bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Heat‐Shock Protein 70 and P53 Proteins in Attenuation of UVC‐lnduced Apoptosis by Thermal Stress in Hepatocellular Carcinoma Cells
Authors: Chen Yen‐Chou , Lin‐Shiau Shoei‐Yn , Lin Jen‐Kun
Published: January 2008

Abstract— Induction of apoptosis is a function of external stimuli and cellular gene expression. Many cells respond to DNA damage by the induction of apoptosis, which depends on a functional p53 protein and is signaled by elevation of p53 levels. In this study, we found that a prior exposure to mild stress (42A°C) can protect HepG2 (p53+/+) ceUs from a subsequent UVC‐induced apoptosis determined by DNA fragmentation and ratio of sub‐Gl peak, but no heat‐enhanced protection was found in Hep3B (p53‐/‐) cells. Although a similar inductive pattern of HSP70 protein and mRNA was detected in the two cell lines under thermal stress, the effect of thermal stress on UVC‐induced apoptosis in HepG2 and Hep3B cells was obviously different. Overexpression of HSP70 by transient trans‐fection of HSP70 expression vector in HepG2 cells significantly inhibited UVC‐induced cell death; however, this inhibitory effect did not occur in transfected‐Hep3B cells. Treatment of HepG2 cells with p53‐specific anti‐sense oligonucleotide could effectively block the antiapo‐ptotic effect of thermal stress on UVC‐induced apoptosis and increase of intracellular wild‐type p53 protein by transfecting wtp53 expression plasmid into Hep3B cells yielded more resistance to UVC irradiation after prior thermal stress exposure. The results reveal an involvement of p53 in the antiapoptotic effect of thermal stress on UVC irradiation. Finally, a p53 protein increase was detected in UVC‐treated HepG2 cells and could be coim‐munoprecipitated with HSP70 after a thermal stress treatment Prolonged p53 binding activity and enhanced expression of p53‐controlled genes such as Gl arrest and DNA damage 45 and wild‐type p53 activation factor 1/ Cdk‐interacting protein 1 by thermal stress are also observed in UVC‐irradiated HepG2 cells. Based on these results, we propose that the antiapoptotic effect of thermal stress is mediated by increasing HSP70 and modulating intracellular p53 function.
Imaging of Spontaneous Canine Mammary Tumors Using Fluorescent Contrast Agents
Authors: Reynolds Jeffery S. , Troy Tamara L. , Mayer Ralf H. , Thompson Alan B. , Waters David J. , Cornell Karen K. , Snyder Paul W. , Sevick‐Muraca Eva M.
Published: January 2008

Abstract— We present near‐infrared frequency‐domain photon migration imaging for the lifetime sensitive detection and localization of exogenous fluorescent contrast agents within tissue‐simulating phantoms and actual tissues. We employ intensity‐modulated excitation light that is expanded and delivered to the surface of a tissue or tissue‐simulating phantom. The intensity‐modulated fluorescence generated from within the volume propagates to the surface and is collected using a gain‐modulated image‐intensified charge‐coupled device camera. From the spatial values of modulation amplitude and phase of the detected fluorescent light, micromolar volumes of dieth‐ylthiatricarbocyanine iodide (π= 1.17 ns) and indocyanine green (ICG) (π= 0.58 ns) embedded 1.0 cm deep in a tissue phantom are localized and discriminated on the basis of their lifetime differences. To demonstrate the utility of frequency‐domain fluorescent measurements for imaging disease, we image the fluorescence emitted from the surface of in vivo and ex vivo canine mammary gland tissues containing lesions with preferential uptake of ICG. Pathology confirms the ability to detect spontaneous mammary tumors and regional lymph nodes amidst normal mammary tissue and fat as deep as 1.5 cm from the tissue surface.
Estimation of the Annual Solar UVR Exposure Dose of Infants and Small Children in Tropical Queensland, Australia
Authors: Moise A. F. , Gies H. P. , Harrison S. L.
Published: January 2008

Abstract— The solar ultraviolet radiation (UVR) exposure of infants and small children was measured for 1 week using UVR‐sensitive polysulfone film attached to the shoulder and chest of the subjects. For the infant study, shoulder and chest badges received similar exposures, while the 2%‐year‐olds received higher exposures on the shoulder than on the chest. Also, the 2Vi‐year‐olds generally received higher exposures than the infants. The median total daily exposures for both groups were 39 and 92 J/m2. The maximum total daily exposures measured were 640 J/m2 (chest) and 240 J/m2 (shoulder) for the infants and 2060 J/m2 (shoulder) and 840 J/m2 (chest) for the 2% year‐olds. Using this exposure data, monthly and annual exposure doses were calculated for both groups and compared to similar data from the UK. The annual exposure dose for infants is 8.4 kj/m2 or 84 standard erythemal dose (SED) for both shoulder and chest. The annual exposure dose for 2V2 year‐old children is 39.4 kj/m2 or 394 SED for the shoulder and 28.8 kj/m2 or 288 SED for the chest. Apart from the generally higher annual exposure doses experienced by the infants and 2% year‐old children in Townsville, the main difference to the UK is the almost nonexistent drop in monthly exposure doses between summer and winter in Townsville compared to the UK. In the UK, the winter‐month exposure dose is only 0.5% of the summer‐month dose. However, in Townsville it is around 40%.
Delivery of 9‐Cis Retinal to Photoreceptors from Bovine Serum Albumin
Authors: Li Zhongzeng , Zhuang Jianling , Corson D. Wesley
Published: January 2008

