Notch1 antiapoptotic activity is abrogated by caspase cleavage in dying T lymphocytes.

Excessive signaling via the Notch1 receptor inhibits apoptosis in T lymphocytes. Since several antiapoptotic proteins are cleaved by caspases during cell death, we investigated whether Notch1 was a caspase substrate. Results demonstrate that the intracellular domain of Notch1 (NICD) is cleaved into six fragments during apoptosis in Jurkat cells or peripheral T lymphocytes. Notch1 cleavage is prevented by the caspase inhibitors DEVD-fmk and VEID-fmk or by Bcl-2 expression. Caspase-3 and caspase-6 cleave the NICD into six fragments using sites located within the NF-kappaB binding domain, the ankyrin repeats and the transactivation domain. Notch1 cleavage correlates with the loss of HES-1 expression in apoptotic T cells. Notch1 fragments cannot inhibit activation-induced cell death in a T-cell hybridoma, confirming the abrogation of Notch1 antiapoptotic activity by caspases. The ability of the NICD but not the fragments to antagonize Nur77 activity supports a role for this factor in Notch1 antiapoptotic function.

Aging in place: a proposal for rural community-based care for frail elders.

Rural health care is hampered by uneven development of services and multiple definitions of rurality. One of the most vulnerable populations in this setting is older residents. This article explores existing models of long-term care for the older population and proposes a model for rural community-based care. This model emphasizes the contributions of advanced practice nurses as coordinators of a collaborative system of care for the targeted population. Issues of cost and quality, as well as the strengths and limitations of the model, are addressed.

The concept of interpersonal pattern in Peplau's theory of nursing.

Interpersonal pattern, defined as the category in which separate interpersonal acts are linked by similar, defining characteristics, is a critical, data-rich concept that is used in the therapeutic nurse-client relationship. If this concept is studied within the framework of aesthetic knowing in nursing, the processes of deliberate involvement that form the crux of the nurse-client relationship become illuminated. An illustrative case study brings this concept to life by demonstrating the use of interpersonal pattern as the focus of intervention with a frail, elderly woman. In this case study, the nurse practices aesthetic knowing to assess and respond to the woman, and ultimately assist in her forward movement towards optimum function and health. Linkages of this type of intervention to all of nursing practice are made by integrating the use of interpersonal pattern with aesthetic knowing in nursing.

Enhanced expression of amyloid precursor protein in response to dibutyryl cyclic AMP is not mediated by the transcription factor AP-2.

The gene for amyloid precursor protein (APP) is expressed almost ubiquitously, with high levels of mRNA being detected in brain. The basal expression level of the APP gene can be modulated by physiological stimuli, and in this report we demonstrate that the second messenger cyclic AMP can regulate APP mRNA through transcriptional mechanisms. Northern blot analysis showed a 1.8-fold increase in steady-state levels of APP mRNA when the neuroblastoma x glioma hybrid cell line NG108-15 was treated with dibutyryl cyclic AMP. Although the upstream sequences of the APP gene do not contain a canonical cyclic AMP response element, transient transfection assays in NG108-15 cells using different portions of the APP promoter showed an increase in reporter gene activity mediated by sequences located between -303 to -204 and -488 to -2991. Cotransfection assays carried out in HepG2 cells with AP-2, a cyclic AMP-regulated transcription factor, failed to activate the APP promoter through the AP-2 consensus sequence (GCCNNNCGG) located at position -205. Electrophoretic mobility shift analysis revealed that the AP-2 binding activity present in HeLa nuclear extracts fails to recognize the APP AP-2 consensus sequence. We conclude that increases in cyclic AMP levels can lead to an up-regulation of APP gene transcription through at least two different regions of the APP promoter. This increase does not involve the AP-2 consensus sequence present in the APP promoter located at position -205, and, moreover, this putative site is not recognized by the transcription factor AP-2.

Transcriptional regulation of amyloid precursor protein during dibutyryl cyclic AMP-induced differentiation of NG108-15 cells.

