A TNF‐p100 pathway subverts noncanonical NF‐κB signaling in inflamed secondary lymphoid organs

Lymphotoxin‐beta receptor (LTβR) present on stromal cells engages the noncanonical NF‐κB pathway to mediate RelB‐dependent expressions of homeostatic chemokines, which direct steady‐state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF‐κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection‐inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non‐infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTβR‐stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF‐mediated inhibitions in inflamed SLOs of immunized Nfkb2^?/? mice. In sum, we reveal that an inhibitory TNF‐p100 pathway modulates the adaptive compartment during immune responses.     

Activation of ras signaling pathway by 8-oxoguanine DNA glycosylase bound to its excision product, 8-oxoguanine

8-Oxo-7,8-dihydroguanine (8-oxoG), arguably the most abundant base lesion induced in mammalian genomes by reactive oxygen species, is repaired via the base excision repair pathway that is initiated with the excision of 8-oxoG by OGG1. Here we show that OGG1 binds the 8-oxoG base with high affinity and that the complex then interacts with canonical Ras family GTPases to catalyze replacement of GDP with GTP, thus serving as a guanine nuclear exchange factor. OGG1-mediated activation of Ras leads to phosphorylation of the mitogen-activated kinases MEK1,2/ERK1,2 and increasing downstream gene expression. These studies document for the first time that in addition to its role in repairing oxidized purines, OGG1 has an independent guanine nuclear exchange factor activity when bound to 8-oxoG.     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

Characterization of goat plasma vitronectin

Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4 degrees C resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca(2+) caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity inthe protein.     

Retinal vascularisation in the loach Noemacheilus rupicola rupicola: coexistence of falciform process and vitreal vessels

Synopsis The coexistence of a fully developed falciform process and vitreal vessels, a hitherto unknown situation in teleostean intraocular vascularisation, has been noted in the loach Noemacheilus rupicola rupicola. Possible implications of this vascular development for the species have been highlighted.     

Action of human endonucleases III and VIII upon DNA-containing tandem dihydrouracil

We have shown previously that endonuclease III from Escherichia coli, its yeast homolog Ntg1p and E. coli endonuclease VIII recognize single dihydrouracil (DHU) lesions efficiently. However, these enzymes have limited capacities for completely removing DHU, when the lesion is present on duplex DNA as a tandem lesion. A duplex 30-mer (duplex1920) containing tandem DHU lesions at positions 19 and 20 from the 5' terminus was used as a substrate for human endonuclease III (hNTH) and endonuclease VIII (NEIL1). Two cleavage products, 18beta and 19beta were formed, when duplex1920 was treated with hNTH. The 18beta corresponded to the expected beta-elimination product generated from duplex1920, when the 5'-DHU of the tandem DHU was processed by hNTH. Similarly, 19beta is the beta-elimination product generated, when the 3'-DHU of the tandem DHU was processed by hNTH; 19beta thus still contained a DHU lesion at the 3' terminus. When these hNTH reaction products were further treated with human APE1, a single new product that corresponded to an 18mer was observed. These data suggested that human APE1 can help to process the 3' terminals following the action of hNTH on DHU lesions. Similarly, when duplex1920 was treated with NEIL1, two cleavage products, 18p and 19p were observed. The 18p and 19p corresponded to the expected beta,delta-elimination products derived from NEIL1 induced cleavage at the 5'-DHU and 3'-DHU of the tandem DHU, respectively. The 3'-phosphoryl group present in 18p can be readily removed by T4 polynucleotide kinase (PNK) to yield an 18mer that is suitable for repair synthesis. However, 19p required the participation of both PNK and APE1 to generate the 18mer. Together, we suggest that the processing of DNA-containing tandem DHU lesions, initiated by hNTH and NEIL1 can be channeled into two sub-pathways, the PNK-independent, APE1-dependent and the PNK, APE1-dependent pathways, respectively.