Abstract— Retinoids are labile compounds with less than micro‐molar solubility in aqueous solutions. They are normally associated with a carrier protein, interphotoreceptor retinoid binding protein, in the interstitial spaces of the retina. In the past, experimental retinoids have been delivered to isolated retinas and photoreceptors using either unprotected ethanolic suspensions or phospholipid vesicles that protect but require burdensome preparation. The objective of these experiments was to characterize the protection of retinoids and their delivery to isolated photoreceptor cells at high concentrations using fatty‐acid‐free bovine serum albumin (BSA) as a convenient vehicle and protectant. Our results confirm that BSA is a useful protectant and vehicle for delivery of retinoids to isolated retinas and photoreceptors.
Amplified Degradation of Photosystem II D1 and D2 Proteins under a Mixture of Photosynthetically Active Radiation and UVB Radiation: Dependence on Redox Status of Photosystem II
Authors: Babu Tripuranthakam S. , Jansen Marcel A. K. , Greenberg Bruce M. , Gaba Victor , Malkin Shmuel , Mattoo Autar K. , Edelman Marvin
Published: January 2008

Abstract—Plants exposed to a mixture of photosynthetically active radiation (PAR) and UVB radiation exhibit a marked boost in degradation of the D1 and D2 photosysteni II (PS II) reaction center proteins beyond that predicted by the sum of rates in PAR and UVB alone (amplified degradation). Becausee degradation driven by visible or UVB radiation alone is uncoupled from PS II redox status, it was therefore assumed that the mixed‐light‐amplified component of degradation would behave similarly. Surprisingly, amplified degradation proved to be coupled tightly to the redox status of PS II. We show that inactivation of the PS II water oxidation by heat shock or oxidation of the plastosemiquinone (QA‐) by silicomolybdate nullifies only the amplified component of degradation but not the basic rates of degradation under PAR or UVB alone. The data are interpreted to indicate that formation of plastosemiquinone or an active water‐oxidizing Mn4cluster, is the UVB chromophore involved in amplified degradation of the D1 and D2 proteins. Furthermore, accumulation of QA‐by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea or 2‐bromo‐3‐methyl‐6‐isopropyl‐4‐nitrophenol stimulated the mixed‐light‐amplified degradation component. Thus, amplified degradation of the D1 and D2 proteins in mixed radiance of PAR plus UVB (which simulates naturally occurring radiance) proceeds by a mechanism clearly distinct from that involved in degradation under PAR or UVB alone.
Photoregulation of DMA Photolyases in Broom Sorghum Seedlings
Authors: Hada Megumi , Buchholz Günther , Hashimoto Tohru , Nikaido Osamu , Wellmann Eckard
Published: January 2008

Abstract— Using dark‐grown 3 day‐old Sorghum bicolor seedlings, photorepair of cyclobutane pyrimidine dimers (CPD) by CPD‐photoIyase and pyrimidine‐(6–4)pyrimidinone photoproducts ([6–4]PP) by [6–4]PP‐photolyase was studied in vivo and in vitro. The two types of DNA photoproducts were measured with specific monoclonal antibodies. Sorghum seedlings have different photorepair enzymes for CPD and [6–4]PP. The CPD‐photolyase was found to be increased in response to light with maximal efficiency in the UVA/blue spectral range, whereas [6–4]PP‐photolyase was not influenced by light.
Photoproduct Formation from a Zinc Benzochlorin Iminium Salt Detected by Fluorescence Microscopy
Authors: Amit Ifat , Malik Zwi , Kessel David
Published: January 2008

Abstract— When examined by fluorescence microscopy, tumor cells loaded with a zinc benzochlorin iminium salt showed a very faint deep‐red fluorescence that was rapidly transformed to a substantially brighter red‐orange fluorescence. Fourier transfer spectroscopy analysis with a red‐sensitive detection system revealed that an initial fluorescence at 770 nm was gradually converted to 640 nm fluorescence during excitation. Image analysis showed that photoproduct formation was accompanied by a change in the site of drug localization from the cytoplasm to the nucleus. These studies illustrate the power of interferometry for the characterization of photoproducts and changes in sensitizer localization during photoproduct formation.
Effects of Calmodulin Inhibitors and Blue Light on Rhythmic Movement of Robinia pseudoacacia Leaflets
Authors: Gómez Luis Angel , Moysset Luisa , Simón Esther
Published: January 2008

Abstract— The effect of several calmodulin (CAM) antagonists, blue light and an intracellular calcium inhibitor, on the circadian rhythm of Robinia pseudoacacia leaflet movement has been studied. The CAM antagonists, chlorpromazine (CPZ), trifluoperazine (TFP), calmidazolium and N‐(6‐aminohexyI)‐5‐chloro‐1‐naphthalenesulfonamide (W7) shifted the phase of the circadian rhythmic movement while W5, an inactive analogue of W7, had no effect. Two hour pulses of calmidazolium (10–50 μM) gave rise to a phase‐response curve with maximum advances (up to 9 h) at circadian time (CT) 6 and maximum delays (up to 7 h) at CT 22. No effect was found on transition from subjective day to subjective night and vice versa. The TFP (10–50 μM), applied as 2 h pulses during the circadian cycle, shifted the phase of the circadian leaflet movement and also produced maximum advances in the middle of subjective day. Two hour blue light pulses shifted the phase of leaflet rhythmic movement. The phase‐response curve obtained showed maximum advances (up to 5 h) in the middle of subjective day and maximum delays on transition from subjective day to subjective night. Two hour pulses of 50 μM 8‐(diethylamino)octyl 3,4,5‐trimethoxybenzoate hypochloride (TMB‐8), an intracellular calcium inhibitor, caused the same type of phase‐response curve, with maximum advances and delays occurring at the same time as those produced by blue light. These results indicate that CAM might be involved in controlling the circadian oscillator that drives Robinia leaflet movement. The relationship between CAM and calcium with red and blue light is discussed.
Development and Investigation of a Novel Oxidative Pyrimidine Dimer Splitting Model*
Authors: Huntley James J. A. , Nieman Ronald A. , Rose Seth D.
Published: January 2008

