Infant leukaemia biology, aetiology and treatment

A method for the determination of aspartame (N-L-alpha-aspartyl-L-phenylalanine methyl ester) and its metabolites, applicable on a routine quality assurance basis, is described. Liquid samples (diet Coke, 7-Up, Pepsi, etc.) were injected directly onto a mini-cartridge reversed-phase column on a high-performance liquid chromatographic system, whereas solid samples (Equal, hot chocolate powder, pudding, etc.) were extracted with water. Optimising chromatographic conditions resulted in resolved components of interest within 12 min. The by-products were confirmed by mass spectrometry. Although the method was developed on a two-pump HPLC system fitted with a diode-array detector, it is straightforward and can be transformed to the simplest HPLC configuration. Using a single-piston pump (with damper), a fixed-wavelength detector and a recorder/integrator, the degradation of products can be monitored as they decompose. The results obtained were in harmony with previously reported tedious methods. The method is simple, rapid, quantitative and does not involve complex, hazardous or toxic chemistry.     

Breast micro-opacities of lead origin mimicking microcalcifications

After a surgical excision of focal mammary "microcalcification" we noticed that in fact they represented fine lead deposits (mean diameter: 100 microns) resulting from an attempted suicide (22 long rifle) 10 years previously. The leaded nature of these foreign microbodies was demonstrated by a micro-analysis by energy dispersion on histological slides.     

Production of human normal adult and fetal hemoglobins in Escherichia coli

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha- globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma- globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.      

Membrane topology of the Rickettsia prowazekii ATP/ADP translocase revealed by novel dual pho-lac reporters

Here, we report the construction and characterization of dual reporters, consisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane protein topology by the gene fusion approach. Each of the reporters, when fused to periplasmic domains of polytopic proteins, produces fusions with high AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E. coli strains capable of alpha-complementation. The dual nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of topological data. Deleterious effects resulting from the cell's attempt to export the full-length BG are eliminated in this approach. We describe dual indicator plates that allow for discrimination between colonies bearing cytoplasmic fusions, periplasmic fusions, and no fusions. We have generated a set of fusions to the topologically well-studied lactose permease of E. coli and demonstrated that topological information generated by these new reporters is in good agreement with the existing model. We used this new methodology for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc). Our results were in agreement with the proposed in silico topological model in which Tlc traverses the cytoplasmic membrane of E. coli 12 times with its N and C termini facing the cytoplasm.     

DNA polymerase fidelity: from genetics toward a biochemical understanding

This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful-first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.