Anti-MRC1 monoclonal antibody, clone 26-3

ATR (ATM and Rad3-related) initiates a DNA damage signaling pathway in human cells upon DNA damage induced by UV and UV-mimetic agents and in response to inhibition of DNA replication. Genetic data with human cells and in vitro data with Xenopus egg extracts have led to the conclusion that the kinase activity of ATR toward the signal-transducing kinase Chk1 depends on the mediator protein Claspin. Here we have reconstituted a Claspin-mediated checkpoint system with purified human proteins. We find that the ATR-dependent phosphorylation of Chk1, but not p53, is strongly stimulated by Claspin. Similarly, DNA containing bulky base adducts stimulates ATR kinase activity, and Claspin acts synergistically with damaged DNA to increase phosphorylation of Chk1 by ATR. Mutations in putative phosphorylation sites in the Chk1-binding domain of Claspin abolish its ability to mediate ATR phosphorylation of Chk1. We also find that a fragment of Claspin containing the Chk1-binding domain together with a domain conserved in the yeast Mrc1 orthologs of Claspin is sufficient for its mediator activity. This in vitro system recapitulates essential components of the genetically defined ATR-signaling pathway.

Hemp seeds have a wide variety of chemical compounds which present biological activity. Specifically, the focus on proteins and bioactive peptides are increasing as alternative sources of nutraceutical uses. In the literature, hemp protein products (HPPs) have reported antioxidant and anti-inflammatory properties. This study aimed to determine the inflammation-related modulatory effects of HPPs on lipopolysaccharide (LPS)-activated primary human monocytes. CD14(+) cells were immunomagnetically isolated from buffy coats and the anti-inflammatory activity of hemp protein isolate (HPI) and hydrolysates (HPHs) was evaluated on LPS-stimulated human primary monocytes. The specific markers of inflammation, polarization, and chemoattraction were measured by RT-qPCR and ELISA assays. Our results showed that HPPs decreased the pro-inflammatory mediators (TNF-alpha, IL-1 beta, and IL-6) and increased the anti-inflammatory mediators (IL-10 and IL-4). In addition, M1 polarization marker gene expression (CCR7 and iNOS) was downregulated by HPPs and, M2 polarization marker gene expression (CD200R and MRC1) was upregulated. Finally, the mRNA expression of chemotaxis genes (CCR2 and CCL2) was downregulated by HPPs. In conclusion, this study suggests that HPPs may improve chronic inflammatory states and promote regenerative processes by reprogramming monocytes toward M2 polarization phenotype.