A globin gene of ancient evolutionary origin in lower vertebrates: evidence for two distinct globin families in animals

Hemoglobin, myoglobin, neuroglobin, and cytoglobin are four types of vertebrate globins with distinct tissue distributions and functions. Here, we report the identification of a fifth and novel globin gene from fish and amphibians, which has apparently been lost in the evolution of higher vertebrates (Amniota). Because its function is presently unknown, we tentatively call it globin X (GbX). Globin X sequences were obtained from three fish species, the zebrafish Danio rerio, the goldfish Carassius auratus, and the pufferfish Tetraodon nigroviridis, and the clawed frog Silurana tropicalis. Globin X sequences are distinct from vertebrate hemoglobins, myoglobins, neuroglobins, and cytoglobins. Globin X displays the highest identity scores with neuroglobin (approximately 26% to 35%), although it is not a neuronal protein, as revealed by RT-PCR experiments on goldfish RNA from various tissues. The distal ligand-binding and the proximal heme-binding histidines (E7 and F8), as well as the conserved phenylalanine CD1 are present in the globin X sequences, but because of extensions at the N-terminal and C-terminal, the globin X proteins are longer than the typical eight alpha-helical globins and comprise about 200 amino acids. In addition to the conserved globin introns at helix positions B12.2 and G7.0, the globin X genes contain two introns in E10.2 and H10.0. The intron in E10.2 is shifted by 1 bp in respect to the vertebrate neuroglobin gene (E11.0), providing possible evidence for an intron sliding event. Phylogenetic analyses confirm an ancient evolutionary relationship of globin X with neuroglobin and suggest the existence of two distinct globin types in the last common ancestor of Protostomia and Deuterostomia.     

Characterisation of the laccase-encoding gene abr2 of the dihydroxynaphthalene-like melanin gene cluster of Aspergillus fumigatus

Aspergillus fumigatus is an important pathogen of the immunocompromised host. Previously, it was shown that the polyketide synthase encoded by the pksP (alb1) gene represents a virulence determinant. pksP is part of a gene cluster involved in dihydroxynaphthalene (DHN)-like melanin biosynthesis. Because a putative laccase-encoding gene (abr2) is also part of the cluster and a laccase was found to represent a virulence factor in Cryptococcus neoformans, here, the Abr2 laccase was characterised. Deletion of the abr2 gene changed the gray-green conidial pigment to a brown color and the ornamentation of conidia was reduced compared with wild-type conidia. In contrast to the white pksP mutant, the susceptibility of the Δabr2 mutant against reactive oxygen species (ROS) was not increased, suggesting that the intermediate of DHN-like melanin produced up to the step catalysed by Abr2 already possesses ROS scavenging activity. In an intranasal mouse infection model, the Δabr2 mutant strain showed no reduction in virulence compared with the wild type. In the Δabr2 mutant, overall laccase activity was reduced only during sporulation, but not during vegetative growth. An abr2p-lacZ gene fusion was expressed during sporulation, but not during vegetative growth confirming the pattern of laccase activity due to Abr2.     

Short-term dynamics of bacterial communities in a tidally affected coastal ecosystem

Tidal effects on the composition of free-living (FL) and particle-associated (PA) bacterial communities were studied in a tidal flat ecosystem in the southern North Sea. Denaturing gradient gel electrophoresis targeting the 16S rRNA gene and the 16S rRNA of Bacteria, Bacteroidetes, Alphaproteobacteria and the Roseobacter clade was applied. Despite strong tidal variations in the quantity and, depending on the season, also the quality of suspended matter as well as variations in bacterial activity, the bacterial community composition remained rather stable. FISH showed some variations of the community composition, but these were not related to typical tidal situations. Variations were higher during tidal cycles in May and July compared with November. Bacteroidetes, Alpha- and Gammaproteobacteria constituted the majority of the bacterial communities but relative proportions of the different groups varied considerably. On particles, Betaproteobacteria were also detected to substantial proportions. The Roseobacter clade constituted up to 90% of FL but only 30% of PA Alphaproteobacteria. Banding patterns of the Bacteroidetes-specific amplicons, and in particular those targeting the 16S rRNA, revealed tidally induced effects, as several bands appeared or disappeared at distinct events such as slack water or resuspension. Sequencing of prominent bands revealed predominantly phylotypes reported previously from this ecosystem.     

A fundamental difference between macrobiota and microbial eukaryotes: protistan plankton has a species maximum in the freshwater-marine transition zone of the Baltic Sea

Remane's Artenminimum at the horohalinicum is a fundamental concept in ecology to describe and explain the distribution of organisms along salinity gradients. However, a recent metadata analysis challenged this concept for protists, proposing a species maximum in brackish waters. Due to data bias, this literature-based investigation was highly discussed. Reliable data verifying or rejecting the species minimum for protists in brackish waters were critically lacking. Here, we sampled a pronounced salinity gradient along a west–east transect in the Baltic Sea and analysed protistan plankton communities using high-throughput eDNA metabarcoding. A strong salinity barrier at the upper limit of the horohalinicum and 10 psu appeared to select for significant shifts in protistan community structures, with dinoflagellates being dominant at lower salinities, and dictyochophytes and diatoms being keyplayers at higher salinities. Also in vertical water column gradients in deeper basins (Kiel Bight, Arkona and Bornholm Basin) appeared salinity as significant environmental determinant influencing alpha- and beta-diversity patterns. Importantly, alpha-diversity indices revealed species maxima in brackish waters, that is, indeed contrasting Remane's Artenminimum concept. Statistical analyses confirmed salinity as the major driving force for protistan community structuring with high significance. This suggests that macrobiota and microbial eukaryotes follow fundamentally different rules regarding diversity patterns in the transition zone from freshwater to marine waters.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Exchange of Cytosolic Content between T Cells and Tumor Cells Activates CD4 T Cells and Impedes Cancer Growth