Abstract— Described is the development of a novel pyrimidine dimer splitting system designed for the continued investigation of the mechanisms and intermediates involved in pyrimidine dimer repair. This system consists of the cationic photopolymerization initiator, N‐ethoxyisoquinolinium hexafluorophosphate and the pyrimidine dimers cissyn‐ and transsyn‐N, N'‐dimethylthymine dimer. Irradiation of the initiator at wavelengths above 300 nm generated a radical cation formed by homolytic N‐O bond cleavage. This reactive species is thought to oxidize a dimer, which resulted in splitting. Additional studies performed demonstrate that splitting cannot be attributed to photoinduced electron transfer reactions involving dimer and excited state initiator or dimer and the excited state of initiator decomposition products. The results suggest that splitting is due to a one‐electron oxidation of dimer by the radical cation of isoquinoline. This splitting mechanism is different from previous oxidative pyrimidine dimer splitting pathways in that electron transfer occurs without concomitant generation of a sensitizer‐derived radical anion, which alters the nature of the back electron transfer step. The concentration dependence of the quantum yields of splitting reflected the differences in reactivity of the stereoisomeric dimers. This novel system may facilitate spectroscopic studies aimed at detection of dimer‐derived intermediates in the splitting reaction.
Time‐Resolved Electron Paramagnetic Resonance Study of Photoionization of Tyrosine Anion in Aqueous Solution
Authors: Clancy Christine M. R. , Forbes Malcolm D. E.
Published: January 2008

Abstract— Photoionization of the amino acid tyrosine in basic water was studied by time‐resolved electron paramagnetic resonance (TREPR) at X‐band (9.5 GHz). Photoionization of deprotonated tyrosine leads to a spin‐polarized emissive/absorptive chemically induced dynamic electron polarization (CIDEP) spectrum produced by the radical pair mechanism, with the tyrosyl radical in emission and the solvated electron in absorption, which implies a triplet precursor. The exchange interaction, J, is found to be negative for this radical pair. The triplet photoionization channel is determined to be monophotonic. The singlet channel of photoionization of deprotonated tyrosine is seen only upon addition of the electron acceptor 2‐bro‐mo‐2‐methylpropionic acid (BMPA) to the sample. The singlet channel is isolated by performing TREPR on a sample containing tyrosine, BMPA and a triplet quencher (2,4‐hexadienoic acid). This channel is also found to be monophotonic.
Photophysical Studies of A2‐E, Putative Precursor of Lipofuscin, in Human Retinal Pigment Epithelial Cells
Authors: Cubeddu Rinaldo , Taroni Paola , Hu Dan‐Ning , Sakai Naomi , Nakanishi Koji , Roberts Joan E.
Published: January 2008

Abstract. With age, human retinal pigment epithelial cells accumulate lipofuscin that can absorb photons in the visible range leading to light‐induced damage and impaired vision. A putative precursor of lipofuscin, 2‐[2,6‐dimethyl‐8‐(2,6,6‐trimethyl‐l‐cyclohexen‐l‐yl)‐lE,3E:,5E,7E‐octa‐tetraenyl]‐l‐(2‐hydroxyethyl)‐4‐[4‐methyl‐6‐(2,6,6‐tri‐methyl‐1 ‐cyclohexen‐1 ‐y 1)‐ IE, 3E, 5E‐hexatrienyl] ‐pyridi‐nium (A2‐E), has recently been isolated and characterized from aged human retinal pigment epithelial cells. We have found that A2‐E inhibits the growth of human retinal pigment epithelial cells at concentrations greater than 1 μM. Time‐resolved fluorescence measurements of 1 μM A2‐E in solution, performed under 413 nm excitation, showed that fluorescence wave forms integrated across the spectrum (450–600 nm) were best‐fitted with three decay times in the nanosecond and subnanosecond time scale: 6.6,1.9 and 0.33 ns. Untreated retinal pigment epithelial cells were characterized by three fluorescence lifetimes: 6.3, 1.7 and 0.35 ns. In retinal pigment epithelial cells treated with 1 μM A2‐E, the fluorescence decay was significantly faster, with the marked presence (30%) of a fourth short lifetime (0.12 ns). These fluorescence decay times for A2‐E bound to human retinal pigment epithelial cells are similar to those of lipofuscin granules isolated from aged human retinal pigment epithelial cells. This similarity supports the hypothesis that A2‐E is a precursor of lipofuscin and suggests that A2‐E may play a role in the overall light damage associated with age‐related retinal diseases.
The Soluble Eumelanin Precursor 5,6‐Dihydroxyindole‐2‐carboxylic Acid Enhances Oxidative Damage in Human Keratinocyte DNA after UVA Irradiation‡
Authors: Kipp Christian , Young Antony R
Published: January 2008

