Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

PARATI – a dynamic model for radiological assessments in urban areas

The structure and mathematical model of PARATI, a detailed computer programme developed for the assessment of the radiological consequences of an accidental contamination of urban areas, is described with respect to the scenarios used for the estimation of exposure fields in a village or town, the models for the initial and secondary contamination with the radionuclide ¹³⁷Cs, the concepts for calculating the resulting radiation exposures and the changes with time of the contamination and radiation fields. Kerma rates at various locations in tropical urban areas are given, and the contribution of different contaminated surfaces to these rates after dry or wet deposition are discussed. Received: 12 April 1996 / Accepted in revised form: 30 August 1996     

Separation and Identification of the Polar Lipids of Chromatium Strain D

The polar lipids of the autotrophically grown, obligately anaerobic, photosynthetic bacterium Chromatium strain D were separated by paper chromatography. Four major phospholipids were identified: lysophosphatidylethanolamine, phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin. In addition, three glycolipids were observed and characterized, namely, monoglucosyldiglyceride, which is found in other biological systems, and (mannosyl, glucosyl)-diglyceride and (dimannosyl, glucosyl)-diglyceride, which heretofore have not been observed in nature.     

Entry of [(1,2-13C2)acetyl]-L-carnitine in liver tricarboxylic acid cycle and lipogenesis: a study by 13C NMR spectroscopy in conscious, freely moving rats.

The biochemical pathways involved in acetyl-L-carnitine utilization were investigated in conscious, freely moving rats by 13C NMR spectroscopy. Following 4-h [(1,2-13C2)acetyl]-L-carnitine infusion in fasted animals, the free carnitine levels in serum were increased, and an efflux of unlabelled acetyl-L-carnitine from tissues was observed. [(1,2-13C2)Acetyl]-L-carnitine was found to enter biosynthetic pathways in liver, and the acetyl moiety was incorporated into both cholesterol and 3-hydroxybutyrate carbon skeleton. In accord with the entry of [(1,2-13C2)acetyl]-L-carnitine in the mitochondrial acetylCoA pool associated with tricarboxylic acid cycle, the 13C label was also found in liver glutamate, glutamine, and glutathione. The analysis of the 13C-labelling pattern in 3-hydroxybutyrate and cholesterol carbon skeleton provided evidence that the acetyl-L-carnitine-derived acetylCoA pool used for ketone bodies synthesis in mitochondria was homogeneous, whereas cholesterol was synthesized from two different acetylCoA pools located in the extra- and intramitochondrial compartment, respectively. Furthermore, cholesterol molecules were shown to be preferentially synthesized by the metabolic route involving the direct channelling of CoA-activated mitochondria-derived ketone bodies into 3-hydroxy-3-methylglutarylCoA pathway, prior to equilibration of their acyl groups with extramitochondrial acetylCoA pool via acetoacetylCoA thiolase.     

Isolation and complete primary sequence of a new ovine wild-type beta-lactoglobulin C

A new wild type of beta-lactoglobulin has been identified in the milk of sheep. It has been designated as ovine beta-lactoglobulin C. Its primary structure has been determined by direct protein microsequencing of intact protein and RP-HPLC-derived tryptic peptides. The new beta-lactoglobulin C is a subtype of ovine beta-lactoglobulin A with a single exchange Arg-Gln at position 148. This exchange may influence polymerisation of beta-lactoglobulin since in the crystal structure of orthorhombic bovine beta-lactoglobulin, residues 145-150 constitute a short beta-sheet region involved in dimer formation by pairing of dyad-related strands.     

Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.