RESUMO
We demonstrate the efficient separation of blood cells from millilitre volumes of whole blood in minutes using a simple gravity sedimentation device. Blood cell and plasma separation is often the initial step in clinical diagnostics, and reliable separation techniques have remained a major obstacle for the success of point-of-care or remote diagnostics. Unlike plasma collection devices that rely solely on microchannels that restrict sample volume and throughput, we demonstrate the use of a hybrid micro/mesoscale sedimentation chamber to enable >99% capture of cells from millilitre blood samples in less than two minutes.
Assuntos
Células Sanguíneas/citologia , Separação Celular/métodos , Células Sanguíneas/metabolismo , Separação Celular/instrumentação , DNA/análise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase em Tempo RealRESUMO
NV junctions (nucleus-vacuole junctions) in Saccharomyces cerevisiae are MCSs (membrane contact sites) formed through specific interactions between Vac8p on the vacuole membrane and Nvj1p in the outer nuclear membrane, which is continuous with the perinuclear ER (endoplasmic reticulum). NV junctions mediate a unique autophagic process that degrades portions of the yeast nucleus through a process called 'piecemeal microautophagy of the nucleus' (PMN). Our studies suggest that the lipid composition of NV junctions plays an important role in the biogenesis of PMN structures. NV junctions represent a unique model system for studying the biology of ER MCSs, as well as the molecular mechanism of selective microautophagy.
Assuntos
Núcleo Celular/química , Membranas Intracelulares/química , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Vacúolos/química , Sítios de Ligação/fisiologia , Núcleo Celular/fisiologia , Retículo Endoplasmático/química , Retículo Endoplasmático/fisiologia , Membranas Intracelulares/fisiologia , Saccharomyces cerevisiae/fisiologia , Vacúolos/fisiologiaRESUMO
Vac8p is a vacuolar membrane protein that is required for efficient vacuole inheritance and fusion, cytosol-to-vacuole targeting, and sporulation. By analogy to other armadillo domain proteins, including beta-catenin and importin alpha, we hypothesize that Vac8p docks various factors at the vacuole membrane. Two-hybrid and copurfication assays demonstrated that Vac8p does form complexes with multiple binding partners, including Apg13p, Vab2p, and Nvj1p. Here we describe the surprising role of Vac8p-Nvj1p complexes in the formation of nucleus-vacuole (NV) junctions. Nvj1p is an integral membrane protein of the nuclear envelope and interacts with Vac8p in the cytosol through its C-terminal 40-60 amino acids (aa). Nvj1p green fluorescent protein (GFP) concentrated in small patches or rafts at sites of close contact between the nucleus and one or more vacuoles. Previously, we showed that Vac8p-GFP concentrated in intervacuole rafts, where is it likely to facilitate vacuole-vacuole fusion, and in "orphan" rafts at the edges of vacuole clusters. Orphan rafts of Vac8p red-sifted GFP (YFP) colocalize at sites of NV junctions with Nvj1p blue-sifted GFP (CFP). GFP-tagged nuclear pore complexes (NPCs) were excluded from NV junctions. In vac8-Delta cells, Nvj1p-GFP generally failed to concentrate into rafts and, instead, encircled the nucleus. NV junctions were absent in both nvj1-Delta and vac8-Delta cells. Overexpression of Nvj1p caused the profound proliferation of NV junctions. We conclude that Vac8p and Nvj1p are necessary components of a novel interorganelle junction apparatus.
Assuntos
Proteínas Fúngicas/metabolismo , Lipoproteínas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fracionamento Celular , Núcleo Celular/metabolismo , Proteínas Fúngicas/genética , Membranas Intracelulares/metabolismo , Lipoproteínas/genética , Proteínas de Membrana/genética , Membrana Nuclear/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Vacúolos/metabolismo , Proteínas de Transporte VesicularRESUMO
Heme oxygenase (HO) breaks down heme to iron, biliverdin, and carbon monoxide, and activity of this enzyme increases in many tissues and cell types after exposure to oxidative stress. There is evidence that increased HO activity is involved in long-term protective mechanisms against oxidative stress. We studied the effect of artificially overexpressed HO activity on the cytotoxicity of oxidative ultraviolet A (UVA) radiation after loading human cells with the HO substrate ferric heme (hemin). In contrast to the reported long-term protection attributed to HO activity, cells overexpressing HO activity were hypersensitive to UVA radiation shortly after heme treatment when compared with control cells. Cells overexpressing HO activity showed an increased rate of heme consumption and a higher level of accumulated free chelatable iron when compared with control cells. The hypersensitivity of cells overexpressing HO to UVA radiation after heme treatment was apparently caused by the increased accumulation of chelatable iron, because the iron chelator desferrioxamine strongly reduced the hypersensitivity. One day after the heme treatment, cells overexpressing HO activity were no longer hypersensitive to UVA radiation. We conclude that increased HO activity can temporarily increase the sensitivity of cells to oxidative stress by releasing iron from heme.