Abstract.Soluble melanin precursors are present in serum and may act as skin chromophores contributing to UVR‐in‐duced oxidative damage. Our study aimed to determine whether the soluble eumelanin precursor 5,6‐dihydroxy‐indole‐2‐carboxylic acid (DHICA) photosensitizes DNA damage in human keratinocytes exposed to UVA irradiation. The HaCaT keratinocytes were incubated with and without DHICA, before irradiation with broadband UVA (320‐400 nm). The DNA photodamage was assessed using the comet assay that detects frank single‐strand breaks (SSB) and specific oxidative lesions with the addition of endonuclease III. Without DHICA incubation, there was no significant increase in SSB, compared to unirradiated cells, for doses up to 48.5 J/cm2(<1 minimum erythemal dose). Preincubation with 0.5 μM DHICA caused an increase in SSB at every UVA dose (significant from 12.1 to 48.5 J/cm2), while varying the DHICA concentration (0.125‐2 μM)showed this effect to be concentration dependent such that SSB increased and endonuclease III‐sensitive sites decreased with increasing DHICA concentration. The irradiation of cells in the presence of antioxidants (catalase, mannitol and histidine) suggests that DHICA‐induced photosensitization is mediatedviasinglet oxygen and, to a lesser extent, hydroxyl radicals. These results indicate that DHICA can induce strand breaks with UVA at clinically relevant dosesviaa mechanism involving reactive oxygen species.
Early Induction of Binucleated Cells by Ultraviolet A (UVA) Radiation: A Possible Role of Microfilaments‡
Authors: Bånrud Hilde , Moan Johan , Berg Kristian
Published: January 2008

The effects of UVA (365 nm) radiation on the cellular distribution of F‐actin and formation of binucleated cells have been studied using 3T3 Swiss albino mouse fibroblasts and V79 Chinese hamster fibroblasts. Ultraviolet A at biologically relevant fluences was found to disintegrate the actin filaments in the cells shortly (5 min) after irradiation, concomitant with the formation of cells with two nuclei. In 76–100% of the bi‐ and multinucleated cells the distribution of F‐actin was clearly altered. Cells in G| phase of the cell cycle were most probably involved in the formation of binucleated cells. The disintegration of F‐actin was presumably not due to depolymerization of F‐actin to G‐actin, as the amount of F‐actin in the cells was unaltered after UVA exposure but rather due to direct breakage of the actin filaments. Ultraviolet B (297/ 302 nm) had no effect on the cellular distribution of microfilaments, not even at highly lethal fluences.
Chronic Photodamage in Skin of Mast Cell‐deficient Mice‡
Authors: Gonzalez Salvador , Moran Michael , Kochevar Irene E.
Published: January 2008

Abstract. Solar elastosis is a hallmark of photoaged human skin and a prominent feature in experimentally produced photoaging in murine skin. The products of mast cells have been implicated in the development of photoaged skin. We evaluated whether products from mast cells mediate chronic UVB‐induced changes in murine skin by employing a strain of mast cell‐deficient mice, WWv. The responses in these mice were compared to those in BALB/c, another albino mouse strain. Mice were exposed three times per week to UVB radiation for 11 weeks; the total dose was 18.8 J/cm2. Irradiated WWv mice showed greater epidermal alterations than the irradiated BALB/c mice. In the dermis, a 3.6‐fold increase in elastin content, as measured by desmosine, was produced in the UVB‐treated BALB/c mice; in contrast, no difference was observed in elastin between UVB‐treated and untreated WWv mice. Collagen content was not increased by UVB treatment in either strain, and the glycosaminoglycan content increased a similar amount in UVB‐treated mice in both strains. The number of mast cells increased twofold and the number of neutrophils increased six‐fold in UVB‐treated BALB/c mice compared to age‐matched unirradiated controls. Neutrophils, as well as mast cells, were absent in untreated and UVB‐treated WWv mouse skin. These results suggest that products of mast cells are important in the development of solar elastosis in murine skin either by directly inducing elastin production by fibroblasts or indirectly by mediating the presence of other cell types that produce products that increase fibroblast elastin production.
Molecular Mechanisms of Photosensitization Induced by Drugs XII. Photochemistry and Photosensitization of Rufloxacin: An Unusual Photodegradation Path for the Antibacterials Containing a Fluoroquinolone‐like Chromophore
Authors: Condorelli G. , Guidi G. De , Giuffrida S. , Sortino S. , Chillemi R. , Sciuto S.
Published: January 2008

Abstract The UVA irradiation of 9‐fluoro‐2,3‐dihydro‐10‐4′‐meth‐yl‐l'‐piperazinyl‐7‐oxo‐7H‐pyrido[l,2,3‐de]‐l,4‐benzo‐thiazine‐6‐carboxylic acid, rufloxacin, a fluoroquinolone antibacterial that shows photosensitizing properties toward biological substrates, leads to formation of two main steady photoproducts characterized by a decarboxylation process and an opening of the piperazinyl ring, respectively. The deprotonation of the 10‐piperazinyl group and the dissociation of the 6‐carboxyl group of rufloxacin are strictly pH dependent. The photosensitizing activity was tested toward membranes as biological targets. Red blood cell hemolysis and lipid peroxidation were considered as markers of photosensitization. Ultraviolet A‐induced damage is strongly influenced by the presence of oxygen, it is triggered by transient species, such as singlet oxygen and free radicals, photogenerated via rufloxacin irradiation, whereas no drug photoproduct is involved in the photosensitization process.
Laser Flash Photolysis Studies of the UVA Sunscreen Mexoryl SX
Authors: Cantrell Ann , McGarvey David J. , Mulroy Louise , Truscott T. George
Published: January 2008

