Jakob Nuesch

New Derivatives of Vitamin B12 Show Preferential Targeting of Tumors

Rapidly growing cells show an increased demand for nutrients and vitamins. The objective of our work is to exploit the supply route of vitamin B12 to deliver new derivatives of this vital vitamin to hyperproliferative cells. To date, radiolabeled ((57)Co and (111)In) vitamin B12 derivatives showed labeling of tumor tissue but also undesired high accumulation of radioactivity in normal tissue. By abolishing the interaction of a tailored vitamin B12 derivative to its transport protein transcobalamin II and therefore interrupting transcobalamin II receptor and megalin mediated uptake in normal tissue, preferential accumulation of a radiolabeled vitamin in cancer tissue could be accomplished. We identified transcobalamin I on tumors as a possible new receptor for this preferential accumulation of vitamin-mediated targeting. The low systemic distribution of radioactivity and the high tumor to blood ratio opens the possibility of a more successful clinical application of vitamin B12 for imaging or therapy.

A GENETIC APPROACH TO THE BIOSYNTHESIS OF THE RIFAMYCINCHROMOPHORE IN NOCARDIA MEDITERRANEI

The mutant under study, designated A8, is derived from a Nocardia mediterranei strain, N813, which is a high rifamycin B producer. A8 is auxotrophic for aromatic amino acids and produces much less rifamycin B than the parent. A mixture of pentoses with D (--) ribulose as the main product is accumulated in the fermentation broth of this mutant. It was shown to be affected in its transketolase activity as no formation of D-sedoheptulose -7P from pentose-phosphates could be detected in vitro using crude extracts. The only pathway so far known which is derived from D-sedoheptulose-7P is the shikimate pathway leading to aromatic amino acids and vitamins. Biochemical and genetic investigations with mutant A8, which is defective in both the biosynthesis of rifamycins and the biosynthesis of shikimate pathway products, show that the seven-carbon amino unit of the rifamycin-chromophore must be derived from an intermediate of the shikimate pathway.

A convenient method for permeabilizing the fungus Cephalosporium acremonium.

Abstract After ether treatment the fungus Cephalosporium acremonium is permeable to enzyme substrates which cannot enter the intact cells. Two enzyme systems, hexokinase/glucose 6-phosphate dehydrogenase and RNA polymerases, have been assayed in ether-treated cells. The first two enzymes can only be measured upon addition of ATP and NADP + and are remarkably stable. The DNA transcription requires the four nucleoside triphosphates as substrates and can be stopped by the addition of actinomycin D.

Effect of glucose and oxygen on β-lactam biosynthesis by Cephalosporium acremonium

Abstract The effects of glucose consumption rate ( q s ) and oxygen limitation on the control of cephalosporin C (Ceph C) biosynthesis and the activities of deacetoxycephalosporin C synthetase/hydroxylase (DAOC-SH) and acetyl coenzyme A: deacetylcephalosporin C o -acetyltransferase (DAC-AT) were investigated in cultivations of the highly productive Cephalosporium acremonium strain TR87 under conditions similar to those used in industrial production. A carefully optimised time course of q s during the first part of fed batch cultivations was essential for maximal Ceph C production. The actual glucose concentration in the medium was of secondary importance. A decrease of q s between 20 and 35 h of cultivation was found to induce the early onset of antibiotic synthesis. By subsequently maintaining q s at a relatively low level using a controlled feed of glucose and a limiting amount of phosphate, maximal production rates were obtained. Oxygen starvation after the onset of Ceph C production led to a pronounced increase in penicillin N formation, a reduced Ceph C yield (−30%) and a strongly reduced activity of the two enzymes tested. In general, neither the time course nor the absolute levels of the two enzyme activities directly correlated with the actual production rates of Ceph C. This is the first time where an independent parameter ( q s ) has been demonstrated to be responsible for triggering the synthesis of an antibiotic.

Product inhibition during the rifamycin S fermentation

Mutants of Saccharomyces cerevisiae with an increased sensitivity to the t ryptophan analogue l~Lr5-methylt ryptophan (5MT) were isolated SCI~f~RCH, MIOZZARI and HOTTER, J. Bact. 717, 1131-1140, 1974). A number of these 5MT sensitive mutants were characterized and assigned to one of the following 3 classes: class I, strains with altered act ivi ty and/or feedback sensitivity of anthranilate synthase; class II, strains with elevated uptake of 5MT; class III, mutants with altered regulation of the tryptophan-biosynthetic enzymes which do not exhibit increases in act ivi ty in the presence of 5MT. The object of the following study was to characterize 3 5MT sensitive mutants which do not belong to one of the above mentioned groups. The 3 mutants were shown to possess the following characteristics: They belong to the same complementation group and are considered allelicl although they show slightly different phenotypes. The 5MT inhibition no longer occurs ill the presence of tryptophan, anthranilic acid or indole. Enzymatic analysis revealed changes in chorismate mutase: a) greatly increased act ivi ty of chorismate mutase; b) diminished feedback sensitivity of this enzyme to tyrosine; c) reduction in its ability to be activated by tryptophan. Genetic studies indicate that this mutation is dominant and is localized in the chorismate mutase gene. Two possibilities to explain the phenotype of the mutation are being studied. We are either dealing with a promotor mutation in the chorismate mutase structural gene, or the mutation affects the equilibrium between an inactive (or less active) and an active form of the enzyme. P r o d u c t I n h i b i t i o n D u r i n g the R i f a m y c i n S F e r m e n t a t i o n