Effects antifreeze peptides on the thermotropic properties of a model membrane

In this paper, we report on the effect of short segments of type I antifreeze protein (AFP I) on the thermotropic properties of a model membrane. Two different types of dimyristoylphosphatidylcholine model membranes were used, multilamellar vesicles and small unilamellar vesicles. The membrane properties were studied by differential scanning calorimetry (DSC) and fluorescence anisotropy. With the incorporation of AFP I and its short segments, the order of the model membrane increased both in the gel state and in the liquid crystalline state. The interaction of AFPs with the model membrane caused a shift in the phase transition to lower temperatures, which is accompanied by a broadening of the DSC thermogram. This preferential stabilization to a more ordered phase by the AFPs could be due to ordering the hydrophobic membrane core and separation into domains. Overall, this approach of employing short segments of AFP I simplifies the correlation between antifreeze protein characteristics and the effect of these parameters on the interaction mechanism of AFP with cell membranes. The success of this approach can lead to the identification of short peptides with high antifreeze activity.     

Altered glycosylation of recombinant NKp30 hampers binding to heparan sulfate: a lesson for the use of recombinant immunoreceptors as an immunological tool

NKp30 is a natural cytotoxicity receptor expressed by human NK cells and involved in NK lytic activity. We previously published that membranal heparan sulfate serves as a coligand for human NKp30. In the present study, we complement our results by showing direct binding of recombinant NKp30 to immobilized heparin. The heparan sulfate epitope(s) on target tumor cells and the heparin epitope(s) recognized by NKp30 share similar characteristics. Warren and colleagues (Warren HS, Jones AL, Freeman C, Bettadapura J, Parish CR. 2005. Evidence that the cellular ligand for the human NK cell activation receptor NKp30 is not a heparan sulfate glycosaminoglycan. J Immunol. 175:207-212) published that NKp30 does not bind to membranal heparan sulfate on target cells and that heparan sulfate is not involved in NKp30-mediated lysis. In the current study, we examine the binding of six different recombinant NKp30s to membranal heparan sulfate and conclude that NKp30 does interact with membranal heparan sulfate. Yet, two of the six recombinant NKp30s, including the commercially available recombinant NKp30 (employed by Warren et al.) did not show heparan sulfate-dependent binding. We demonstrate that this is due to an altered glycosylation of these two recombinant NKp30s. Upon removal of its N-linked glycans, heparan sulfate-dependent binding to tumor cells and direct binding to heparin were restored. Overall, our results emphasize the importance of proper glycosylation for analysis of NKp30 binding to its ligand and that membranal heparan sulfate could serve as a coligand for NKp30. At the cellular level, soluble heparan sulfate enhanced the secretion of IFNgamma by NK-92 natural killer cells activated with anti-NKp30 monoclonal antibody. We discuss the involvement of heparan sulfate binding to NKp30 in NKp30-mediated activation of NK cells.     

Dual role of cysteine 172 in redox regulation of ribulose 1,5-bisphosphate carboxylase/oxygenase activity and degradation

Alkylation and oxidation of cysteine residues significantly decrease the catalytic activity and stimulate the degradation of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We analyzed the role of vicinal cysteine residues in redox regulation of RuBisCO from Synechocystis sp. strain PCC 6803. Cys172 and Cys192, which are adjacent to the catalytic site, and Cys247, which cross-links two large subunits, were replaced by alanine. Whereas all mutant cells (C172A, C192A, C172A-C192A, and C247A) and the wild type grew photoautotrophically at similar rates, the maximal photosynthesis rates of C172A mutants decreased 10 to 20% as a result of 40 to 60% declines in RuBisCO turnover number. Replacement of Cys172, but not replacement of Cys192, prominently decreased the effect of cysteine alkylation or oxidation on RuBisCO. Oxidants that react with vicinal thiols had a less inhibitory effect on the activity of either the C172A or C192A enzyme variants, suggesting that a disulfide bond was formed upon oxidation. Thiol oxidation induced RuBisCO dissociation into subunits. This effect was either reduced in the C172A and C192A mutant enzymes or eliminated by carboxypentitol bisphosphate (CPBP) binding to the activated enzyme form. The CPBP effect presumably resulted from a conformational change in the carbamylated CPBP-bound enzyme, as implied from an alteration in the electrophoretic mobility. Stress conditions, provoked by nitrate deprivation, decreased the RuBisCO contents and activities in the wild type and in the C192A and C247A mutants but not in the C172A and C172A-C192A mutants. These results suggest that although Cys172 does not participate in catalysis, it plays a role in redox regulation of RuBisCO activity and degradation.     

