Activation of the gene encoding decay accelerating factor following nerve growth factor treatment of sensory neurons is mediated by promoter sequences within 206 bases of the transcriptional start site

This study was designed to develop a quick methodology to assess the healthcare needs of a rural community and to determine what factors make these communities 'happy' or 'unhappy' with respect to medical service provision. Two rural shires of approximately 4000 people each were chosen from different health regions of Western Australia. The methodology consisted of interviews with healthcare providers and key community informants as well as a community questionnaire. The interviewing process showed that key community informants offered no new information in addition to that already provided by the healthcare providers. Furthermore, all key points would have been covered by interviewing approximately 60% of all healthcare providers in each community. Hand delivery of the community questionnaire yielded the highest response rate. The level of community satisfaction with general practitioner (GP) and hospital services determines whether a community is medically 'happy' or 'unhappy'.     

Self-reported physical activity in a rural county: a New York county health census

We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.     

Telomerase is activated in the prostate and seminal vesicles of the castrated rat

Telomeres, the repetitive non-coding DNA sequences found at the ends of all eukaryotic chromosomes, shorten with each cell division. It has been proposed that telomere shortening may be the counting element of a mitotic clock that keeps track of cell divisions; with shortening to a critical length acting as a senescence signal underlying cellular aging. The enzyme telomerase functions to maintain telomere length, thus allowing unlimited cell division, and has been associated with cellular immortalization and cancer. Stem cells have large, perhaps unlimited, replicative capacities. Since these cells are potentially immortal, we reasoned that they might posses active telomerase. We therefore assayed for telomerase activity in the stem cell enriched pools of the androgen-depleted sex accessory tissues in the castrated male rat. Following castration, the ventral prostate and seminal vesicles of the rat involute, losing approximately 90% of their cells by 21 days. These residual glands persist, and are enriched for stem cells, being capable of fully regenerating these glands if testosterone is re-introduced into the animal. We assayed telomerase activity in extracts from normal, involuted, and regenerating ventral prostate and seminal vesicles. Normal glands were found to be telomerase negative, whereas telomerase activity appeared as these glands involuted following castration. Conversely, telomerase activity disappeared during testosterone-induced regeneration of these residual glands. These results provide strong evidence for the ability of androgen to negatively-regulate telomerase activity in stem cell populations of the rat ventral prostate and seminal vesicles. and represent the first in vivo model system for the modulation of telomerase activity.     

Uptake and intracellular trafficking of asialoglycoprotein-polylysine-DNA complexes in isolated rat hepatocytes

Receptor-mediated gene delivery has been reported for a number of different receptor systems although the intracellular fate of such systems has not been systematically investigated. In this study, we have determined the fate of a commonly used asialoglycoprotein (ASGP)-dependent DNA delivery system in isolated rat hepatocytes. ASPG-polylysine (PLL296) was ionically complexed with pSV-CAT DNA at a molar ratio of 10:1. The resulting complex inhibited 125I-ASGP binding to rat hepatocytes but ASGP only partially inhibited the binding of complex. The ASGP-independent binding was due to the interaction of the PLL component of the complex with plasma membranes and could be minimised by replacing PLL296 with PLL19. Following internalisation, ASGP was cleaved from the complex and translocated to the lysosomes where it was degraded. The DNA, however, remained in an intracellular compartment that cosedimented with plasma membranes in Percoll density gradients. This study shows first that hepatocytes do not process DNA internalised as ASGP complexes in a manner similar to ASGP itself, and second that the differential sorting of the two cleaved molecules leads to a rapid intracellular compartmentalisation of the DNA. Controlled release from this compartment may be a means for prolonged gene expression in gene therapy protocols.     

Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Methylation analysis of individual cytosines in genomic DNA can be determined quantitatively by bisulphite treatment and PCR amplification of the target DNA sequence, followed by restriction enzyme digestion or sequencing. Methylated and unmethylated molecules, however, have different sequences after bisulphite conversion. For some sequences this can result in bias during the PCR amplification leading to an inaccurate estimate of methylation. PCR bias is sequence dependent and often strand-specific. This study presents a simple method for detection and measurement of PCR bias for any set of primers, and investigates parameters for overcoming PCR bias.     

Receptive field changes after strokelike cortical ablation: a role for activation dynamics

The reorganization of neural activity that takes place after stroke is of paramount importance in producing functional recovery. Experimental stroke models have suggested that this reorganization may have two phases, but physiology alone cannot fully resolve what causes each phase. Computer modeling suggests that these phases might involve an initial change in dynamics occurring immediately, followed by synaptic plasticity. We combined physiological recording from macaque middle temporal cortex (area MT) with a neural network computer model to examine this first phase of altered cortical function after a small, experimentally induced cortical lesion. Major receptive field (RF) changes seen in the first few days postlesion included both expansion and contraction of receptive fields. Although only expansion could be reproduced in an initial model, addition of inhibitory interneuron loss in a ring around the primary ablation, suggested by immunohistochemical examination, permitted contraction to be replicated as well. We therefore predict that this immunochemical observation reflects an immediate extension of the lesion rather than a late response. Additionally our model successfully predicted a correlation between increased firing rate and RF size. Our model suggests that activation dynamics alone, without anatomic remodeling, can cause the large receptive field changes that allow the rapid behavioral recovery seen after middle temporal lesions.