Trafficking and gating of hyperpolarization-activated cyclic nucleotide-gated channels are regulated by interaction with tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) and cyclic AMP at distinct sites

Ion channel trafficking and gating are often influenced by interactions with auxiliary subunits. Tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b) is an auxiliary subunit for neuronal hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. TRIP8b interacts directly with two distinct sites of HCN channel pore-forming subunits to control channel trafficking and gating. Here we use mutagenesis combined with electrophysiological studies to define and distinguish the functional importance of the HCN/TRIP8b interaction sites. Interaction with the last three amino acids of the HCN1 C terminus governed the effect of TRIP8b on channel trafficking, whereas TRIP8b interaction with the HCN1 cyclic nucleotide binding domain (CNBD) affected trafficking and gating. Biochemical studies revealed that direct interaction between TRIP8b and the HCN1 CNBD was disrupted by cAMP and that TRIP8b binding to the CNBD required an arginine residue also necessary for cAMP binding. In accord, increasing cAMP levels in cells antagonized the up-regulation of HCN1 channels mediated by a TRIP8b construct binding the CNBD exclusively. These data illustrate the distinct roles of the two TRIP8b-HCN interaction domains and suggest that TRIP8b and cAMP may directly compete for binding the HCN CNBD to control HCN channel gating, kinetics, and trafficking.     

Ammonia transformations and abundance of ammonia oxidizers in a clay soil underlying a manure pond

Unlined manure ponds are constructed on clay soil worldwide to manage farm waste. Seepage of ammonia-rich liquor into underlying soil layers contributes to groundwater contamination by nitrate. To identify the possible processes that lead to the production of nitrate from ammonia in this oxygen-limited environment, we studied the diversity and abundance of ammonia-transforming microorganisms under an unlined manure pond. The numbers of ammonia-oxidizing bacteria and anammox bacteria were most abundant in the top of the soil profile and decreased significantly with depth (0.5 m), correlating with soil pore-water ammonia concentrations and soil ammonia concentrations, respectively. On the other hand, the numbers of ammonia-oxidizing archaea were relatively constant throughout the soil profile (10(7) amoA copies per g(soil)). Nitrite-oxidizing bacteria were detected mainly in the top 0.2 m. The results suggest that nitrate accumulation in the vadose zone under the manure pond could be the result of complete aerobic nitrification (ammonia oxidation to nitrate) and could exist as a byproduct of anammox activity. While the majority of the nitrogen was removed within the 0.5-m soil section, possibly by combined anammox and heterotrophic denitrification, a fraction of the produced nitrate leached into the groundwater.     

Satellite tracking of the winter migration of Magellanic Penguins Spheniscus magellanicus breeding in the Falkland Islands

Magellanic penguin populations in the Falkland Islands may have decreased over the past decade. The post-breeding migration may be the period in which the birds are most vulnerable. To investigate this we equipped ten Magellanic Penguins after their moult at Seal Bay (51̀38'S, 58̀03'W), East Falkland, with satellite transmitters. The movements of the penguins were tracked for between 15 and 99 days until transmission ceased. All birds initially migrated to the northwest. However, four birds entered Argentinean coastal waters, then headed northeast following the coastline, The other penguins remained offshore, but also changed to a northeasterly course. Two birds migrated beyond 36̀S with a maximum distance to the colony of more than 1800 km, while the minimum distance travelled was up to 2700 km. Initially, the birds migrated quickly but their speed was reduced and became more variable as time progressed. A high concentration of positional fixes, associated with low travelling speeds, indicated at least four different areas where birds were assumed to forage efficiently. The relevance of these areas is discussed with respect to the diet of Magellanic Penguins and possible interactions with human activities.     

Grapevine fanleaf virus (GFLV)-specific antibodies confer GFLV and Arabis mosaic virus (ArMV) resistance in Nicotiana benthamiana

Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL₃, which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T₁ and T₂ progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance.     

Prediction of retention volumes and UV spectra of peptides in reversed phase HPLC

A method for calculating retention volumes of linear peptides with known primary structures and the values of their UV absorption at chosen wavelengths in reversed phase HPLC are described. These parameters are calculated for every peptide on the basis of the contributions of its amino acid residues determining its degree of retention and its UV spectrum. The contribution values are experimentally found from chromatograms of the free amino acids obtained by multiwavelength photometric detection under the conditions of the peptide chromatography. Thirty peptides have been chromatographed for the evaluation of the proposed method, and the correlation coefficients between the calculated and the experimental retention volumes have been found to be 0.95.     

Enkephalins and Opiate Antagonists Control Calmodulin Distribution in Neuroblastoma-Glioma Cells

The calcium binding protein calmodulin and the opiate receptor binding sites are unevenly distributed in various subcellular fractions of neuroblastoma-glioma NG108-15 cells. The crude mitochondrial-membrane fraction of these cells contains two membrane fractions that are separable by sucrose gradient centrifugation. These two differ in the content of both calmodulin and opiate receptors. Leucine enkephalin and D-Ala2-methionine enkephalinamide decrease the amount of membrane-bound calmodulin in the NC108-15 cells in a time- and dose-dependent manner, whereas the opiate antagonists naloxone and levallorphan have an opposite effect. Naloxone blocks the effect of leucine enkephalin and dextrallorphan has no significant effect. The opiate alkaloids entorphine and phenazocine induce changes similar to that of the enkephalins whereas morphine is inactive even at high concentrations. The alteration in the amount of membrane-bound calmodulin after a short incubation (15 min) with the enkephalins or with naloxone is reflected as an opposite change in the amount of calmodulin in the cell cytosol. Naloxone and levallorphan also increase the number of opiate receptors in NG108-15 cells but dextrallorphan has no such effect. Modulation of the intracellular distribution of calmodulin by opioid peptides and alkaloids may control the activity of various membrane-bound and cytosolic systems that are calmodulin- and/or calcium-dependent.     

