Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.     

Climatic sensitivity of species’ vegetative and reproductive phenology in a Hawaiian montane wet forest

Understanding how tropical tree phenology (i.e., the timing and amount of seed and leaf production) responds to climate is vital for predicting how climate change may alter ecological functioning of tropical forests. We examined the effects of temperature, rainfall, and photosynthetically active radiation (PAR) on seed phenology of four dominant species and community-level leaf phenology in a montane wet forest on the island of Hawaiʻi using monthly data collected over ~ 6 years. We expected that species phenologies would be better explained by variation in temperature and PAR than rainfall because rainfall at this site is not limiting. The best-fit model for all four species included temperature, rainfall, and PAR. For three species, including two foundational species of Hawaiian forests (Acacia koa and Metrosideros polymorpha), seed production declined with increasing maximum temperatures and increased with rainfall. Relationships with PAR were the most variable across all four species. Community-level leaf litterfall decreased with minimum temperatures, increased with rainfall, and showed a peak at PAR of ~ 400 μmol/m²s⁻¹. There was considerable variation in monthly seed and leaf production not explained by climatic factors, and there was some evidence for a mediating effect of daylength. Thus, the impact of future climate change on this forest will depend on how climate change interacts with other factors such as daylength, biotic, and/or evolutionary constraints. Our results nonetheless provide insight into how climate change may affect different species in unique ways with potential consequences for shifts in species distributions and community composition.     

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.     

The rise in testicular androgens during the first days of treatment with an LHRH agonist in the dog can be blocked by aminoglutethimide or ketoconazole

Up to day 6 of treatment of adult dogs, daily subcutaneous administration of 50 micrograms of the LHRH agonist [D-Trp6, des-Gly-NH2-10]LHRH ethylamide causes up to a 3-fold increase in serum testosterone (T) concentration which is followed by a progressive decrease to castration levels (less than or equal to 0.2 ng/ml) at later time intervals (up to 21 days, the last time interval studied). Both aminoglutethimide and ketoconazole, two inhibitors of steroid biosynthesis, cause a 30-40% rise in serum T when administered alone. However, either drug administered in combination with the LHRH agonist completely blocks the transient rise in serum T observed when the LHRH agonist is administered alone. On the other hand, the LHRH agonist prevents the secondary rise in steroid secretion observed when either of the two inhibitors of steroid secretion is used alone. Administration of the pure antiandrogen Flutamide alone or in combination with LHRH-A and an inhibitor of steroid biosynthesis does not influence serum T levels. When the serum levels of pregnenolone, 17-OH-pregnenolone, progesterone, 17-OH-progesterone, dehydroepiandrosterone (DHEA), androstenedione (delta 4-dione), androst-5-ene-3 beta, 17 beta-diol (delta 5-diol), T, dihydrotestosterone (DHT), androstane-3 alpha, 17 beta-diol, androstane-3 beta. 17 beta-diol and 17 beta-estradiol (E2) are analyzed in detail, it can be seen that both aminoglutethimide and ketoconazole not only prevent the rise in serum steroids observed during the first 8 days of treatment with the LHRH agonist but that both compounds enhance the inhibitory effect of the LHRH agonist at later time intervals. A predominant inhibitory effect of ketoconazole is exerted on 17,20-desmolase activity. Aminoglutethimide has little influence on the loss of serum LH bioactivity induced by the LHRH agonist while ketoconazole stimulates the concentration of serum bioactive LH in the absence or presence of simultaneous treatment with the LHRH agonist. The present data clearly demonstrate that aminoglutethimide or ketoconazole can prevent the rise in serum androgens accompanying the first days of treatment with an LHRH agonist in the dog. Moreover, after 3 weeks of treatment, the inhibitory effect of the LHRH agonist on serum androgen levels is enhanced by addition of aminoglutethimide or ketoconazole. Moreover, Flutamide does not interfere with the inhibitory action of the LHRH agonist, aminoglutethimide or ketoconazole, thus suggesting that maximal inhibition of androgen action is likely to be achieved by a combination of these drugs.     

Characteristics of dynamic postural reactions in the locust hindleg

Summary The use of the locust (Schistocerca americana) hindleg in postural control was examined in animals that stood on a repeatedly swayed vertical substrate. Myograms were recorded from leg muscles and the angle of the femoro-tibial joint was monitored photographically. Two discrete strategies were observed,; in compensatory reactions the hindleg was held in place, while in flexion reactions, the leg was moved, most often to complete flexion of the femoro-tibial joint. Tightly coupled, rhythmic bursting occurred in the flexor and levator muscles of the leg during compensatory reactions. Bursting was initiated repeatedly when the substrate was being pulled away from the animal. Bursting was correlated with subsequent decreases in the rate of change of the femorotibial joint angle. Compensatory and flexion reactions occurred preferentially in different ranges of joint angles: most often, compensatory reactions occurred in the midrange, while flexion reactions were elicited in the extremes of joint angle. These differences may be due to the mechanical advantages of the tibial muscles and the leg may be moved to full flexion because of a locking mechanism of the flexor muscle tendon. These reactions are compared with known reflexes of hindleg proprioceptors and contrasted with similar responses of vertebrates.