Factors in the rat submaxillary gland that stimulate growth of cultured glioma cells: identification and partial characterization

The effect of rat submaxillary extract on the growth of rat C6 glioma cells in serum-free culture has been examined. Extracts (10-15 microgram/ml) of submaxillary glands from both male and female rats markedly enhanced the growth of serum-deprived C6 cells and, in combination with insulin, transferrin, and NIH-LH (a source of fibroblast growth factor), were able to stimulate C6 cell growth to an extent comparable to that achieved with an optimal amount of fetal calf serum. The mitogenic activity of rat submaxillary extracts was found to be heat-labile, acid-stable, and partially inactivated by protease and 2-mercaptoethanol. Under our assay conditions, biologically active preparations of purified mouse submaxillary gland epidermal growth factor (EGF) or nerve growth factor (NGF) were not mitogenic for C6 cells, nor was the mitogenic activity of rat submaxillary extracts inhibited by antiserum to these mouse submaxillary gland growth factors. These results suggest that the active component(s) of rat submaxillary extracts is unrelated to either EGF or NGF. The growth-enhancing effect also appears unrelated to esteropeptidase activity present in these extracts since the mitogenic activity was unaffected by several protease inhibitors. Moreover, two purified mouse submaxillary gland arginylesteropeptidases, EGF-binding protein and gamma-subunit of 7 S NGF, were unable to elicit a comparable growth response even when added to cell culture medium at unreasonably high concentrations. The C6 cell mitogenic activity of crude submaxillary extracts could be separated into two biologically similar components by either gel filtration on Sephadex G-100, preparative isoelectric focusing in a pH gradient of 3-10, or adsorption to DEAE-cellulose followed by elution with a sodium chloride gradient. One of the active components was acidic in nature and had an apparent molecular weight of 40,000, while the other was near neutral in charge and possessed a molecular weight of approximately 20,000. The relationship between these two C6 cell mitogenic components and the rat submaxillary gland component responsible for stimulating Balb/c-3T3 cell growth in serum-free, factor supplemented medium (McClure et al., 1979, J. Cell Biol. 83:96a) is also discussed.     

Purification and characterization of a new ribopolynucleotide synthesizing enzyme from Escherichia coli.

A new type of ribopolynucleotide-synthesizing enzyme was found both on cytoplasmic membranes and in protein-DNA complexes isolated from Escherichia coli. The enzyme was purified by exploiting a specific, reversible enzyme aggregation with ATP and spermidine. The purified enzyme (more than 90% pure) was free from other enzymatic activities such as ATPase and polynucleotide phosphorylase. The enzyme (molecular weight 270,000 ± 15%) contains two kinds of polypeptide chain (molecular weights 91,000 ± 10%, and 45,000 ± 10%) and these polypeptides are not common with those of DNA-dependent RNA polymerase. The enzyme catalyses the synthesis of ribopolynucleotides from nucleoside triphosphates in the presence of 1 mm-MgCl₂. Rifampicin and streptolydigin do not affect the enzyme reaction.     

On-line estimation of kLa by an analog computer

A new method was developed for estimating the volumetric oxygen transfer coefficient, k_La, in a fermentor. Various methods were investigated for the on-line estimation of k_La with an analog computer employing a steepest-descent calculation technique. The method by which k_La is estimated (by minimizing the error residue of the model) was found to be very applicable. A method for the simultaneous estimation of the volumetric oxygen transfer coefficient and respiration rate in biological systems is also presented.