Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

The effect of extracellular osmolality on cell volume and resting skeletal muscle metabolism

The purpose of the current investigation was to establish an in-l'itro skeletal muscle model to study acute alterations in resting skeletal muscle cell volume. Isolated. whole muscle (SOL and EDL) was dissected from Long Evans rats and incubated for 60 min in Sigma Medium-199 (resting tension (lg). bubbled with 95:5% 02:C02, 30 ± 2°C, and pH 7.4). Media osmolality was altered to simulate hypo-osmotic (190 ± 10 Osm) (HYPO) or hyper-osmotic conditions (400 ± 10 Osm) (HYPER) while an iso-osmotic condition (290± 1 0 Osm) (CON) served as a control (n= 17.19.17). Following incubation, relative muscle water content decreased with HYPER and increased with HYPO in both muscle types (p<0.05). The cross-sectional area of HYPO SOL type I and type II fibres increased (p<0.05) while the EDL type 11 fibre area decreased in HYPER and increascd from HYPO exposure. Furthermore, HYPER exposure in both muscles lead to decreased ATP and phosphocreatine (p<0.05) and increased creatine and lactate (p<0.05) compared to CON. This isolated skeletal muscle model proved viable and demonstrated that altering extracellular osmolality could cause acutc alterations in muscle water content and resting muscle metabolism.

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.

TIME COURSE OF SIGNALING PROCESSES INVOLVED WITH EXTRACELLULAR OSMOTIC - INDUCED GLUCOSE UPTAKE IN MAMMALIAN SKELETAL MUSCLE

Extracellular hyper-osmotic (HYPER) stress increases glucose uptake to defend cell volume, when compared to iso-osmotic (ISO) conditions in skeletal muscle. The purpose of this study was to determine a time course for changes in common signaling proteins involved in glucose uptake during acute hyper-osmotic stress in isolated mammalian skeletal muscle. Rat extensor digitorum longus (EDL) muscles were excised and incubated in a media formulated to mimic ISO (290 ± 10 mmol/kg) or HYPER (400 ± 10 mmol/kg) extracellular condition (Sigma Media-199). Signaling mechanisms were investigated by determining the phosphorylation states of Akt, AMPK, AS160, cPKC and ERK after 30, 45 and 60 minutes of incubation. AS160 was found to be significantly more phosphorylated in HYPER conditions compared to ISO after 30 minutes (p<0.01). It is speculated that AS160 phosphorylation increases glucose transporter 4 (GLUT4) content at the cell surface thereby facilitating an increase in glucose uptake under hyper-osmotic stress.

Investigation of the Time Dependent Influence of Extracellular Osmotic Stress on Protein Turnover in Skeletal Muscle Cells

Acute alterations in cell volume can substantively modulate subsequent metabolism of substrates. However, how such alterations in skeletal muscle modulate protein metabolism is limited. The purpose of this study was to determine the time dependent influence of extracellular osmotic stress on protein turnover in skeletal muscle cells. L6 cells were incubated in hyperosmotic (HYPER; 425.3 ± 1.8mmol/kg), hypo-osmotic (HYPO; 235.4 ± 1.0mmol/kg) or control (CON; 333.5 ± 1.4mmol/kg) media for 4, 8, 12, or 24hrs. During the final 4hrs, incorporation of L-[ring-3,5-3H]-tyrosine was measured to estimate protein synthesis. Western blotting measured markers of protein synthesis and degradation. No differences were observed in any outcomes except p70S6K phosphorylation whereby HYPO was lower (p<0.05) than CON and HYPER; which remained similar except for a large increase at 8hrs for HYPER. These findings suggest that regardless of duration, extracellular osmotic stress does not significantly affect protein metabolism in L6 cells.

INFLUENCE OF INCREASED EXTRACELLULAR LEUCINE ON THE PROTEIN METABOLIC RESPONSES DURING OSMOTIC STRESS IN SKELETAL MUSCLE

The purpose of this study was to examine the effects of increased extracellular leucine concentration on protein metabolism in skeletal muscle cells when exposed to 3 different osmotic stresses. L6 skeletal muscle cells were incubated in either a normal or supplemental leucine (1.5mM) medium set to hypo-osmotic (230 ± 10 Osm), iso-osmotic (330 ± 10 Osm) or hyper-osmotic (440 ± 10 Osm) conditions. 3H-tyrosine was used to quantify protein synthesis. Western blotting analysis was performed to determine the activation of mTOR, p70S6k, ubiquitin, actin, and μ-calpain. Hypo-osmotic stress resulted in the greatest increase in protein synthesis rate under the normal-leucine condition while iso-osmotic stress has the greatest increase under the elevated-leucine condition. Elevated-leucine condition had a decreased rate in protein degradation over the normal condition within the ubiquitin proteasome pathway (p<0.05). Leucine and hypo-osmotic stress therefore creates a favourable environment for anabolic events to occur.