Other ways of financing your company.

The public markets aren't what they used to be and venture capitalists are seeking investments with shorter timelines. But the good news is several new sources of financing are becoming available.

Reflections on the cot death cases.

Sally Clark, Angela Cannings and Trupti Patel not only suffered the tragic sudden deaths of two or more babies. They were then accused, and in the first two cases convicted, of their murder. The misuse of statistics at Sally Clark's trial had profound consequences, especially for her, but also for the subsequent cases. Ray Hill reflects on the statistical issues involved.

Autoradiography in opioid triple knockout mice reveals opioid and opioid receptor like binding of naloxone benzoylhydrazone.

Naloxone benzoylhydrazone (NalBzoH) is a ligand used to study opioid receptors. It has been suggested to act at a novel kappa3 receptor but also appears to bind to classical opioid receptors, and possibly the ORL1 receptor. We have used opioid receptor triple knockout mice, deficient in genes coding for the mu, delta and kappa-receptor, to characterise the relative contributions of opioid and ORL1 activity to the binding of this ligand, by carrying out receptor autoradiography with [3H]NalBzoH. As competing ligands we have used diprenorphine and nociceptin at 1 microM, alone or in combination, to determine the contribution of opioid and ORL1 receptor binding. At 4 nM [3H]NalBzoH showed labelling in wild-type brains indicative of broad spectrum classical opioid receptor binding. In the triple knockout brains all labelling was completely absent, suggesting that at this concentration there is no binding to ORL1 sites. However at 50 nM [3H]NalBzoH showed labelling in triple knockout brains with a distribution pattern indicative of ORL1 labelling. Quantitative analysis showed that nociceptin displaced typically 30% of the residual labelling in knockout brains whilst diprenorphine had relatively little effect. The data show that at 50 nM NalBzoH no binding was detected other than to classical opioid receptors or to ORL1 in an approximate ratio of 2:1.

Autoradiography of opioid and ORL1 ligands in opioid receptor triple knockout mice.

Three genes for the opioid receptors ( micro, delta and kappa) and a gene coding for a related receptor (ORL1) have been cloned but pharmacological studies suggest that further subtypes exist that remain poorly understood. To determine if there are other classically defined opioid binding sites we have carried out homogenate binding and section autoradiography with [3H]naloxone in mice that lack all three opioid genes and are hyperalgesic in a thermal nociceptive test. We have also examined [3H]bremazocine labelling in triple knockout brain and spinal cord as this ligand has been proposed to label novel kappa-receptors. No receptor labelling for either ligand was detected in the brains or spinal cord of knockout mice demonstrating that all binding is the product of the three known receptors and that there is no cross-labelling of the ORL1 receptor. Nociceptin (1 micro m) caused marked displacement of [3H]bremazocine in wild-type brains indicating that nociceptin at high concentrations can displace classical opioid binding. As a number of studies have proposed a close association between the classical opioid receptors and the ORL1 system we also hypothesized that loss of all of the classical opioid receptors might lead to compensatory changes in ORL1 receptors. Labelling of the ORL1 receptor with [3H]nociceptin showed region-dependent quantitative increases in triple knockout brains indicating a close relationship between the two systems in specific brain areas.