Background

T cells are known to participate in the response to tumor cells and react with cytotoxicity and cytokine release. At the same time tumors established versatile mechanisms for silencing the immune responses. The interplay is far from being completely understood. In this study we show contacts between tumor cells and lymphocytes revealing novel characteristics in the interaction of T cells and cancer cells in a way not previously described.

Methods/ Findings

Experiments are based on the usage of a hydrophilic fluorescent dye that occurs free in the cytosol and thus transfer of fluorescent cytosol from one cell to the other can be observed using flow cytometry. Tumor cells from cell lines of different origin or primary hepatocellular carcinoma (HCC) cells were incubated with lymphocytes from human and mice. This exposure provoked a contact dependent uptake of tumor derived cytosol by lymphocytes – even in CD4⁺ T cells and murine B cells – which could not be detected after incubation of lymphocytes with healthy cells. The interaction was a direct one, not requiring the presence of accessory cells, but independent of cytotoxicity and TCR engagement.Electron microscopy disclosed 100-200nm large gaps in the cell membranes of connected cells which separated viable and revealed astonishing outcome. While the lymphocytes were induced to proliferate in a long term fashion, the tumor cells underwent a temporary break in cell division. The in vitro results were confirmed in vivo using a murine acute lymphoblastic leukemia (ALL) model. The arrest of tumor proliferation resulted in a significant prolonged survival of challenged mice.

Conclusions

The reported cell-cell contacts reveal new characteristics i.e. the enabling of cytosol flow between the cells including biological active proteins that influence the cell cycle and biological behaviour of the recipient cells. This adds a completely new aspect in tumor induced immunology.     

Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19

Autophagy is the major pathway for the delivery of cytoplasmic material to the vacuole or lysosome. Selective autophagy is mediated by cargo receptors, which link the cargo to the scaffold protein Atg11 and to Atg8 family proteins on the forming autophagosomal membrane. We show that the essential kinase Hrr25 activates the cargo receptor Atg19 by phosphorylation, which is required to link cargo to the Atg11 scaffold, allowing selective autophagy to proceed. We also find that the Atg34 cargo receptor is regulated in a similar manner, suggesting a conserved mechanism.     

Neuroprotection from stroke in the absence of MHCI or PirB

Recovery from stroke engages mechanisms of neural plasticity. Here we examine a role for MHC class I (MHCI) H2-Kb and H2-Db, as well as PirB receptor. These molecules restrict synaptic plasticity and motor learning in the healthy brain. Stroke elevates neuronal expression not only of H2-Kb and H2-Db, but also of PirB and downstream signaling. KbDb knockout (KO) or PirB KO mice have smaller infarcts and enhanced motor recovery. KO hippocampal organotypic slices, which lack an intact peripheral immune response, have less cell death after in?vitro ischemia. In PirB KO mice, corticospinal projections from the motor cortex are enhanced, and the reactive astrocytic response is dampened after MCAO. Thus, molecules that function in the immune system act not only to limit synaptic plasticity in healthy neurons, but also to exacerbate brain injury after ischemia. These results suggest therapies for stroke by targeting MHCI and PirB.     

Dealing with Uncertainty in Spatially Explicit Population Models

It has been argued that spatially explicit population models (SEPMs) cannot provide reliable guidance for conservation biology because of the difficulty of obtaining direct estimates for their demographic and dispersal parameters and because of error propagation. We argue that appropriate model calibration procedures can access additional sources of information, compensating the lack of direct parameter estimates. Our objective is to show how model calibration using population-level data can facilitate the construction of SEPMs that produce reliable predictions for conservation even when direct parameter estimates are inadequate. We constructed a spatially explicit and individual-based population model for the dynamics of brown bears (Ursus arctos) after a reintroduction program in Austria. To calibrate the model we developed a procedure that compared the simulated population dynamics with distinct features of the known population dynamics (=patterns). This procedure detected model parameterizations that did not reproduce the known dynamics. Global sensitivity analysis of the uncalibrated model revealed high uncertainty in most model predictions due to large parameter uncertainties (coefficients of variation CV 0.8). However, the calibrated model yielded predictions with considerably reduced uncertainty (CV 0.2). A pattern or a combination of various patterns that embed information on the entire model dynamics can reduce the uncertainty in model predictions, and the application of different patterns with high information content yields the same model predictions. In contrast, a pattern that does not embed information on the entire population dynamics (e.g., bear observations taken from sub-areas of the study area) does not reduce uncertainty in model predictions. Because population-level data for defining (multiple) patterns are often available, our approach could be applied widely.