Protection by isoprene against singlet oxygen in leaves

Isoprene (2-methyl-1,3-butadiene) protection against effects of singlet oxygen was investigated in Myrtus communis and Rhamnus alaternus. In M. communis, singlet oxygen produced in the leaves by Rose Bengal (RB) led to a 65% decrease in net assimilation rates within 3 h, whereas isoprene emission rates showed either a 30% decrease at ambient CO2 concentrations or a 70% increase under high CO2. In both cases, these changes led to an increase in calculated internal isoprene concentrations. The isoprene protection effect was directly demonstrated by fumigation of young (non-emitting) leaves, treated with RB or bromoxynil (simulating photoinhibition). There was 42% and 29% reduction in the damage to net assimilation compared with non-fumigated leaves for RB or bromoxynil, respectively. In R. alaternus, similar effects of RB on net assimilation were observed, and additional fluorescence measurements showed a significantly smaller decrease in Fv/Fm in isoprene-fumigated young leaves treated with RB (from 0.78 to 0.52), compared with non-fumigated leaves (from 0.77 to 0.27). The internal isoprene concentrations used in this study and possible rate of 1O2 production in leaves indicate that the protective effects observed should be beneficial also under natural conditions.     

SherLoc: high-accuracy prediction of protein subcellular localization by integrating text and protein sequence data

MOTIVATION: Knowing the localization of a protein within the cell helps elucidate its role in biological processes, its function and its potential as a drug target. Thus, subcellular localization prediction is an active research area. Numerous localization prediction systems are described in the literature; some focus on specific localizations or organisms, while others attempt to cover a wide range of localizations. RESULTS: We introduce SherLoc, a new comprehensive system for predicting the localization of eukaryotic proteins. It integrates several types of sequence and text-based features. While applying the widely used support vector machines (SVMs), SherLoc's main novelty lies in the way in which it selects its text sources and features, and integrates those with sequence-based features. We test SherLoc on previously used datasets, as well as on a new set devised specifically to test its predictive power, and show that SherLoc consistently improves on previous reported results. We also report the results of applying SherLoc to a large set of yet-unlocalized proteins. AVAILABILITY: SherLoc, along with Supplementary Information, is available at: http://www-bs.informatik.uni-tuebingen.de/Services/SherLoc/     

Wounding by bombardment yields highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.)

Highly efficient Agrobacterium-mediated transformation of carnation (Dianthus caryophyllus L.) was obtained by first wounding stem explants via microprojectile bombardment. When this was followed by cocultivation with disarmed Agrobacterium in the dark, the transformation frequency-based on transient GUS expression-increased to over 10-fold that of explants wounded by other means and cocultivated under constant light. Two cycles of regeneration/selection on kanamycin were employed to generate stably transformed carnation plants and eliminate chimeras: first, plantlets were regenerated from inoculated stem explants and then leaves from these plantlets were used to generate transgenes in a second selection cycle of adventitious shoot regeneration. Agrobacterium strain AGLO, carrying the binary vector pCGN7001 containing uidA and nptII genes, was used in the stable transformation experiments. The combination of wounding via bombardment, cocultivation in the dark and two cycles of kanamycin selection yielded an overall transformation efficiency of 1–2 transgenes per 10 stem explants for the three carnation varieties analyzed. Histochemical and molecular analyses of marker genes in T₀ and T₁ generations confirmed the transgenic nature of the selected plants.     

Breast cancer is marked by specific,Public T-cell receptor CDR3 regions shared by mice and humans

The partial success of tumor immunotherapy induced by checkpoint blockade, which is not antigen-specific, suggests that the immune system of some patients contain antigen receptors able to specifically identify tumor cells. Here we focused on T-cell receptor (TCR) repertoires associated with spontaneous breast cancer. We studied the alpha and beta chain CDR3 domains of TCR repertoires of CD4 T cells using deep sequencing of cell populations in mice and applied the results to published TCR sequence data obtained from human patients. We screened peripheral blood T cells obtained monthly from individual mice spontaneously developing breast tumors by 5 months. We then looked at identical TCR sequences in published human studies; we used TCGA data from tumors and healthy tissues of 1,256 breast cancer resections and from 4 focused studies including sequences from tumors, lymph nodes, blood and healthy tissues, and from single cell dataset of 3 breast cancer subjects. We now report that mice spontaneously developing breast cancer manifest shared, Public CDR3 regions in both their alpha and beta and that a significant number of women with early breast cancer manifest identical CDR3 sequences. These findings suggest that the development of breast cancer is associated, across species, with biomarker, exclusive TCR repertoires.