the concentrations usually achieved in overt lactic acidosis, are able per se to inhibit human lipolysis and to redistribute beta-adrenoceptors from cell surface to a domain not accessible to hydrophilic ligands.     

Characterization and molecular cloning of Bifidobacterium longum cryptic plasmid pMB1

The small cryptic plasmid pMB1 (1.9 kb), previously isolated from Bifidobacterium longum, has been characterized by physical mapping. Two cloning vectors, pMR3 and pDG7, carrying chloramphenicol and ampicillin resistances derived from pJH101, have been electroporated in Escherichia coli.     

Transformation of NIH 3T3 cells by overexpression of the normal coding sequence of the rat neu gene.

While the normal human erbB-2 gene is potently transforming when overexpressed in NIH 3T3 cells, its rat homolog, the neu gene, seems to acquire transforming properties only upon alteration of its coding sequence. In this study, we compared the effects of different levels of expression of normal erbB-2 and neu in NIH 3T3 cells. Our results revealed that the normal rat neu gene acts as a potent oncogene when sufficiently overexpressed in NIH 3T3 cells.