Abstract The results of a nanosecond laser flash photolysis investigation of the UVA sunscreen Mexoryl* SX in various solvent environments and within a commercial sunscreen formulation are reported. To the best of our knowledge this is the first laser flash photolysis study of a commercial suncare formulation. In each of these environments kinetic UV‐visible absorption measurements following nanosecond 355 nm laser excitation reveals a short‐lived species with a solvent‐dependent absorption maximum around 470–500 nm and a solvent‐dependent lifetime of ?50–120 ns. This transient absorption is attributed to the triplet state of Mexoryl* SX on the basis that it is quenched by molecular oxygen leading to the formation of singlet oxygen in acetonitrile. The singlet oxygen quantum yield (φΔ), determined by comparative time‐resolved near‐infrared luminescence measurements and extrapolated to the limit of complete triplet state quenching, is estimated as 0.09 ± 0.03 in acetonitrile. In aqueous solution the shorter triplet state lifetime combined with lower ambient oxygen concentrations precludes significant triplet state quenching. For the commercial sunscreen formulation there was no observable difference in the measured triplet lifetime between samples exposed to oxygen or argon, suggesting that the singlet oxygen quantum yield in such environments is likely to be orders of magnitude lower than that measured in acetonitrile.
Nitrate and Nitrite Ultraviolet Actinometers
Authors: Jankowski Joseph J. , Kieber David J. , Mopper Kenneth
Published: January 2008

Abstract We developed nitrate and nitrite actinometers to determine radiant fluxes from 290 to 410 nm. These actinometers are based on the reaction of the photochemically generated OH radical with benzoic acid to form salicylic acid (SA) and p‐hydroxybenzoic acid (pHBA). Actinom‐eter development included determination of the temperature and wavelength dependence of the quantum yield for formation of SA and pHBA from nitrate and nitrite photolysis in air‐saturated solutions. Quantum yields (at 25°C) for SA production from nitrate photolysis ranged from 0.00146 to 0.00418 between 290 and 350 nm, and from 0.00185 to 0.00633 for nitrite photolysis between 290 and 405 nm. The quantum yields for SA production were approximately 50–60% greater than quantum yields for pHBA production from nitrate and nitrite photolysis. For both actinometers, SA and pHBA formation was temperature dependent, increasing by approximately a factor of 2.2 from 0 to 35°C. Activation energies for SA formation varied with wavelength, ranging from 14.7 to 16.5 kj mol ‐1 between 290 and 330 nm for the nitrate actinometer and 12.3 to 17.8 kj mol‐1 between 310 and 390 nm for the nitrite actinometer. Activation energies for pHBA formation were 2–11% higher. Wavelength‐dependent changes in the quantum yield and activation energy for SA and pHBA formation from nitrate photolysis suggest multiple electronic transitions for nitrate from 290 to 350 nm. Quantum yields for OH radical formation from nitrate and nitrite photolyses were estimated from SA and pHBA quantum yields at 25°C. Wavelength‐dependent OH quantum yields ranged from 0.007 to 0.014 for nitrate photolysis between 290 and 330 nm and from 0.024 to 0.078 for nitrite photolysis between 298 and 390 nm. The nitrate and nitrite actinometers can maintain initial rate conditions for hours, are insensitive to laboratory lighting, easy to use and extremely sensitive; the minimum radiant energy that can be detected in our irradiation system is approximately 10‐9 einsteins.
The Mode of Cell Death Induced by Photodynamic Treatment Depends on Cell Density
Authors: Dahle Jostein , Steen Harald B. , Moan Johan
Published: January 2008

Abstract Madison Darby canine kidney II (MDCK II) cells were seeded out at two different densities and incubated with 125 μ/mL of the photosensitizer me50‐tetra(4‐sulfona‐tophenyUporphine (TPPS4) for 18 h, washed and irradiated with blue light. Four hours later the cells were studied by fluorescence microscopy. Apoptotic cells were detected by virtue of the distinct condensation and fragmentation of chromatin, and necrotic cells were detected by uptake of propidium iodide. In addition apoptosis was measured by the TdT assay. The fraction of apoptotic cells and the fraction of necrotic cells were determined for both cell densities at various levels of survival. With <55% total cell death the apoptotic fraction was significantly higher for cells in confluent monolayers than for cells growing in microcolonies at equitoxic doses. Confluent cells were 2.9 times more sensitive than cells in microcolonies partly due to a 1.5 times higher uptake of TPPS4 in monolayer cells. The difference in mode of cell death for the different cell densities was not related to any observable difference in subcellular localization pattern of TPPS4 at equitoxic doses of photodynamic treatment.
Interaction of Acrylodan with Human Serum Albumin. A Fluorescence Spectroscopic Study
Authors: Moreno Fermin , Cortijo Manuel , González‐Jiménez José
Published: January 2008

The binding of the fluorescent probe acrylodan (AC) to human serum albumin (HSA) was studied by fluorescence spectroscopy. The binding isotherms could be fitted to two types of sites. Competition experiments using io‐doacetamide suggested that AC binds tightly on HSA by the cysteine‐34. Attempts were made to find the location of the second site using high concentrations of warfarin, phenylbutazone, diazepam, indomethacin, palmitic acid or bilirubin in order to displace the bound AC to the HSA. Bilirubin was the only ligand able to displace the bound AC. This result suggests that AC, which is a very hydrophobic molecule also capable of labeling lysine residues, should also bind the human albumin in the primary site of bilirubin, but with less affinity than to the cysteine‐34.
Development of Intracellular Calcium Measurement by Time‐resolved Photon‐counting Fluorescence
Authors: Schoutteten Laetitia , Denjean Patrick , Joliff‐Botrel Gouenael , Bernard Christine , Pansu Danielle , Pansu Robert B.
Published: January 2008

