Structural basis for the coiled-coil architecture of human CtIP.

The DNA repair factor CtIP has a critical function in double-strand break (DSB) repair by homologous recombination, promoting the assembly of the repair apparatus at DNA ends and participating in DNA-end resection. However, the molecular mechanisms of CtIP function in DSB repair remain unclear. Here, we present an atomic model for the three-dimensional architecture of human CtIP, derived from a multi-disciplinary approach that includes X-ray crystallography, small-angle X-ray scattering (SAXS) and diffracted X-ray tracking (DXT). Our data show that CtIP adopts an extended dimer-of-dimers structure, in agreement with a role in bridging distant sites on chromosomal DNA during the recombinational repair. The zinc-binding motif in the CtIP N-terminus alters dynamically the coiled-coil structure, with functional implications for the long-range interactions of CtIP with DNA. Our results provide a structural basis for the three-dimensional arrangement of chains in the CtIP tetramer, a key aspect of CtIP function in DNA DSB repair.

Fusion suppression and sub-barrier breakup of weakly bound nuclei.

The mechanism for the large suppression of complete fusion in the 9Be+208Pb reaction has been investigated through measurement of sub-barrier breakup of 9Be. Excluding breakup through the 8Be ground state, whose lifetime is too long, a prompt breakup component remains, having sufficient probability to explain the observed suppression of complete fusion. This appears to be associated with interactions at the nuclear surface. The fusion suppression is predicted to be almost proportional to the charge of the target nucleus, making it most significant in reactions with heavy nuclei.

Unexpected inhibition of fusion in nucleus-nucleus collisions.

Unstable heavy atomic nuclei not found in nature can be created by fusing two stable nuclei, in a process analogous to colliding charged droplets of liquid. Recently, the formation of a handful of super-heavy nuclei with atomic numbers 114 (ref. 1) and 116 (ref. 2) has been achieved by fusion of heavy nuclei. The electrostatic energy of such systems is very large (which is the reason super-heavy nuclei are unstable), so although the two nuclei may initially be captured by the nuclear potential, rather than fusing, they almost always separate after transfer of mass to the lighter nucleus. This process, called quasi-fission, can inhibit fusion by many orders of magnitude. Understanding this inhibition may hold the key to forming more super-heavy elements. Theoretically, inhibition is predicted (ref. 5 and references therein) when the product Z1Z2 of the charges of the projectile and target nuclei is larger than about 1,600. Here we report measurements of three fusion reactions with Z1Z2 around half this value, each forming 216 88Ra. We find convincing model-independent evidence both of inhibition of fusion, and of the presence of quasi-fission. These results defy interpretation within the standard picture of nuclear fusion and fission.

Long-term depression of C-fibre-evoked spinal field potentials by stimulation of primary afferent A delta-fibres in the adult rat.

Long-term potentiation (LTP) of spinal C-fibre-evoked field potentials can be induced by brief electrical stimulation of afferent C-fibres, by natural noxious stimulation of skin or by acute nerve injury. Here, we report that in urethane anaesthetized, adult rats prolonged high frequency burst stimulation of the sciatic nerve at Adelta-fibre strength produced long-term depression (LTD) of C-fibre-evoked field potentials, and also depressed the increased amplitudes of C-fibre-evoked field potentials recorded after LTP had been established (depotentiation). Electrical stimulation of Abeta-fibres failed to induce LTD or depotentiation. In spinalized rats, prolonged Adelta-fibre conditioning stimulation induced LTP rather than LTD of C-fibre-evoked field potentials. Thus, tonic descending inhibition may determine the direction of plastic changes in C-fibre-mediated synaptic transmission. Spinal application of the N-methyl-D-aspartic acid receptor antagonist D-APV blocked induction of LTD in intact rats and LTP in spinalized rats. The presently described LTD and the depotentiation of established LTP of C-fibre-evoked field potentials in spinal dorsal horn may underlie some forms of prolonged analgesia induced by peripheral nerve stimulation procedures.

Modulation of cutaneous nociceptor activity by electrical stimulation in the brain stem does not inhibit the nociceptive excitation of dorsal horn neurons.

In anesthetized cats, recordings were obtained from single lumbar dorsal horn neurons and from primary afferent fibers of the posterior tibial nerve excited by controlled noxious radiant heating of glabrous hindpaw skin. Electrical stimulation in four brain stem regions (periaqueductal gray and lateral reticular formation in the midbrain, raphe and reticular formation in the medulla) during noxious skin heating markedly reduced the nociceptive excitation of the dorsal horn neurons. In contrast, such brain stem stimulation had small and variable effects upon the noxious heat-evoked activity in the primary afferent fibers; both increases and decreases were observed. The brain stimulation also produced transient changes in blood pressure, suggesting that circulatory effects may underlie the mechanism of nociceptor modulation. It is concluded that brain stem stimulation can modulate cutaneous nociceptor activity, but that this modulatory effect on nociceptor inflow is too small and inconsistent to explain the marked descending inhibition of the nociceptive excitation of dorsal horn neurons.