Early expression of amyloid precursor protein (APP) during development of the nervous system suggests that this protein may play an important role first in axogenesis and later in synaptogenesis. To study regulation of APP mRNA expression in neuronal cells, NG108-15 neuroblastoma x glioma cells were induced to differentiate in the presence of dibutyryl cyclic AMP. Steady-state levels of APP mRNA and APP isoforms increased gradually, concomitantly with the appearance of differentiated phenotype. Northern blot analysis showed a three-fold increase in APP expression at day 6 of dibutyryl cyclic AMP treatment. Nuclear run-on assays and transient transfections performed using APP promoter/reporter constructs confirmed a twofold increase in the rate of APP gene transcription. The stability of the mRNA was unchanged, with differentiated and nondifferentiated cells having the same half-life of about 21 h. These results strongly suggest that APP mRNA induction in the differentiated NG108-15 cells is due to an increase in the rate of transcription of the gene.

The helix-loop-helix transcription factor USF interacts with the basal promoter of human amyloid precursor protein.

Nuclear factors from HeLa, PC12, NG108-15 and SK-N-SH cell lines recognized an oligonucleotide (-56 to -37: APP-E1) containing an E box (CANNTG) which had previously been characterized as a DNase I-protected sequence in the basal promoter of the human amyloid precursor protein (APP) gene. Binding to APP-E1 was competed with an oligonucleotide encompassing the recognition site of the transcription factor USF. Antibodies directed against USF interacted with the APP-E1-protein complex and in vitro synthesized USF could bind APP-E1. Co-expression of USF cDNA transactivated a human APP-reporter gene construct. These results suggest that USF may play a role in the expression of the APP gene.

Identification of active-site residues of the adenovirus endopeptidase.

Multiple sequence alignment of the 12 adenovirus endopeptidases known to date identified a number of conserved residues which might be important for enzyme activity. Eleven mutants were created in the cloned gene by site-directed mutagenesis to identify the active site of this thiol endopeptidase. Analysis of the proteolytic activity in a crude system using viral precursor proteins, as well as in a purified system with activated proteinases using a new chromophoric octapeptide substrate, yielded results consistent with Cys-104 and His-54 being two members of the active site. This result was confirmed by the carboxymethylation of the reactive Cys-104 and its prevention by the active-thiol-specific agent E64. Although Cys-122 and Cys-126 were also reactive cysteines, mutation of these residues did not affect enzyme activity. Replacement of the active-site Cys-104 by serine converted the enzyme into a serine-like proteinase, sensitive to serine proteinase inhibitors. The absence of homology to other proteinases, particularly at the active-site cysteine, coupled with the requirement for activation by a substrate cleavage fragment, indicates that the adenovirus endoproteinase may represent a new subclass of cysteine proteinases.

Expression of amyloid precursor protein in a neuronal cell line: functional activity of proximal regulatory elements.

To study cell type-specific regulation of the gene for amyloid precursor protein (APP), we have analysed the human APP promoter for transcriptional activity and interaction with nuclear proteins in the neuronal cell line NG108-15. Sequences from -203 to +104 were sufficient for promoter function and regulatory elements within this region (-128 to -63) were identified by DNase I protection experiments. These results suggest that essentially the same proximal elements are recognized by nuclear factors from both neuronal and nonneuronal cells.

Isolation and properties of adenovirus type 2 proteinase.

We have cloned and expressed the human adenovirus type 2 proteinase gene in Escherichia coli. The expressed proteinase was isolated by a four-step chromatographic procedure. Purity and identity of the recombinant protein was established by two-dimensional gel electrophoresis, N-terminal sequencing, and specific antisera. The pure enzyme did not contain disulfide bridges, and it consisted of one subunit with a pI of 10.2. It did not show any sign of autocleavage. Labeled iodoacetate bound the pure enzyme while labeled diisopropyl fluorophosphate did not. The protease readily cleaved the viral pVII protein, ovalbumin, fibrin, and actin but had no effect on synthetic penta-, octa-, or nonapeptides carrying the consensus sequence for cleavage. The inhibitory profile of the isolated proteinase and the affinity labeling clearly indicate that the human adenovirus type 2 proteinase is a cysteine rather than a serine proteinase as previously believed. The most likely candidate for an active site residue is one of the two conserved cysteines, Cys-104 or Cys-122.