Efficient Removal of Platelets from Peripheral Blood Progenitor Cell Products Using a Novel Micro-Chip Based Acoustophoretic Platform

Excessive collection of platelets is an unwanted side effect in current centrifugation-based peripheral blood progenitor cell (PBPC) apheresis. We investigated a novel microchip-based acoustophoresis technique, utilizing ultrasonic standing wave forces for the removal of platelets from PBPC products. By applying an acoustic standing wave field onto a continuously flowing cell suspension in a micro channel, cells can be separated from the surrounding media depending on their physical properties

Supplementary data for the article: Apostolovic, D.; Sánchez-Vidaurre, S.; Waden, K.; Curin, M.; Grundström, J.; Gafvelin, G.; Cirkovic Velickovic, T.; Grönlund, H.; Thomas, W. R.; Valenta, R.; et al. The Cat Lipocalin Fel d 7 and Its Cross-Reactivity with the Dog Lipocalin Can f 1. Allergy: European Journal of Allergy and Clinical Immunology 2016, 71 (10), 1490–1495. https://doi.org/10.1111/all.12955

Supplementary material for: [https://doi.org/10.1111/all.12955]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/2337]Related to accepted version: [http://cherry.chem.bg.ac.rs/handle/123456789/3650

The transcription factor E2A can bind to and cleave single-stranded immunoglobulin heavy chain locus DNA

Class switch recombination (CSR) changes the constant region of the immunoglobulin heavy chain (IgH), and somatic hypermutation (SH) introduces point mutations in the variable regions of the antibody genes. Both these processes that optimize antibody responses of B lymphocytes are initiated by the enzyme Activation Induced cytidine Deaminase (AID). Here we have searched for CSR or SH coupled activities of the transcription factor E2A, since E2A is in a complex with AID and the transcription factors PAX5, ETS1 and IRF4 on key sequences of the Igh locus in B lymphocytes activated to CSR and SH. We report that E2A in contrast to other described transcription factors binds sequence specifically also to single-stranded DNA. The binding of E2A to single-stranded DNA has a strong sequence preference for one strand of a site in the intronic enhancer of the Igh locus. Furthermore, E2A was also found to cleave single-stranded DNA. The sequence profile of substrates cleaved by E2A is coupled to the sequences of substrates and products of AID, suggesting that E2A has a role not only in targeting of AID to switch regions and SH parts of antibody genes but also in cleavage of DNA at these sites

Regulated localization of an AID complex with E2A, PAX5 and IRF4 at the Igh locus

Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates somatic hypermutation (SH) and class switch recombination (CSR) by deaminating cytosine to uracil. The targeting of AID and therefore SH and CSR to Ig genes is a central process of the immune system, but the trans-acting factors mediating the specific targeting have remained elusive. Here we show that defective calmodulin inhibition of the transcription factor E2A after activation of the B cell receptor (BCR) leads to reduced BCR, IL4 plus CD40 ligand stimulated CSR to IgE and instead CSR to other Ig classes. AID that initiates CSR is shown to be in a complex with the transcription factors E2A, PAX5 and IRF4 on key sequences of the Igh locus. Calmodulin shows proximity with each of them after BCR stimulation. BCR signaling reduces binding of the proteins to some of the target sites on the Igh locus, and calmodulin resistance of E2A blocks these reductions. AID binds directly to the bHLH domain of E2A and to the PD domain of PAX5. E2A, AID, PAX5 and IRF4 are components of a CSR complex that is redistributed on the Igh locus by BCR signaling through calmodulin binding

Nucleotide and calcium-induced conformational changes in histone H1

AbstractThe principal constituents of chromatin, histoneH1 (H1) and the nucleosome have essential roles in regulation of eukaryotic gene expression. However, mechanisms for the H1-dependent inactivation and for the ATP-dependent chromatin remodeling upon activation are largely unelucidated. Using circular dichroism (CD) analysis we show that ATP and other nucleotides and Ca2+ induce structural changes in H1. ATP and Ca2+ also induce changes when H1 is interacting with DNA, and the changes in H1 are accompanied by alterations in its DNA interaction. These results suggest that nucleotide and Ca2+ binding may be important for H1-mediated chromatin changes

Regulation of c-Rel Nuclear Localization by Binding of Ca(2+)/Calmodulin

The NF-κB/Rel family of transcription factors participates in the control of a wide array of genes, including genes involved in embryonic development and regulation of immune, inflammation, and stress responses. In most cells, inhibitory IκB proteins sequester NF-κB/Rel in the cytoplasm. Cellular stimulation results in the degradation of IκB and modification of NF-κB/Rel proteins, allowing NF-κB/Rel to translocate to the nucleus and act on its target genes. Calmodulin (CaM) is a highly conserved, ubiquitously expressed Ca(2+) binding protein that serves as a key mediator of intracellular Ca(2+) signals. Here we report that two members of the NF-κB/Rel family, c-Rel and RelA, interact directly with Ca(2+)-loaded CaM. The interaction with CaM is greatly enhanced by cell stimulation, and this enhancement is blocked by addition of IκB. c-Rel and RelA interact with CaM through a similar sequence near the nuclear localization signal. Compared to the wild-type protein, CaM binding-deficient mutants of c-Rel exhibit increases in both nuclear accumulation and transcriptional activity on the interleukin 2 and granulocyte macrophage colony-stimulating factor promoters in the presence of a Ca(2+) signal. Conversely, for RelA neither nuclear accumulation nor transcriptional activity on these promoters is increased by mutation of the sequence interacting with CaM. Our results suggest that CaM binds c-Rel and RelA after their release from IκB and can inhibit nuclear import of c-Rel while letting RelA translocate to the nucleus and act on its target genes. CaM can therefore differentially regulate the activation of NF-κB/Rel proteins following stimulation