Identification and iterative combinatorial mutagenesis of a new naringinase-producing strain, Aspergillus tubingensis MN589840.

Naringinase was mainly obtained by microbial fermentation, and mutagenesis was a major way for obtaining excellent mutants. The aim of this study was to screen out a high naringinase yielding mutant to enhance the potential application value of its industrialization and compare the effects of different mutagenic methods on the enzyme activity of the strain. A novel producing naringinase strain, Aspergillus tubingensis MN589840, was isolated from mildewed pomelo peel, later subjected to mutagenesis including UV, ARTP and UV-ARTP. After five rounds iterative mutagenesis, the mutants U1, A6 and UA13 were screened out with 1448·49, 1848·71, 2475·16 U mg-1 enzyme activity, the naringinase productivity raised by 79·08, 123·56 and 206%, respectively. In addition, the naringinase activity of three mutants rose after each round of iterative mutagenesis. These results indicated that the mutagenesis efficiency of UV-ARTP was higher than that of single ARTP, and both are better than UV. In summary, the iterative UV-ARTP mutagenesis is an effective strategy for screening high naringinase-producing strains.

The I-Abm12 mutation, which confers resistance to experimental myasthenia gravis, drastically affects the epitope repertoire of murine CD4+ cells sensitized to nicotinic acetylcholine receptor.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG), which is induced in mice by injection of purified Torpedo nicotinic acetylcholine receptor (TAChR), is influenced by the I-A locus products, which restrict presentation of AChR Th epitopes. The bm12 mutation of the I-Ab molecule in the C57BL/6 strain, which is highly susceptible to EAMG, yields the EAMG resistant mutant B6.C-H-2bm12 (bm12). We investigated here the consequences of the bm 12 mutation on the CD4+ response to the TAChR alpha subunit. Upon immunization with TAChR, CD4+ cells became sensitized to TAChR and anti-AChR antibodies were produced in both bm12 and C57BL/6 strains. Overlapping synthetic peptides, corresponding to the complete sequence of TAChR alpha subunit, were used to identify Th epitopes. CD4+ cells from C57BL/6 mice recognized peptides T alpha 150-169, T alpha 181-200, and T alpha 360-378. CD4+ cells from bm12 mice did not respond to any synthetic sequence. Upon injection of the three C57BL/6 Th epitope peptides, either individually or as a pool, CD4+ cells from C57BL/6 mice recognized each peptide and TAChR. Therefore they recognized epitopes similar or identical to those originated from TAChR processing. CD4+ cells from bm12 mice injected with the same peptides responded to T alpha 360-378 strongly, to a lesser extent to T alpha 181-200, never to peptide T alpha 150-169. Only CD4+ cells sensitized against the T epitope peptide T alpha 181-200 responded to TAChR. We tested if lack of response to T alpha 150-169, and the low response to T alpha 181-200, was due to inability of the I-Abm12 molecule to present the T epitope peptides. bm12 and C57BL/6 APC were used to present the T epitope peptides to specifically sensitized CD4+ cells from C57BL/6 mice. All T epitope peptides were presented by bm12 APC, although T alpha 150-169 was presented less efficiently than by C57BL/6 APC. Resistance to EAMG induced by the bm12 mutation may be due to the change in the epitope repertoire of AChR-specific Th cells, and lack of recognition of otherwise immunodominant Th epitopes. For at least one epitope this might be due to absence of potentially reactive, specific CD4+ clones.

Immunofluorescent studies on microtubules of normal and malignant human esophageal cells.

Anti-tubulin antibody indirect immunofluorescence microscopy is used in this report. Difference of intracellular microtubule fluorescent staining patterns between normal and malignant human esophageal cells has been observed. Primarily cultured human esophageal epithelial cells display an elaborate array of fluorescent cytoplasmic microtubule filaments, the cytoplasmic microtubule complex (CMTC), in interphase cells and mitotic spindle microtubules in mitotic cells. Malignant esophageal cells from human esophageal carcinoma, ECa109 cells, contain very few cytoplasmic microtubules in interphase cells, but there is no difference of mitotic spindle microtubule fluorescence between normal and malignant cells. The results presented in this report suggest that the diminution of interphase cytoplasmic microtubules in tumor cells is probably due to the deficiency of microtubule organizing mechanism in interphase tumor cells.