A visceral pain pathway in the dorsal column of the spinal cord

A limited midline myelotomy at T10 can relieve pelvic cancer pain in patients. This observation is explainable in light of strong evidence in support of the existence of a visceral pain pathway that ascends in the dorsal column (DC) of the spinal cord. In rats and monkeys, responses of neurons in the ventral posterolateral thalamic nucleus to noxious colorectal distention are dramatically reduced after a lesion of the DC at T10, but not by interruption of the spinothalamic tract. Blockade of transmission of visceral nociceptive signals through the rat sacral cord by microdialysis administration of morphine or 6-cyano-7-nitroquinoxaline-2,3-dione shows that postsynaptic DC neurons in the sacral cord transmit visceral nociceptive signals to the gracile nucleus. Retrograde tracing studies in rats demonstrate a concentration of postsynaptic DC neurons in the central gray matter of the L6-S1 spinal segments, and anterograde tracing studies show that labeled axons ascend from this region to the gracile nucleus. A similar projection from the midthoracic spinal cord ends in the gracile and cuneate nuclei. Behavioral experiments demonstrate that DC lesions reduce the nocifensive responses produced by noxious stimulation of the pancreas and duodenum, as well as the electrophysiological responses of ventral posterolateral neurons to these stimuli. Repeated regional blood volume measurements were made in the thalamus and other brain structures in anesthetized monkeys in response to colorectal distention by functional MRI. Sham surgery did not reduce the regional blood volume changes, whereas the changes were eliminated by a DC lesion at T10.

Pain perception: Is there a role for primary somatosensory cortex?

Anatomical, physiological, and lesion data implicate multiple cortical regions in the complex experience of pain. These regions include primary and secondary somatosensory cortices, anterior cingulate cortex, insular cortex, and regions of the frontal cortex. Nevertheless, the role of different cortical areas in pain processing is controversial, particularly that of primary somatosensory cortex (S1). Human brain-imaging studies do not consistently reveal pain-related activation of S1, and older studies of cortical lesions and cortical stimulation in humans did not uncover a clear role of S1 in the pain experience. Whereas studies from a number of laboratories show that S1 is activated during the presentation of noxious stimuli as well as in association with some pathological pain states, others do not report such activation. Several factors may contribute to the different results among studies. First, we have evidence demonstrating that S1 activation is highly modulated by cognitive factors that alter pain perception, including attention and previous experience. Second, the precise somatotopic organization of S1 may lead to small focal activations, which are degraded by sulcal anatomical variability when averaging data across subjects. Third, the probable mixed excitatory and inhibitory effects of nociceptive input to S1 could be disparately represented in different experimental paradigms. Finally, statistical considerations are important in interpreting negative findings in S1. We conclude that, when these factors are taken into account, the bulk of the evidence now strongly supports a prominent and highly modulated role for S1 cortex in the sensory aspects of pain, including localization and discrimination of pain intensity.

The genetic mediation of individual differences in sensitivity to pain and its inhibition

The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a combination of “top-down” and “bottom-up” strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents.

Structural basis for fibroblast growth factor receptor 2 activation in Apert syndrome

Apert syndrome (AS) is characterized by craniosynostosis (premature fusion of cranial sutures) and severe syndactyly of the hands and feet. Two activating mutations, Ser-252 → Trp and Pro-253 → Arg, in fibroblast growth factor receptor 2 (FGFR2) account for nearly all known cases of AS. To elucidate the mechanism by which these substitutions cause AS, we determined the crystal structures of these two FGFR2 mutants in complex with fibroblast growth factor 2 (FGF2) . These structures demonstrate that both mutations introduce additional interactions between FGFR2 and FGF2, thereby augmenting FGFR2–FGF2 affinity. Moreover, based on these structures and sequence alignment of the FGF family, we propose that the Pro-253 → Arg mutation will indiscriminately increase the affinity of FGFR2 toward any FGF. In contrast, the Ser-252 → Trp mutation will selectively enhance the affinity of FGFR2 toward a limited subset of FGFs. These predictions are consistent with previous biochemical data describing the effects of AS mutations on FGF binding. Alterations in FGFR2 ligand affinity and specificity may allow inappropriate autocrine or paracrine activation of FGFR2. Furthermore, the distinct gain-of-function interactions observed in each crystal structure provide a model to explain the phenotypic variability among AS patients.

