Tracing of labile zinc in live fish hepatocytes using FluoZin-3

Intracellular zinc levels are homeostatically regulated and although most is bound, a pool of labile Zn(II) is present in cells. We show here that the zinc probe FluoZin-3 is useful to monitor zinc fluxes during fluorescent imaging of the trout hepatic cell line D11. Nuclei and bulk cytosol appeared to lack detectable labile zinc, while the punctuate staining pattern colocalized with a lysosome-specific probe. Applying extracellular zinc alone resulted in vesicular sequestration of the metal ion. Together with Na-pyrithione a delayed and toxic rise in cellular fluorescence was triggered. When using another ionophore, 4-Br A23187, a zinc buffering effect of the vesicular pools was evident. Secondly, N-ethylmaleimide induced a homogeneous fluorescence rise, which was strongly enhanced by addition of Zn-pyrithione and disappeared after TPEN washing. This suggests the involvement of thiol residues in controlling available cytosolic zinc. Our observations have implications for the interpretation of calculated intracellular Zn²⁺ concentrations.     

Firefly Femmes Fatales: A Case Study in the Semiotics of Deception

Mimicry and deception are two important issues in studies about animal communication. The reliability of animal signs and the problem of the benefits of deceiving in sign exchanges are interesting topics in the evolution of communication. In this paper, we intend to contribute to an understanding of deception by studying the case of aggressive signal mimicry in fireflies, investigated by James Lloyd. Firefly femmes fatales are specialized in mimicking the mating signals of other species of fireflies with the purpose of attracting responding males to become their prey. These aggressive mimics are a major factor in the survival and reproduction of both prey and predator. It is a case of deception through active falsification of information that leads to efficient predation by femmes fatales fireflies and triggered evolutionary processes in their preys’ communicative behaviors. There are even nested coevolutionary interactions between these fireflies, leading to a remarkable system of deceptive and counterdeceptive signaling behaviors. We develop here a semiotic model of firefly deception and also consider ideas advanced by Lloyd about the evolution of communication, acknowledging that deception can be part of the explanation of why communication evolves towards increasing complexity. Increasingly complex sign exchanges between fireflies evolve in an extremely slow pace. Even if deceptive maneuvers are played out time and time again between particular firefly individuals, the evolution of the next level of complexity—and thus the next utterance in the dialogue between species—is likely to take an immense amount of generations.     

N-Cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells

Specific cell–cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human–human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and β-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.     

In vivo biotinylated recombinant influenza A virus hemagglutinin for use in subtype-specific serodiagnostic assays

There is an urgent need for robust subtype-specific serological tests to diagnose influenza A virus infections in poultry and mammals, including humans. Such assays require reliable subtype-specific sources of soluble and authentically folded seroreactive hemagglutinin (HA), one of the integral membrane proteins that determine the serological subtype of influenza viruses. To this purpose, a bigenic pFastBacDual baculovirus transfer vector allowing efficient in vivo biotinylation of soluble HA homo-oligomers expressed via the secretory pathway was developed. An Avi-Tag allowed site-specific biotinylation by a coexpressed genetically modified BirA biotin ligase retained in the endoplasmic reticulum (ER). Highly seroreactive mono-biotinylated HA of recent H5 and H7 influenza A subtypes was secreted from recombinant baculovirus infected High-Five insect cells at levels sufficient to directly load streptavidin-coated enzyme-linked immunosorbent assay (ELISA) matrices, thereby avoiding any purification steps. The recombinant antigens retained authentic antigenicity, including conformation-dependent epitopes involved in hemagglutination inhibition as detected by monoclonal antibodies. This is the first bigenic in vivo biotinylation system established for use in insect cells with secretable recombinant membrane proteins biotinylated by an ER-retained variant of BirA biotin ligase. The proposed technique is expected to significantly increase flexibility in the design of subtype-specific assays, thereby expanding the power of influenza A virus serodiagnosis.     

Overexpression of manganese superoxide dismutase does not increase clonogenic cell survival despite effect on apoptosis in irradiated lymphoblastoid cells

Gene therapy-mediated overexpression of superoxide dismutases (SOD) appears to be a promising strategy for modulating radiosensitivity based on detoxification of superoxide radicals and suppression of apoptosis. Using recombinant lentiviral-based vectors, the effects of SOD overexpression on both were tested in human lymphoblastoid cells (TK6) that are sensitive to radiation-induced apoptosis. TK6 cells were transduced with vectors containing CuZnSOD, MnSOD or inverted MnSOD (MSODi) cDNA. Gene transfer efficiency, SOD activity, superoxide-radical resistance, apoptosis and clonogenic survival were determined. A six- to eightfold increase in SOD activity was observed after transduction, rendering MnSOD-overexpressing TK6 cells significantly more resistant to paraquat-induced superoxide radical production than controls. Although significant differences in sensitivity to apoptosis were observed for MnSOD, no differences in clonogenic survival after irradiation were detected between any groups. Our data show that efficient cellular SOD overexpression, an increased superoxide radical detoxifying ability and, for MnSOD, decreased apoptosis did not result in increased clonogenic survival after irradiation. This strengthens the hypothesis of differences in the radiation-modulating effects of SOD on normal and malignant cells (protective and nonprotective, respectively), thereby showing its potential to increase the therapeutic index in future clinical SOD-based radioprotection approaches.