Effects of hypoxia on oxygen consumption,swimming velocity and gut evacuation in southern bluefin tuna (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

Of the few measurements of the behavioural and physiological responses of tuna to hypoxia, most are restricted to shallow diving tropical species. Furthermore, when wild tuna experience low dissolved oxygen, they are likely to have an increased oxygen demand associated with the metabolic cost of food digestion and assimilation (specific dynamic action). However the response of postprandial tuna to hypoxia has never been examined. This study focuses on the metabolic and behavioural responses of both fasted and postprandial southern bluefin tuna (Thunnus maccoyii) to low dissolved oxygen. Fasted T. maccoyii were exposed to dissolved oxygen levels of 4.44, 3.23, 2.49 and 1.57 mg·l⁻¹ for 20–21 h. In moderate hypoxia (4.44 and 3.23 mg·l⁻¹), swimming speed was enhanced (1.5 and 1.3 times normoxic speed, respectively) presumably to increase ventilation volume. Routine metabolic rate (R _r ) was similarly elevated (1.3 and 1.2 times normoxic R _r , respectively), most likely due to increased metabolic demand of faster swimming. At 2.49 mg·l⁻¹, swimming speed increased to over double the normoxic speed, possibly as an escape response. At 1.57 mg·l⁻¹, both swimming speed and R _r were reduced (0.8 and 0.9 times normoxic level, respectively), and tuna failed to survive the entire 20 h exposure period. This reveals that the critical oxygen level of T. maccoyii is between 1.57 and 2.49 mg·l⁻¹, demonstrating that they are remarkably well adapted to low dissolved oxygen. Feeding did not greatly influence their hypoxia tolerance with tuna surviving exposure to dissolved oxygen levels of 2.96 and 1.81 mg·l⁻¹ for 21 h, after ingesting a ration of 6.7% body weight of sardines (Sardinops sagax). In a subsequent experiment to determine the effects of hypoxia on digestion rate, T. maccoyii were fed to satiation and exposed to a dissolved oxygen level of 2.84 mg·l⁻¹ for 6.5–8 h. There was no significant difference in swimming speed, R _r and gastric evacuation rates of tuna in hypoxia compared to those in normoxia. This demonstrates that in moderate to severe hypoxia, T. maccoyii are still capable of aerobically supporting maintenance metabolism, routine swimming and specific dynamic action. It is hypothesized that adaptations which support the large metabolic scope of tuna are also likely to be beneficial for oxygen extraction and delivery in conditions of hypoxia.     

Automated cryoEM data acquisition and analysis of 284742 particles of GroEL

One of the goals in developing our automated electron microscopy data acquisition system, Leginon, was to improve both the ease of use and the throughput of the process of acquiring low dose images of macromolecular specimens embedded in vitreous ice. In this article, we demonstrate the potential of the Leginon system for high-throughput data acquisition by describing an experiment in which we acquired images of more than 280,000 particles of GroEL in a single 25 h session at the microscope. We also demonstrate the potential for an automated pipeline for molecular microscopy by showing that these particles can be subjected to completely automated procedures to reconstruct a three-dimensional (3D) density map to a resolution better than 8 A. In generating the 3D maps, we used a variety of metadata associated with the data acquisition and processing steps to sort and select the particles. These metadata provide a number of insights into factors that affect the quality of the acquired images and the resulting reconstructions. In particular, we show that the resolution of the reconstructed 3D density maps improves with decreasing ice thickness. These data provide a basis for assessing the capabilities of high-throughput macromolecular microscopy.     

Structural basis for cargo regulation of COPII coat assembly

Using cryo-electron microscopy, we have solved the structure of an icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) coassembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice-forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is well positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 beta-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo, including procollagen and chylomicrons, that is sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.     

CRAF autophosphorylation of serine 621 is required to prevent its proteasome-mediated degradation

The CRAF protein kinase regulates proliferative, differentiation, and survival signals from activated RAS proteins to downstream effectors, most often by inducing MEK/ERK activation. A well-established model of CRAF regulation involves RAS-mediated translocation of CRAF to the plasma membrane, where it is activated by a series of events including phosphorylation. Here we have discovered a new mode of regulation that occurs prior to this step. By creating a kinase-defective version of CRAF in mice or by use of the RAF inhibitor sorafenib, we show that CRAF must first undergo autophosphorylation of serine 621 (S621). Autophosphorylation occurs in cis, does not involve MEK/ERK activation, and is essential to ensure the correct folding and stability of the protein. In the absence of S621 phosphorylation, CRAF is degraded by the proteasome by mechanisms that do not uniquely rely on the E3 ubiquitin ligase CHIP.     

B cells promote resistance to heterosubtypic strains of influenza via multiple mechanisms

Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.     

Développement de la partie antérieure de la mandibule de Salmo trutta fario L. (Pisces, Teleostei, Salmonidae)

A study of the origin and development of the bony complex ("dentaire" s.l.) later forming the anterior part of the mandible of Salmo fario leads to the following conclusions: (1) various modes of ossifications occur in the ontogeny of this bony complex, (2) the main component is the dermal (dento-splénial) whereas the enchondral one (mentomeckelien) is much reduced, (3) the proposed term "dento-splénio-mentomeckelien" focuses attention, at the same time, on the order of appearance and relative importance of the two components.     

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild‐type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.     

Allosteric Regulation of DNA Cleavage and Sequence-Specificity through Run-On Oligomerization

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