L-Tryptophan Production by Auxotrophic and Analogue Resistant Mutants of Aureobacterium flavescens

A number of tyrosine plus phenylalanine double auxotrophic mutants were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) treatment of a locally isolated strain of Aureobacterium flavescens of which 11A39 and 11A17 were selected on the basis of their tryptophan production in a mineral salt medium over other isolated mutant strains. The mutational block in the aromatic amino acid biosynthetic pathway of the selected double auxotrophs were determined. By controlling pH of the production medium to near neutrality, the active growth period could be extended up to 72 h and more tryptophan was accumulated compared to pH unregulated culture where the active growth ceased after 48 h. Further improvement of the tryptophan production has been achieved by stepwise isolation of a mutant strain resistant to the tryptophan analogues p-fluorotryptophan (FT) and 5-methyl tryptophan (MT) from the 11A39. Demand for L-tryptophan as food additive and therapeutic agent is increasing day by day throughout the World, particularly in the underdeveloped and developing countries like India. Still to date India depends on other countries for L-tryptophan. The aim of this work is to develop a potent high yielding, feed back insensitive mutant strain and optimization of its medium pH for maximum production of tryptophan.

Carcinogenesis of the upper gastrointestinal tract induced by N-methyl-N'-nitro-nitrosoguanidine and reflux of duodenal contents in the rat

PURPOSE: To investigate the combined effects of reflux of duodenal contents through the pylorus and treatment with N-methyl-N'-nitro-nitrosoguanidine (MNNG) on the development of lesions in the glandular stomach, at the gastrojejunal anastomosis and in the forestomach of rats. METHODS: Eighty Male Wistar rats were divided into 4 groups: G1: MNNG + Reflux, G2: Reflux, G3: MNNG and G4: Gastrostomy. MNNG was given in the drinking water (100 mg/ml) for 12 weeks and then two groups (G1 and G2) were submitted to a gastrojejunal anastomosis followed by section of the afferent loop and suture of both stumps to allow reflux of duodenal contents through the pylorus. The animals were sacrificed 18 and 36 weeks after surgery. The lesions obtained in the antral mucosa, at the gastrojejunal anastomosis and in the forestomach were analysed histologically. RESULTS: Duodenal reflux induced proliferative lesions at both glandular and squamous mucosa of the stomach. In the antrum, adenomatous hyperplasia (AH) was observed in 20% and 50% of the animals at the 18th and 36th weeks respectively. Aditionally 85% of the animals presented AH at the gastrojejunal anastomosis and 60% developed squamous hyperplasia at the squamous portion of the stomach. MNNG treatment plus duodenal reflux enhanced the development of malignant tumors at both glandular and squamous mucosa, since there were 30% of antral adenocarcinomas and 45% of squamous carcinomas at the 18th week and the frequency of these malignant tumors rose to 50% in the antrum and 65% in the squamous mucosa at the 36th week. CONCLUSION: The reflux of duodenal contents through the pylorus enhanced the development of proliferative lesions, benign and malignant, in the glandular stomach and in the forestomach of rats.

BCR/ABL Kinase Disrupts Formation of Mismatch Repair Complex To Induce Genomic Instability.

Among different genotoxic agents, BCR/ABL cells are more resistant to N-methyl-N’-nitro-N-nitrosoguanine (MNNG), which methylates 06 position of guanine. 06 -MeG bases pair with either T or C during replication giving rise to mismatches. Mismatch repair (MMR) proteins are responsible for detecting and removing misincorporated nucleotides, which escaped proofreading activity of DNA polymerases. MMR proteins assembled on the mismatch can signal to repair or apoptosis. Defects in expression of MMR genes leads to drug resistance and mutator phenotype, observed in several different solid tumors. The role of MMR system in drug resistance and/or genomic instability of leukemic cells remains poorly understood. Parental cells and BCR/ABL expressing clones were incubated with MNNG for 4 weeks resulting in their MNNG-resistant derivatives, which may accumulate mutations in their genomic DNA resulting from methylating activity of the drug. To investigate the mutation rate and phenotype, ouabain-resistance test was employed. Cells become resistant to ouabain, a glycoside that inhibits ATP1A1 subunit of Na/K ATP-ase, when mutations arise in the gene fragment encoding this subunit. The clonogenic assay revealed over 5 times more ouabain resistant colonies in MNNG-resistant BCR/ABL-positive cells than in parental counterparts. To investigate the type of mutations, a fragment of Na/K ATP-ase gene encoding the ATP1A1 subunit was sequenced from MNNG-resistant BCR/ABL-positive and parental cells. The dominating mutation in BCR/ABL MNNG-resistant cells was C to T, while A to G mutations were prevalent in parental cells. In order to check the status of MMR proteins, Western blotting and immunofluorescence studies were performed. Expression of MMR proteins in BCR/ABL transformed cells was similar to parental cells, however immunofluorescence visualized dramatic changes after DNA damage in the nuclear co-localization of MMR proteins in BCR/ABL-transformed cells (CML patient cells and leukemic cell lines) in comparison to normal cells. Co-localization of MSH2 and MSH6 proteins, forming a heterodimer homologous to bacterial MutS, remained similar in parental and leukemia cells upon MNNG treatment. However, co-localization of MLH1 and PMS2 proteins, which form a heterodimer homologous to bacterial MutL, was detected in non-transformed cells, but not in BCR/ABL leukemia cells. In addition, MLH1 and MSH2 co-localized in normal, but not BCR/ABL-positive cells after MNNG treatment. The defects in interaction of MMR proteins in leukemia cells were reversed by inhibition of BCR/ABL kinase by STI571. Thus, the assembly of MMR proteins on mismatched bases and subsequent signaling to repair and/or apoptosis may be impaired in BCR/ABL leukemia cells. These results suggests a novel mechanism of regulation of DNA damage response proteins by oncogenic tyrosine kinase that can lead to genomic instability and drug resistance of leukemic cells.