Isolation and characterization of a chitinase-secreting mutant of mortierella pusilla with altered interaction with the mycoparasite piptocephalis virginiana

Mortierella pusilla is a susceptible host and supports good growth of the mycoparasite, Piptocephalis virginiana. Uninucleate spores of M. pusilla were sUbjected to N-methyl-N'-nitro-nitrosoguanidine (MNNG). To attain a high mutation frequency , a 1o-minute exposure to 10 mg/ml MNNG was used and lead to the survival of about 10 % of the spores. The exposed spores then were plated on chitin or milk plates. Approximately 30,000 colonies were examined after mutagenesis on the screening media. A strain, MUT23 , with abnormal slow growth morphology was found to delay parasitism by £. virginiana. The particular morphology was not due to auxotrophy, because this strain displayed normal hyphae when glucose was used as the sole carbon source. One interesting phenomenon was that MUT23 showed an extensive clearing zone around the colony on colloidal chitin agar after 20-25 d. On the same conditions, wild type strain did not show this phenotype. In addition, the MUT23 strain produced the same normal hypha as the wild type strain when it was grown on colloidal chitin agar. The MUT23 was also able to produce more spores on colloidal chitin agar than on malt-yeast extract and minimal media. The parasite germ tubes formed appressoria at the point of contact on the cell surface of wild type and MUT23 grown for 6 days cell surface but not on the cel surface of MUT23 grown for 2 days. Thus, interaction between MUT23 strain and the mycoparasite was dependent on MUT23 age. The effect of MUT23 filtrate on germination of the parasite was tested. Lysis of germinated spores of the parasite were observed in concentrated MUT23 filtered solution. MUT23 was compared to the wild type strain for their chitinase production in sUbmerged culture. The chitinase isozymes of both wild type and MUT23 were shown by immunoblotting. Eight distinct chitinase molecules were detected. MUT23 showed markedly higher chitinase activity than the wild type cultured in chitin-containing medium. Maximum chitinase activities of MUT23 were 13.5 fold higher at 20 day of the culture then that of wild type.

Protein Turnover in Rat Skeletal Muscle During In Vitro Hypo-Osmotic Stress

Hypo-osmolality influences tissue metabolism, but research on protein turnover in skeletal muscle is limited. The purpose of this investigation was to examine the effects of hypo-osmotic stress on protein turnover in rat skeletal muscle. We hypothesized increased protein synthesis and reduced degradation following hypo-osmotic exposure. EDL muscles (n=8/group) were incubated in iso-osmotic (290 Osm/kg) or hypo-osmotic (190 Osm/kg) modified medium 199 (95% O2, 5% CO2, pH 7.4, 30±2 °C) for 60 min, followed by 75 min incubations with L-U[14C]phenylalanine or cycloheximide to determine protein synthesis and degradation. Immunoblotting was performed to assess signalling pathways involved. Phenylalanine uptake and incorporation were increased by 199% and 169% respectively in HYPO from ISO (p < 0.05). This was supported by elevated phosphorylation of mTOR Ser2448 (+12.5%) and increased Thr389 phosphorylation on p70s6 kinase (+23.6%) (p < 0.05). Hypo-osmotic stress increased protein synthesis and potentially amino acid uptake. Future studies should examine the upstream mechanisms involved.