Secretory carrier membrane protein 2 regulates exocytic insertion of NKCC2 into the cell membrane

The renal-specific Na-K-2Cl co-transporter, NKCC2, plays a pivotal role in regulating body salt levels and blood pressure. NKCC2 mutations lead to type I Bartter syndrome, a life-threatening kidney disease. Regulation of NKCC2 trafficking behavior serves as a major mechanism in controlling NKCC2 activity across the plasma membrane. However, the identities of the protein partners involved in cell surface targeting of NKCC2 are largely unknown. To gain insight into these processes, we used a yeast two-hybrid system to screen a kidney cDNA library for proteins that interact with the NKCC2 C terminus. One binding partner we identified was SCAMP2 (secretory carrier membrane protein 2). Microscopic confocal imaging and co-immunoprecipitation assays confirmed NKCC2-SCAMP2 interaction in renal cells. SCAMP2 associated also with the structurally related co-transporter NCC, suggesting that the interaction with SCAMP2 is a common feature of sodium-dependent chloride co-transporters. Heterologous expression of SCAMP2 specifically decreased cell surface abundance as well as transport activity of NKCC2 across the plasma membrane. Co-immunolocalization experiments revealed that intracellularly retained NKCC2 co-localizes with SCAMP2 in recycling endosomes. The rate of NKCC2 endocytic retrieval, assessed by the sodium 2-mercaptoethane sulfonate cleavage assay, was not affected by SCAMP2. The surface-biotinylatable fraction of newly inserted NKCC2 in the plasma membrane was reduced by SCAMP2, demonstrating that SCAMP2-induced decrease in surface NKCC2 is due to decreased exocytotic trafficking. Finally, a single amino acid mutation, cysteine 201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, which is believed to regulate exocytosis, abolished SCAMP2-mediated down-regulation of the co-transporter. Taken together, these data are consistent with a model whereby SCAMP2 regulates NKCC2 transit through recycling endosomes and limits the cell surface targeting of the co-transporter by interfering with its exocytotic trafficking.     

Manipulation of flavour and aroma compound sequestration and release using a glycosyltransferase with specificity for terpene alcohols

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP‐glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)‐hex‐3‐enol, contribute strongly to the ‘grassy‐green’ aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over‐expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan‐3‐ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over‐expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over‐expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3‐methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening.     

Fuel economy,woodland management and adaptation strategies in a Classic Maya city: applying anthracology to urban settings in high biodiversity tropical forests

Vegetation History and Archaeobotany - Fuelling ancient Maya cities and industries has been identified for some time now as a critical concern for the pre-Columbian Maya, especially since there is...     

Stable Isotope and Signature Fatty Acid Analyses Suggest Reef Manta Rays Feed on Demersal Zooplankton

Assessing the trophic role and interaction of an animal is key to understanding its general ecology and dynamics. Conventional techniques used to elucidate diet, such as stomach content analysis, are not suitable for large threatened marine species. Non-lethal sampling combined with biochemical methods provides a practical alternative for investigating the feeding ecology of these species. Stable isotope and signature fatty acid analyses of muscle tissue were used for the first time to examine assimilated diet of the reef manta ray Manta alfredi, and were compared with different zooplankton functional groups (i.e. near-surface zooplankton collected during manta ray feeding events and non-feeding periods, epipelagic zooplankton, demersal zooplankton and several different zooplankton taxa). Stable isotope δ¹⁵N values confirmed that the reef manta ray is a secondary consumer. This species had relatively high levels of docosahexaenoic acid (DHA) indicating a flagellate-based food source in the diet, which likely reflects feeding on DHA-rich near-surface and epipelagic zooplankton. However, high levels of ω6 polyunsaturated fatty acids and slightly enriched δ¹³C values in reef manta ray tissue suggest that they do not feed solely on pelagic zooplankton, but rather obtain part of their diet from another origin. The closest match was with demersal zooplankton, suggesting it is an important component of the reef manta ray diet. The ability to feed on demersal zooplankton is likely linked to the horizontal and vertical movement patterns of this giant planktivore. These new insights into the habitat use and feeding ecology of the reef manta ray will assist in the effective evaluation of its conservation needs.     

