Kinetic control of mitochondrial ATP synthesis

In order to gain a clearer understanding of the kinetic control of ATP synthesis, rat liver and rat heart mitochondria were incubated under conditions that resulted in various rates of net ATP synthesis or ATP hydrolysis. Radiolabeled phosphate was included in the incubation media, and exchange rates between phosphate and ATP were determined as a function of rates of net ATP synthesis. Since ATP synthase is a highly reversible enzyme, the catalyzed reaction was expected to approach equilibrium especially at low rates of respiration and net ATP synthesis. Thus ADP + Pi V1 in equilibrium V2 ATP. If V1 is the rate of incorporation of radiolabeled phosphate into ATP, then net ATP synthesis (or hydrolysis) is V1 - V2. Since V1 and V1 - V2 could be measured, it was possible to calculate V2. V1 doubled in the transition from zero to maximal net ATP synthesis, whereas V2 decreased by over 90% when the rate of ATP synthesis was high due to high-media ADP. In heart mitochondria at 37 degrees C when respiration increased from 104 +/- 10 to 842 +/- 51 nanoatoms of O2/(min X mg), incorporation of [33P]phosphate into ATP (V1) increased from 1,100 +/- 60 to 1,978 +/- 121 and V2 decreased from 1,100 to near zero. These data demonstrate that mitochondrial ATP synthesis does not occur near equilibrium under physiological conditions and relatively high rates of ATP synthesis. A reaction with a high ratio of forward to reverse flux is obviously not near equilibrium. The important most sensitively controlled reaction appears to be V2, ATP hydrolysis. Possible mechanisms of kinetic control of V2 are discussed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermittent illumination increases biophotolytic hydrogen yield by Anabaena cylindrica.

Intermittent illumination increased H2 and C2H4 yields per unit of light from growing cells and from nitrogren-starved cells by 1.7- and 1.35-fold, respectively, as compared with continuous illumination.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

Status,trends, and equilibrium abundance estimates of the translocated sea otter population in Washington State

Sea otters (Enhydra lutris kenyoni) historically occurred in Washington State, USA, until their local extinction in the early 1900s as a result of the maritime fur trade. Following their extirpation, 59 sea otters were translocated from Amchitka Island, Alaska, USA, to the coast of Washington, with 29 released at Point Grenville in 1969 and 30 released at La Push in 1970. The Washington Department of Fish and Wildlife has outlined 2 main objectives for sea otter recovery: a target population level and a target geographic distribution. Recovery criteria are based on estimates of population abundance, equilibrium abundance (K), and geographic distribution; therefore, estimates of these parameters have important management implications. We compiled available survey data for sea otters in Washington State since their translocation (1977–2019) and fit a Bayesian state-space model to estimate past and current abundance, and equilibrium abundance at multiple spatial scales. We then used forward projections of population dynamics to explore potential scenarios of range recolonization and as the basis of a sensitivity analysis to evaluate the relative influence of movement behavior, frontal wave speed, intrinsic growth, and equilibrium density on future population recovery potential. Our model improves upon previous analyses of sea otter population dynamics in Washington by partitioning and quantifying sources of estimation error to estimate population dynamics, by providing robust estimates of K, and by simulating long-term population growth and range expansion under a range of realistic parameter values. Our model resulted in predictions of population abundance that closely matched observed counts. At the range-wide scale, the population size in our model increased from an average of 21 independent sea otters (95% CI = 13–29) in 1977 to 2,336 independent sea otters (95% CI = 1,467–3,359) in 2019. The average estimated annual growth rate was 12.42% and varied at a sub-regional scale from 6.42–14.92%. The overall estimated mean K density of sea otters in Washington was 1.71 ± 0.90 (SD) independent sea otters/km² of habitat (1.96 ± 1.04 sea otters/km², including pups), and estimated densities within the current range correspond on average to 87% of mean sub-regional equilibrium values (range = 66–111%). The projected value of K for all of Washington was 5,287 independent sea otters (95% CI = 2,488–8,086) and 6,080 sea otters including pups (95% CI = 2,861–9,300), assuming a similar range of equilibrium densities in currently un-occupied habitats. Sensitivity analysis of simulations of sea otter population growth and range expansion suggested that mean K density estimates in currently occupied sub-regions had the largest impact on predicted future population growth (r² = 0.52), followed by the rate of southward range expansion (r² = 0.26) and the mean K density estimate of currently unoccupied sub-regions to the south of the current range (r² = 0.04). Our estimates of abundance and sensitivity analysis of simulations of future population abundance and geographic range help determine population status in relation to population recovery targets and identify the most influential parameters affecting future population growth and range expansion for sea otters in Washington State.