Calcium green I, a ratiometric probe based on fluorescence lifetime measurements, was used to monitor intracellular calcium activity ([Ca2+]1) in RINm5F cells using a time‐resolved fluorescence confocal microscope. The probe affinity constant has been recalibrated in single cells using ionomycin as a calcium ionophore and eth‐ylenebis(oxyethylenenitrilo)tetraacetic acid as a calcium buffer; KA was found to equal 150 nmol/L. The kinetics of ionomycin equilibration showed that the calcium release from calcium stores occurs before equilibration with extracellular calcium. The response to the muscarinic agonist carbachol, measured on 17 cells receiving three consecutive applications was characterized both by a [Ca2+]1 peak lasting 50 s without any trailing plateau and by desensitization with a 30% decrease in the response. The dose‐dependent response was obtained for a carbachol concentration from 5 μmol/L to 0.5 mmol/L. The ability of our set‐up to obtain a value every 10 ms enabled us to record asynchronous spikes of [Ca2+]1 in the RINm5F cells. The spikes, lasting less than 1 s, are significantly bigger than the noise, and they are not observed in the colonic HT29 cells.
Parabolic Quantitative Structure‐Activity Relationships and Photodynamic Therapy: Application of a Three‐Compartment Model with Clearance to the In Vivo Quantitative Structure‐Activity Relationships of a Congeneric Series of Pyropheophorbide Derivatives Used as Photosensitizers for Photodynamic Therapy
Authors: Potter W. R. , Henderson B. W. , Bellnier D. A. , Pandey R. K. , Vaughan L. A. , Weishaupt K. R. , Dougherty T. J.
Published: January 2008

An open three‐compartment pharmacokinetic model was applied to the in vivo quantitative structure‐activity relationship (QSAR) data of a homologous series of pyropheophorbide photosensitizers for photodynamic therapy (PDT). The physical model was a lipid compartment sandwiched between two identical aqueous compartments. The first compartment was assumed to clear irreversibly at a rate K0+. The measured octanol‐water partition coefficients, P1 (where i is the number of carbons in the alkyl chain) and the clearance rate K0 determined the clearance kinetics of the drugs. Solving the coupled differential equations of the three‐compartment model produced clearance kinetics for each of the sensitizers in each of the compartments. The third compartment was found to contain the target of PDT. This series of compounds is quite lipophilic. Therefore these drugs are found mainly in the second compartment. The drug level in the third compartment represents a small fraction of the tissue level and is thus not accessible to direct measurement by extraction. The second compartment of the model accurately predicted the clearance from the serum of mice of the hexyl ether of pyropheophorbide a, one member of this series of compounds. The diffusion and clearance rate constants were those found by fitting the pharmacokinetics of the third compartment to the QSAR data. This result validated the magnitude and mechanistic significance of the rate constants used to model the QSAR data. The PDT response to dose theory was applied to the kinetic behavior of the target compartment drug concentration. This produced a pharmacokinetic‐based function connecting PDT response to dose as a function of time postinjection. This mechanistic dose‐response function was fitted to published, single time point QSAR data for the pheophorbides. As a result, the PDT target threshold dose together with the predicted QSAR as a function of time postinjection was found.
p53 Mutations in Basal Cell Carcinomas Arising in Routine Users of Sunscreens
Authors: Rosenstein Barry S. , Phelps Robert G. , Weinstock Martin A. , Bernstein Jonine L. , Gordon Marsha L. , Rudikoff Donald , Kantor Irwin , Shelton Ronald , Lebwohl Mark G.
Published: January 2008

Sun exposure histories were obtained from a series of patients age 35 or younger following diagnosis and removal of a basal cell carcinoma (BCC). The DNA was extracted from tumor biopsy samples derived from BCC of 10 patients who reported that they did not use sunscreens during youth (age 18 or younger) and 10 patients who routinely employed sunscreens during this age period. Exons 5–9 of the p53 gene were then amplified in three fragments from these samples using a nested polymerase chain reaction (PCR) approach and screened for mutations using an RNA heteroduplex assay. All PCR products displaying evidence of a mutation were sequenced. It was found that 6 of the 10 patients who were not routine sunscreen users displayed mutations in these pS3 exons. All of the mutations were located at dipyr‐imidine sites, five of the six were C ← T transitions and one mutation was a tandem double mutation, consistent with a role for solar UVB in BCC formation. In contrast, only one p53 mutation was detected in the group of 10 patients who routinely employed sunscreens during childhood and adolescence. Hence, a significantly (P= 0.029) lower level of p53 mutations was detected in the BCC obtained from sunscreen users compared with tumors derived from nonusers. These findings suggest that the mechanisms involved in the etiology of skin carcinogenesis differ in sunscreen users compared with people who did not routinely employ sunscreens. These data are also indicative of a protective effect associated with sunscreen use against the formation of p53 mutations. It is possible that the patients who were diagnosed with BCC despite their use of sunscreens possessed a genetic susceptibility for skin cancer formation and developed BCC through a p53‐independent pathway. Alternatively, solar U V A wavelengths, that were generally not blocked by the suncare products employed by the sunscreen users, may have played a significant role in BCC development through induction of a mutation(s) in an oncogene and/ or a tumor suppressor gene, other than p53, for these patients.
Measurement of In Vivo Sunscreen Immune Protection Factors in Humans
Authors: Damian Diona L. , Barnetson Ross StC. , Halliday Gary M.
Published: January 2008

