Activation of ion transport pathways by changes in cell volume.

Swelling-activated K+ and Cl- channels, which mediate RVD, are found in most cell types. Prominent exceptions to this rule include red cells, which together with some types of epithelia, utilize electroneutral [K(+)-Cl-] cotransport for down-regulation of volume. Shrinkage-activated Na+/H+ exchange and [Na(+)-K(+)-2 Cl-] cotransport mediate RVI in many cell types, although the activation of these systems may require special conditions, such as previous RVD. Swelling-activated K+/H+ exchange and Ca2+/Na+ exchange seem to be restricted to certain species of red cells. Swelling-activated calcium channels, although not carrying sufficient ion flux to contribute to volume changes may play an important role in the activation of transport pathways. In this review of volume-activated ion transport pathways we have concentrated on regulatory phenomena. We have listed known secondary messenger pathways that modulate volume-activated transporters, although the evidence that volume signals are transduced via these systems is preliminary. We have focused on several mechanisms that might function as volume sensors. In our view, the most important candidates for this role are the structures which detect deformation or stretching of the membrane and the skeletal filaments attached to it, and the extraordinary effects that small changes in concentration of cytoplasmic macromolecules may exert on the activities of cytoplasmic and membrane enzymes (macromolecular crowding). It is noteworthy that volume-activated ion transporters are intercalated into the cellular signaling network as receptors, messengers and effectors. Stretch-activated ion channels may serve as receptors for cell volume itself. Cell swelling or shrinkage may serve a messenger function in the communication between opposing surfaces of epithelia, or in the regulation of metabolic pathways in the liver. Finally, these transporters may act as effector systems when they perform regulatory volume increase or decrease. This review discusses several examples in which relatively simple methods of examining volume regulation led to the discovery of transporters ultimately found to play key roles in the transmission of information within the cell. So, why volume? Because it's functionally important, it's relatively cheap (if you happened to have everything else, you only need some distilled water or concentrated salt solution), and since it involves many disciplines of experimental biology, it's fun to do.     

Law as Clinical Evidence: A New ConstitutiveModel of Medical Education and Decision-Making

Over several decades, ethics and law have been applied to medical education and practice in a way that reflects the continuation during the twentieth century of the strong distinction between facts and values. We explain the development of applied ethics and applied medical law and report selected results that reflect this applied model from an empirical project examining doctors’ decisions on withdrawing/withholding treatment from patients who lack decision-making capacity. The model is critiqued, and an alternative “constitutive” model is supported on the basis that medicine, medical law, and medical ethics exemplify the inevitable entanglement of facts and values. The model requires that ethics and law be taught across the medical education curriculum and integrated with the basic and clinical sciences and that they be perceived as an integral component of medical evidence and practice. Law, in particular, would rank as equal in normative authority to the relevant clinical scientific “facts” of the case, with graduating doctors having as strong a basic command of each category as the other. The normalization of legal knowledge as part of the clinician’s evidence base to be utilized in practice may provide adequate consolation for clinicians who may initially resent further perceived incursions on their traditional independence and discretion.     

Hosts do not simply outsource pathogen resistance to protective symbionts

Microbial symbionts commonly protect their hosts from natural enemies, but it is unclear how protective symbionts influence the evolution of host immunity to pathogens. One possibility is that ‘extrinsic’ protection provided by symbionts allows hosts to reduce investment in ‘intrinsic’ immunological resistance mechanisms. We tested this idea using pea aphids (Acyrthosiphon pisum) and their facultative bacterial symbionts that increase host resistance to the fungal pathogen Pandora neoaphidis. The pea aphid taxon is composed of multiple host plant associated populations called biotypes, which harbor characteristic communities of symbionts. We found that biotypes that more frequently carry protective symbionts have higher, rather than lower, levels of intrinsic resistance. Within a biotype there was no difference in intrinsic resistance between clones that did and did not carry a protective symbiont. The host plant on which an aphid feeds did not strongly influence intrinsic resistance. We describe a simple conceptual model of the interaction between intrinsic and extrinsic resistance and suggest that our results may be explained by selection favoring both the acquisition of protective symbionts and enhanced intrinsic resistance in habitats with high pathogen pressure. Such combined protection is potentially more robust than intrinsic resistance alone.     

