Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine, acting through the TNF-R1 and TNF-R2 receptors. The two receptors have been proposed to mediate distinct TNF-alpha effects in the CNS, TNF-R1 contributing to neuronal damage and TNF-R2 being neuroprotective. Whether TNF-alpha and its receptors play any role for neurogenesis in the adult brain is unclear. Here we used mouse models with loss of TNF-R1 and TNF-R2 function to establish whether signaling through these receptors could influence hippocampal neurogenesis in vivo under basal conditions, as well as after status epilepticus (SE), which is associated with inflammation and elevated TNF-alpha levels. Notably, in the intact brain, the number of new, mature hippocampal neurons was elevated in TNF-R1(-/-) and TNF-R1/R2(-/-) mice, whereas no significant changes were detected in TNF-R2(-/-) mice. Also after SE, the TNF-R1(-/-) and TNF-R1/R2(-/-) mice produced more new neurons. In contrast, the TNF-R2(-/-) mice showed reduced SE-induced neurogenesis. Cell proliferation in the dentate subgranular zone was elevated in TNF-R1(-/-) and TNF-R1/R2(-/-) mice both under basal conditions and after SE. The TNF-R2(-/-) mice either showed no change or minor decrease of cell proliferation. TNF-R1 and TNF-R2 receptors were expressed by hippocampal progenitors, as assessed with reverse transcription-PCR on sorted or cultured cells and immunocytochemistry on cultures. Our data reveal differential actions of TNF-R1 and TNF-R2 signaling in adult hippocampal neurogenesis and identify for the first time TNF-R1 as a negative regulator of neural progenitor proliferation in both the intact and pathological brain.

https://www.jneurosci.org/content/jneuro/26/38/9703.full.pdf

Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis.

The proliferating cell nuclear antigen PCNA functions at multiple levels in directing DNA metabolic pathways. Unbound to DNA, PCNA promotes localization of replication factors with a consensus PCNA-binding domain to replication factories. When bound to DNA, PCNA organizes various proteins involved in DNA replication, DNA repair, DNA modification, and chromatin modeling. Its modification by ubiquitin directs the cellular response to DNA damage. The ring-like PCNA homotrimer encircles double-stranded DNA and slides spontaneously across it. Loading of PCNA onto DNA at template-primer junctions is performed in an ATP-dependent process by replication factor C (RFC), a heteropentameric AAA+ protein complex consisting of the Rfc1, Rfc2, Rfc3, Rfc4, and Rfc5 subunits. Loading of yeast PCNA (POL30) is mechanistically distinct from analogous processes in E. coli (beta subunit by the gamma complex) and bacteriophage T4 (gp45 by gp44/62). Multiple stepwise ATP-binding events to RFC are required to load PCNA onto primed DNA. This stepwise mechanism should permit editing of this process at individual steps and allow for divergence of the default process into more specialized modes. Indeed, alternative RFC complexes consisting of the small RFC subunits together with an alternative Rfc1-like subunit have been identified. A complex required for the DNA damage checkpoint contains the Rad24 subunit, a complex required for sister chromatid cohesion contains the Ctf18 subunit, and a complex that aids in genome stability contains the Elg1 subunit. Only the RFC-Rad24 complex has a known associated clamp, a heterotrimeric complex consisting of Rad17, Mec3, and Ddc1. The other putative clamp loaders could either act on clamps yet to be identified or act on the two known clamps.

The PCNA–RFC Families of DNA Clamps and Clamp Loaders

The proliferating cell nuclear antigen (PCNA) is an essential protein for DNA replication and damage repair. How its function is controlled remains an important question. Here, we show that the chromatin-bound PCNA protein is phosphorylated on Tyr 211, which is required for maintaining its function on chromatin and is dependent on the tyrosine kinase activity of EGF receptor (EGFR) in the nucleus. Phosphorylation on Tyr 211 by EGFR stabilizes chromatin-bound PCNA protein and associated functions. Consistently, increased PCNA Tyr 211 phosphorylation coincides with pronounced cell proliferation, and is better correlated with poor survival of breast cancer patients, as well as nuclear EGFR in tumours, than is the total PCNA level. These results identify a novel nuclear mechanism linking tyrosine kinase receptor function with the regulation of the PCNA sliding clamp.

Tyrosine phosphorylation controls PCNA function through protein stability.

Tight junctions and the regulation of gene expression.

