Post-translational Modifications of Recombinant Human Lysyl Oxidase-like 2 (rhLOXL2) Secreted from Drosophila S2 Cells

Human lysyl oxidase-like 2 (hLOXL2) is highly up-regulated in metastatic breast cancer cells and tissues and induces epithelial-to-mesenchymal transition, the first step of metastasis/invasion. hloxl2 encodes four N-terminal scavenger receptor cysteine-rich domains and the highly conserved C-terminal lysyl oxidase (LOX) catalytic domain. Here, we assessed the extent of the post-translational modifications of hLOXL2 using truncated recombinant proteins produced in Drosophila S2 cells. The recombinant proteins are soluble, in contrast to LOX, which is consistently reported to require 2–6 m urea for solubilization. The recombinant proteins also show activity in tropoelastin oxidation. After phenylhydrazine derivatization and trypsin digestion, we used mass spectrometry to identify peptides containing the derivatized lysine tyrosylquinone cross-link at Lys-653 and Tyr-689, as well as N-linked glycans at Asn-455 and Asn-644. Disruption of N-glycosylation by site-directed mutagenesis or tunicamycin treatment completely inhibited secretion so that only small quantities of inclusion bodies were detected. The N-glycosylation site at Asn-644 in the LOX catalytic domain is not conserved in human LOX (hLOX), although the LOX catalytic domain of hLOX shares ∼50% identity and ∼70% homology with hLOXL2. The catalytic domain of hLOX was not secreted from S2 cells using the same expression system. These results suggest that the N-glycan at Asn-644 of hLOXL2 enhances the solubility and stability of the LOX catalytic domain.     

Evidence for MR1 antigen presentation to mucosal-associated invariant T cells

The novel class Ib molecule MR1 is highly conserved in mammals, particularly in its alpha1/alpha2 domains. Recent studies demonstrated that MR1 expression is required for development and expansion of a small population of T cells expressing an invariant T cell receptor (TCR) alpha chain called mucosal-associated invariant T (MAIT) cells. Despite these intriguing properties it has been difficult to determine whether MR1 expression and MAIT cell recognition is ligand-dependent. To address these outstanding questions, monoclonal antibodies were produced in MR1 knock-out mice immunized with recombinant MR1 protein, and a series of MR1 mutations were generated at sites previously shown to disrupt the ability of class Ia molecules to bind peptide or TCR. Here we show that 1) MR1 molecules are detected by monoclonal antibodies in either an open or folded conformation that correlates precisely with peptide-induced conformational changes in class Ia molecules, 2) only the folded MR1 conformer activated 2/2 MAIT hybridoma cells tested, 3) the pattern of MAIT cell activation by the MR1 mutants implies the MR1/TCR orientation is strikingly similar to published major histocompatibility complex/alphabetaTCR engagements, 4) all the MR1 mutations tested and found to severely reduce surface expression of folded molecules were located in the putative ligand binding groove, and 5) certain groove mutants of MR1 that are highly expressed on the cell surface disrupt MAIT cell activation. These combined data strongly support the conclusion that MR1 has an antigen presentation function.     

A Lectin-like Molecule is Discharged from Mucocysts of Tetrahymena pyriformis in the Presence of Insulin

ABSTRACT. By use of a monoclonal antibody directed against purified lectin from the sponge Geodia cydonium it was demonstrated that the mucocysts of Tetrahymena pyriformis contain a substance immunologically similar to that found in G. cydonium . In extracts of T. pyriformis the monoclonal antibody recognizes a 36 kDa protein; binding could be abolished by adsorption of the antibody with (i) crude extract, (ii) purified lectin from G. cydonium and (iii) a 29 aa long peptide. In addition the data show that 10^-6 M of insulin causes first the release of mucocyst material, which reacts with the lectin antibody, and second its subsequent redistribution on the surface of the somatic cilia and the oral field.     

Plasmodium berghei: inhibited incorporation of AMP-8- 3 H into nucleic acids of erythrocyte-free malarial parasites by acridines, phenanthridines, and 8-aminoquinolines

The ability of several acridine derivatives and various other heterocyclic compounds to inhibit incorporation of radioactivity from exogenous AMP-8-³H into the DNA and RNA of erythrocyte-free malarial parasites (Plasmodium berghei) was studied. The most potent acridine derivative was acriflavin (50% inhibition of RNA between 10⁻⁶ and 10⁻⁵ M). Acridine orange (50% inhibition of RNA at nearly 10⁻⁴ M) and acridine red (50% inhibition of RNA at nearly 10⁻³ M) were less potent than acriflavin by about 10-and 100-fold, respectively. Another heterocycle, methylene blue NN, approached the potency of acriflavin (50% inhibition of RNA at slightly greater than 10⁻⁵ M). Three phenanthridine heterocycles were tested and M &; B 1502 (dimidium butoxide) was found to be the most potent, exerting 50% inhibition of RNA at slightly greater than 10⁻⁵ M. Another derivative, M &; B 4250A exerted 50% inhibition of RNA at 10⁻⁴ M while a dose midway between 10⁻⁴ and 10⁻³ M was required of M &; B 4180A to cause this magnitude of inhibition. Two 8-aminoquinolines were also studied to determine their inhibitory potency against plasmodial nucleic acid anabolism. Both of these two-ringed heterocycles were relatively lacking in potency, exerting 50% inhibition of RNA at doses somewhat greater than 10⁻⁴ M.     

