DNA minor groove recognition by bis-benzimidazole analogues of Hoechst 33258: insights into structure-DNA affinity relationships assessed by fluorescence titration measurements.
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Fluorescence titration measurements have been used to examine the binding interaction of a number of analogues of the bis -benzimidazole DNA minor groove binding agent Hoechst 33258 with the decamer duplex d(GCAAATTTGC)2. The method of continuous variation in ligand concentration (Job plot analysis) reveals a 1:1 binding stoichiometry for all four analogues; binding constants are independent of drug concentration (in the range [ligand] = 0.1-5 microM). The four analogues studied were chosen in order to gain some insight into the relative importance of a number of key structural features for minor groove recognition, namely (i) steric bulk of the N -methylpiperazine ring, (ii) ligand hydrophobicity, (iii) isohelicity with the DNA minor groove and (iv) net ligand charge. This was achieved, first, by replacing the bulky, non-planar N -methylpiperazine ring with a less bulky planar charged imidazole ring permitting binding to a narrower groove, secondly, by linking the N -methylpiperazine ring to the phenyl end of the molecule to give the molecule a more linear, less isohelical conformation and, finally, by introducing a charged imidazole ring in place of the phenolic OH making it dicationic, enabling the contribution of the additional electrostatic interaction and extended conformation to be assessed. Delta G values were measured at 20 degrees C in the range -47.6 to -37.5 kJ mol-1 and at a number of pH values between 5.0 and 7.2. We find a very poor correlation between Delta G values determined by fluorescence titration and effects of ligand binding on DNA melting temperatures, concluding that isothermal titration methods provide the most reliable method of determining binding affinities. Our results indicate that the bulky N -methylpiperazine ring imparts a large favourable binding interaction, despite its apparent requirement for a wider minor groove, which others have suggested arises in a large part from the hydrophobic effect. The binding constant appears to be insensitive to the isohelical arrangement of the constituent rings which in these analogues gives the same register of hydrogen bonding interactions with the floor of the groove. (+info)
Topical all-trans retinoic acid augments ultraviolet radiation-induced increases in activated melanocyte numbers in mice.
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We have previously shown that daily application of 0.05% retinoic acid to the backs of lightly pigmented, hairless HRA:Skh-2 mice increases melanogenesis resulting from exposure to solar-simulated ultraviolet radiation. In this study we show that as early as 1 wk following commencement of treatment, there is a 2- fold increase in the number of epidermal 3,4-dihydroxyphenylalanine positive melanocytes in retinoic acid and ultraviolet radiation treated HRA:Skh-2 mice compared with mice that received ultraviolet radiation only. This increased to a 2.9-fold difference by 6 wk. Retinoic acid also augmented ultraviolet radiation-stimulated melanogenesis, with a 4-fold increase being observed after only 2 wk. These findings were also seen in C57BL mice. Ultraviolet radiation and retinoic acid needed to be applied to the same skin site for the augmentation in melanocyte activation to occur. Ultraviolet B rather than ultraviolet A was mainly responsible for melanogenesis and the retinoic acid primarily increased ultraviolet B-induced melanogenesis. Furthermore, retinoic acid on it's own, in the absence of ultraviolet radiation caused a small but statistically significant increase in 3,4-dihydroxyphenylalanine positive melanocyte numbers and melanogenesis. Thus topical retinoic acid is a potent modulator of melanocyte activation. Alone it is able to increase the number of activated epidermal melanocytes and make melanocytes more sensitive to activation by ultraviolet B. (+info)
Spectrum of p53 gene mutations suggests a possible role for ultraviolet radiation in the pathogenesis of advanced cutaneous lymphomas.
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There is evidence that the incidence of primary cutaneous lymphoma, like other forms of non-Hodgkin's lymphoma, is increasing, yet little is known of the pathogenetic events involved in this group of disorders. In this study we examine the frequency and spectrum of P53 gene mutations in a large series of primary cutaneous lymphomas, with particular emphasis on tumor stage mycosis fungoides, as it is in these cases that p53 overexpression has previously been reported. Sixty-six samples from 55 patients with primary cutaneous B cell and T cell lymphomas were analyzed for mutations in exons 5-9 of the P53 gene using polymerase chain reaction/single strand conformational polymorphism, and subsequent cloning and sequencing of genomic DNA. Fourteen separate P53 mutations were identified in blood, skin, and lymph node samples in 13 patients (24%). Twelve of 14 mutations occurred at dipyrimidine sites, eight resulting in C-->T transitions and one in a CC-->TT tandem base transition, a mutation spectrum strikingly similar to that reported in nonmelanoma skin cancer and characteristic of DNA damage caused by ultraviolet B radiation. In the subset of patients with mycosis fungoides, P53 mutations were identified in six of 17 patients with tumor-stage but in none of 12 patients with plaque-stage disease (Fisher's exact test p = 0.027). These data suggest a role for ultraviolet radiation in the pathogenesis of primary cutaneous lymphomas and a possible ultraviolet B-related step in the progression of mycosis fungoides from plaque to tumor-stage disease. (+info)
In vivo UVA-1 and UVB irradiation differentially perturbs the antigen-presenting function of human epidermal Langerhans cells.
