Expression of tyrosine hydroxylase (TH) is regulated in a tissue-specific manner by multiple mechanisms. In catecholaminergic cells, the expression of TH-mRNA is up-regulated by forskolin (FK) and is ...suppressed by retinoic acid (RA). We have previously provided evidence that, in N-18 cells, the expression of TH-mRNA is suppressed by DNA methylation of the TH gene itself. In the present study, using a catecholaminergic cell line, N1E-115, we performed deletional and mutational analyses on the 5′-flanking region of the mouse TH gene. The results indicate that a cAMP response element (CRE) mediates constitutive transcription of the TH gene, as well as responsiveness to FK and RA. Using bisulfite sequencing methods, we analyzed the methylation status of the TH gene 5′-flanking region in various cell lines and rat tissues. We found that three cytosine residues in the domain surrounding the CRE of the TH gene promoter were specifically methylated in N-18 cells and TH non-expressing rat tissues. In contrast, these cytosines were undermethylated in TH expressing cell lines and tissues. The inverse correlation between the frequency of cytosine methylation at these specific sites and the levels of TH expression supports a role for DNA methylation in the regulation of tissue-specific gene expression.
Electrophysiological studies of dorsal root ganglion (DRG) neurons, and the results of PCR, Northern blot and in situ hybridization analyses have demonstrated the molecular diversity of Na+ channels ...that operate in sensory neurons. Several subtypes of alpha-subunit have been detected in DRG neurons and transcripts encoding all three beta-subunits are also present. Interestingly, one alpha subunit, Na(v)1.8, is selectively expressed in C-fibre and Adelta fibre associated sensory neurons that are predominantly involved in damage sensing. Another channel, Na(v).3, is selectively up regulated in a variety of models of neuropathic pain. In this review we focus on Na+ channels that are selectively expressed in DRG neurons as potential analgesic drug targets. In the absence of subtype specific inhibitors, the production of null mutant mice provides useful information on the specialized functions of particular Na+ channels. A refinement of this approach is to delete Na+ channel genes flanked by lox-P sites in the sensory ganglia of adult animals, using viruses to deliver the bacteriophage Cre recombinase enzyme.
Voltage-gated sodium channels initiate and propagate action potentials in excitable cells. The tetrodotoxin-resistant Na
+ channel (Na
V1.8/SNS) is expressed in damage-sensing neurons (nociceptors) ...and plays an important role in pain pathways. Expression of high levels of functional Na
V1.8 in heterologous cells has proved problematic, even in the presence of known sodium channel accessory β-subunits. This suggests that other regulatory proteins are required for normal levels of Na
V1.8 expression. Here we report the use of a yeast two-hybrid system and a rat dorsal root ganglion cDNA library to identify 28 different clones encoding proteins which interact with intracellular domains of Na
V1.8. Many clones are expressed at high levels in small diameter DRG neurons as judged by in situ hybridization. Interacting proteins include cytoplasmic elements and linker proteins (e.g. β-actin and moesin), enzymes (e.g. inositol polyphosphate 5-phosphatase and TAO2 thousand and one protein kinase), channels and membrane-associated proteins (voltage-dependent anion channel VDAC3V and tetraspanin), as well as motor proteins (dynein intermediate and light chain) and transcripts encoding previously undescribed proteins. Immunoprecipitation (pull-down) assays confirm that some of the proteins interact with, and may hence regulate, Na
V1.8 in vivo.
Potent analgesic effects of GDNF in neuropathic pain states. (King's College London, London, United Kingdom) Science 2000;290:124–127.
Neuropathic pain arises as a debilitating consequence of nerve ...injury. The etiology of such pain is poorly understood, and existing treatment is largely ineffective. We demonstrate here that glial cell line‐derived neurotrophic factor (GDNF) both prevented and reversed sensory abnormalities that developed in neuropathic pain models, without affecting pain‐related behavior in normal animals. GDNF reduces ectopic discharges within sensory neurons after nerve injury. This may arise as a consequence of the reversal by GDNF of the injury‐induced plasticity of several sodium channel subunits. Together these findings provide a rational basis for the use of GDNF as a therapeutic treatment for neuropathic pain states.
Comment by Marshall Devor, PhD.
Nerve injury frequently causes sensory neurons to become electrically hyperexcitable. The resulting ectopic afferent firing is thought to be the immediate cause of ongoing pain, movement‐evoked pain, and allodynia in neuropathic pain states. But why do the neurons become hyperexcitable? A popular hypothesis has it that when the axon is cut, the sensory cell body in the dorsal root ganglion (DRG) is starved of essential neurotrophic molecules that are normally produced in the periphery and transported to the DRG via axoplasmic transport. If so, then providing an exogenous source of the critical neurotrophin should prevent, or even reverse, the hyperexcitability and the pain. Boucher et al tried this, administering 1 of 3 candidate neurotrophins (NGF, NT‐3, or GDNF) by the intrathecal (i.t.) route. One of them, GDNF, worked dramatically. The authors believe that i.t. GDNF acts by diffusing into the DRG (rather than the spinal cord) where it alters the expression of the channel proteins that are responsible for electrical hyperexcitability.
Altered expression of voltage-gated sodium, calcium and potassium channels has been associated with neuropathic pain conditions. In addition, roles for the ligand-gated P2X3 and NMDA receptors, as ...well as pacemaker HCN channels have also been invoked in the pathogenesis of neuropathic pain. In this chapter, evidence of an important role for post-translational regulation of Nav1.9 in setting pain thresholds is presented. Despite the importance of tactile allodynia and mechanical hyperalgesia in chronic pain, we remain ignorant of the molecular nature of mechanosensors present in sensory neurons. A number of candidate mechanosensor genes, identified because of their structural similarity with mechanosensors in Caenorbabditis elegans and Drosophila melanogaster have been identified. Acid-sensing ion channels (ASICs) are structurally related to putative mechanosensors in C. elegans, whilst transient receptor potential channels (TRPs) have been implicated in mechanosensation in the Drosophila acoustic system. Evidence against a role for ASICs as primary transducers of mechanosensation is provided here, and recent evidence implicating TRP channels is reviewed. Finally, the use of sensory neuron-specific gene deletion approaches to unravel the significance of individual ion channels in the regulation of sensory neuron excitability and the induction of pain will be described.
The application is considered of adaptive robust estimation procedures to the problem of calibrating small-range measuring instruments. Results are reported from simulation of the adaptive method ...developed for verification of the given class of measuring instruments.PUBLICATION ABSTRACT
Hepatitis B virus (HBV) reactivation in patients with resolved HBV infection was found in 23 (4%) of 552 newly hepatitis B surface antigen-positive patients in Japan. Because one-fourth of cases ...develop into fulminant hepatic failure and mortality is 100%, management of HBV reactivation in patients with resolved HBV infection should be discussed.
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