Sequences in the proximal 5′ flanking region of the rat neuron-specific enolase (NSE) gene are sufficient for cell type-specific reporter gene expression

We investigated the regulation of the rat neuron-specific enolase gene using a transient transfection approach. Recent transgenic mouse studies have shown that a 1.8-kb segment of the ratNSE gene 5′ flanking region, including the first (noncoding) exon but not the first intron, is able to drive expression of a reporter gene in parallel with endogenousNSE. These data suggest thatcis-acting elements responsible for the spatial and temporal pattern ofNSE gene expression are located within the proximal 1.8 kb of the 5′ flanking sequence. To further investigate this region, we joined the 1.8-kb regulatory cassette to thecat reporter gene and generated a number of constructs in which the flanking sequence was progressively deleted from the 5′ end. These constructs were tested by transient transfection into neuronal and nonneuronal cells, followed by an assay for CAT activity. We found that as little as 255 bp of 5′ flanking sequence was able to confer cell type-specificity on the reporter gene. Further truncation to 120 bp of 5′ sequence resulted in a sharp downregulation of reporter activity in PC12 cells but a significant rise in both Neuro-2A neuroblastoma cells and nonneuronal Ltk- cells, indicating thatcis-acting elements controlling the regulation ofNSE in Ltk-, Neuro-2A, and PC12 cells may lie within the 135 bp region covered by this deletion. This region contains an AP-2 site and an element similar in sequence and position to a motif identified in the proximal promoter region of the neuron-specific peripherin gene. Reduction to 95 bp of 5′ sequence resulted in a slight downregulation of CAT activity in all cell lines tested, and further truncation to 65 bp of 5′ sequence caused a universal reduction to background levels of CAT activity, concomitant with the disruption of the basalNSE promoter. Our results show that the 5′ flanking region of theNSE gene is capable of conferring cell type-specificity on a heterologous gene in transfected cells and that elements responsible for this are located within the proximal 255 bp.     

A Truly Knotted Cord

Summary: A case of 4 true knots in an umbilical cord, which did not cause any detectable harm, is presented. Careful examination of the placenta, membranes and umbilical cord continues to be encouraged.     

Ultrasound and neonatal hip screening. A prospective study of 'high risk' babies

We describe a simple, quick ultrasound screening test for CDH, and its use in a prospective study of babies with a 'high risk' factor, over one year from January 1987. From a birth population of 3,879, 812 hip scans were performed on 406 babies and 98 babies were abnormal. So far, there have been no late cases of CDH. Family history, breech malposition, and postural foot deformities were confirmed to be important risk factors, but babies with a simple click were equally at risk. Our early results indicate that a large proportion of the potential late cases are contained within our extended high-risk group.     

Renal function during onset of carbachol-induced hypertension in conscious rats

This study evaluated the changes in renal function that occur during the early phases of chronic infusion of carbachol into the lateral ventricle in conscious rats. Infusion of 1.0 micrograms/h of carbachol i.c.v. resulted in a prompt pressor response with mean arterial pressure rising 20 mm Hg within 15 min. The pressure remained elevated for the duration of a 2-hour infusion. Carbachol infusion at 0.5 micrograms/h induced a similar elevation in blood pressure, but the onset was delayed, reaching significance only after 30-60 min. The higher dose of carbachol was associated with a marked and sustained natriuresis, with sodium excretion increasing from 2.1 +/- 0.3 to 5.3 +/- 1.0 microEq/100 g min after 2 h, compared to 2.0 +/- 0.5 and 1.8 +/- 0.3 microEq/100 g min in vehicle-infused control animals. Sodium excretion did not change significantly in animals infused with carbachol at 0.5 microgram/h. There were no significant changes in glomerular filtration rate in any of the groups. Plasma levels of atrial natriuretic peptide (ANP) were not altered significantly by ventricular infusion of carbachol (188 +/- 99 before vs. 83 +/- 17 pg/ml after infusion). It is concluded that the pressor response to central carbachol infusion is not dependent on retention of sodium and water. The natriuresis observed with carbachol infusion can be dissociated from the pressor response, and is not mediated by ANP.     

Calcium Concentration-dependent Mechanisms through which Ketamine Relaxes Canine Airway Smooth Muscle

Background: Ketamine is a potent bronchodilator that, in clinically used concentrations, relaxes airway smooth muscle in part by a direct effect. This study explored the role of calcium concentration (Ca2+) in this relaxation.

Methods: Canine trachea smooth muscle strips were loaded with the fluorescent probe fura-2 and mounted in a spectrophotometric system to measure force and intracellular calcium concentration ([Ca2+]i) simultaneously. Calcium influx was estimated using a manganese quenching technique. Cyclic nucleotides in the airway smooth muscle strips were measured by radioimmunoassay.

Results: In smooth muscle strips stimulated with submaximal (0.1 micro Meter) and maximal (10 micro Meter) concentrations of acetylcholine, ketamine caused a concentration-dependent decrease in force and [Ca2+]i. The sensitivity of the force response to ketamine significantly decreased as the intensity of muscarinic receptor stimulation increased; the median effective concentration for relaxation induced by ketamine was 59 micro Meter and 850 micro Meter for tissue contracted by 0.1 micro Meter or 10 micro Meter acetylcholine, respectively (P < 0.05). In contrast, the sensitivity of the [Ca2+] sub i response did not depend on the intensity of muscarinic receptor stimulation. Ketamine at 1 mM significantly inhibited calcium influx. Ketamine did not significantly increase cyclic nucleotide concentrations.