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Ultraviolet B (UVB, 290-320 nm) radiation is known to suppress the immune function of epidermal Langerhans cells. We have recently described that in vitro UVB irradiation perturbs the antigen-presenting cell function of Langerhans cells by inhibiting their expression of functional B7 costimulatory molecules (B7-1, B7-2). The aim of this study was to determine wavelength-specific UV effects on Langerhans cells function in vivo, specifically UVB and UVA-1. To address this issue, volunteers were irradiated on the sun protected volar aspects of their forearms with 3 x minimal erythema dose of UVB (Philips TL-12) and UVA-1 (UVASUN 5000 Mutzhaas). Langerhans cells were isolated from suction blister roofs immediately following irradiation. Langerhans cells isolated from UVB- but not from UVA-1-irradiated skin failed to activate naive resting allogeneic T cells (mixed epidermal cell leukocyte reaction) or primed tetanus toxoid reactive autologous T cells. Langerhans cells isolated from sham-irradiated or UVA-1-irradiated skin strongly upregulated B7-2 molecules during short-term tissue culture. By contrast, Langerhans cells from UVB-irradiated skin did not upregulate B7-2 molecules. Furthermore, exogenous stimulation of the B7 pathway by anti-CD28 stimulatory monoclonal antibodies restored the capacity of UVB-irradiated Langerhans cells to activate both alloreactive and tetanus toxoid-reactive T cells, implying suppressed antigen-presenting cell activities and perturbed B7 expression of Langerhans cells isolated from UVB-irradiated skin are related. Those studies demonstrate that in vivo UVB, but not UVA-1, interferes with the activation-dependent upregulation of B7 molecules on Langerhans cells, which in turn is of functional relevance for the perturbed antigen-presenting cell function of Langerhans cells within UVB- but not UVA-1-irradiated skin. (+info)
Preferential release of 11-cis-retinol from retinal pigment epithelial cells in the presence of cellular retinaldehyde-binding protein.
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In photoreceptor cells of the retina, photoisomerization of 11-cis-retinal to all-trans-retinal triggers phototransduction. Regeneration of 11-cis-retinal proceeds via a complex set of reactions in photoreceptors and in adjacent retinal pigment epithelial cells where all-trans-retinol is isomerized to 11-cis-retinol. Our results show that isomerization in vitro only occurs in the presence of apo-cellular retinaldehyde-binding protein. This retinoid-binding protein may drive the reaction by mass action, overcoming the thermodynamically unfavorable isomerization. Furthermore, this 11-cis-retinol/11-cis-retinal-specific binding protein potently stimulates hydrolysis of endogenous 11-cis-retinyl esters but has no effect on hydrolysis of all-trans-retinyl esters. Apo-cellular retinaldehyde-binding protein probably exerts its effect by trapping the 11-cis-retinol product. When retinoid-depleted retinal pigment epithelial microsomes were preincubated with different amounts of all-trans-retinol to form all-trans-retinyl esters and then [3H]all-trans-retinol was added, as predicted, the specific radioactivity of [3H]all-trans-retinyl esters increased during subsequent reaction. However, the specific radioactivity of newly formed 11-cis-retinol stayed constant during the course of the reaction, and it was largely unaffected by expansion of the all-trans-retinyl ester pool during the preincubation. The absence of dilution establishes that most of the ester pool does not participate in isomerization, which in turn suggests that a retinoid intermediate other than all-trans-retinyl ester is on the isomerization reaction pathway. (+info)
Is arcA3 a possible mediator in the signal transduction pathway during agonist cell cycle arrest by salicylic acid and UV irradiation?
