Andrea M. Moerman

Characterization of a neuronal κB-binding factor distinct from NF-κB

Abstract Transcription factors that bind κB enhancer elements have begun to garner wide attention in neurobiology. Data suggest that activation of κB-binding factors in neurons can be protective against various neurotoxins, but other data have connected NF-κB to cell death. In electrophoretic mobility shift assays of κB-binding activity, we have found that the predominant activity in rat brain tissue, in primary neurons, and in neuronal cell lines has a mobility inconsistent with that of bona fide NF-κB (RelA-p50 heterodimer). We have tentatively termed this activity neuronal κB-binding factor (NKBF). Competition assays with various DNA probes distinguished NKBF from NF-κB. Probes that efficiently bind the p50 homodimer were able to compete with a conventional NF-κB probe for NKBF binding, but NKBF did not react with antibodies to p50 (or any other known Rel family members). Furthermore, UV-crosslinking indicated that NKBF is composed of two polypeptides of 82 kDa and 27 kDa. Although NKBF activity can be elevated in a manner independent of new macromolecular synthesis, it does not appear to be modulated by IκB. Finally, no NF-κB was induced by glutamate in highly enriched neuronal cultures, although it was induced in neuron-glia cocultures. These data suggest that the primary κB-binding transcription factor in neurons is a novel protein complex distinct from NF-κB.

Insulin-like growth factor binding protein-3 is overexpressed in senescent and quiescent human fibroblasts.

Cellular insulin-like growth factor binding protein-3 (IGFBP-3) mRNA and IGFBP-3 levels in conditioned medium were consistently higher in cultures of late passage normal (old) fibroblasts and prematurely senescent fibroblasts derived from Werner syndrome (WS) during quiescence induced by serum depletion and during the renewed growth ensuing after serum repletion, compared to cultures of early passage normal (young) fibroblasts. Molar ratios of IGFBP-3/IGF-II were always higher in senescent cultures and maintained a hierarchy of old > WS > young human diploid fibroblasts. Transfection into fibroblasts of the normal full-length IGFBP-3 cDNA in an expression vector resulted in a significant reduction in colony formation compared to cells transfected with an empty expression vector (no cDNA) or with IGFBP-3 cDNA altered by a 273 base pair (bp) deletion. Addition to old and young cultures of recombinant human IGFBP-3 and IGF-I at 1:1 or 5:1 molar ratios inhibited IGF-I-mediated DNA synthesis by approximately 70-80%. These data indicate that IGFBP-3 may play an important role in the quiescent and senescent growth arrest of HDF.

Dictyostelium discoideum Hsp32 is a resident nucleolar heat-shock protein

Abstract. Hsp32 is a small shock protein in Dictyostelium discoideum that is expressed in growing cells in the absence of heat shock. Here we show that Hsp32 is an Ag-NOR-staining protein capable of binding DNA with high affinity. Hsp32 is also shown to be a resident nucleolar protein both under normal growth conditions and during heat stress. In unstressed cells, Hsp32 localizes to the nucleolar periphery in a pattern reminiscent of the rDNA in this organism. During the first several hours of heat shock, the peripheral localization of Hsp32 is not altered, although rDNA transcription is arrested. Prolonged heat shock causes a condensation of the rDNA. Under these conditions, Hsp32 is no longer predominantly associated with the rDNA, but is instead distributed over the entire nucleolus. Hsp32 therefore retains ist nucleolar localization under prolonged heat shock conditions by associating with nucleolar components other than the rDNA or rRNA.

Inhibition of AMPA responses by mutated presenilin 1

The inheritance of Alzheimers disease in some families, as well as ablation/rescue genetics in mice, suggest that point mutations in the presenilin‐1 (PS1) gene can cause disease through an unknown gain‐of‐function. While mutations associated with familial Alzheimers can alter apoptotic rates and β‐amyloid precursor processing, it is possible that other physiological effects contribute to pathogenesis. We have begun to explore effects on neurotransmission by monitoring responses of the neuropotent Ntera‐2 cell line expressing wild‐type PS1 or a FAD mutant thereof. Although no differences were initially apparent in calcium responses of metabotropic receptors, responses to glutamate were dampened in cells expressing the L286V mutant of PS1. Analysis of ionotropic agonists demonstrated that AMPA receptor alterations were responsible for this effect, whereas NMDA responses were unaltered. These data suggest that PS1 mutation could lead to cognitive deficits through subtoxic physiological effects. J. Neurosci. Res. 57:962–967, 1999.