Characterization of growth and product formation by a thermophilic streptomycete grown in a particulate rapemeal-derived liquid medium

Streptomyces thermoviolaceus was grown in a glutamate salts medium and the applicability of DNA and a number of intracellular dehydrogenases as indicators for growth in a particulate repemeal salts medium were assessed. Only NADH dehydrogenase proved unsuitable but analysis of the rates of increase of activities of this enzyme during batch culture suggested that energy metabolism was an important factor in defining the onset of secondary metabolism and as a causal element of the biphasic pattern of growth exhibited by this streptomycete. Growth, substrate utilization and production of extracellular products could readily be analysed in the particulate rapemeal salts medium. Free sugars and carbohydrates were utilized predominantly in early exponential phase but thereafter the rapemeal proteins were metabolized as observed by rises in protease activities and levels of ammoniacal nitrogen. Growth in rapemeal-derived media produced titres of the antibiotic granaticin which were at least as high (usually higher) as most of the growth substrates tested suggesting that rapemeal may have considerable potential for the exploitation of natural products. Particulate rapemeal medium also resulted in high yields of commercially important extracellular enzymes.     

Structure-function analysis of epidermal growth factor: site directed mutagenesis and nuclear magnetic resonance

The role of leucine-47 in determining the structure and activity of human epidermal growth factor was examined using site-directed mutagenesis. Wild type protein and four variants in which Leu47 was replaced by valine, glutamate, aspartate and alanine were produced from yeast. 1H NMR experiments demonstrated that substitution of Leu47 had little effect on the protein structure. The observed reduction in receptor binding affinity caused by the substitutions could thus be attributed to perturbation of a residue directly involved in receptor interactions.     

Nitrosating activity in Escherichia coli

Nitrosation activity was measured in Escherichia coli isolates and a range of nitrite reductase (nir) mutants. Activity was only detected in intact cells and could be inhibited by a number of treatments such as sonication and osmotic shock. Aerobically-grown cells had highest nitrosation activity compared to oxygen-limited ones. Inclusion of nitrite in growth media induced high activities of nitrite reductase and for some isolates, nitrosation. Analysis of nir mutants identified two which were unable to nitrosate. This result suggested that NADH-dependent nitrite reductase was implicated either directly or indirectly in nitrosation.     

Deletion mapping of the medulloblastoma locus on chromosome 17p

Isochromosome 17q has previously been observed consistently in cytogenetic studies of medulloblastoma, the most common posterior fossa neoplasm in children. We performed a restriction fragment length polymorphism (RFLP) investigation of medulloblastoma which showed a loss of chromosome 17p sequences in 45% of these tumors. This finding was predictive of a poor clinical response to treatment. A contiguous panel of markers permitted mapping of the deletion to 17p12-p13.1, the same chromosomal region for which loss of alleles has been shown in tumor specimens from patients with colon cancer, and the same region to which the p53 gene has been mapped. This suggests that medulloblastoma is associated with a recessive oncogene on chromosome 17p that may be involved in the genesis of several embryologically unrelated neoplasms and that the absence of this gene in tumor tissue has prognostic significance.     

Analysis of a transformed cell line using antisense c-fos RNA

Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c-fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c-fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c-fos antisense-RNA expression vector, pSVsof, or control plasmids. pSVsof-transfected cells exhibit markedly decreased c-fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c-fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.     

The "Pardridge" hypotheses relating to the role of hormone binding proteins in hormone delivery: a critique

Though the physiological role of specific binding proteins in serum is unknown, and the validity of the "free hormone hypothesis" has been challenged by several authors (including ourselves), we regard Pardridge's widely publicised objections to this hypothesis (and his recent suggestion of the existence of complex biochemical mechanisms causing the release of individual hormones in particular tissues) as deriving from an incorrect analysis of the effects of serum binding proteins on the kinetics of hormone transport, and therefore without foundation. This presentation is intended to substantiate this view.     

Human glyceraldehyde-3-phosphate dehydrogenase: mRNA levels and enzyme activity in developing muscle.

Analysis of human glyceraldehyde-3-phosphate dehydrogenase mRNA revealed that levels in adult skeletal muscle are 12-fold greater per microgram of polyadenylated RNA than in fetal skeletal muscle, whereas in cardiac muscle RNA levels were about equal in fetal and adult tissue. The mRNA levels correlate well with glyceraldehyde 3-phosphate dehydrogenase enzyme activities. There was no evidence for fetus- or tissue-specific forms.