Assuntos
Heme Oxigenase (Desciclizante)/fisiologia , Heme/efeitos da radiação , Ferro/fisiologia , Raios Ultravioleta , Sobrevivência Celular , Desferroxamina/farmacologia , Células HeLa/efeitos dos fármacos , Células HeLa/metabolismo , Células HeLa/efeitos da radiação , Heme/química , Heme/farmacologia , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase (Desciclizante)/genética , Hemina/metabolismo , Humanos , Quelantes de Ferro/farmacologia , Oxirredução , Estresse Oxidativo , Fotoquímica , Tolerância a Radiação/efeitos dos fármacos , Proteínas Recombinantes de Fusão/fisiologia , TransfecçãoRESUMO
Highly pigmented, dark skin is more resistant to the harmful effects of solar ultraviolet radiation than light-colored human skin. The extent to which tanning protects skin from harmful effects including induction of skin cancer is not known, however. We have investigated whether the skin pigment, melanin, sensitizes or protects isolated DNA or nuclear DNA in melanoma cells from the induction of the premutagenic oxidative DNA base damage, 8-hydroxy-deoxyguanosine, by ultraviolet A irradiation. Synthetic eumelanin sensitized isolated DNA to induction of the oxidative DNA base damage by ultraviolet A, but it also induced the oxidative DNA base damage in the dark. To study the role of natural melanin in mammalian melanoma cells in the induction of oxidative DNA base damage, melanin synthesis was modulated 5-7-fold in the human melanoma cells GLL19 and IGR1 (which contain both pheomelanin and eumelanin) as well as in the mouse melanoma cells B16 (which contain mainly eumelanin). Increased melanin synthesis clearly did not protect against ultraviolet A-induced oxidative DNA base damage in cells. On the contrary, the human melanoma cells with high melanin content accumulated two times more 8-hydroxy-deoxyguanosine after ultraviolet A irradiation than cells with low melanin content. Furthermore, preirradiation of the human melanoma cells, IGR1, with ultraviolet A 4 h before a second ultraviolet A exposure produced an altered amount of induced 8-hydroxy-deoxyguanosine dependent on the melanin content of the cells. We conclude that stimulation of melanin synthesis, but probably not melanin itself, increases the susceptibility of human melanoma cells to induction of premutagenic oxidative DNA base damage by ultraviolet A irradiation.
Assuntos
Melaninas/fisiologia , Melanoma/patologia , Raios Ultravioleta , Animais , Dano ao DNA , Humanos , Melaninas/farmacologia , Melanoma/química , Camundongos , Óptica e Fotônica , Oxirredução , Fatores de Tempo , Células Tumorais Cultivadas/efeitos da radiaçãoRESUMO
The level of endogenous photosensitiser, protoporphyrin IX (PPIX), can be enhanced in the cells by 5-aminolevulinic acid (ALA). We investigated the effect of critical parameters such as growth state of the cells and availability of intracellular iron in modulating the level of PPIX, in human primary cultured skin fibroblasts (FEK4) maintained either in exponentially growing or growth-arrested phase, following treatment with ALA. The addition of ALA to exponentially growing cells increased the level of PPIX 6-fold relative to control cells; however, in growth-arrested cells the same treatment increased the level of PPIX up to 34-fold. The simultaneous addition of the hydrophilic iron-chelator Desferal with ALA, boosted the level of PPIX up to 47-fold in growing cells and up to 42-fold in growth-arrested cells, suggesting that iron is limiting under the latter conditions. The strict dependence of PPIX enhancement on free available iron levels was examined by the level of activation of iron regulatory protein in band shift assays. This analysis revealed that the basal level of iron regulatory protein in growth-arrested cells was 6-fold higher than in growing cells, reflecting the influence of the free available iron pool in exponentially growing cells. Interestingly, the same ratio was found between the basal level concentration of PPIX in growing and growth-arrested cells. We propose that iron regulatory protein activation could serve as a marker for developing photodynamic therapy protocols because it identifies cells and tissues with a propensity to accumulate PPIX and it is therefore likely to predict the effectiveness of such therapies.
