Extracellular HspBP1 inhibits formation of a cytotoxic Tag7-Hsp70 complex in vitro and in human serum

Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.     

Mass spectrometric characterization of human N-acylethanolamine-hydrolyzing acid amidase

N-Acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal enzyme that primarily degrades palmitoylethanolamine (PEA), a lipid amide that inhibits inflammatory responses. We developed a HEK293 cell line stably expressing the NAAA pro-enzyme (zymogen) and a single step chromatographic purification of the protein from the media. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF MS analysis of the zymogen (47.7 kDa) treated with peptide-N-glycosidase F (PNGase F) identified 4 glycosylation sites, and acid cleavage of the zymogen into α- and β-subunits (14.6 and 33.3 kDa) activated the enzyme. Size exclusion chromatography estimated the mass of the active enzyme as 45 ± 3 kDa, suggesting formation of an α/β heterodimer. MALDI-TOF MS fingerprinting covered more than 80% of the amino acid sequence, including the N-terminal peptides, and evidence for the lack of a disulfide bond between subunits. The significance of the cysteine residues was established by their selective alkylation resulting in almost complete loss of activity. The purified enzyme was kinetically characterized with PEA and a novel fluorogenic substrate, N-(4-methyl coumarin) palmitamide (PAMCA). The production of sufficient quantities of NAAA and a high throughput assay could be useful in discovering novel inhibitors and determining the structure and function of this enzyme.     

Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture: analysis of H9 escape mutants

We used a panel of monoclonal antibodies to H9 hemagglutinin to select 18 escape mutants of mouse-adapted influenza A/Swine/Hong Kong/9/98 (H9N2) virus. Cross-reactions of the mutants with the antibodies and the sequencing of hemagglutinin genes revealed two minimally overlapping epitopes. We mapped the amino acid changes to two areas of the recently reported three-dimensional structure of A/Swine/Hong Kong/9/98 hemagglutinin. The grouping of the antigenically relevant amino acid positions in H9 hemagglutinin differs from the pattern observed in H3 and H5 hemagglutinins. Several positions in site B of H3 hemagglutinin are distributed in two sites of H9 hemagglutinin. Unlike any subtype analyzed so far, H9 hemagglutinin does not contain an antigenic site corresponding to site A in H3 hemagglutinin. Positions 145 and 193 (H3 numbering), which in H3 hemagglutinin belong to sites A and B, respectively, are within one site in H9 hemagglutinin. This finding is consistent with the peculiarity of the three-dimensional structure of the H9 molecule, that is, the absence from H9 hemagglutinin of the lateral loop that forms site A in H3 and the equivalent site in H5 hemagglutinins. The escape mutants analyzed displayed phenotypic variations, including decreased virulence for mice and changes in affinity for sialyl substrates. Our results demonstrate a correlation between intersubtype differences in three-dimensional structure and variations among subtypes in the distribution of antigenic areas. Our findings also suggest that covariation and pleiotropic effects of antibody-selected mutations may be important in the evolution of H9 influenza virus, a possible causative agent of a future pandemic.     

Innate-Like Control of Human iNKT Cell Autoreactivity via the Hypervariable CDR3β Loop

Invariant Natural Killer T cells (iNKT) are a versatile lymphocyte subset with important roles in both host defense and immunological tolerance. They express a highly conserved TCR which mediates recognition of the non-polymorphic, lipid-binding molecule CD1d. The structure of human iNKT TCRs is unique in that only one of the six complementarity determining region (CDR) loops, CDR3β, is hypervariable. The role of this loop for iNKT biology has been controversial, and it is unresolved whether it contributes to iNKT TCR:CD1d binding or antigen selectivity. On the one hand, the CDR3β loop is dispensable for iNKT TCR binding to CD1d molecules presenting the xenobiotic alpha-galactosylceramide ligand KRN7000, which elicits a strong functional response from mouse and human iNKT cells. However, a role for CDR3β in the recognition of CD1d molecules presenting less potent ligands, such as self-lipids, is suggested by the clonal distribution of iNKT autoreactivity. We demonstrate that the human iNKT repertoire comprises subsets of greatly differing TCR affinity to CD1d, and that these differences relate to their autoreactive functions. These functionally different iNKT subsets segregate in their ability to bind CD1d-tetramers loaded with the partial agonist α-linked glycolipid antigen OCH and structurally different endogenous β-glycosylceramides. Using surface plasmon resonance with recombinant iNKT TCRs and different ligand-CD1d complexes, we demonstrate that the CDR3β sequence strongly impacts on the iNKT TCR affinity to CD1d, independent of the loaded CD1d ligand. Collectively our data reveal a crucial role for CDR3β for the function of human iNKT cells by tuning the overall affinity of the iNKT TCR to CD1d. This mechanism is relatively independent of the bound CD1d ligand and thus forms the basis of an inherent, CDR3β dependent functional hierarchy of human iNKT cells.     

