Cleavage of E2F-1-regulating proteins and activation of E2F-1 during CD95-induced death of thymocytes.

The CD95 death receptor activates caspases that cleave a variety of intracellular substrates, including cell cycle control proteins. However, the significance of this cleavage for the induction of apoptosis is unclear. In this study, CD95-induced cleavage of the G1/S checkpoint regulator proteins, retinoblastoma protein (pRb) and murine-double-minute-2 (mdm-2), was associated with an increased protein concentration of a key transcription factor, E2F-1, which is regulated by both of them. Furthermore, DNA-binding activity to E2F sites is increased. In thymocytes, CD95-induced apoptosis was associated with increased E2F-1 DNA-binding activity, while thymocytes that lacked E2F-1 were less susceptible to CD95-induced apoptosis. We conclude that the G1/S checkpoint is an important target of CD95 signalling. CD95-activated caspases cleave regulator proteins to increase E2F-1 activity, and inappropriate activation of E2F-1 is part of the mechanism of CD95-induced apoptosis.

The types of neuron in spinal dorsal horn which possess neurokinin-1 receptors.

In order to provide further information about the types of spinal neuron which possess neurokinin-1 receptors, we have carried out pre-embedding immunocytochemistry on sections of rat lumbar spinal cord with an antiserum raised against a synthetic peptide corresponding to part of the sequence of the receptor, and combined this with post-embedding immunocytochemistry to detect GABA and glycine. Numerous neuronal cell bodies showing neurokinin-1 receptor-immunoreactivity were seen in lamina I, laminae III-VI, the lateral spinal nucleus and the area around the central canal. Most of the cells observed in lamina III were small and had relatively restricted dendritic trees which could often not be followed into lamina II, however some larger cells in laminae III and IV had dendrites which extended through lamina II and into lamina I. Cells of the latter type are likely to represent a major target of substance P released from small-diameter primary afferents in the superficial dorsal horn. The great majority (255 out of 283) of spinal neurons which possessed neurokinin-1 receptor-immunoreactivity, including all of those in lamina I, were not GABA- or glycine-immunoreactive, however a few cells in the deep part of the dorsal horn and the lateral spinal nucleus and several cells near the central canal were GABA-immunoreactive, and some of these were also glycine-immunoreactive. These results suggest that substance P acts through neurokinin-1 receptors mainly on excitatory neurons within the spinal cord.