Assuntos
Ácido Aminolevulínico/farmacologia , Fotoquimioterapia , Protoporfirinas/biossíntese , Pele/efeitos dos fármacos , Células Cultivadas , Desferroxamina/farmacologia , Humanos , Proteínas Reguladoras de Ferro , Proteínas Ferro-Enxofre/fisiologia , RNA/metabolismo , Proteínas de Ligação a RNA/fisiologia , Pele/metabolismo , Raios UltravioletaRESUMO
Induction of heme oxygenase 1 transcription and enzymatic activity is a common response after exposure of cells to various forms of oxidative stress including ultraviolet A radiation (UVA) and hydrogen peroxide. We now show that UVA irradiation or hydrogen peroxide treatment of human skin fibroblasts leads to an immediate release of the heme oxygenase substrate, heme, from microsomal hemeproteins. The release of heme by UVA apparently involves cyclooxygenase activity because it is inhibited by the cyclooxygenase inhibitor indomethacin. We also demonstrate a high degree of correlation between the amount of heme released and the degree of subsequent induction of heme oxygenase 1 transcription following UVA and hydrogen peroxide treatment. We propose that release of heme from microsomal hemeproteins determines the degree of induction of heme oxygenase 1 transcription in human fibroblasts after oxidative stress.
Assuntos
Heme Oxigenase (Desciclizante)/genética , Heme/metabolismo , Hemeproteínas/metabolismo , Peróxido de Hidrogênio/farmacologia , Microssomos/metabolismo , Prostaglandina-Endoperóxido Sintases/metabolismo , Transcrição Gênica , Raios Ultravioleta , Hidroxitolueno Butilado/farmacologia , Linhagem Celular , Inibidores de Ciclo-Oxigenase/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase-1 , Humanos , Indometacina/farmacologia , Cinética , Proteínas de Membrana , Microssomos/efeitos dos fármacos , Microssomos/efeitos da radiação , Estresse Oxidativo , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiaçãoRESUMO
Heme oxygenase (HO) activity oxidizes heme, releasing carbon monoxide; heme iron; and biliverdin, which is converted to bilirubin by biliverdin reductase. Inducible HO-I expression is a marker of oxidative stress in mammalian cells, while noninducible HO-II contributes to basal HO activity. HO-I and HO-II activities are implicated in cellular antioxidant defense mechanisms. We describe a microassay for HO activity in cultured human cells, using high-performance liquid chromatography of biliverdin and bilirubin. The assay is sufficiently sensitive to quantify basal and inducible HO activity in various human cell types. We have established human cell lines overexpressing heme oxygenase-II activity in microsomes using a metallothionein promoter-regulated expression system. Stable transformants treated with ZnCl2 express up to ninefold induction of HO activity. We have constructed human cell lines overexpressing HO-II protein and activity (5-15-fold) in the absence of tetracycline, using the HtTA-1 cell line transfected with tetracycline-regulated expression vectors (Gossen et al., Proc. Natl. Acad. Sci. USA 89, 1992). Functional HO-II overexpressing clones will be useful in investigating anti- or pro-oxidant effects of HO activity during cellular oxidative stress.
