The effects of inflammation and inflammatory mediators on nociceptive behaviour induced by ATP analogues in the rat.
(17/12422)
1. We have studied the behavioural effects of intraplantar injections of adenosine 5'-triphosphate (ATP) and related compounds in freely moving rats and investigated whether these nociceptive effects are augmented in the presence of inflammatory mediators. 2. We find that in normal animals ATP and analogues produce dose-dependent nocifensive behaviour (seen as bursts of elevation of the treated hindpaw), and localized thermal hyperalgesia. The rank order of potency was: alpha,beta-methyleneadenosine 5'-triphosphate (alpha,beta-methylene ATP) > 2-methylthioadenosine triphosphate (2-methylthio ATP) > ATP. After neonatal treatment with capsaicin, to destroy small calibre primary sensory neurones, nocifensive behaviour was largely absent. 3. The effects of ATP analogues were assessed in three models of peripheral sensitization: 2 h after dilute intraplantar carrageenan (0.25% w v(-1)); 24 h after irradiation of the hindpaw with ultraviolet (U.V.) B; immediately following prostaglandin E2 (PGE2) treatment. In all models the effect of alpha,beta-methylene ATP was greatly augmented. After carrageenan, significant hindpaw-lifting behaviour activity was induced by injection of only 0.05 nmol of alpha,beta-methylene ATP, some 100 times less than necessary in normal skin. 4. Our data suggest that it is much more likely that endogenous levels of ATP will reach levels capable of exciting nociceptors in inflamed versus normal skin. Our data also suggest the involvement of P2X3 receptor subunits in ATP-induced nociception. (+info)
End group of naturally terminated and UV lesion terminated T7 in vitro RNA.
(18/12422)
The 3' terminal nucleosides of RNA transcribed in vitro by E. coli RNA polymerase from T7 DNA and UV irradiated TN DNA were determined. The 3' terminal nucleoside of naturally terminated (t1 termination site) RNA cytidine. In the case of RNA terminated at UV lesions, it is cytidine in 0 per cent of the molecules and adenosine in the remaining 30 per cent. Cytidine trialcohols are labile in high concentrations of KOH and at high temperature and appear to convert to uridine. (+info)
UV-A-induced decrease in nuclear factor-kappaB activity in human keratinocytes.
(19/12422)
Previous reports have demonstrated an increase in nuclear factor-kappaB (NF-kappaB) activity in response to UV radiation. These studies have essentially focused on the DNA-damaging fraction of solar UV radiation (UV-B and UV-C). In contrast, the effects of UV-A radiation (320-400 nm) on NF-kappaB are not well known. In this study, we present evidence that UV-A radiation induces a marked decrease in NF-kappaB DNA-binding activity in NCTC 2544 human keratinocytes. In addition, NCTC 2544 keratinocytes pretreated with UV-A fail to respond to NF-kappaB inducers. Moreover, UV-A radiation induces a decrease in NF-kappaB-driven luciferase reporter gene expression in NCTC 2544 keratinocytes. The expression of the gene encoding IkappaBalpha (IkappaB is the NF-kappaB inhibitor), which is closely associated with NF-kappaB activity, is also reduced (3-fold) upon UV-A treatment. Our results indicate that the UV-A-induced decrease in NF-kappaB DNA-binding activity is associated with a decrease in the levels of the p50 and p65 protein subunits. This is the first evidence that an oxidative stress, such as UV-A radiation, may induce a specific decrease in NF-kappaB activity in mammalian cells, probably through degradation of NF-kappaB protein subunits. These findings suggest that UV-A could modulate the NF-kappaB-dependent gene expression. (+info)
Stress- and cell type-dependent regulation of transfected c-Jun N-terminal kinase and mitogen-activated protein kinase kinase isoforms.
(20/12422)
The cJun N-terminal kinases (JNKs) are encoded by three genes generating ten protein kinase polypeptides and are activated in settings of cell stress, mitogenesis, differentiation and morphogenesis. The specific role of the JNK family members in these diverse cell programmes is largely undefined. In this study, we tested the hypothesis that individual JNK isoforms would exhibit distinct patterns of regulation within cells. The cDNAs encoding five haemagglutinin (HA)-tagged JNK isoforms (p46JNK1alpha, p54JNK2alpha, p54JNK2beta, p46JNK3 and p54JNK3) were expressed in cultured rat PC12 phaeochromocytoma cells and human small-cell lung cancer (SCLC) cells by retrovirus-mediated gene transfer. In addition, HA-tagged forms of the dual-specificity mitogen-activated protein kinase kinases (MKKs), MKK4 and MKK7, which are specific activators of the JNK enzymes, were similarly expressed. Reverse transcription and PCR revealed that JNK3 is endogenously expressed in SCLC cells, but not in either chromaffin or neuronally differentiated PC12 cells. MKK4 and MKK7 were endogenously expressed in both PC12 cells and SHP77 cells. Immunoprecipitation and analysis of the JNKs expressed in SCLC cells revealed strong stimulation of all five JNK isoforms by UV radiation. Hypertonic stress, elicited by mannitol, also significantly stimulated these same JNKs, although the JNK3 isoforms were most strongly activated. In PC12 cell transfectants, however, selective and equal activation of p54JNK2alpha and p54JNK3 by UV and osmotic stress was observed, with little or no activation of JNK1alpha or JNK2beta. In contrast with the broad activation of the JNK enzymes by UV in SCLC cells, only HA-MKK4 was stimulated by UV exposure in these cells, whereas osmotic stress stimulated both HA-MKK4 and HA-MKK7. These findings indicate selective activation of JNK and MKK isoforms in a manner that is dependent upon the specific cell stress and the cell type. (+info)
Reverse genetic analysis of Caenorhabditis elegans presenilins reveals redundant but unequal roles for sel-12 and hop-1 in Notch-pathway signaling.