Temperature effects on fish production across a natural thermal gradient

Global warming is widely predicted to reduce the biomass production of top predators, or even result in species loss. Several exceptions to this expectation have been identified, however, and it is vital that we understand the underlying mechanisms if we are to improve our ability to predict future trends. Here, we used a natural warming experiment in Iceland and quantitative theoretical predictions to investigate the success of brown trout as top predators across a stream temperature gradient (4–25 °C). Brown trout are at the northern limit of their geographic distribution in this system, with ambient stream temperatures below their optimum for maximal growth, and above it in the warmest streams. A five‐month mark‐recapture study revealed that population abundance, biomass, growth rate, and production of trout all increased with stream temperature. We identified two mechanisms that contributed to these responses: (1) trout became more selective in their diet as stream temperature increased, feeding higher in the food web and increasing in trophic position; and (2) trophic transfer through the food web was more efficient in the warmer streams. We found little evidence to support a third potential mechanism: that external subsidies would play a more important role in the diet of trout with increasing stream temperature. Resource availability was also amplified through the trophic levels with warming, as predicted by metabolic theory in nutrient‐replete systems. These results highlight circumstances in which top predators can thrive in warmer environments and contribute to our knowledge of warming impacts on natural communities and ecosystem functioning.     

Tapirus balkanicus nov. sp., nouveau tapir (Perissodactyla, Mammalia) du Turolien de Bulgarie

A new species of Tapiridae, Tapirus balkanicus, is etablished for new material of the Bulgarian Upper Miocene. The comparison with other Neogene tapirids leads us to consider the upper jaw as more characteristic than the lower one for taxonomic determination and study of phyletic relations. A new subgenus, Meyeriscus, is created for Tapirus pannonicus, the most evolved European tapirid.

Résumé

Une nouvelle espèce de tapir est décrite, Tapirus balkanicus du Miocène supérieur de Bulgarie. La comparaison avec les espèces européennes du Miocène moyen (Tapirus teilen) au Villafranchien (Tapirus arvernensis) amènent à privilégier la denture supérieure sur l'inférieure pour la détermination et les rapports phylétiques éventuels entre les espèces. Pour l'espèce la plus évoluée de tous les Tapiridés européens, Tapirus pannonicus est proposé un nom de sous-genre particulier, Meyeriscus nov.     

Test of dynamic closed-loop baroreflex and autoregulatory control of total peripheral resistance in intact and conscious sheep

This is the first study able to examine and delineate the actual actions of the physiological mechanisms responsible for the dynamic couplings between cardiac output (CO), arterial pressure (Pa), right atrial pressure (PRA), and total peripheral resistance (TPR) in an individual subject without altering the underlying regulatory mechanisms. Eight conscious male sheep were used, where both types of baroreceptors were independently exposed to simultaneous beat-to-beat pressure perturbations under intact closed-loop conditions while CO, Pa, PRA, and TPR were measured. We applied the cardiovascular system identification method proposed in a companion paper (4) to quantitatively characterize the dynamic closed-loop transfer relations CO-->Pa, PRA-->Pa, Pa-->TPR, and PRA-->TPR from the measured signals. To validate the dynamic properties of the estimated transfer relations, the essential parts of the linear dynamics of the model were independently and comprehensively evaluated via error model cross-validation, and the overall model's steady-state behavior was compared with a separate random effects regression approach. In addition to numerous physiological findings, we found that the cardiovascular system identification results were exceptionally consistent with the analytically derived solutions previously discussed in Ref. 4. In conclusion, this study presents the first time validation of a cardiovascular system identification method by means of experimentally acquired animal data in the intact and conscious animal and offers a set of powerful quantitative tools essential to advancing our knowledge of cardiovascular regulatory physiology.