Analysis of Diffusion of Ras2 in Saccharomyces cerevisiae Using Fluorescence Recovery after Photobleaching

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane localized fluorophore with the cytosol during Fluorescence Recovery after Photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma-membrane localized wild type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 micron x 1 micron bleach region-of-interest (ROI) and a 0.5 micron x 0.5 micron bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane associated fluorophores using FRAP on commercial confocal laser scanning microscopes.Comment: Accepted for publication in Physical Biology (2010). 28 pages, 7 figures, 3 table

Hydrology, Water Chemistry, and Vegetation Characteristics of a Tamarack Bog in Bath Township, Ohio: Towards Restoration and Enhancement

Author Institution: Dept. of Biology, University of Akron, OHAuthor Institution: Dept. of Botany, University of British Columbia, Vancouver, BC, CanadaAuthor Institution: Dept. of Natural Resource Science, Thompson Rivers University, Kamloops, BC, CanadaThe current state of the Bath Tamarack Bog has raised concern about the health and function of the system. Only 6 tamarack (Larix laricina) trees remain, while deciduous trees, particularly red maple (Acer rubrum) and invasive species such as glossy buckthorn (Rhamnus frangula) and multiflora rose (Rosa multiflora), dominate the bog. Our purpose was to assess the physical, chemical, and biological properties of the tamarack bog. Environmental and biological properties of Bath Tamarack Bog were measured from May 2001 through November 2002. In 2001, the center of the bog experienced water levels below those typically found in bogs, yet experienced normal water levels in the following year. Water chemistry results indicate the pH is much greater than that characteristic of a typical bog, ranging from 5.94 to 7.41. Nutrient levels fluctuated and were generally higher for calcium, potassium, and phosphate than a typical bog, while nitrogen levels remained low. These results indicate that the bog is not functioning normally and is in decline. The degradation of the bog is most likely due to anthropogenic activity. Ditching occurred between 1963 and 1969 and seems to have induced the progression of red maple trees and invasive species into the bog by lowering water levels. Since 1938, the first aerial photo we have record of, the bog has reduced to approximately a third of its size, which is approximately 1.99 hectares. The bog appears to be in a late successional stage, rapidly changing to a forested wetland. We discuss possible management and restoration efforts needed to restore or enhance the tamarack bog, including 1) planting Sphagnum mats, 2) introducing tamarack seedlings, 3) controlling invasive species, and 4) maintaining the hydrology close to the soil surface. All of these measures are suggested in association with educational outreach

Test of models for replication of simian virus 40 DNA following ultraviolet irradiation.

When mammalian cells are irradiated with ultraviolet light, semiconservative DNA replication is inhibited and the length of newly synthesized daughter strands is reduced. We have used the simian virus 40 (SV40) viral system to examine the molecular mechanism by which this inhibition of DNA replication occurs immediately following ultraviolet irradiation. We tested two models for DNA replication-inhibition by using a procedure first developed by Danna, K. J., and D. Nathans (1972, Proc. Natl. Acad. Sci. USA, 69:3097-3100) in which the distribution of 3H-label in segments of newly completed SV40 form-I molecules is measured after short pulse labeling with 3H-thymidine. Our experimental results were compared with those predicted by mathematical models that describe two possible molecular mechanisms of replication inhibition. Our data are best fit by a "blockage" model in which any pyrimidine dimer encountered by the replication fork prevents complete replication of the SV40 genome. An alternative model called "slowdown" in which DNA damage causes a generalized slowdown of replication fork movement on all genomes has more adjustable parameters but does not fit the data as well as the blockage model

Testing the fit of a quantal model of neurotransmission.

Many studies of synaptic transmission have assumed a parametric model to estimate the mean quantal content and size or the effect upon them of manipulations such as the induction of long-term potentiation. Classical tests of fit usually assume that model parameters have been selected independently of the data. Therefore, their use is problematic after parameters have been estimated. We hypothesized that Monte Carlo (MC) simulations of a quantal model could provide a table of parameter-independent critical values with which to test the fit after parameter estimation, emulating Lilliefors's tests. However, when we tested this hypothesis within a conventional quantal model, the empirical distributions of two conventional goodness-of-fit statistics were affected by the values of the quantal parameters, falsifying the hypothesis. Notably, the tests' critical values increased when the combined variances of the noise and quantal-size distributions were reduced, increasing the distinctness of quantal peaks. Our results support two conclusions. First, tests that use a predetermined critical value to assess the fit of a quantal model after parameter estimation may operate at a differing unknown level of significance for each experiment. Second, a MC test enables a valid assessment of the fit of a quantal model after parameter estimation

Optimal design of multioutput sampling schedules - Software and applications to endocrine-metabolic and pharmacokinetic models

This paper describes a program for computing optimal sampling schedules for multiinput-multioutput experiments designed for parameter estimation of physiological systems models. Theory of the algorithm and details of its implementation are given. Practical applications of the software to models of glucose-insulin regulation, ketone body, and insulin kinetics are presented. Results document the potentiality of the software for designing experiments, and show that optimal design can considerably reduce the number of samples withdrawn from a patient in in vivo clinical studies