Baclofen and the Release of Neuropeptides in the Cat Spinal Cord.

The antibody microprobe technique was used to study the effect of baclofen on the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of pentobarbitone-anaesthetized spinalized cats. Both peptides were released in the region of the substantia gelatinosa during ipsilateral noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of baclofen suppressed the excitation of lumbar dorsal horn neurons, but did not produce detectable alterations of the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial grey matter dorsal to these neurons. The results suggest that the antinociceptive action of baclofen does not involve a reduction of the intraspinal release of substance P or calcitonin gene-related peptide from the central terminals of nociceptive sensory fibres.

Intraspinal release of substance P and calcitonin gene-related peptide during opiate dependence and withdrawal.

The antibody microprobe technique was used to study the release of immunoreactive substance P and immunoreactive calcitonin gene-related peptide within the lower lumbar spinal cord of anaesthetized spinalized cats pretreated twice daily for 3.5 days with increasing doses of morphine hydrochloride (2-20 mg/kg, i.p.). Both peptides were released in the region of the substantia gelatinosa during noxious cutaneous thermal stimulation or high-intensity electrical stimulation of a hind limb nerve. Intravenous administration of naloxone increased the nociceptive excitation of lumbar dorsal horn neurons, but did not alter the evoked release of immunoreactive substance P or immunoreactive calcitonin gene-related peptide in the superficial gray matter dorsal to these neurons. In addition, the release of both peptides was not significantly different to that detected under similar experimental conditions in opioid-naive cats. The results suggest that alterations in neuropeptide release from the central terminals of nociceptive primary afferent neurons do not occur during states of opiate dependence and withdrawal, and thus do not contribute to the characteristic signs of these phenomena in dependent animals.

Dynorphin A: in vivo release in the spinal cord of the cat.

The antibody microprobe technique was used to study the release of immunoreactive dynorphin A within the lower lumbar spinal cord of anaesthetised cats. A basal release was observed in the dorsal horn, centered in the region of lamina I, but was abolished by spinal cord transection at the thoracolumbar junction. Release of dynorphin A in the lamina I region was evoked by high-frequency electrical stimulation of unmyelinated primary afferent fibres, whereas stimulation of myelinated (including A delta) afferents was ineffective. Evidence was also obtained for release in laminae V-VI and at the spinal cord surface. These results suggest that in the lumbar spinal cord of the cat, dynorphin A is released in the superficial dorsal horn by impulses in descending pathways and in somatic unmyelinated primary afferent fibres.

Morphine does not reduce the intraspinal release of calcitonin gene-related peptide in the cat.

In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive calcitonin gene-related peptide (irCGRP) in the lumbar dorsal horn during stimulation of non-nociceptive or nociceptive primary afferent fibres. Release of irCGRP was detected in the substantia gelatinosa, where immunostaining for CGRP was subsequently observed. Intravenous administration of morphine did not affect release of irCGRP detected during either non-nociceptive or nociceptive afferent stimulation. The results suggest that the analgesic action of morphine does not involve reduced release of CGRP from the central terminals of nociceptive primary afferents.

Morphine and substance P release in the spinal cord.

In anaesthetised cats, antibody microprobes were used to measure the release of immunoreactive substance P (irSP) in the lumbar dorsal horn during noxious cutaneous stimulation or high-intensity electrical stimulation of a hind limb nerve. The major region of irSP release detected was centered on the substantia gelatinosa, with lesser release at the dorsal cord surface. Release at these sites was unchanged by systemic administration of morphine, or of morphine followed by naloxone. During superfusion of the dorsal cord surface with high concentrations of morphine, irSP release in the substantia gelatinosa region was slightly reduced and surface release was not observed, effects not reversed by systemic naloxone administration. The results suggest that the analgesic action of morphine does not involve reduced release of SP in the spinal cord.

Somatostatin: evidence for a role in thermal nociception.

In barbiturate-anaesthetized spinalized cats, antibody microprobes were used to investigate the release of immunoreactive somatostatin (irSS) in the lumbar dorsal horn in response to cutaneous stimuli. In the absence of applied stimulation, a significant basal release of irSS was present in the region of the substantia gelatinosa. Such release was not increased by innocuous or noxious cutaneous mechanical stimuli nor by innocuous thermal stimuli, but was increased by noxious thermal stimulation. The magnitude of this noxious heat-evoked release was estimated by comparing in vivo microprobes with those used to detect known concentrations of somatostatin in vitro. Pairs of microprobes were used to detect simultaneous release of both irSS and immunoreactive substance P in the substantia gelatinosa. The results support the putative role of somatostatin in the spinal transmission of thermal nociceptive information.