Structure of the NDH-2 – HQNO inhibited complex provides molecular insight into quinone-binding site inhibitors

Type II NADH:quinone oxidoreductase (NDH-2) is a proposed drug-target of major pathogenic microorganisms such as Mycobacterium tuberculosis and Plasmodium falciparum. Many NDH-2 inhibitors have been identified, but rational drug development is impeded by the lack of information regarding their mode of action and associated inhibitor-bound NDH-2 structure. We have determined the crystal structure of NDH-2 complexed with a quinolone inhibitor 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO). HQNO is nested into the slot-shaped tunnel of the Q-site, in which the quinone-head group is clamped by Q317 and I379 residues, and hydrogen-bonds to FAD. The interaction of HQNO with bacterial NDH-2 is very similar to the native substrate ubiquinone (UQ₁) interactions in the yeast Ndi1–UQ₁ complex structure, suggesting a conserved mechanism for quinone binding. Further, the structural analysis provided insight how modifications of quinolone scaffolds improve potency (e.g. quinolinyl pyrimidine derivatives) and suggests unexplored target space for the rational design of new NDH-2 inhibitors.     

Implications of high animal by-product feed inputs in life cycle assessments of farmed Atlantic salmon

Purpose

Animal by-products may be increasingly relied upon to satisfy nutritional requirements of salmonids and other fed aquaculture species as demand for fish meal outpaces supply. Previous studies of aquaculture supply chains have included either no animal by-product inputs or small inputs of poultry by-products. Australian Atlantic salmon production includes high use of feed inputs derived from poultry and mammalian by-products and provides a case study to explore the environmental implications and methodological challenges associated with these inputs.

Methods

Life cycle assessment was carried out on a vertically integrated salmon production system in Tasmania, representing approximately 40% of Australian Atlantic salmon production. The system included feed production, smolt/juvenile production, farm grow-out, processing and packaging, and distribution of head-on gutted salmon to market. Impacts from animal production were allocated to by-products on a gross chemical energy basis. Scenario analyses were conducted to determine the extent to which changes in feed conversion ratio, feed composition, and other variables affect results. Sensitivity analysis was carried out on the allocation method for fishery and animal by-products.

Results and discussion

Environmental impacts associated with Tasmanian salmon fed high quantities of animal by-products were markedly higher than those of previously assessed systems. All impacts were driven by feed production with the exception of eutrophication potential, which was driven equally by feed production and nutrient loss during grow-out. Animal by-products accounted for the majority of all impacts from feed production. Adopting a feed composition without animal by-products would result in dramatic improvements, including a 70% decrease in greenhouse gas emissions. Allocation choice had a clear effect on results, with biophysical allocation methods placing much more burden from animal production on fed systems than economic or no-impact allocation methods.

Conclusions

The use of animal by-product inputs in aquaculture feeds has a substantial effect on the environmental profile of farmed salmon products. The magnitude of this effect is dependent on the allocation method chosen for the treatment of products and by-products in upstream systems. The high impact of such systems recognizes the environmental cost of future aquaculture production that may rely more on intensive and high-impact animal production inputs as more efficient fishery inputs become increasingly limited relative to demand.

     

Studies of modern Italian dog populations reveal multiple patterns for domestic breed evolution

Through thousands of years of breeding and strong human selection, the dog (Canis lupus familiaris) exists today within hundreds of closed populations throughout the world, each with defined phenotypes. A singular geographic region with broad diversity in dog breeds presents an interesting opportunity to observe potential mechanisms of breed formation. Italy claims 14 internationally recognized dog breeds, with numerous additional local varieties. To determine the relationship among Italian dog populations, we integrated genetic data from 263 dogs representing 23 closed dog populations from Italy, seven Apennine gray wolves, and an established dataset of 161 globally recognized dog breeds, applying multiple genetic methods to characterize the modes by which breeds are formed within a single geographic region. Our consideration of each of five genetic analyses reveals a series of development events that mirror historical modes of breed formation, but with variations unique to the codevelopment of early dog and human populations. Using 142,840 genome‐wide SNPs and a dataset of 1,609 canines, representing 182 breeds and 16 wild canids, we identified breed development routes for the Italian breeds that included divergence from common populations for a specific purpose, admixture of regional stock with that from other regions, and isolated selection of local stock with specific attributes.     

Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.     

Differential Regulation of Histamine- and Bradykinin-Stimulated Phospholipase C in Adrenal Chromaffin Cells: Evidence for Involvement of Different Protein Kinase C Isoforms

Abstract: In this report we investigate the isoforms of protein kinase C (PKC) present in cultured adrenal chromaffin cells with respect to their modulation by treatment with phorbol ester and their possible differential involvement in the regulation of responses to histamine and bradykinin. The presence of individual isoforms of PKC was investigated by using eight isoform specific antisera, as a result of which PKC-α, ε, and ζ were identified. To characterize down-regulation of these enzymes, cells were incubated for 6–48 h with 1 µM phorbol myristate acetate (PMA). PKC-ε down-regulated more rapidly than PKC-α. At 12 h, PMA pretreatment, for example, PKC-ε was maximally down-regulated (23 ± 4% of controls), whereas PKC-α was unchanged. PKC-α showed partial down-regulation by 24 h of PMA pretreatment. PKC-ζ did not down-regulate at any of the times tested. Translocation from cytosol to membrane in response to PMA was also more rapid for PKC-ε than for PKC-α. The accumulation of total ³H-inositol (poly)phosphates in response to bradykinin or histamine was essentially abolished by prior treatment with 10-min PMA treatment (1 µM). However, with 12-h exposure to PMA, the bradykinin response was restored to the level seen with no prior PMA exposure. The histamine response showed no recovery by 12 h of PMA, but showed partial recovery by 24 h of PMA pretreatment. These observations showed that the restoration of the response to bradykinin corresponds to the loss of PKC-ε, whereas the restoration of the histamine response corresponds to the loss of PKC-α. This picture was confirmed with further studies on cytosolic Ca²⁺. The results show that chromaffin cells exhibit an unusual pattern of down-regulation of PKC isoforms on prolonged exposure to PMA, and that there is a differential effect of exposure to PMA on the histamine and bradykinin responses, suggesting that different PLC-linked receptors in chromafin cells are differentially regulated by PKC isoforms.     

Phytosiderophore release in relation to micronutrient metal deficiencies in barley

Phytosiderophore release occurs under both iron and zinc deficiencies in representative Poaceae and has been speculated to be a general adaptive response to enhance the acquisition of micronutrient metals. To test this hypothesis, phytosiderophore (PS) release rates from barley (Hordeum vulgare cv. CM72) subjected to deficiencies of Fe, Zn, Mn, and Cu were compared using chelator-buffered nutrient solutions. PS release rates were determined at two day intervals during onset and development of deficiency symptoms. Plant dry matter yields and nutrient concentrations, measured at three time points were used to construct growth curves for calculation of PS release per unit root mass and estimation of critical internal nutrient levels associated with PS release. In comparison to trace metal-sufficient control plants, dry matter production was markedly reduced in the Zn, Mn, and Cu deficiency treatments, with final relative yields of 49, 61, and 34%, respectively. Relative yields for Fe-deficient plants grown at three suboptimal Fe levels ranged from 95 to 33% of control, and provided a basis for comparison of PS release rates by Zn-, Mn-, and Cu-deficient plants at similar levels of growth inhibition. Under Fe deficiency, PS release increased with severity of the deficiency as measured by foliar Fe concentration, yield reduction, and chlorosis. Changes in PS release rates over time suggested a cyclical pattern that may be regulated by Fe concentration in the plant shoot. The highest rate of PS release (35 mol g^–1 root dw 2 h^–1) was measured after 10 days of growth at pFe 19, whereas control plants adapted for growth at pFe 17 released only 2 to 3 mol g^–1 root dw 2 h^–1. In a second experiment, maximum PS release rates for barley subjected to Zn, Mn, and Cu deficiencies were only 2.6, 2.5 and 1 mol g^–1 2 h^–1, respectively and were only slightly elevated over those of the control plants (ca. 1 mol g^–1 root dw 2 h^–1) grown at pFe 16.5. Moreover, enhanced PS release under Zn deficiency occurred much later, after the deficiency had already caused severely reduced growth. The results suggest that phytosiderophore release in this barley cultivar is a specific response to Fe deficiency and is not significantly induced in response to deficiencies of other trace metals.