p73 is a growth-regulated protein in vascular smooth muscle cells and is present at high levels in human atherosclerotic plaque.

p73 is a newly described homologue of the tumour suppressor p53 that was cloned serendipitously and subsequently shown to possess considerable homology in the most evolutionarily conserved p53 domains. Yet despite the fact that p53 and p73 have extensive structural similarities, their functions are proving to be quite different. We now show that p73 is a growth-regulated protein in the vasculature, being markedly increased in cultured vascular smooth muscle (VSM) cells stimulated with 10% serum, with no significant change in p73 mRNA levels. Stability of p73 is increased after serum stimulation and, probably contributing to this increase in p73 stability, the c-Abl oncogene protein displays a higher molecular weight species and is probably phosphorylated and activated in serum-stimulated VSM cells. The serum-mediated induction of p73 is not altered when the cells are incubated with inhibitors of the MAP/ERK pathway or tyrosine kinases, and is not stimulated by PDGF-BB, demonstrating that the mechanism of the increase in p73 does not involve this classical receptor tyrosine kinase growth factor signalling cascade. p73 is markedly increased in plaque tissue taken from atherosclerotic human carotid arteries, but not in comparable intimal scrapings from normal human arteries. Our data indicate that the tumour suppressor homologue p73 probably plays a role in VSM cell cycle progression, being mediated by a specific, as yet unidentified, serum component, and identifies a new function for this protein as being important in the pathogenesis of human atherosclerosis as well as other vascular diseases.

Description and demonstration of a CMOS amplifier-based-system with measurement and stimulation capability for bioelectrical signal transduction.

An extracellular recording system incorporating an electrode array and an amplifier/stimulator CMOS chip is described and characterized. Important features of this custom VLSI chip include 16 instrumentation amplifiers with a gain of 50 and the incorporation of a cross-point array allowing designation of an extracellular microelectrode as either a stimulator or sensor. The planar array consisted of 32 microelectrodes, 14 microns in diameter, and four larger reference electrodes. Microelectrodes, interconnecting traces, and bond pads were patterned with a 500-nm layer of gold. The interconnecting traces were passivated with a 1-micron thick layer of silicon nitride to provide chemical and electrical insulation and microelectrode impedance was lowered utilizing electrode position of platinum black. The amplifier exhibited a nearly flat frequency response with high pass and low pass corner frequencies of 0.7 Hz and 50 kHz, respectively. The input referred noise over the 50 kHz bandwidth was 12-16 microVRMS, well below the magnitude of previously reported extracellular potentials. Crosstalk between neighboring channels resulted in an output signal below the amplifier noise level, even for relatively large extracellular potentials. Using this system, extracellular recording were demonstrated yielding typical peak-to-peak biopotentials of magnitude 0.9-2.1 mV and 100-400 microV for chick cardiac myocytes and rat spinal cord neurons, respectively. The key components of this extracellular recording system can be manufactured using industry standard thin film photolithographic techniques.

Comparison of impedance at the microelectrode-saline and microelectrode-culture medium interface.

The impedance characteristics of gold-plated indium-tin-oxide microelectrodes immersed in culture medium (MEM) are described and compared with the impedance characteristics observed when those microelectrodes are immersed in isotonic saline. For microelectrode areas of approximately 100 microns2, applied voltage levels of 5, 50, and 100 mV, and for frequencies of from 100 Hz to 10 kHz the resistance, capacitance, capacitive reactance, and total impedance are given as a function of frequency both in culture medium and in saline. The results, which hold for current densities ranging from 0.45 to 700 pA/microns2, are compared. Also given are the alpha and K values determining the frequency characteristics of the interface resistance and capacitance in medium and in saline.