Neuroprotection by donepezil against glutamate excitotoxicity involves stimulation of ��7 nicotinic receptors and internalization of NMDA receptors

BACKGROUND AND PURPOSE

Glutamate excitotoxicity may be involved in ischaemic injury to the CNS and some neurodegenerative diseases, such as Alzheimer''s disease. Donepezil, an acetylcholinesterase (AChE) inhibitor, exerts neuroprotective effects. Here we demonstrated a novel mechanism underlying the neuroprotection induced by donepezil.

EXPERIMENTAL APPROACH

Cell damage in primary rat neuron cultures was quantified by lactate dehydrogenase release. Morphological changes associated with neuroprotective effects of nicotine and AChE inhibitors were assessed by immunostaining. Cell surface levels of the glutamate receptor sub-units, NR1 and NR2A, were analyzed using biotinylation. Immunoblot was used to measure protein levels of cleaved caspase-3, total NR1, total NR2A and phosphorylated NR1. Immunoprecipitation was used to measure association of NR1 with the post-synaptic protein, PSD-95. Intracellular Ca²⁺ concentrations were measured with fura 2-acetoxymethylester. Caspase 3-like activity was measured using enzyme substrate, 7-amino-4-methylcoumarin (AMC)-DEVD.

KEY RESULTS

Levels of NR1, a core subunit of the NMDA receptor, on the cell surface were significantly reduced by donepexzil. In addition, glutamate-mediated Ca²⁺ entry was significantly attenuated by donepezil. Methyllycaconitine, an inhibitor of α7 nicotinic acetylcholine receptors (nAChR), inhibited the donepezil-induced attenuation of glutamate-mediated Ca²⁺ entry. {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a phosphatidyl inositol 3-kinase (PI3K) inhibitor, had no effect on attenuation of glutamate-mediated Ca²⁺ entry induced by donepezil.

CONCLUSIONS AND IMPLICATIONS

Decreased glutamate toxicity through down-regulation of NMDA receptors, following stimulation of α7 nAChRs, could be another mechanism underlying neuroprotection by donepezil, in addition to up-regulating the PI3K-Akt cascade or defensive system.     

A multigene RT-PCR assay used to predict recurrence in early breast cancer: two presentations with contradictory results

During the 2003 San Antonio Breast Cancer Symposium two studies were presented that were designed to validate a recurrence score, derived from a 21-gene RT-PCR assay, in patients with axillary node-negative breast cancer. This recurrence score was highly predictive for the risk of recurrence in 668 patients treated in a large multicenter trial with adjuvant tamoxifen. However, no prognostic value was found in a small group of patients who were retrospectively selected in a single institution and who did not receive any adjuvant systemic therapy. Further validation is needed to establish the prognostic and predictive role of this assay in clinical management.     

High-frequency oscillatory ventilation in children: a single-center experience of 53 cases

Introduction  

The present article reports our experience with high-frequency oscillatory ventilation (HFOV) in pediatric patients who deteriorated on conventional mechanical ventilation.     

Apnoea detection: human performance and reliability of a computer algorithm

We examined the consistency of apnoea recognition between three human experts. The hypothesis was that computer detection of apnoea could emulate human expert apnoea recognition. The aim was to detect apnoeas with the highest possible accuracy from a single breathing signal, by both human experts and computer. Three human experts independently examined recordings of breathing waveform from overnight sleep studies from 10 infants aged 3-17 weeks. All apnoeas of 5 s or more were identified and reviewed. However, there still remained 10% disagreement. A computer apnoea detector was implemented. An algorithm analysed statistical properties of the signal to find breathing pauses. Optimal performance was 1 % missed apnoeas (compared with the agreed apnoeas identified by the three experts) and 29% false detections. This computer algorithm reliably identified most apnoeas but did not replace the human expert. Algorithm, apnoea, breathing, detection, expert     

Accuracy of self-reported body weight,height and waist circumference in a Dutch overweight working population

Background  

In population studies, body mass index (BMI) is generally calculated from self-reported body weight and height. The self-report of these anthropometrics is known to be biased, resulting in a misclassification of BMI status. The aim of our study is to evaluate the accuracy of self-reported weight, height and waist circumference among a Dutch overweight (Body Mass Index [BMI] ≥ 25 kg/m²) working population, and to determine to what extent the accuracy was moderated by sex, age, BMI, socio-economic status (SES) and health-related factors.     

A rapid method for the measurement of the oxoreductase activity of 11beta-hydroxysteroid dehydrogenase in granulosa-lutein cells from patients undergoing in-vitro fertilization

The presence of 11beta-hydroxysteroid dehydrogenase (11beta-HSD; EC1.1.1.146), the enzyme responsible for the interconversion of cortisol and cortisone, in granulosa-lutein (GL) cells is associated with a poor outcome in in-vitro fertilization (IVF). We have developed a simple method of assessing the reductase component of 11beta-HSD in these cells which is sufficiently rapid to provide data on the enzyme's activity prior to embryo replacement. Cells were pooled from follicular aspirates and challenged with cortisone within 2 h of aspiration. Cortisol secretion was then measured by radioimmunoassay. Conversion of cortisone to cortisol was linear for up to 3 h and was completely inhibited by glycyrrhetinic acid, a specific 11beta-HSD inhibitor. Initial velocity rates were determined for eight cortisone concentrations (range 0.1-8 micromol/l), and the apparent Km calculated (1.6 +/- 0.4 micromol/l). There was no evidence of substrate/product inhibition and conversion of cortisone to cortisol was <2% in all experiments. In subsequent work, cells were challenged with cortisone (6 micromol/l) for 2 h. Cells challenged for 2 h immediately following purification from follicular aspirates produced varying amounts of cortisol (range 25-150 nmol/pooled follicles from each patient, n = 10 patients), while basal outputs were <6 nmol/l. Enzyme activity was also examined in cells on a per follicle basis from individual patients and found to vary considerably (e.g. 19, 53 and 36 nmol/l cortisol/1000 cells, three follicles). Having established the method for assessing 11beta-reductase activity within GL cells, we performed a small prospective study on a series of 20 patients examining the enzyme activity within 110 individual follicles. 11Beta-reductase activity varied greatly from patient to patient and from follicle to follicle ranging from <0.024-0.57 nmol cortisol/microg DNA but at present low patient numbers preclude a meaningful correlation between enzyme activity and pregnancy rate. In summary, we have developed a simple, rapid (<8 h) assay for detecting the reductase activity of 11beta-HSD in GL cells isolated from pooled or individual follicles. This procedure is sufficiently quick to aid in the choice of embryo for replacement.      

Predicted maximal oxygen consumption of indigenous Siberians

This study presents physical fitness data on two indigenous Siberian populations, the Evenki and Keto. The Canadian Aerobic Test of Fitness (CATF) was utilized to provide estimates of maximal oxygen consumption (V?O₂ max) for a sample of 44 subjects (30 males, 14 females) as baseline data for further studies on changing fitness levels and the health problems associated with acculturation. Estimates of V?O₂ max average 46.2 ml kg^?1 min^?1 for males and 33.9 ml kg^?1 min^?1 for females. These values are comparable to those previously reported for other semisubsistence, cold adapted populations. The Siberian groups are below the Canadian norms in the 15–19 year age range, and thereafter track at about the 50th percentile throughout adulthood. This suggests that the cardiorespiratory systems of adult Evenki and Keto are functionally comparable to the average adult Canadian. © 1994 Wiley-Liss, Inc.