(21/12422)
Mutations in the human presenilin genes PS1 and PS2 cause early-onset Alzheimer's disease. Studies in Caenorhabditis elegans and in mice indicate that one function of presenilin genes is to facilitate Notch-pathway signaling. Notably, mutations in the C. elegans presenilin gene sel-12 reduce signaling through an activated version of the Notch receptor LIN-12. To investigate the function of a second C. elegans presenilin gene hop-1 and to examine possible genetic interactions between hop-1 and sel-12, we used a reverse genetic strategy to isolate deletion alleles of both loci. Animals bearing both hop-1 and sel-12 deletions displayed new phenotypes not observed in animals bearing either single deletion. These new phenotypes-germ-line proliferation defects, maternal-effect embryonic lethality, and somatic gonad defects-resemble those resulting from a reduction in signaling through the C. elegans Notch receptors GLP-1 and LIN-12. Thus SEL-12 and HOP-1 appear to function redundantly in promoting Notch-pathway signaling. Phenotypic analyses of hop-1 and sel-12 single and double mutant animals suggest that sel-12 provides more presenilin function than does hop-1. (+info)
Control of corynebacteriophage reproduction by heteroimmune repression.
(22/12422)
Corynebacteriophages beta and gamma are closely related but heteroimmune; hence, gamma reproduces in C7(beta). A series of gamma mutants, designated gamma-bin (beta-inhibited), has been isolated. They reproduce in only 2 to 14% of infected C7(beta) cells, and, as a result, plaque with an efficiency of 10(-4) to 10(-5) on this strain. The proportion of C7(beta) cells in which gamma-bin phage can replicate is increased to 30 to 80% when immunity is lifted by UV induction of C7(beta) or by heat induction of C7(beta-tsr3). The gamma-bin mutants carry out a normal vegetative or lysogenic cycle in strain C7 and thus do not appear to be defective in any essential phage function. Infection of C7(beta) by gamma-bin results in cell killing whether the infection is productive or nonproductive. The data support the hypothesis that inhibition of gamma-bin is due to the direct or indirect action of a beta prophage gene. The simplest hypothesis is that gamma-bin phages have sustained mutations in an operator site and that beta repressor now combines with the mutated operator to inhibit normal replication in a significant proportion of infected cells. (+info)
Regulation of JNK signaling by GSTp.
(23/12422)
Studies of low basal Jun N-terminal kinase (JNK) activity in non-stressed cells led us to identify a JNK inhibitor that was purified and identified as glutathione S-transferase Pi (GSTp) and was characterized as a JNK-associated protein. UV irradiation or H2O2 treatment caused GSTp oligomerization and dissociation of the GSTp-JNK complex, indicating that it is the monomeric form of GSTp that elicits JNK inhibition. Addition of purified GSTp to the Jun-JNK complex caused a dose-dependent inhibition of JNK activity. Conversely, immunodepleting GSTp from protein extracts attenuated JNK inhibition. Furthermore, JNK activity was increased in the presence of specific GSTp inhibitors and a GSTp-derived peptide. Forced expression of GSTp decreased MKK4 and JNK phosphorylation which coincided with decreased JNK activity, increased c-Jun ubiquitination and decreased c-Jun-mediated transcription. Co-transfection of MEKK1 and GSTp restored MKK4 phosphorylation but did not affect GSTp inhibition of JNK activity, suggesting that the effect of GSTp on JNK is independent of the MEKK1-MKK4 module. Mouse embryo fibroblasts from GSTp-null mice exhibited a high basal level of JNK activity that could be reduced by forced expression of GSTp cDNA. In demonstrating the relationships between GSTp expression and its association with JNK, our findings provide new insight into the regulation of stress kinases. (+info)
Oxidized derivatives of 7-dehydrocholesterol induce growth retardation in cultured rat embryos: a model for antenatal growth retardation in the Smith-Lemli-Opitz syndrome.
(24/12422)
7-Dehydrocholesterol accumulates in fetuses affected by the Smith-Lemli-Opitz syndrome as a result of a deficit in the ultimate step of cholesterol synthesis catalyzed by Delta7 reductase. Rat embryos explanted at gestation day 10 and cultured for 48 h in the presence of the Delta7 reductase inhibitor AY 9944 were used as a model to discriminate between the beneficial effect of supplementation with cholesterol and the deleterious effect of supplementation with 7-dehydrocholesterol. Cholesterol supplementation in the form of mixed cholesterol/lecithin liposomes added to serum serving as the culture medium restores the growth of embryos which is markedly decreased in the presence of the inhibitor. 7-Dehydrocholesterol under identical conditions does not restore growth and impairs the beneficial effect of cholesterol added simultaneously. UV-photooxidation of 7-dehydrocholesterol-supplemented culture medium enhances its embryotoxicity, which suggests uptake by the embryo of toxic by-products formed from 7-dehydrocholesterol. By contrast photooxidation of cholesterol-supplemented culture medium does not induce embryotoxicity. alpha-Tocopherol reduces the toxicity of photooxidized 7-dehydrocholesterol supplementing the culture medium. We conclude that 7-dehydrocholesterol does not fulfill the cholesterol requirement of the developing embryos and exerts an additional embryotoxic effect probably via oxidized by-products. This could explain the antenatal growth retardation of SLOS by a blockage of the maternal compensatory cholesterol influx. (+info)