EPR and multi-field magnetisation of reduced forms of the binuclear iron centre in ribonucleotide reductase from mouse

Mouse ribonucleotide reductase is composed of a 1?:?1 complex of two homodimeric subunits and catalyses the first unique step on the biochemical pathway to DNA synthesis. The small subunit, protein R2, contains dinuclear iron-oxygen clusters and a tyrosyl free radical required for catalytic activity. We have studied the mixed valent and fully reduced forms of the diiron oxygen cluster from mouse R2 protein by low-temperature EPR. EPR signals of the mixed-valent states of proteins R2 reconstituted with ferrous iron and oxygen in normal and deuterated water, using the same buffers, show apparent g values of 1.92, 1.73, and 1.60 for the mixed-valent state in H₂O and 1.93, 1.73, and 1.62 in D₂O. These g values are typical for diiron-oxygen proteins, while the effect of D₂O is unprecedented for this class of proteins. We estimate the coupling constant J for the Heisenberg exchange (H?=?2J*S₁*S₂) to be J?=?–7.5±1?cm^–1 for the mixed-valent form. The diferrous R2 protein shows an integer spin EPR signal in the presence of azide or 20% glycerol. Variable temperature variable field saturation magnetisation measurements show that only in the azide-complexed R2 protein does a weak ferromagnetic coupling occur (J?=?0.26±0.05?cm^–1), while R2 protein in the absence or presence of 20% glycerol contains non-coupled mononuclear ferrous iron (S?=?2) sites.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

Computer controlled fed-batch fermentation of the methylotroph Pseudomonas AM1

Summary A simple on-line computer control strategy based on dissolved oxygen level readings has been developed to control methanol addition during a fermentation of the methylotroph Pseudomonas AM1. This strategy has led to significant and reproducible improvements in the performance of the fermentation.     

Sorption of Cadmium by Microorganisms in Competition with Other Soil Constituents

The fate of cadmium in soil is influenced to a great extent by microbial activity. Microorganisms were compared with abiotic soil components for their ability to sorb Cd from a liquid medium. When the same amount (on a dry weight basis) of bacterial cells (Serratia marcescens and Paracoccus sp.), clay (montmorillonite), or sand was separately incubated in 0.05 M phosphate buffer, pH 7.2, containing 10 ppm of Cd (10 μg/ml), bacterial cells removed the largest quantity of Cd. Dead cells sorbed much more Cd from the medium than live cells. A comparative study of Cd removal from the medium by seven soil bacteria and four fungi did not indicate appreciable differences. With increasing microbial biomass, the relative efficiency of 0.1 M NaOH as an extractant of sorbed Cd increased, whereas the extraction efficiency of 0.005 M DTPA (diethylenetriaminepentaacetic acid) decreased. It appeared that NaOH and DTPA extracted different chemical forms of Cd. This assumption was supported by vastly different correlation coefficients in the relative amount of Cd extracted by the two solvents.     

Influence of inoculum age on hybridoma culture kinetics

To determine the influence of the inoculum age on the kinetics of hybridoma growth and metabolism, spinner flasks have been inoculated with cells previously propagated in T flasks for 43, 52, 62 and 71 hr respectively. Increasing the age of the inoculum is found to result in a longer lag phase, in a lower maximum specific growth rate and in a reduced maximal cell density. During the growth phase specific rates of glucose and glutamine uptake and of ammonia and lactate production are similar. However, with the older inoculum, much higher metabolic activities are observed during the lag phase. The production of antibodies is delayed with increasing inoculum age, but the final antibody concentrations are similar, which indicates a higher specific antibody production rate when inoculating with older cells.     

Lipopolysaccharide receptor on rabbit peritoneal macrophages. I. Binding characteristics

The Bordetella pertussis endotoxin, labeled with tritium ((3H)-LPS), bound irreversibly and nonspecifically to rabbit lung macrophages, but bound reversibly and specifically to both resident and elicited rabbit peritoneal macrophages. The specific binding capacity of the macrophages was saturated with about 3 X 10(4) LPS molecules per cell. The binding was inhibited with the homologous unlabeled endotoxin, but not at all with endotoxin from Proteus mirabilis, thus assessing ligand specificity. Endotoxins from other bacteria gave intermediate inhibition value. Binding of tritium-labeled pertussis endotoxin was significantly inhibited by one of the two polysaccharides (PS-1) present in this endotoxin, but neither the other polysaccharide (PS-2) nor the Lipid A fragment exhibited such activity. These results strongly suggest the presence of a lectin-like receptor for LPS on the membrane of rabbit peritoneal macrophages.     

Presence of interleukin 3-like activity in the supernatants of lipopolysaccharide-stimulated mouse splenocytes

An Interleukin 3 (IL 3) activity was found in the supernatants of mouse spleen cells stimulated by lipopolysaccharide (LPS). The IL 3 activity was maximum in the supernatants of 5 to 7 days cultures. IL 3 activity was assessed by the capacity of the spleen cell supernatants to allow the growth of the FDC-P2 cell line, a strictly IL 3 dependent cell line. Polymyxin B could inhibit the IL 3 induction by LPS.     

Yarrowia lipolytica: A model and a tool to understand the mechanisms implicated in lipid accumulation

The oleaginous yeast Yarrowia lipolytica is known to inhabit various lipid-containing environments. One of the most striking features in this yeast is the presence of several multigene families involved in the metabolic pathways of hydrophobic substrate utilization. The complexity and the multiplicity of these genes give Y. lipolytica a wide capability range towards hydrophobic substrate (HS) utilization and storage. The combination of the increasing knowledge of this yeast's metabolism and the development of more efficient genetic tools is offering new perspectives in using Y. lipolytica as a model organism to study the mechanisms involved in lipid metabolism associated to fat uptake, storage, deposition, mobilization and regulation. Nutrient status and culture conditions seem to play a major role in obesity.