Transcription factor TFIIH and DNA endonuclease Rad2 constitute yeast nucleotide excision repair factor 3: implications for nucleotide excision repair and Cockayne syndrome.

Nucleotide excision repair (NER) of ultraviolet light-damaged DNA in eukaryotes requires a large number of highly conserved protein factors. Recent studies in yeast have suggested that NER involves the action of distinct protein subassemblies at the damage site rather than the placement there of a "preformed repairosome" containing all the essential NER factors. Neither of the two endonucleases, Rad1-Rad10 and Rad2, required for dual incision, shows any affinity for ultraviolet-damaged DNA. Rad1-Rad10 forms a ternary complex with the DNA damage recognition protein Rad14, providing a means for targeting this nuclease to the damage site. It has remained unclear how the Rad2 nuclease is targeted to the DNA damage site and why mutations in the human RAD2 counterpart, XPG, result in Cockayne syndrome. Here we examine whether Rad2 is part of a higher order subassembly. Interestingly, we find copurification of Rad2 protein with TFIIH, such that TFIIH purified from a strain that overexpresses Rad2 contains a stoichiometric amount of Rad2. By several independent criteria, we establish that Rad2 is tightly associated with TFIIH, exhibiting an apparent dissociation constant < 3.3 x 10(-9) M. These results identify a novel subassembly consisting of TFIIH and Rad2, which we have designated as nucleotide excision repair factor 3. Association with TFIIH provides a means of targeting Rad2 to the damage site, where its endonuclease activity would mediate the 3' incision. Our findings are important for understanding the manner of assembly of the NER machinery and they have implications for Cockayne syndrome.

A monoclonal IgG anticardiolipin antibody from a patient with the antiphospholipid syndrome is thrombogenic in mice.

Antiphospholipid antibodies, including anticardiolipin antibodies (ACA), are strongly associated with recurrent thrombosis in patients with the antiphospholipid syndrome (APS). To date, reports about the binding specificities of ACA and their role(s) in causing and/or sustaining thrombosis in APS are conflicting and controversial. The plasmas of patients with APS, usually containing a mixture of autoantibodies, vary in binding specificity for different phospholipids/cofactors and vary in in vitro lupus anticoagulant activity. Although in vivo assays that allow assessment of the pathogenic procoagulant activity of patient autoantibodies have recently been developed, the complex nature of the mixed species prevented determination of the particular species responsible for in vivo thrombosis. We have generated two human IgG monoclonal ACA from an APS patient with recurrent thrombosis. Both bound to cardiolipin in the presence of 10% bovine serum, but not in its absence, and both were reactive against phosphatidic acid, but were nonreactive against purified human beta-2 glycoprotein 1, DNA, heparan sulfate, or four other test antigens. Both monoclonal autoantibodies lacked lupus anticoagulant activity and did not inhibit prothrombinase activity. Remarkably, one of the monoclonal antibodies has thrombogenic properties when tested in an in vivo mouse model. This finding provides the first direct evidence that a particular antiphospholipid antibody specificity may contribute to in vivo thrombosis.

Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.

Gene targeting approaches to complex genetic diseases: atherosclerosis and essential hypertension.

Gene targeting allows precise, predetermined changes to be made in a chosen gene in the mouse genome. To date, targeting has been used most often for generation of animals completely lacking the product of a gene of interest. The resulting "knockout" mice have confirmed some hypotheses, have upset others, but have rarely been uninformative. Models of several human genetic diseases have been produced by targeting--including Gaucher disease, cystic fibrosis, and the fragile X syndrome. These diseases are primarily determined by defects in single genes, and their modes of inheritance are well understood. When the disease under study has a complex etiology with multiple genetic and environmental components, the generation of animal models becomes more difficult but no less valuable. The problems associated with dissecting out the individual genetic factors also increases substantially and the distinction between causation and correlation is often difficult. To prove causation in a complex system requires rigorous adherence to the principle that the experiments must allow detection of the effects of changing only a single variable at one time. Gene targeting experiments, when properly designed, can test the effects of a precise genetic change completely free from the effects of differences in any other genes (linked or unlinked to the test gene). They therefore allow proofs of causation.