Emerging areas of nanotechnology hold the promise of overcoming the limitations of existing technologies for intracellular manipulation. These new developments provide approaches for the creation of chemical–biological hybrid nanocomposites that can be introduced into cells and subsequently used to initiate intracellular processes or biochemical reactions. Such nanocomposites would advance medical biotechnology, just as they are improving microarray technology and imaging in biology and medicine, and introducing new possibilities in chemistry and material sciences. Here we describe the behaviour of 45-A nanoparticles of titanium dioxide semiconductor combined with oligonucleotide DNA into nanocomposites in vivo and in vitro. These nanocomposites not only retain the intrinsic photocatalytic capacity of TiO2 and the bioactivity of the oligonucleotide DNA (covalently attached to the TiO2 nanoparticle), but also possess the chemically and biologically unique new property of a light-inducible nucleic acid endonuclease, which could become a new tool for gene therapy.

Biology of TiO2-oligonucleotide nanocomposites.

Proliferating cell nuclear antigen (PCNA) protein is one of the central molecules responsible for decisions of life and death of the cell. The PCNA gene is induced by p53, while PCNA protein interacts with p53-controlled proteins Gadd45, MyD118, CR6 and, most importantly, p21, in the process of deciding cell fate. If PCNA protein is present in abundance in the cell in the absence of p53, DNA replication occurs. On the other hand, if PCNA protein levels are high in the cell in the presence of p53, DNA repair takes place. If PCNA is rendered non-functional or is absent or present in low quantities in the cell, apoptosis occurs. The evolution from prokaryotes to eukaryotes involved a change of function of PCNA from a 'simple' sliding clamp protein of the DNA polymerase complex to an executive molecule controlling critical cellular decision pathways. The evolution of multicellular organisms led to the development of multicellular processes such as differentiation, senescence and apoptosis. PCNA, already an essential molecule in the life of single cellular organisms, then became a protein critical for the survival of multicellular organisms.

/pdf/proliferating-cell-nuclear-antigen-pcna-ringmaster-of-the-4to48z1ylo.pdf

Proliferating Cell Nuclear Antigen (PCNA): Ringmaster of the Genome

Purpose : Previous work from the authors' group and others has demonstrated that some of the effects of UV irradiation on gene expression are modulated in response to the addition of salicylic acid to irradiated cells. The presumed effector molecule responsible for this modulation is NF-kappaB. In the experiments described here, differential-display RT-PCR was used to identify those cDNAs that are differentially modulated by UV radiation with and without the addition of salicylic acid. Materials and methods : Differential-display RT-PCR was used to identify differentially expressed genes. Results : Eight such cDNAs are presented: lactate dehydrogenase (LDH-beta), nuclear encoded mitochondrial NADH ubiquinone reductase 24kDa (NDUFV2), elongation initiation factor 4B (eIF4B), nuclear dots protein SP100, nuclear encoded mitochondrial ATPase inhibitor (IF1), a cDNA similar to a subunit of yeast CCAAT transcription factor HAP5, and two expressed sequence tags (AA187906 and AA513156). Conclusions : Sequences of...

Identification of genes regulated by UV/salicylic acid.

In the process of identifying genes that are differentially regulated in cells exposed to ultraviolet radiation (UV), we identified a transcript that was repressed following the exposure of cells to a combination of UV and salicylate, a known inhibitor of NF-kappaB. Sequencing this band determined that it has identify to lactate dehydrogenase, and Northern blots confirmed the initial expression pattern. Analysis of the sequence of the LDH 5` region established the presence of NF-kappaB, Sp1, and two Ap-2 elements; two partial AP- 1; one partial RE, and two halves of E-UV elements were also found. Electromobility shift assays were then performed for the AP-1, NF- kappaB, and E-UV elements. These experiments revealed that binding to NF-kappaB was induced by UV but repressed with salicylic acid; UV did not affect AP-1 binding, but salicylic acid inhibited it alone or following UV exposure; and E-UV binding was repressed by UV, and salicylic acid had little effect. Since the binding of no single element correlated with the expression pattern of LDH, it is likely that multiple elements govern UV/salicylate-mediated expression.

/pdf/radiation-induced-gene-responses-47owezsn6z.pdf

J. Oryhon

Papers

Proliferating Cell Nuclear Antigen (PCNA): Ringmaster of the Genome

Identification of genes regulated by UV/salicylic acid.

Radiation-induced gene responses