The structures of COPI-coated vesicles reveal alternate coatomer conformations and interactions

Transport between compartments of eukaryotic cells is mediated by coated vesicles. The archetypal protein coats COPI, COPII, and clathrin are conserved from yeast to human. Structural studies of COPII and clathrin coats assembled in vitro without membranes suggest that coat components assemble regular cages with the same set of interactions between components. Detailed three-dimensional structures of coated membrane vesicles have not been obtained. Here, we solved the structures of individual COPI-coated membrane vesicles by cryoelectron tomography and subtomogram averaging of in vitro reconstituted budding reactions. The coat protein complex, coatomer, was observed to adopt alternative conformations to change the number of other coatomers with which it interacts and to form vesicles with variable sizes and shapes. This represents a fundamentally different basis for vesicle coat assembly.     

Effects of establishment method and fall stocking rate of wheat pasture on forage mass, forage chemical composition, and performance of growing steers

Stocking rate is a fundamental variable for managing pastures, and there is a distinct relationship between stocking rate and animal performance for each forage type. This research was conducted to determine the effects of fall stocking rate (SR) and method of establishment of wheat pasture planted into dedicated crop fields on animal performance during the fall and subsequent spring. There was a factorial arrangement of tillage methods used in the establishment of wheat pasture and fall stocking rates. Tillage treatments included 1) CT, seed sown into a prepared seedbed, 2) RT, a single pass with a light disk followed by broadcasting of seed, or 3) NT, direct seeding into the undisturbed stubble of the grazed-out wheat pasture from the previous year. The fixed SR during the fall were 1.9, 2.5, and 3.7 growing beef steers (Bos taurus L.)/ha. In the spring all pastures were grazed at the same fixed SR by steers for graze out. Data were analyzed using the mixed procedure of SAS as a randomized complete block design with field as the experimental unit and year as the block. Forage mass, forage nutritive composition, and animal performance during the fall or spring were not affected (P ≥ 0.14) by tillage method. During the fall grazing season, with increasing SR there were linear (P < 0.01) decreases in BW of steers upon removal from pasture, BW gain per steer, and ADG, whereas grazing-day per hectare and BW gain per hectare increased linearly (P < 0.01) with increasing SR. The carryover effects of fall SR into the spring grazing season decreased (P< 0.01) grazing-day per hectare and tended (P ≤ 0.09) to produce quadratic changes in BW upon removal from pasture and BW gain per hectare. Across the fall and spring grazing seasons, grazing-day per hectare increased linearly (P < 0.01) with greater SR, and BW gain per hectare increased quadratically (P = 0.02) with increased fall SR. A tillage treatment by fall SR interaction (P = 0.10) indicates that although there was no difference (P ≥ 0.12) due to tillage treatment in BW gain per hectare at 1.9 or 2.5 SR, NT fields produced (P ≤ 0.04) more BW gain per hectare than CT or RT at the 3.7 SR. Although increasing SR of growing steers leads to reduced animal performance in the fall and reduced carrying capacity in the spring, NT appears to be capable of withstanding greater fall SR with less impact on total production per hectare than CT or RT.     

Estrus cycle effect on muscle tyrosine kinase activity in bitches

Estrus cycle is a well recognized cause of insulin resistance in bitches. The insulin receptor (IR) as well as the insulin-like growth factor-I receptor belong to the same subfamily of tyrosine kinase (TK) receptors. The objective of this study was to evaluate basal TK activity in muscle tissue of bitches during the estrus cycle. Twenty-four bitches were used in the study (7 in anestrus, 7 in estrus, and 10 in diestrus). Muscle samples, taken after spaying surgery to determine TK activity, were immediately frozen in liquid nitrogen and then stored at −80°C until the membranes were prepared by sequential centrifugation after being homogenized. TK activity was determined by Poly (Glu 4:Tyr 1) phosphorylation and expressed in cpm/μg of protein. TK activity was significantly lower (P < 0.001) in the animals in estrus (104.5 ± 11.9 cpm/μg of protein) and diestrus (94.5 ± 16.9 cpm/μg of protein) when compared with bitches in anestrus (183.2 ± 39.2 cpm/μg of protein). These results demonstrate, for the first time, lower basal TK activity in the muscle tissue of female dogs during estrus and diestrus, which may represent lower insulin signaling capacity, opening a new field of investigation into the molecular mechanisms of insulin resistance in dogs.     

Coaching and feedback: enhancing communication teaching and learning in veterinary practice settings

Communication is a critical clinical skill closely linked to clinical reasoning, medical problem solving, and significant outcomes of care such as accuracy, efficiency, supportiveness, adherence to treatment plans, and client and veterinarian satisfaction. More than 40 years of research on communication and communication education in human medicine and, more recently, in veterinary medicine provide a substantive rationale for formal communication teaching in veterinary education. As a result, veterinary schools are beginning to invest in communication training. However, if communication training is to result in development of veterinary communication skills to a professional level of competence, there must be follow-through with effective communication modeling and coaching in practice settings. The purpose of this article is to move the communication modeling and coaching done in the "real world" of clinical practice to the next level. The development of skills for communication coaching and feedback is demanding. We begin by comparing communication coaching with what is required for teaching other clinical skills in practice settings. Examining both, what it takes to teach others (whether DVM students or veterinarians in practice for several years) and what it takes to enhance one's own communication skills and capacities, we consider the why, what, and how of communication coaching. We describe the use of teaching instruments to structure this work and give particular attention to how to engage in feedback sessions, since these elements are so critical in communication teaching and learning. We consider the preconditions necessary to initiate and sustain communication skills training in practice, including the need for a safe and supportive environment within which to implement communication coaching and feedback. Finally we discuss the challenges and opportunities unique to coaching and to building and delivering communication skills training in practice settings.     

The Impact of Mitochondrial and Thermal Stress on the Bioenergetics and Reserve Respiratory Capacity of Fish Cell Lines

Abstract

Various stressors affect the health of wild and cultured fish and can cause metabolic disturbances that first manifest at the cellular level. Here, we sought to further our understanding of cellular metabolism in fish by examining the metabolic responses of cell lines derived from channel catfish Ictalurus puntatus (CCO), white bass Morone chrysops (WBE), and fathead minnow Pimephales promelas (EPC) to both mitochondrial and thermal stressors. Using extracellular flux (EF) technology, we simultaneously measured the oxygen consumption rate (OCR; a measure of mitochondrial function) and extracellular acidification rate (ECAR; a surrogate of glycolysis) in each cell type. We performed a mitochondrial function protocol whereby compounds modulating different components of mitochondrial respiration were sequentially exposed to cells. This provided us with basal and maximal OCR, OCR linked to ATP production, OCR from ion movement across the mitochondrial inner membrane, the reserve capacity, and OCR independent of the electron transport chain. After heat shock, EPC and CCO significantly decreased OCR and all three cell lines modestly increased ECAR. After heat shock, the reserve capacity, the mitochondrial energetic reserve used to cope with stress and increased bioenergetic demand, was unaffected in EPC and CCO and completely abrogated in WBE. These findings provide proof-of-concept experimental data that further highlight the utility of fish cell lines as tools for modeling bioenergetics.

Received April 12, 2012; accepted August 5, 2012