Resonance energy transfer study of membrane-bound aggregates of the sarcoplasmic reticulum calcium ATPase

The aggregation of the membrane-bound calcium ATPase from sarcoplasmic reticulum has been studied by resonance energy transfer. The temperature dependence of resonance energy transfer from a fluorescent membrane lipid donor to an acceptor covalently linked to the Ca2+ ATPase was observed for the native sarcoplasmic reticulum vesicles and for purified protein reconstituted into phospholipid vesicles. The efficiency of energy transfer in these systems increases as the size of protein aggregates decrease. This is due to the increased exposure of the protein in the lipid domain that results in the shortening of distances between donors and acceptors. The degree of aggregation was observed to decrease with increasing temperature. Aggregates rea h a limiting size at low temperature (5 degrees C) but not a high temperatures (45 degrees C). For the reconstituted system, the aggregate size showed a continuous, smooth decrease with increasing temperature. Sarcoplasmic reticulum vesicles showed a decrease in aggregation except for a region from 20 to 30 degrees C in which no change occurred. Arrhenius plots of the calcium transport activities for both systems do not reflect these differences, but instead show similar discontinuities and activation energies. A theoretical model is used to analyze the resonance energy transfer results for the reconstituted vesicles. The average radius of the ATPase aggregate is obtained from this analysis. The limiting, low temperature value of the aggregate radius is consistent with the formation of a tetramer. This structure breaks down to smaller, functional units at higher temperatures.     

An ultra-pure in vitro phase synchrony method employing centrifugal elutriation and viable flow cytometric cell sorting

We present a method of synchronizing cells in G1-, S-, and G2M-phases employing sequential centrifugal elutriation and viable flow cytometric cell sorting of Hoechst-33342 stained Chinese hamster ovary cells. G1- and S-phase cells can be separated to greater than 99% homogeneity and G2-M to 70% purity. Most of the 30% contamination in the G2-M fraction was due to S-phase cells, whose reproductive integrity could be eliminated through the use of high specific activity 3H-TdR. There were minimal toxic effects or perturbations to growth following the selection procedures. The most significant limitation of this technique appears to be the rate of cell sorting, which, with current equipment, is approximately 3,000 cells per second.     

Interactions of thyrotropin releasing hormone and morphine sulfate in rodents.

Thyrotopin releasing hormone (TRH) produces “wet dog shakes” in rats similar to those observed during morphine withdrawal. The shaking behavior precipitated by morphine abstinence can be exacerbated by TRH administration while the other components of the morphine withdrawal syndrome remain unchanged. Morphine, chlorpromazine, apomorphine, and Δ⁹-tetrahydrocannabinol effectively block shakes induced by either TRH administration or morphine withdrawal. These results suggest the possibility that endogenous TRH may be associated with the “wet dog shakes” observed as a portion of morphine's abstinence syndrome in rats. However, TRH is unable to alter the stereospecific binding of morphine $\text{in}$$\text{vivo}$ or $\text{in}$$\text{vitro}$, and naloxone fails to potentiate the number of TRH-induced shakes. TRH has no antinociceptive properties, and it cannot alter those of morphine. These data suggest that more than one neuromechanism may be responsible for shaking behavior in rats.     

Prion gene sequence variation within diverse groups of U.S. sheep,beef cattle,and deer

Prions are proteins that play a central role in transmissible spongiform encephalopathies in a variety of mammals. Among the most notable prion disorders in ungulates are scrapie in sheep, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer. Single nucleotide polymorphisms in the sheep prion gene (PRNP) have been correlated with susceptibility to natural scrapie in some populations. Similar correlations have not been reported in cattle or deer; however, characterization of PRNP nucleotide diversity in those species is incomplete. This report describes nucleotide sequence variation and frequency estimates for the PRNP locus within diverse groups of U.S. sheep, U.S. beef cattle, and free-ranging deer (Odocoileus virginianus and O. hemionus from Wyoming). DNA segments corresponding to the complete prion coding sequence and a 596-bp portion of the PRNP promoter region were amplified and sequenced from DNA panels with 90 sheep, 96 cattle, and 94 deer. Each panel was designed to contain the most diverse germplasm available from their respective populations to facilitate polymorphism detection. Sequence comparisons identified a total of 86 polymorphisms. Previously unreported polymorphisms were identified in sheep (9), cattle (13), and deer (32). The number of individuals sampled within each population was sufficient to detect more than 95% of all alleles present at a frequency greater than 0.02. The estimation of PRNP allele and genotype frequencies within these diverse groups of sheep, cattle, and deer provides a framework for designing accurate genotype assays for use in genetic epidemiology, allele management, and disease control.     

Regulation to Create Environments Conducive to Physical Activity: Understanding the Barriers and Facilitators at the Australian State Government Level

Introduction

Policy and regulatory interventions aimed at creating environments more conducive to physical activity (PA) are an important component of strategies to improve population levels of PA. However, many potentially effective policies are not being broadly implemented. This study sought to identify potential policy/regulatory interventions targeting PA environments, and barriers/facilitators to their implementation at the Australian state/territory government level.

Methods

In-depth interviews were conducted with senior representatives from state/territory governments, statutory authorities and non-government organisations (n = 40) to examine participants'': 1) suggestions for regulatory interventions to create environments more conducive to PA; 2) support for preselected regulatory interventions derived from a literature review. Thematic and constant comparative analyses were conducted.