From peptide-bond formation to cotranslational folding: dynamic, regulatory and evolutionary aspects

Ribosomes are ribozymes exerting substrate positioning and promoting substrate-mediated catalysis. Peptide-bonds are formed within a symmetrical region, thus suggesting that ribosomes evolved by gene-fusion. Remote interactions dominate substrate positioning at stereochemistry suitable for peptide-bond formation and elaborate architectural-design guides the processivity of the reaction by rotatory motion. Nascent proteins are directed into the exit tunnel at extended conformation, complying with the tunnel's narrow entrance. Tunnel dynamics facilitate its interactive participation in elongation, discrimination, cellular signaling and nascent-protein trafficking into the chaperon-aided folding site. Conformational alterations, induced by ribosomal-recycling factor, facilitate subunit dissociation. Remarkably, although antibiotics discrimination is determined by the identity of a single nucleotide, involved also in resistance, additional nucleotides dictate antibiotics effectiveness.     

Ribosomal crystallography: a flexible nucleotide anchoring tRNA translocation, facilitates peptide-bond formation, chirality discrimination and antibiotics synergism

The linkage between internal ribosomal symmetry and transfer RNA (tRNA) positioning confirmed positional catalysis of amino-acid polymerization. Peptide bonds are formed concurrently with tRNA-3' end rotatory motion, in conjunction with the overall messenger RNA (mRNA)/tRNA translocation. Accurate substrate alignment, mandatory for the processivity of protein biosynthesis, is governed by remote interactions. Inherent flexibility of a conserved nucleotide, anchoring the rotatory motion, facilitates chirality discrimination and antibiotics synergism. Potential tRNA interactions explain the universality of the tRNA CCA-end and P-site preference of initial tRNA. The interactions of protein L2 tail with the symmetry-related region periphery explain its conservation and its contributions to nascent chain elongation.     

Human mast cells release metalloproteinase-9 on contact with activated T cells: juxtacrine regulation by TNF-alpha

Mast cells, essential effector cells in allergic inflammation, have been found to be activated in T cell-mediated inflammatory processes in accordance with their residence in close physical proximity to T cells. We have recently reported that mast cells release granule-associated mediators and TNF-alpha upon direct contact with activated T cells. This data suggested an unrecognized activation pathway, where mast cells may be activated during T cell-mediated inflammation. Herein, we show that this cell-cell contact results in the release of matrix metalloproteinase (MMP)-9 and the MMP inhibitor tissue inhibitor of metalloproteinase 1 from HMC-1 human mast cells or from mature peripheral blood-derived human mast cells. The expression and release of these mediators, as well as of beta-hexosaminidase and several cytokines, were also induced when mast cells were incubated with cell membranes isolated from activated, but not resting, T cells. Subcellular fractionation revealed that the mature form of MMP-9 cofractionated with histamine and tryptase, indicating its localization within the secretory granules. MMP-9 release was first detected at 6 h and peaked at 22 h of incubation with activated T cell membranes, while TNF-alpha release peaked after only 6 h. Anti-TNF-alpha mAb inhibited the T cell membrane-induced MMP-9 release, indicating a possible autocrine regulation of MMP release by mast cell TNF-alpha. This cascade of events, whereby mast cells are activated by T cells to release cytokines and MMP-9, which are known to be essential for leukocyte extravasation and recruitment to affected sites, points to an important immunoregulatory function of mast cells within the context of T cell-mediated inflammatory processes.     

Short-term changes in eating patterns explain the effects of condensed tannins on feed intake in heifers

Ingestion of condensed tannins decreases feed intake in ruminants. Polyethylene glycol (PEG) forms high-affinity complexes with tannins. In two experiments carried out on Holstein heifers, quebracho (Q) from the Aspidosperma quebracho served as source of condensed tannins. The aims of the study were (i) to quantify the effect of Q on feed intake and eating behaviour in cattle fed complete mixed diets (CMDs); (ii) to clarify if changes induced in ingestive behaviour and feed intake by Q in cattle can be reversed by feeding PEG; and (iii) to clarify if the decrease in feed intake is associated with short-term (astringency, post-ingestive malaise) or longer-term effects. In experiment 1, 500 g/day of Q was found to be the minimal dose that decreased feed intake in heifers. A ratio of PEG:Q equal to 1:12.5 did not fully restore feed intake. In experiment 2, four heifers received a random sequence of four rations in a Latin-square design with feeding cycles of ca. 7 days: CMD containing no supplements (C), or supplemented with 625 g/day of Q without PEG (Q), with 625 g/day of Q and 250 g/day of PEG (Q-PEG), or with 250 g/day of PEG without Q (PEG). Individual rations were continuously weighed in the trough and the behaviour of heifers was observed for 180 min after distribution of CMD. Overall, feeding Q was associated with lowered feed intake and shorter duration of eating bouts, mainly of the first eating bout, immediately after distribution of the diet. A larger portion of the diet was consumed subsequent to 180 min after distribution in Q-fed heifers. Eating rate and the water to food ratio were not affected by Q. The effects of Q on feed intake were attenuated by feeding PEG. Heifers adapted effectively to condensed tannins by increasing the number of eating bouts and the portion of diet consumed subsequent to 180 min after distribution, so that no differences in feed intake were noted on the last day of each feeding cycle. Data are interpreted to show that: (i) negative effects of Q on feed intake derive from astringency of CT and short-term post-ingestive malaise; (ii) the increased number of eating bouts and their wider partition throughout the day are means to preserve the ruminal environment in Q-fed heifers; (iii) PEG has the potential to neutralize negative effects of condensed tannins in cattle.