Carole Rickards

A structure for the porcine TSH receptor

Affinity purified, detergent‐solubilised porcine TSH receptors have been crosslinked to a 125I‐labelled photoactive derivative of TSH and analysed by gel electrophoresis, gel filtration and sucrose density gradient centrifugation. Our studies suggest that the porcine TSH receptor is made up of a hydrophilic A subunit with an M r about 45000 linked to an amphiphilic B subunit (M rapprox. 25 000) by a disulphide bridge(s). The A subunit forms the binding site for TSH on the outside of the cell membrane. The B subunit appears to penetrate the membrane and form the site for interaction with adenylate cyclase either in the lipid bilayer or close to the cytoplasmic surface of the membrane.

Differential responses of rat cerebral somatostatinergic and cholinergic cells to glutamate agonists

Reductions in cortical somatostatin (SRIH) and choline acetyl-transferase (ChAT) are major biochemical deficits in Alzheimer disease (AD). SRIH and ChAT were measured in fetal rat cerebral neurons after exposure to the glutamate agonists N-methyl-D-aspartate (NMDA), kainate (KA), and quisqualate (Q). NMDA (96 h incubation) stimulated SRIH release and content in a dose-dependent manner with a Bmax of 10(-5)M and EC50 of 2-3 x 10(-6)M. KA showed a small stimulation in SRIH levels at 10(-5)M, but produced marked inhibition at 10(-4)M. Q decreased both intracellular and secreted SRIH. KA (51-76% of basal) and Q (27-56% of basal) but not NMDA (91-114% of basal) also inhibited the incorporation of [35S]methionine into proteins. In similar experiments 10(-4)M Q (23 +/- 9% of basal) and KA (20 +/- 3% of basal) but not NMDA (80 +/- 16% of basal) reduced ChAT levels in hypothalamic/septal cultures. These inhibitory actions on ChAT activity by KA and Q were reversed by gamma-glutamyltaurine (GT) but not by 2-amino-5-phosphonopentanoic acid (AP5). Chronic NMDA exposure partially inhibited muscarinic acetylcholine receptor (mAChR) mediated inositol phospholipid (PI) turnover, whereas it was abolished after KA and Q pretreatment. These findings suggest that in cerebral cell cultures, NMDA has a stimulatory action on somatostatinergic neurons and non-NMDA receptor agonism could play an important role in EAA-mediated neural damage.

Prolonged exposure to N-methyl-D-aspartate increases intracellular and secreted somatostatin in rat cortical cells.

Somatostatin (SRIF) release from fetal rat cortical cells was stimulated by exposure to 10(-5) M N-methyl-D-aspartate (NMDA) (250 +/- 20% of basal at 96 h). A similar but much less potent effect was seen with kainate (KA) but not with quisqualate (Q) which inhibited SRIF release (KA 150 +/- 13%, Q 65 +/- 18% of basal at 96 h). Similar data were obtained for intracellular levels of SRIF. Dose-dependent experiments showed that the EC50 for the stimulatory action of NMDA was 2-3 x 10(-6) M with a Bmax of around 10(-5) M. At 10(-4) M KA and Q but not NMDA reduced tissue content and release of SRIF (KA: 47 +/- 14, 67 +/- 17%; Q: 36 +/- 13, 42 +/- 6% of basal for content and release, respectively). These findings indicate that cortical SRIF content and release is enhanced by exposure to NMDA but not by KA or Q. We suggest that SRIF-containing neurones are sensitive to glutamate damage through the activation of non-NMDA rather than NMDA receptors.

Production of replication-deficient adenovirus recombinants.

In essence, replication-deficient (RD) adenovirus (Ad) vectors can be considered to function as an extremely efficient DNA transfection system capable of providing transgene expression in up to 100% of cells both in vitro or in vivo. As researchers continue to realize the full potential of this vector system, discover novel applications, and further develop and enhance systems, the use of this vector system has increased exponentially. The exploitation of Ad recombinants in HCV research is encouraged by demonstration that the virus will efficiently infect and express transgenes in hepatocytes and that following iv inoculation, transgene expression can readily be detected in hepatocytes in the liver (1-5). A number of HCV proteins have now been expressed in Ad recombinants (6-8) whereas these vectors have also been used to deliver antisense and ribozyme molecules as prototype HCV therapeutics (9).