This study investigates the level of protection provided by sunscreens against solar‐simulated UV radiation‐induced immunosuppression in humans. The in vivo immune protection factors (IPF) of two broad‐spectrum sunscreens were determined by assessing their ability to prevent UV‐induced suppression of nickel contact hypersensitivity (CHS) in 15 nickel‐allergic volunteers. Each volunteer was irradiated on unprotected skin of the back with different doses of UV daily for 4 days. Multiples of these UV doses were concurrently delivered to sunscreen‐treated sites on the contralateral back. Nickel patches were then applied to both irradiated sites and adjacent, unirradiated control sites. Nickel‐induced erythema at each site was measured 72 h later with a reflectance spectrometer. Comparison of the nickel reactions of irradiated and unirradiated skin revealed linear UV dose–responses for immunosuppression in both unprotected and sunscreen‐treated skin. The minimum level of immunosuppression that can be reliably detected with this method is 20%. Therefore, the UV dose that reduces mean nickel CHS by 20% is the minimal immune suppression dose (MISD). Sunscreen IPF were determined by dividing the mean MISD of sunscreen‐treated skin by that of unprotected skin. The sunscreens, with sun protection factors of 9 and 24, had IPF of 6.5 and >25, respectively.
The Phototumorigenic Fluoroquinolone Lomefloxacin Photosensitizes Pyrimidine Dimer Formation in Human Keratinocytes In Vitro
Authors: Traynor Nicola J. , Gibbs Neil K.
Published: January 2008

The fluoroquinolone antibiotic lomefloxacin is phototoxic, photogenotoxic, photomutagenic and photosensitizes tu‐morigenesis in mouse skin. We have used T4 endonucle‐ase V to demonstrate that lomefloxacin photosensitizes pyrimidine dimer formation in a human keratinocyte line (HaCaT). A possible mechanism for this effect would be triplet‐triplet energy transfer. However, there is indirect evidence that the lomefloxacin triplet yield is very low, making this reaction less likely. The finding that lomefloxacin photosensitizes production of highly mutagenic pyrimidine dimers correlates with its ability to initiate skin tumor formation in mice. Until the potential of other fluoroquinolones to photosensitize dimer formation is explored it may be unadvisable to prescribe these antibiotics to patients with defective DNA repair capacity (e.g. xeroderma pigmentosum).
Heparin-binding epidermal growth factor and Src family kinases in proliferation of renal epithelial cells
Authors: Zhuang Shougang , Kinsey Gilbert R. , Rasbach Kyle , Schnellmann Rick G.
Published: January 2008

Our recent studies have shown that proliferation of renal proximal tubular cells (RPTC) in the absence of growth factors requires activation of the epidermal growth factor (EGF) receptor. We sought to identify the endogenous EGF receptor ligand and investigate the mechanism(s) by which RPTC proliferate in different models. RPTC expressed both pro- and cleaved forms of heparin-binding epidermal growth factor (HB-EGF) and several metalloproteinases (MMP-2, -3, -9, and ADAM10, ADAM17) that have been reported to cleave HB-EGF. Treatment of RPTC with CRM 197, an inhibitor of HB-EGF binding to the EGF receptor, or downregulation of HB-EGF with small interfering RNA inhibited RPTC proliferation following plating. Furthermore, GM6001 (pan-MMP inhibitor), tumor-necrosis factor protease inhibitor-1 (TAPI-1; MMP and ADAM17 inhibitor), and GW280264X (ADAM10 and -17 inhibitor), but not GI254023X (ADAM10 inhibitor), attenuated the proliferation after plating. Although EGF receptor activation is required for RPTC proliferation after oxidant injury, CRM197, GM6001, and TAPI-1 did not block this response. In contrast, inhibition of Src with PP1 blocked EGF receptor activation and RPTC proliferation after oxidant injury. In addition, PP1 treatment attenuated HB-EGF-enhanced RPTC proliferation. We suggest that RPTC proliferation after plating is mediated by HB-EGF produced through an autocrine/paracrine mechanism and RPTC proliferation following oxidant injury is mediated by Src without involvement of HB-EGF.
Transcriptome Profile of Human Colorectal Adenomas
Authors: Sabates-Bellver Jacob , Van der Flier Laurens G. , de Palo Mariagrazia , Cattaneo Elisa , Maake Caroline , Rehrauer Hubert , Laczko Endre , Kurowski Michal A. , Bujnicki Janusz M. , Menigatti Mirco , Luz Judith , Ranalli Teresa V. , Gomes Vito , Pastorelli Alfredo , Faggiani Roberto , Anti Marcello , Jiricny Josef , Clevers Hans , Marra Giancarlo
Published: January 2008

Colorectal cancers are believed to arise predominantly from adenomas. Although these precancerous lesions have been subjected to extensive clinical, pathologic, and molecular analyses, little is currently known about the global gene expression changes accompanying their formation. To characterize the molecular processes underlying the transformation of normal colonic epithelium, we compared the transcriptomes of 32 prospectively collected adenomas with those of normal mucosa from the same individuals. Important differences emerged not only between the expression profiles of normal and adenomatous tissues but also between those of small and large adenomas. A key feature of the transformation process was the remodeling of the Wnt pathway reflected in patent overexpression and underexpression of 78 known components of this signaling cascade. The expression of 19 Wnt targets was closely correlated with clear up-regulation of KIAA1199, whose function is currently unknown. In normal mucosa, KIAA1199 expression was confined to cells in the lower portion of intestinal crypts, where Wnt signaling is physiologically active, but it was markedly increased in all adenomas, where it was expressed in most of the epithelial cells, and in colon cancer cell lines, it was markedly reduced by inactivation of the β-catenin/T-cell factor(s) transcription complex, the pivotal mediator of Wnt signaling. Our transcriptomic profiles of normal colonic mucosa and colorectal adenomas shed new light on the early stages of colorectal tumorigenesis and identified KIAA1199 as a novel target of the Wnt signaling pathway and a putative marker of colorectal adenomatous transformation. (Mol Cancer Res 2007;5(12):1263–75)
Basal Caspase Activity Promotes Migration and Invasiveness in Glioblastoma Cells
Authors: Gdynia Georg , Grund Kerstin , Eckert Anika , Böck Barbara C. , Funke Benjamin , Macher-Goeppinger Stephan , Sieber Sebastian , Herold-Mende Christel , Wiestler Benedict , Wiestler Otmar D. , Roth Wilfried
Published: January 2008