Quantifying turbulent wall shear stress in a stenosed pipe using large eddy simulation

Large eddy simulation was applied for flow of Re=2000 in a stenosed pipe in order to undertake a thorough investigation of the wall shear stress (WSS) in turbulent flow. A decomposition of the WSS into time averaged and fluctuating components is proposed. It was concluded that a scale resolving technique is required to completely describe the WSS pattern in a subject specific vessel model, since the poststenotic region was dominated by large axial and circumferential fluctuations. Three poststenotic regions of different WSS characteristics were identified. The recirculation zone was subject to a time averaged WSS in the retrograde direction and large fluctuations. After reattachment there was an antegrade shear and smaller fluctuations than in the recirculation zone. At the reattachment the fluctuations were the largest, but no direction dominated over time. Due to symmetry the circumferential time average was always zero. Thus, in a blood vessel, the axial fluctuations would affect endothelial cells in a stretched state, whereas the circumferential fluctuations would act in a relaxed direction.     

Response of green alder (Alnus viridis subsp. fruticosa) patch dynamics and plant community composition to fire and regional temperature in north‐western Canada

Aim Feedbacks between climate warming and fire have the potential to alter Arctic and sub‐Arctic vegetation. In this paper we assess the effects and interactions of temperature and wildfire on plant communities across the transition between the Arctic and sub‐Arctic. Location Mackenzie Delta region, Northwest Territories, Canada. Methods We sampled air temperatures, green alder (Alnus viridis ssp. fruticosa) cover, growth, reproduction and age distributions, and overall plant community composition on burned and unburned sites across a latitudinal gradient. Results Mean summer temperature across the study area decreased by 3 °C per degree of increasing latitude (6 °C across the study area). In the northern part of the study area, where seed viability was low, alder was less dominant than at southern sites where seed viability was high. The age structure of alder populations across the temperature gradient was highly variable, except in the northern part of the forest–tundra transition, where populations were dominated by young individuals. Alder growth and reproduction were significantly greater on burned sites (38–51 years following fire) than on unburned sites. North to south across the temperature gradient, vegetation changed from a community dominated by dwarf shrubs and fruticose lichens to one characterized by black spruce (Picea mariana), alder and willows (Salix spp.). Regardless of the position along the temperature gradient, burned sites were dominated by tall shrubs. Main conclusions Temperature limitation of alder abundance and repro‐duction, combined with evidence of recent recruitment on unburned sites, indicates that alder is likely to respond to increased temperature. Elevated alder growth and reproduction on burned sites shows that wildfire also has an important influence on alder population dynamics. The magnitude of alder’s response to fire, combined with observations that burns at the southern margin of the low Arctic are shrub dominated, suggest that increases in the frequency of wildfire have the potential to alter northern vegetation on decadal scales. By creating new seedbeds, fire provides opportunities for colonization that may facilitate the northward movement of tall shrubs. Feedbacks between the global climate system and low Arctic vegetation make understanding the long‐term impact of increasing fire frequency critical to predicting the response of northern ecosystems to global change.     

MAIT Cells Detect and Efficiently Lyse Bacterially-Infected Epithelial Cells

Mucosal associated invariant T cells (MAIT) are innate T lymphocytes that detect a large variety of bacteria and yeasts. This recognition depends on the detection of microbial compounds presented by the evolutionarily conserved major-histocompatibility-complex (MHC) class I molecule, MR1. Here we show that MAIT cells display cytotoxic activity towards MR1 overexpressing non-hematopoietic cells cocultured with bacteria. The NK receptor, CD161, highly expressed by MAIT cells, modulated the cytokine but not the cytotoxic response triggered by bacteria infected cells. MAIT cells are also activated by and kill epithelial cells expressing endogenous levels of MRI after infection with the invasive bacteria Shigella flexneri. In contrast, MAIT cells were not activated by epithelial cells infected by Salmonella enterica Typhimurium. Finally, MAIT cells are activated in human volunteers receiving an attenuated strain of Shigella dysenteriae-1 tested as a potential vaccine. Thus, in humans, MAIT cells are the most abundant T cell subset able to detect and kill bacteria infected cells.     

MAIT Cell Recognition of MR1 on Bacterially Infected and Uninfected Cells

Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant αβ TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.     

Risk of tumorigenicity in mesenchymal stromal cell–based therapies—Bridging scientific observations and regulatory viewpoints

In the past decade, the therapeutic value of mesenchymal stromal cells (MSCs) has been studied in various indications, thereby taking advantage of their immunosuppressive properties. Easy procurement from bone marrow, adipose tissue or other sources and conventional in vitro expansion culture have made their clinical use attractive. Bridging the gap between current scientific knowledge and regulatory prospects on the transformation potential and possible tumorigenicity of MSCs, the Cell Products Working Party and the Committee for Advanced Therapies organized a meeting with leading European experts in the field of MSCs. This meeting elucidated the risk of potential tumorigenicity related to MSC-based therapies from two angles: the scientific perspective and the regulatory point of view. The conclusions of this meeting, including the current regulatory thinking on quality, nonclinical and clinical aspects for MSCs, are presented in this review, leading to a clearer way forward for the development of such products.