Detection of a quaternary organization into dimer of trimers of Corynebacterium ammoniagenes FAD synthetase at the single-molecule level and at the in cell level

Biochemical characterization of Corynebacterium ammoniagenes FADS (CaFADS) pointed to certain confusion about the stoichiometry of this bifunctional enzyme involved in the production of FMN and FAD in prokaryotes. Resolution of its crystal structure suggested that it might produce a hexameric ensemble formed by a dimer of trimers. We used atomic force microscopy (AFM) to direct imaging single CaFADS molecules bound to mica surfaces, while preserving their catalytic properties. AFM allowed solving individual CaFADS monomers, for which it was even possible to distinguish their sub-molecular individual N- and C-terminal modules in the elongated enzyme. Differences between monomers and higher stoichiometries were easily imaged, enabling us to detect formation of oligomeric species induced by ligand binding. The presence of ATP:Mg^2 + particularly induced the appearance of the hexameric assembly whose mean molecular volume resembles the crystallographic dimer of trimers. Finally, the AFM results are confirmed in cross-linking solution, and the presence of such oligomeric CaFADS species detected in cell extracts. All these results are consistent with the formation of a dimer of trimers during the enzyme catalytic cycle that might bear biological relevance.     

Peptide antibiotics in action: Investigation of polypeptide chains in insoluble environments by rotational-echo double resonance

Rotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus.     

Study of p53 expression and post‐transcriptional modifications after GSM‐900 radiofrequency exposure of human amniotic cells

The potential effects of radiofrequency (RF) exposure on the genetic material of cells are very important to determine since genome instability of somatic cells may be linked to cancer development. In response to genetic damage, the p53 protein is activated and can induce cell cycle arrest allowing more time for DNA repair or elimination of damaged cells through apoptosis. The objective of this study was to investigate whether the exposure to RF electromagnetic fields, similar to those emitted by mobile phones of the second generation standard, Global System for Mobile Communications (GSM), may induce expression of the p53 protein and its activation by post‐translational modifications in cultured human cells. The potential induction of p53 expression and activation by GSM‐900 was investigated after in vitro exposure of human amniotic cells for 24 h to average specific absorption rates (SARs) of 0.25, 1, 2, and 4 W/kg in the temperature range of 36.3–39.7 °C. The exposures were carried out using a wire‐patch cell (WPC) under strictly controlled conditions of temperature. Expression and activation of p53 by phosphorylation at serine 15 and 37 were studied using Western blot assay immediately after three independent exposures of cell cultures provided from three different donors. Bleomycin‐exposed cells were used as a positive control. According to our results, no significant changes in the expression and activation of the p53 protein by phosphorylation at serine 15 and 37 were found following exposure to GSM‐900 for 24 h at average SARs up to 4 W/kg in human embryonic cells. Bioelectromagnetics 34:52–60, 2013. © 2012 Wiley Periodicals, Inc.     

Synthesis of a human insulin gene. VII. Synthesis of preproinsulin-like human DNA, its cloning and expression in M13 bacteriophage

A 74-bp DNA sequence coding for the pre sequence of human preproinsulin and containing EcoRI termini was synthesized by the chemical enzymatic method, joined with previously synthesized proinsulin DNA, and cloned in the M 13mp8 vector. A clone pNB82 -121 was identified by DNA sequence which confirmed the correct orientation of the pre sequence to the proinsulin DNA. The EcoRI site at the junction of pre- and proinsulin DNA was eliminated by removing a triplet ATT using a synthetic 19-mer primer. To simplify preproinsulin isolation and to study its expression in the M 13 system, a 25-bp affinity leader sequence coding for (glu)7 was inserted at the remaining EcoRI site; this put the preproinsulin DNA in a correct reading frame with the AUG initiation codon of beta-galactosidase. Preproinsulin was expressed under lac promoter control as analyzed by a radioimmunoassay (RIA) against C-peptide.     

Determination of erythrocyte surface sialic acid residues by a new colorimetric method.

The 3-methyl-2-benzothiazolone hydrazone method has been applied to the determination of erythrocyte membrane sialic acid residues. This method requires a mild oxidation of sialic acid which results in the formation of analogs. Their separation by chromatography, after labeling, allows the choice of the best conditions for this oxidation. The concomitant liberated formaldehyde is determined. This method requires no prior release of sialic acid as opposed to the periodate-thiobarbituric method of Warren (1959, J. Biol. Chem., 234, 1971–1975). These two methods have been compared.     

Negatively supercoiled DNA from plants infected with a single-stranded DNA virus

A method for isolating covalently closed circular double-stranded DNA from plants infected with the geminivirus, tomato golden mosaic virus, is described. Ethidium bromide titration showed this DNA to be negatively supercoiled with a superhelical density of -0.062. The presence of S1 nuclease-sensitive secondary structure in the supercoiled DNA was demonstrated by its conversion to the open circular and linear DNA forms on treatment with this enzyme.