Glioblastomas, the most malignant of all brain tumors, are characterized by cellular resistance to apoptosis and a highly invasive growth pattern. These factors contribute to the poor response of glioblastomas to radiochemotherapy and prevent their complete neurosurgical resection. However, the driving force behind the distinct motility of glioma cells is only partly understood. Here, we report that in the absence of cellular stress and proapoptotic stimuli, human glioblastoma cells exhibit a constitutive activation of caspases in vivo and in vitro. The inhibition of caspases by various peptide inhibitors decreases the migration of cells in scrape motility assays and the invasiveness of cells in spheroid assays. Similarly, specific small interfering RNA– or antisense-mediated down-regulation of caspase-3 and caspase-8 results in an inhibition of the migratory potential of glioma cells. The constitutive caspase-dependent motility of glioblastoma cells is independent of CD95 activation and it is not mediated by mitogen-activated protein/extracellular signal-regulated kinase kinase signaling. The basal caspase activity is accompanied by a constant cleavage of the motility-associated gelsolin protein, which may contribute to the caspase-mediated promotion of migration and invasiveness in glioblastoma cells. Our results suggest that the administration of low doses of caspase inhibitors that block glioma cell motility without affecting the execution of apoptotic cell death may be exploited as a novel strategy for the treatment of glioblastomas. (Mol Cancer Res 2007;5(12):1232–40)
Comparative Study of Circadian Oscillatory Network Models of Drosophila
Authors: Ogawa Yukino , Arakawa Kazuharu , Kaizu Kazunari , Miyoshi Fumihiko , Nakayama Yoichi , Tomita Masaru
Published: January 2008

The circadian clock of Drosophila is a model pathway for research in biological clock mechanisms, both with traditional experimental approaches and with emerging systems biology approaches utilizing mathematical modeling and in silico computer simulation. Dynamic diurnal oscillations are achieved by the complex interaction of components as a system, and mathematical reconstruction has proven to be an invaluable means of understanding such systematic behavior. In this study, we implemented eight published models of the Drosophila circadian clock in Systems Biology Markup Language (SBML) for comparative systems biology studies using E-Cell Simulation Environment version 3, to examine the system-level requirements for the clock mechanism to be robust, by calculating the period and amplitude sensitivity coefficients with simulation experiments. While all models were generally robust as determined by the network topology of the oscillatory feedback loop structure, existing models place relatively strong emphasis on transcription regulation, although this is a limitation on robustness. We suggest that more comprehensive modeling including protein phosphorylation, polymerization, and nuclear transport with regard to amplitude sensitivity will be necessary for understanding the light entrainment and temperature compensation of circadian clocks.
Race, Education, and the Politics of Fear
Authors: Jackson Barbara Loomis
Published: January 2008

This article explores the legacies of the 1954 Brown v. Board of Education Supreme Court decision within the historical context of race relations in the United States. The pursuit by African Americans to exercise their rights of citizenship is described as influenced by the changing face of fear. The Supreme Court decisions that determined the status of African Americans provide the framework for the description of the strategies (e.g. litigation, community protest, and electoral politics that were used in the past). The last section looks to the future, assessing the current changes in the social and economic status of African Americans. Can education still make a difference and keep the promise of social justice, equity, and equality—the fundamental values on which the nation was founded—alive? Will new fears emerge even as some progress is made toward a more integrated society?
The environmental toxicant 2,3,7,8-tetrachlorodibenzo-p-dioxin disrupts morphogenesis of the rat pre-implantation embryo
Authors: Hutt Karla J , Shi Zhanquan , Albertini David F , Petroff Brian K
Published: January 2008

Background

Environmental toxicants, whose actions are often mediated through the aryl hydrocarbon receptor (AhR) pathway, pose risks to the health and well-being of exposed species, including humans. Of particular concern are exposures during the earliest stages of development that while failing to abrogate embryogenesis, may have long term effects on newborns or adults. The purpose of this study was to evaluate the effect of maternal exposure to the AhR-specific ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the development of rat pre-implantation embryos with respect to nuclear and cytoskeletal architecture and cell lineage allocation.

Results

We performed a systematic 3 dimensional (3D) confocal microscopy analysis of rat pre-implantation embryos following maternal exposure to environmentally relevant doses of TCDD. Both chronic (50 ng/kg/wk for 3 months) and acute (50 ng/kg and 1 μg/kg at proestrus) maternal TCDD exposure disrupted morphogenesis at the compaction stage (8–16 cell), with defects including monopolar spindle formation, f-actin capping and fragmentation due to aberrant cytokinesis. Additionally, the size, shape and position of nuclei were modified in compaction stage pre-implantation embryos collected from treated animals. Notably, maternal TCDD exposure did not compromise survival to blastocyst, which with the exception of nuclear shape, were morphologically similar to control blastocysts.

Conclusion

We have identified the compaction stage of pre-implantation embryogenesis as critically sensitive to the effects of TCDD, while survival to the blastocyst stage is not compromised. To the best of our knowledge this is the first in vivo study to demonstrate a critical window of pre-implantation mammalian development that is vulnerable to disruption by an AhR ligand at environmentally relevant doses.