Memory-enhancing effects of secreted forms of the β-amyloid precursor protein in normal and amnestic mice

When administered intracerebroventricularly to mice performing various learning tasks involving either short-term or long-term memory, secreted forms of the β-amyloid precursor protein (APPs751 and APPs695) have potent memory-enhancing effects and block learning deficits induced by scopolamine. The memory-enhancing effects of APPs were observed over a wide range of extremely low doses (0.05-5,000 pg intracerebroventricularly), blocked by anti-APPs antisera, and observed when APPs was administered either after the first training session in a visual discrimination or a lever-press learning task or before the acquisition trial in an object recognition task. APPs had no effect on motor performance or exploratory activity. APPs695 and APPs751 were equally effective in the object recognition task, suggesting that the memory-enhancing effect of APPs does not require the Kunitz protease inhibitor domain. These data suggest an important role for APPss on memory processes.

Activities and response to DNA damage of latent and active sequence-specific DNA binding forms of mouse p53

The mouse p53 protein generated by alternative splicing (p53as) has amino acid substitutions at its C terminus that result in constitutively active sequence-specific DNA binding (active form), whereas p53 protein itself binds inefficiently (latent form) unless activated by C-terminal modification. Exogenous p53as expression activated transcription of reporter plasmids containing p53 binding sequences and inhibited growth of mouse and human cells lacking functional endogenous p53. Inducible p53as in stably transfected p53 null fibroblasts increased p21WAF1/Cip-1/Sdi and decreased bcl-2 protein steady-state levels. Endogenous p53as and p53 proteins differed in response to cellular DNA damage. p53 protein was induced transiently in normal keratinocytes and fibroblasts whereas p53as protein accumulation was sustained in parallel with induction of p21WAF1/Cip-1/Sdi protein and mRNA, in support of p53as transcriptional activity. Endogenous p53 and p53as proteins in epidermal tumor cells responded to DNA damage with different kinetics of nuclear accumulation and efficiencies of binding to a p53 consensus DNA sequence. A model is proposed in which C-terminally distinct p53 protein forms specialize in functions, with latent p53 forms primarily for rapid non-sequence-specific binding to sites of DNA damage and active p53 forms for sustained regulation of transcription and growth.

“Switch I” mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by σ54-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of σ54-holoenzyme—residues 206–220—is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrCA216V and NtrCG219K, have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrCA216C and NtrCG219C, are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix–turn–helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with σ54-holoenzyme. The conserved region in which these amino acid substitutions occur (206–220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21ras.

Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-β receptor–IgG1 fusion protein

Early in ontogeny, the secondary lymphoid organs become populated with numerous cells of mesodermal origin which forms both the lymphoid and stromal elements. The critical receptor/ligand interactions necessary for lymphoid organogenesis to occur are for the most part unknown. Although lymphotoxin-α (LTα) has been shown to be required for normal lymph node, Peyer’s patch, and splenic development, it is unclear if soluble LTα3, and/or cell-bound lymphotoxin-αβ (LTαβ) mediate these developmental events. Here we report that blocking LTαβ/lymphotoxin-β receptor (LTβR) interaction in vivo by generating mice which express a soluble LTβR–Fc fusion protein driven by the human cytomegalovirus promoter results in an array of anatomic abnormalities affecting both the spleen and Peyer’s patches, but not the lymph nodes. These results demonstrate that surface LTαβ ligand plays a critical role in normal lymphoid organ development.

A mutant RNA polymerase that forms unusual open promoter complexes

We describe a mutant Escherichia coli RNA polymerase (RNAP) that forms stable open promoter complexes even at −20°C but with a shortened melted region that extends downstream to only position −7. In the presence of initiating transcription substrates, the mutant RNAP undergoes a temperature-dependent isomerization, resulting in a promoter complex that is indistinguishable from the wild-type RNAP–promoter complex, with the melted region extended downstream to position +4. We propose that the open complex formed by the mutant RNAP represents an intermediate on the normal promoter-opening pathway and that our results support earlier findings that initial promoter opening occurs in the upstream region of the −10 promoter consensus element and subsequently extends downstream to encompass the transcription start site.

Core binding factor beta-smooth muscle myosin heavy chain chimeric protein involved in acute myeloid leukemia forms unusual nuclear rod-like structures in transformed NIH 3T3 cells.

Patients with the M4Eo subtype of acute myeloid leukemia almost invariably are found to have an inversion of chromosome 16 in their leukemic cells, which results in a gene fusion between the transcription factor called core binding factor beta (CBFbeta) on 16q and a smooth muscle myosin heavy chain (SMMHC) gene on 16p. Subcellular localizations of the wild-type CBFbeta and the CBFbeta-SMMHC fusion protein were determined by immunofluorescence of NIH 3T3 cells that overexpress wild-type or fusion protein. Normal CBFbeta showed an unexpected perinuclear pattern consistent with primary localization in the Golgi complex. The CBFbeta-SMMHC fusion protein had a very different pattern. Nuclear staining included rod-like crystalline structures as long as 11 microm. The heterodimeric partner of CBFbeta, CBFalpha, formed part of this complex. Cytoplasmic staining included stress fibers that colocalized with actin, probably as a consequence of the myosin heavy chain component of the fusion protein. Deletion of different regions of the CBFbeta portion of the fusion protein showed that binding to CBFalpha was not required for nuclear translocation. However, deletion of parts of the SMMHC domain of the fusion protein involved in myosin-mediated filament formation resulted in proteins that did not form rod-like structures. These observations confirm previous indirect evidence that the CBFbeta-SMMHC fusion protein is capable of forming macromolecular nuclear aggregates and suggests possible models for the mechanism of leukemic transformation.

Alternative translation initiation site usage results in two functionally distinct forms of the GATA-1 transcription factor.

GATA-1 is a zinc-finger transcription factor that plays a critical role in the normal development of hematopoietic cell lineages. In human and murine erythroid cells a previously undescribed 40-kDa protein is detected with GATA-1-specific antibodies. We show that the 40-kDa GATA-1 (GATA-1s) is produced by the use of an internal AUG initiation codon in the GATA-1 transcript. The GATA-1 proteins share identical binding activity and form heterodimers in erythroleukemic cells but differ in their transactivation potential and in their expression in developing mouse embryos.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.