Two-dimensional NMR investigation of iron-sulfur cluster electronic and molecular structure of oxidized Clostridium pasteurianum ferredoxin. Interpretability of contact shifts in terms of cysteine orientation.

A two-dimensional NMR study has been carried out on the four-iron clusters of a bacterial oxidized ferredoxin for the purpose of investigating the relationship between contact shift patterns and the orientation of the individual coordinated cysteines. The ferredoxin from Clostridium pasteurianum, CpFdox, was selected because of its extensive sequence homology, and likely close structural similarity, to the crystallographically characterized ferredoxin from Peptococcus aerogenes, Pa Fdox (Adman, E.T., Sieker, L.C., and Jensen, L. H. (1973) J. Biol. Chem. 248, 3987-3996). Rapid data collection rates with minimal but adequate acquisition time allowed the detection of numerous CpFdox cross-peaks from the contact-shifted and strongly relaxed coordinated cysteinyl C beta H protons in the resolved 10-20 ppm window. Relatively strong magnitude COSY cross peaks from the resolved eight cysteinyl C beta H resonance unambiguously locate the geminal C beta H partner for each residue; weaker cross-peaks locate the C alpha Hs from three of the residues. The geminal nature of the magnitude-COSY detected partners to the resolved C beta H peaks is confirmed by strong NOESY cross-peaks. The NOESY spectra, moreover, assign an additional two cysteinyl C alpha H resonances. The present results confirm some previous one-dimensional NOE assignments, revise others, and locate resonances previously undetected (Bertini, I., Briganti, F., Luchinat, C., and Scozzafara, A. (1990) Inorg. Chem. 29, 1874-1880). A striking pairwise pseudo-symmetry in cysteinyl contact shift patterns is observed which is attributed to the previously recognized pseudo-symmetry in the crystal of PaFdox. A detailed analysis of the structural/electronic determinants of the coordinated cysteine C beta H contact shift pattern is made, and the NMR data necessary for unique interpretation are identified. It is shown that analysis of the relaxation properties of cysteine beta-methylene protons provides the stereospecific assignments necessary for comparison of shift ratios with crystallographic structural data. The available structural data on PaFdox (Backes, G., Mino, Y., Loehr, T., Meyer, T., Cusanovich, M., Sweeney, W., Adman, E., and Sanders-Loehr, J. (1991) J. Am. Chem. Soc. 13, 2055-2064) are qualitatively but not quantitatively consistent with the observed cysteinyl contact shift pattern, with the NMR data reflecting more asymmetry than previous studies. A tentative assignment of a single pair of symmetry-related cysteines is proposed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tetracycline‐controlled expression of glycosylated porcine interferon‐gamma in mammalian cells

Abstract

Tetracycline‐controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon‐γ in the RK‐13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon‐γ (rGPoIFN‐γ) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN‐γ cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN‐γ (up to 16 μg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of ³⁵S‐Methionine, labelled rGPoIFN‐γ with natural leukocytic IFN‐γ after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN‐γ species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N‐glycosydase F. The biological activity of rGPoIFN‐γ was in the same range as that of natural leukocytic PoIFN‐γ (2 × 10⁶ U/mg). Eventually, this recombinant mammalian IFN‐γ should constitute a very useful substitute for leukocyte PoIFN‐γ in in vitro or in vivo experiments.     

Processing of naked 45-S ribosomal RNA precursor in vitro by an RNA-associated endoribonuclease

A processing endoribonuclease was isolated from the cytoplasm of chick embryos. The enzyme was easily obtained using an RNA extraction procedure based on a mild deproteinization with Sarkosyl and cold phenol/chloroform. This technique assured the recovery of several proteins and the endoribonuclease in association with the RNA. It was demonstrated that this endoribonuclease was capable of promoting, in vitro, a precise processing of naked 45-S ribosomal RNA precursor to molecules resembling the intermediates as well as the 28-S and 18-S cytoplasmic RNAs found in vivo. The presence of magnesium ions was required for the correct processing function of the enzyme. In addition, under the same conditions, the mature ribosomal RNA substrates were degraded at a slower rate by this RNA-associated RNase. It was possible to fractionate the enzymatic preparation into two different populations by means of a sucrose gradient: one associated and the other partially free of an RNA component. The effect of the intrinsic RNA associated with the endoribonuclease on the enzymatic activity was tested by analyzing both the enzymatic populations and the total enzymatic preparation treated with pronase or with immobilized pancreatic RNase. In all cases in which the RNA component was present, the enzyme showed processing activity. On the other hand, when the RNA component was absent or at least partially degraded the enzyme proved to be more active in processing precursor molecules and in promoting extensive degradation of mature RNA species. Although the presence of RNA in association with the enzyme was demonstrated, its role in the regulation of the enzymatic activity is yet not clear.     

Intracellular Ca2+ concentration and the antidiuretic hormone-induced increase in water permeability: effects of ionophore A23187 and quinidine

The hydroosmotic responses induced by oxytocin and 8-bromo-cyclic AMP, in frog and toad urinary bladders, were recorded minute by minute. 3HHO and 45Ca unidirectional fluxes as well as prostaglandin B2 liberation were also measured. It was observed that: (1) Addition of the calcium ionophore A23187 or quinidine to the serosal bath inhibited the response to oxytocin, but not to 8-bromo-cyclic AMP, while increasing prostaglandin E1 liberation into the serosal but not into the mucosal bath. (2) Addition of A23187 to the mucosal bath induced a transient and temperature-dependent inhibition of the response elicited by 8-bromo-cyclic AMP. The time-course of this reduction in water permeability and its sensitivity to medium temperature were similar to those observed after the withdrawal of agonist, but clearly different of those observed after intracellular acidification. (3) The hydroosmotic response was also transitorily inhibited when the Ca2+ concentration was step-changed in the mucosal bath. (4) When added to the mucosal or to the serosal baths, the ionophore increased either the apical or the laterobasal Ca2+ permeabilities. It is concluded that manipulation of intracellular Ca2+ interferes with the hydroosmotic response at two different levels. (1) A first target point located 'pre-cyclic-AMP production'. This effect would be mediated by prostaglandin liberation. (2) A second target point located after cyclic AMP production and before the 'temperature-dependent rate-limiting step'. This effect is probably related to the mechanism controlling the insertion and removal of water channels.     

The Caenorhabditis elegansRAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes

In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule. Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms. We have cloned the homolog of RAD51 in Caenorhabditis elegans. The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively. We discuss the evolutionary implications of these findings. Received: 26 May 1998 / Accepted: 18 August 1998     

Defibrotide decreases cholesterol amount in hypercholesterolemic rabbit aorta, with no modification of plasma or lipoprotein cholesterol

Defibrotide, (D) an antithrombotic agent, when administered i.v. to cholesterol-fed rabbits decreased cholesterol in the aorta without changing total plasma cholesterol, triglyceride or phospholipid, nor the cholesterol, triglyceride, phospholipid and protein of plasma lipoproteins. Platelet aggregation was decreased in rabbits treated with D. There were fewer vascular lesions in the hearts and kidneys of animals treated with D than in animals fed cholesterol and treated with placebo. These data suggest that the antithrombotic activity of D and its ability to reduce platelet sensitivity could help to reduce the amount of cholesterol in the cardiovascular system in atherosclerosis-prone situations.