Release of sensory neuropeptides in the spinal cord: studies with calcitonin gene-related peptide and galanin.

In anaesthetized cats, antibody microprobes were used to investigate the release of immunoreactive calcitonin gene-related peptide and galanin in the lower lumbar spinal cord. In the absence of applied stimulation, a basal release of both peptides was detected at the level of the substantia gelatinosa. This release of calcitonin gene-related peptide was not altered by innocuous thermal cutaneous stimulation nor by electrical stimulation of low-threshold myelinated primary afferent fibres, but was increased by noxious thermal or noxious mechanical cutaneous stimuli and by electrical stimulation of unmyelinated primary afferents. A simultaneous release of both calcitonin gene-related peptide and substance P was detected in the substantia gelatinosa region by the use of pairs of microprobes. In contrast, none of the peripheral stimulation procedures increased intraspinal galanin release. The results suggest that the spinal transmission of nociceptive information may involve the simultaneous release and action of several neuropeptides within the superficial layers of the dorsal horn.

Release of immunoreactive somatostatin in the spinal dorsal horn of the cat.

Antibody microprobes were used to investigate the possible release of immunoreactive somatostatin (irSS) within the lumbar spinal cord of anaesthetized cats. A basal release of irSS was detected in the region of the substantia gelatinosa of the dorsal horn. By comparison with in vitro standards the concentration of SS detected in this region was estimated at 10(-7) M. This release of irSS was not significantly altered by electrical stimulation of large myelinated primary afferent fibres but was increased when unmyelinated afferents were additionally stimulated. Release of irSS was also detected at the spinal cord surface. The results support a role for somatostatin in nociceptive transmission in the spinal cord.

Inhibition of nociceptive responses of lumbar dorsal horn neurones by remote noxious afferent stimulation in the cat.

In cats anaesthetized with nitrous oxide and sodium pentobarbital, multireceptive lumbar dorsal horn neurones excited by controlled noxious radiant heating of glabrous hind paw skin were recorded by extracellular microelectrodes. These noxious heat responses were inhibited by concomitant noxious stimulation of the ipsilateral forepaw or pinna, or repetitive electrical stimulation of the ipsilateral forelimb deep radial nerve. Similar extents of inhibition were produced by noxious peripheral stimulation and by deep radial nerve stimulation in repetitive trains at intensities sufficient to excite small myelinated fibres or unmyelinated fibres. A greater inhibitory effect was produced by continuous repetitive high-intensity stimulation of the deep radial nerve. With a constant frequency (5 Hz) of continuous deep radial nerve stimulation, graded increases in stimulation intensity revealed the threshold for inhibition in the small myelinated fibre range, and an additional increment of the inhibitory effect when unmyelinated fibres were also activated. When suprathreshold for unmyelinated fibres, the efficacy of continuous deep radial nerve stimulation increased with graded increases in stimulation frequency, with a threshold frequency for inhibition between 0.5 and 1 Hz and maximal effect at 5 Hz. Two nociceptive-specific neurones studied were also inhibited by deep radial nerve stimulation. The results indicate that 'diffuse noxious inhibitory controls' (DNIC) occur in the cat and can be activated by remote electrical or natural noxious stimulation.

Cutaneous stimuli releasing immunoreactive substance P in the dorsal horn of the cat.

In barbiturate-anaesthetized spinal cats, antibody microprobes were used to examine immunoreactive substance P (irSP) release at sites within the spinal cord following cutaneous stimuli. A basal level of irSP release was detected in the region of the substantia gelatinosa of the lumbar spinal cord. No increase in this irSP release was produced by non-noxious thermal or mechanical cutaneous stimulation. Noxious thermal, mechanical or chemical cutaneous stimuli all increased release of irSP in the region of the substantia gelatinosa and in the overlying pia mater. The results support a role for SP in the transmission of information from nociceptors to spinal neurones.

The preparation and use of antibody microprobes.

A new method of detecting release of neuropeptides in the central nervous system is described. Glass micropipettes are treated with gamma-aminopropyltriethoxysilane resulting in a fine outer coating of a siloxane polymer containing free amino groups. Glutaraldehyde is then used to covalently couple protein A which in turn binds antibodies to a particular peptide. Following use in the central nervous system, microprobes are incubated in a radiolabelled form of the peptide being studied and release is detected on autoradiographs as localized zones of inhibition of binding of the labelled peptide. The spatial resolution of the method is at least 100 micron. Necessary tests of the validity of the technique are also described.

Analysis of antibody microprobe autoradiographs by computerized image processing.

A computerized digitizing method to analyze the autoradiographs derived from the antibody microprobe technique is described. The method enables the averaging of groups of microprobes that have been placed in similar locations in the central nervous system during the same physiological stimulation. Control and experimental groups of microprobes can be compared, and a level of significance for the release of a neuropeptide obtained. In addition estimates can be made of the concentration of peptide present in regions of release.