Results

Policy interventions most commonly suggested by participants fell into two areas: 1) urban planning and provision of infrastructure to promote active travel; 2) discouraging the use of private motorised vehicles. Of the eleven preselected interventions presented to participants, interventions relating to walkability/cycling and PA facilities received greatest support. Interventions involving subsidisation (of public transport, PA-equipment) and the provision of more public transport infrastructure received least support. These were perceived as not economically viable or unlikely to increase PA levels. Dominant barriers were: the powerful ‘road lobby’, weaknesses in the planning system and the cost of potential interventions. Facilitators were: the provision of evidence, collaboration across sectors, and synergies with climate change/environment agendas.

Conclusion

This study points to how difficult it will be to achieve policy change when there is a powerful ‘road lobby’ and government investment prioritises road infrastructure over PA-promoting infrastructure. It highlights the pivotal role of the planning and transport sectors in implementing PA-promoting policy, however suggests the need for clearer guidelines and responsibilities for state and local government levels in these areas. Health outcomes need to be given more direct consideration and greater priority within non-health sectors.     

Maximizing antibody production in a targeted integration host by optimization of subunit gene dosage and position

Historically, therapeutic protein production in Chinese hamster ovary (CHO) cells has been accomplished by random integration (RI) of expression plasmids into the host cell genome. More recently, the development of targeted integration (TI) host cells has allowed for recombination of plasmid DNA into a predetermined genomic locus, eliminating one contributor to clone-to-clone variability. In this study, a TI host capable of simultaneously integrating two plasmids at the same genomic site was used to assess the effect of antibody heavy chain and light chain gene dosage on antibody productivity. Our results showed that increasing antibody gene copy number can increase specific productivity, but with diminishing returns as more antibody genes are added to the same TI locus. Random integration of additional antibody DNA copies in to a targeted integration cell line showed a further increase in specific productivity, suggesting that targeting additional genomic sites for gene integration may be beneficial. Additionally, the position of antibody genes in the two plasmids was observed to have a strong effect on antibody expression level. These findings shed light on vector design to maximize production of conventional antibodies or tune expression for proper assembly of complex or bispecific antibodies in a TI system.     

Effects of Partial Replacement of Corn with Glycerin on Ruminal Fermentation in a Dual-Flow Continuous Culture System

The objective of this study was to evaluate the effects of partially replacing dry ground corn with glycerin on ruminal fermentation using a dual-flow continuous culture system. Six fermenters (1,223 ± 21 ml) were used in a replicated 3x3 Latin square arrangement with three periods of 10 d each, with 7 d for diet adaptation and 3 d for sample collections. All diets contained 75% concentrate and three dietary glycerin levels (0, 15, and 30% on dry matter basis), totaling six replicates per treatment. Fermenters were fed 72 g of dry matter/d equally divided in two meals/d, at 0800 and 2000 h. Solid and liquid dilution rates were adjusted daily to 5.5 and 11%/h, respectively. On d 8, 9, and 10, samples of 500 ml of solid and liquid digesta effluent were mixed, homogenized, and stored at -20°C. Subsamples of 10 ml were collected and preserved with 0.2 mL of a 50% H₂SO₄ solution for later determination of NH₃-N and volatile fatty acids. Microbial biomass was isolated from fermenters for chemical analysis at the end of each experimental period. Data were analyzed using the MIXED procedure in SAS with α = 0.05. Glycerin levels did not affect apparent digestibility of DM (P _Lin. = 0.13; P _Quad. = 0.40), OM (P _Lin. = 0.72; P _Quad. = 0.15), NDF (P _Lin. = 0.38; P _Quad. = 0.50) and ADF (P _Lin. = 0.91; P _Quad. = 0.18). Also, glycerin inclusion did not affect true digestibility of DM (P _Lin. = 0.35; P _Quad. = 0.48), and OM (P _Lin. = 0.08; P _Quad. = 0.19). Concentrations of propionate (P < 0.01) and total volatile fatty acids (P < 0.01) increased linearly and concentrations of acetate (P < 0.01), butyrate (P = 0.01), iso-valerate (P < 0.01), and total branched-chain volatile fatty acids, as well as the acetate: propionate ratio (P < 0.01) decreased with glycerin inclusion. Linear increases on NH₃-N concentration in digesta effluent (P < 0.01) and on NH₃-N flow (P < 0.01) were observed due to glycerin inclusion in the diets. Crude protein digestibility (P = 0.04) and microbial N flow (P = 0.04) were greater in the control treatment compared with the other treatments and responded quadratically with glycerin inclusion. Furthermore, the inclusion of glycerin linearly decreased (P = 0.02) non-ammonia N flow. Glycerin levels did not affect the flows of total N (P _Lin. = 0.79; P _Quad. = 0.35), and dietary N (P _Lin. = 0.99; P _Quad. = 0.07), as well as microbial efficiency (P _Lin. = 0.09; P _Quad. = 0.07). These results suggest that partially replacing dry ground corn with glycerin may change ruminal fermentation, by increasing total volatile fatty acids, and propionate concentration without affecting microbial efficiency, which may improve glucogenic potential of beef cattle diets.