Characterization of ferritin in murine erythroleukaemia cells

Murine erythroleukaemic cells were studied to determine whether different isoferritins have different functions. The cells were labelled with radioactive iron and the pattern of isoferritins was analysed by chromatofocussing. No change was found after iron-loading the cells but after inducing erythroid differentiation with dimethyl sulphoxide (DMSO), iron was incorporated into both more basic and more acidic isoferritins. This was compared to ferritin subunit synthesis; DMSO induced the synthesis of a third, minor subunit whereas iron-loading had no effect. The fate of murine erythroleukaemic cell ferritin iron was followed after incubations in iron-deficient medium containing DMSO; some, but not all, of the ferritin iron was mobilized and used for haem synthesis, and the remaining iron was found amongst the more basic isoferritins. Finally, sequential radioactive iron labels were used to demonstrate that the movement of iron from ferritin to haem was compatible with the 'last-in-first-out' principle, but this could not be related to different isoferritins. These results show firstly that DMSO changes the pattern of isoferritins and ferritin subunits in murine erythroleukaemic cells. Secondly, iron associated with more basic isoferritins seems to be less easily mobilized for haem synthesis. These results support the concept that different isoferritins have different functions.     

The effects of salts and amino group modification on the iron binding domains of transferrin

The origins of the effects of salts on the properties of the iron binding sites of transferrin have been investigated. The chaotropically distinct salts NaCl and NaClO4 each induce characteristic changes in the EPR lineshapes of the N- and C-terminal Fe3+ binding domains, respectively. To a good approximation the perturbed EPR spectrum of diferric transferrin in the presence of salts is the sum of the EPR spectra of the N- and C-terminal monoferric proteins. Acetylation of amino groups causes spectral and kinetic changes in the protein similar to those induced by NaClO4. Thus, both acetylation and NaClO4 cause a loss of structure in the g' = 4.3 EPR signal of the N-terminal domain, and both retard iron removal from this domain. In contrast, iron removal from the C-terminal domain is accelerated by acetylation or the presence of NaClO4. These observations are ascribed to charge effects of lysine residues which are probably in the vicinity of the iron binding sites.     

Substrate concentration control by dialysis in fermentations of Escherichia coli

A substrate control system based upon dialysis culture techniques has been developed. In fermentations of Escherichia coli where phosphate or sulfate concentrations were controlled, the relation between the apparent specific growth rate, mu, and the phosphate concentration followed Monod kinetics, while the relation between mu and sulfate showed a sharp optimun. The pH of fermentations of E. coli was controlled by dialysis culture techniques without the fluctuations in pH associated with control by direct addition of acid or base.     

Family outbreak of gastroenteritis due to Yersinia enterocolitica serotype 0:3 from well water

Yersinia enterocolitica serotype 0:3, biotype 4, has been isolated from two family members with diarrhea and from the well used as a source of their drinking water.     

Mathematical model for human myeloma relating growth kinetics and drug resistance

We present a computer-based mathematical model that can simulate characteristic features of the clinical time course of human myeloma. It asserts that therapy resistance in myeloma cells is an inherited trait associated with the longer inter-mitotic times of some cells and that the strength of this trait affects tumour growth characteristics. These kinetic differences within the malignant cell clone may also influence therapeutic efficacy. In the model, the same total therapy, administered in different time-dose fractions, could be 'curative' or 'minimally effective' depending on kinetic properties. For example, as others have shown, in myeloma pulsed intermittent therapy is often more effective than low dose continuous therapy. According to our model this finding is compatible with a high coefficient of inheritability of resistance from one cell generation to the next. The model also suggests that if there are subclones of varying resistance, a therapy must have some effect on each of them if it is to be employed in a curative fashion. While many aspects of the model are not yet clinically testable, exploration of its concepts might increase knowledge about fundamental neoplastic mechanisms.     

Secretion of a heterologous protein from Bacillus subtilis with the aid of protease signal sequences.

Secretion vectors based on the genes from Bacillus amyloliquefaciens P for alkaline protease (aprBamP) and neutral protease (nprBamP) were constructed. With both aprBamP and nprBamP, a unique restriction site was introduced 3' of the predicted signal coding region by using the technique of oligonucleotide-directed mutagenesis. The new sites enabled us to fuse a heterologous gene to the expression and secretion elements. We used the protein A gene (spa) from Staphylococcus aureus as a heterologous gene. Bacillus subtilis cells carrying the resulting apr-spa or npr-spa gene fusions synthesized the fusion protein. B. subtilis cells were also capable of removing the signal peptide from the fusion protein, as indicated by the appearance of processed protein A into the growth medium. In addition, these gene fusions allowed us to identify the signal processing site of both the APR-SPA and NPR-SPA proteins.     

Localization of the ganglioside-binding site of fibronectin

It has been demonstrated via biological assays that fibronectin possesses a receptor for gangliosides that is involved in cell adhesion and restoration of the normal morphology of transformed cells. In this study, fluorescence polarization has been employed to monitor the binding of ganglioside oligosaccharide to fibronectin. Parameters involved in ganglioside oligosaccharide binding to fibronectin are described and compared to the interaction of heparin with fibronectin. A Kd of 1.4 X 10(-8) mol/liter has been calculated, and it is demonstrated that labeled ganglioside oligosaccharides can be eluted from fibronectin with either unlabeled ganglioside oligosaccharides or 4 M urea. Using the fluorescence polarization assay developed in this study for measurement of ganglioside binding to fibronectin, it is demonstrated that gangliosides bind to the 31,000-dalton amino terminal heparin-binding domain of fibronectin. A ganglioside-Sepharose affinity column has been constructed which specifically binds the 31,000-dalton amino terminal fragment of fibronectin. The localization of the ganglioside receptor to the amino terminal domain of fibronectin indicates that the ganglioside receptor is distinct from the putative fibronectin cell surface receptor which is located near the center of the fibronectin molecule.     

Is vegetation in equilibrium with climate? How to interpret late-Quaternary pollen data

Current methods for estimating past climatic patterns from pollen data require that the vegetation be in dynamic equilibrium with the climate. Because climate varies continuously on all time scales, judgement about equilibrium conditions must be made separately for each frequency band (i.e. time scale) of climatic change. For equilibrium conditions to exist between vegetation and climatic changes at a particular time scale, the climatic response time of the vegetation must be small compared to the time scale of climatic variation to which it is responding. The time required for vegetation to respond completely to climatic forcing at a time scale of 10⁴ yr is still unknown, but records of the vegetational response to climatic events of 500-to 1000-yr duration provide evidence for relatively short response times. Independent estimates for the possible patterns and timing of late-Quaternary climate changes suggest that much of the vegetational evidence previously interpreted as resulting from disequilibrium conditions can instead be interpreted as resulting from the individualistic response of plant taxa to the different regional patterns of temperature and precipitation change. The differences among taxa in their response to climate can lead a) to rates and direction of plant-population movements that differ among taxa and b) to fossil assemblages that differ from any modern assemblage. An example of late-Holocene vegetational change in southern Quebec illustrates how separate changes in summer and winter climates may explain the simultaneous expansion of spruce (Picea) populations southward and beech (Fagus) populations northward.