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Progression of BY-2 tobacco cells through the cell cycle was followed after treatments with ultra violet (UV) and salicylic acid (SA) used as a potent inhibitor of the octadecanoid pathway which can mediate response to UV irradiation. Cells in S phase were more sensitive than G0/G1 or G2 cells to UV irradiation. Although SA efficiently blocked cells in G0/G1 or G2, it did not block S phase synchronized cells. UV and SA applied simultaneously to cells in G0/G1 delayed the cell cycle progression more than each one separately. Therefore UV irradiation and SA act as agonists to arrest BY-2 cells at cell cycle entry. To further investigate the signalling pathway mediating UV response, we complemented a UV-sensitive Escherichia coli strain with a Nicotiana xanthi cDNA expression library. A cDNA (arcA3) whose coding sequence is identical to the 2,4-D induced arcA cDNA cloned by Ishida et al. (1993) was isolated. We show that arcA3 transcription is induced at cell cycle entry but not directly by the 2,4-D treatment. Moreover, arcA3 transcription is induced prior to the restriction point as shown with the CDK inhibitor roscovitine. The arcA3 transcription level is increased by UV irradiation but prevented by SA. Indeed, addition of SA prior to UV irradiation blocks the induction of arcA3 transcription. This suggests that arcA3 gene is modulated in both UV and SA responses, the SA effect preceding the UV step. Since arcA3 is 67% similar to RACK1 (functional homology), a rat intracellular receptor for protein kinase C, and possesses identical PKC fixation motifs, it is hypothesised that the arcA3 gene is involved in UV and SA cell cycle arrest. (+info)
Formate-induced inhibition of photoreceptor function in methanol intoxication.
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Formic acid is the toxic metabolite responsible for the retinal and optic nerve toxicity produced in methanol intoxication. Previous studies in our laboratory have documented formate-induced retinal dysfunction and histopathology in a rodent model of methanol intoxication. The present studies define the time and concentration dependence of formate-induced retinal toxicity in methanol-intoxicated rats. Retinal function was assessed 24, 48, and 72 h after the initial dose of methanol by flicker electroretinographic measurements. Retinal histopathology was assessed at the same time intervals. Rod- and cone-mediated electroretinogram (ERG) responses were attenuated in a formate concentration- and time-dependent manner, and both retinal sensitivity and maximal responsiveness to light were diminished. Attenuation of UV-cone-mediated responses was temporally delayed in comparison to the functional deficits observed in the 15 Hz/510 nm responses, which have a rod-mediated component and occurred at significantly higher formate concentrations. Both 15 Hz/510 nm and UV-cone-mediated ERG responses were undetectable by 72 h; however, if light intensity was increased, a retinal ERG response could be recorded, indicating that photoreceptor function was profoundly attenuated, but not abolished, under these intoxication conditions. Functional changes preceded structural alterations. Histopathological changes were most pronounced in the outer retina with evidence of inner segment swelling, photoreceptor mitochondrial disruption, and the appearance of fragmented photoreceptor nuclei in the outer nuclear layer. The nature of both the functional and structural alterations observed are consistent with formate-induced inhibition of mitochondrial energy production, resulting in photoreceptor dysfunction and pathology. (+info)
Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome.
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UV cross-linking analyses were performed in an attempt to determine cellular protein-viral RNA interactions with the 3' untranslated region (3' UTR) of the hepatitis C virus RNA genome. Two cellular proteins, with estimated molecular masses of 58 kDa (p58) and 35 kDa (p35), respectively, were found to specifically bind to the 3' UTR. The p58 protein was determined to be the polypyrimidine tract-binding protein. In addition to binding to the conserved 98 nucleotides (nt) of the 3' UTR, p58 also binds to the poly(U) tract of the 3' UTR. The p35 protein was found to interact only with the poly(U) tract of the 3' UTR. These conclusions are supported by the following findings: (1) p58, and not p35, binds to the 3' end conserved 98 nt, (2) both p58 and p35 bind to a 3' UTR RNA with a deletion of the conserved 98 nt, (3) the 98-nt deletion mutant 3' UTR competed out both p58 and p35 binding, (4) a poly(U) homopolymer competed out both p58 and p35 binding, (5) a 3' UTR RNA with deletion of the poly(U) tract competed out only p58 binding but not p35 binding, and (6) an RNA containing the variable region of the 3' UTR with a deletion of both poly(U) tract and 98 nt failed to compete for binding of either p58 or p35. Interaction of these cellular proteins with the HCV 3' UTR is probably involved in regulation of translation and/or replication of the HCV RNA genome. (+info)