Opsonin-dependent phagocytosis of bacteria by murine macrophage-like cells

We have investigated the phagocytic properties of the macrophage-like cell line DCH-7, derived from fusion of mouse macrophages with a mouse T-lymphoma cell line. These cells phagocytosed opsonized bacteria. IgG appeared to be the major opsonin forStaphylococcus aureus Wood 46 as well as for threeEscherichia coli strains; complement components were not required as opsonins. Intracellular bacteria survived to a large extent. This model system should be a useful tool for studying the process of phagocytosis and phagocytic killing of bacteria.     

Clonotype and repertoire changes drive the functional improvement of HIV-specific CD8 T cell populations under conditions of limited antigenic stimulation

Persistent exposure to cognate Ag leads to the functional impairment and exhaustion of HIV-specific CD8 T cells. Ag withdrawal, attributable either to antiretroviral treatment or the emergence of epitope escape mutations, causes HIV-specific CD8 T cell responses to wane over time. However, this process does not continue to extinction, and residual CD8 T cells likely play an important role in the control of HIV replication. In this study, we conducted a longitudinal analysis of clonality, phenotype, and function to define the characteristics of HIV-specific CD8 T cell populations that persist under conditions of limited antigenic stimulation. Ag decay was associated with dynamic changes in the TCR repertoire, increased expression of CD45RA and CD127, decreased expression of programmed death-1, and the emergence of polyfunctional HIV-specific CD8 T cells. High-definition analysis of individual clonotypes revealed that the Ag loss-induced gain of function within HIV-specific CD8 T cell populations could be attributed to two nonexclusive mechanisms: 1) functional improvement of persisting clonotypes; and 2) recruitment of particular clonotypes endowed with superior functional capabilities.     

Fcγ receptors inhibit mouse and human basophil activation

Besides high-affinity IgE receptors (FcεRI), human basophils express activating (FcγRIIA) and inhibitory (FcγRIIB) low-affinity IgG receptors. IgG receptors (FcγR) were also found on mouse basophils, but not identified. We investigated in this study FcγR and the biological consequences of their engagement in basophils of the two species. We found the following: 1) that mouse basophils also express activating (FcγRIIIA) and inhibitory (FcγRIIB) low-affinity FcγR; 2) that activating FcγR can activate both human and mouse basophils, albeit with different efficacies; 3) that negative signals triggered by inhibitory FcγR are dominant over positive signals triggered by activating FcγR, thus preventing both human and mouse basophils from being activated by IgG immune complexes; 4) that the coengagement of FcεRI with inhibitory and activating FcγR results in a FcγRIIB-dependent inhibition of IgE-induced responses of both human and mouse basophils; 5) that FcγRIIB has a similar dominant inhibitory effect in basophils from virtually all normal donors; and 6) that IL-3 upregulates the expression of both activating and inhibitory FcγR on human basophils from normal donors, but further enhances FcγRIIB-dependent inhibition. FcγR therefore function as a regulatory module, made of two subunits with antagonistic properties, that prevents IgG-induced and controls IgE-induced basophil activation in both mice and humans.     

Crystal structures and magnetic properties of Ni-bisdithiolene complexes with decamethylmetallocenium cations

Three new compounds of formula (1), (2), and (3) have been synthesised, and structurally and magnetically characterised (dmit²⁻ = 1,3-dithiol-2-thione-4,5-dithiolato; dmid²⁻ = 1,3-dithiol-2-one-4,5-dithiolato). Their structural features and magnetic behaviours are compared with those of and . The result of this is that the interactions between the Ni(dmit)₂ units in 1 are of ferromagnetic-type, as suggested previously for . The change from acetonitrile to acetone when going from to 2 results in stronger ferromagnetic intermolecular interactions. This better cooperativity is due to a significant increase of the number of contacts between the various moieties within the . On the contrary, the inclusion of larger solvents such as benzonitrile in this type of complexes results in a totally different structural arrangement, which leads to an antiferromagnetic behaviour for 3.