Assuntos
Heme Oxigenase (Desciclizante)/biossíntese , Linhagem Celular , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Doxiciclina/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Fibroblastos/enzimologia , Heme Oxigenase (Desciclizante)/genética , Humanos , Rim , Metalotioneína/genética , Regiões Promotoras Genéticas/genética , Pirróis/análise , Pele , Tetraciclina/farmacologia , Tetrapirróis , TransfecçãoRESUMO
The pre-mutagenic oxidative DNA base damage of 8-hydroxy-guanine is present in DNA isolated from cells and the amount present increases with exposure of cells to oxidative stress. The oxidative DNA base damage may be present before isolation of DNA or it may be produced during isolation and processing of DNA. We have found that the amount of oxidative base damage measured in DNA can be reduced to a stable lower level by adding increasing concentrations of the antioxidants desferrioxamine, histidine and reduced glutathione immediately before cell lysis. Inclusion of these antioxidants after cell lysis did not affect the level of DNA damage. Oxidative DNA base damage produced by ultraviolet A irradiation of human cells was also reduced by adding antioxidants after irradiation and before cell lysis. Thus, unidentified oxidants induced by ultraviolet A irradiation may damage DNA significantly during extractions of DNA from cells subsequent to ultraviolet A irradiation.
Assuntos
Dano ao DNA , 8-Hidroxi-2'-Desoxiguanosina , Antioxidantes/farmacologia , Células Cultivadas , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análise , Humanos , OxirreduçãoRESUMO
The premutagenic oxidative DNA base damage, 7,8-dihydro-8-oxoguanine, is induced in human skin fibroblasts by monochromatic radiation ranging from a UVB wavelength (312 nm) up to wavelengths in the near visible (434 nm). The oxidative damage is not generated by absorption of radiation in DNA but rather by activation of photosensitizers generating genotoxic singlet oxygen species. The spectrum for the yield of the oxidative damage in confluent, non-growing, primary skin fibroblasts shows that it is UVA (above 334 nm) and near visible radiations which cause almost all of this guanine oxidation by natural sunlight in the fibroblast model. We estimate that the total amount of oxidation of guanine induced by sunlight in fibroblasts in the epidermis of the skin equals or exceeds the amount of the major type of direct DNA damage, cyclobutane pyrimidine dimers. In rapidly dividing lymphoblastoid cells, no oxidative guanine damage was induced. However, in melanoma cells almost as much damage as in non-growing fibroblasts (1.1 per 10(4) guanine bases after 1200 kJ/m2 UVA) was found. We conclude that oxidative DNA base damage can probably contribute to the induction of both non-melanoma and melanoma skin cancer by sunlight.
Assuntos
Dano ao DNA/efeitos da radiação , Desoxiguanosina/análogos & derivados , Pele/efeitos da radiação , 8-Hidroxi-2'-Desoxiguanosina , Células Cultivadas , Desoxiguanosina/metabolismo , Relação Dose-Resposta à Radiação , Fibroblastos , Humanos , Melanoma/patologia , Oxirredução , Dímeros de Pirimidina , Neoplasias Cutâneas/patologia , Análise Espectral , Raios UltravioletaRESUMO
Human NHIK 3025 cells were sensitized to light by incubation with Photofrin II (PII) and aluminum phthalocyanine tetrasulphonate (AlPCS4). Light exposure of sensitized cells at 1 degree C induced DNA strand breaks causing unwinding of DNA in alkali. Subsequent to light exposure, cells were treated with deoxyribonuclease I (DNAse I) which produces DNA strand breaks in active chromatin. The combination of photo-damage, killing > 95% of the cells, and treatment with DNAse I caused the same degree of DNA unwinding as DNAse I treatment alone. This was not the case for X-irradiation. Thus, the photosensitized DNA strand breaks were selectively induced in or close to DNAse I-sensitive sites.
Assuntos
Cromatina/metabolismo , Cromatina/efeitos da radiação , Dano ao DNA , DNA/efeitos dos fármacos , DNA/efeitos da radiação , Desoxirribonuclease I/metabolismo , Éter de Diematoporfirina/farmacologia , Indóis/farmacologia , Compostos Organometálicos/farmacologia , Radiossensibilizantes/farmacologia , Células Cultivadas , Cromatina/enzimologia , DNA/metabolismo , Humanos , Cinética , FotoquímicaRESUMO
Human REH cells were incubated with the photosensitizers meso-tetra(4-sulfonatophenyl)porphyrin (TSPP = TPPS4) or meso-tetra(3-hydroxyphenyl)porphyrin (3-THPP). The relatively hydrophilic TSPP was partly found in the cytoplasm and partly in the nuclei, whereas the lipophilic 3-THPP was found apparently in membranes and not inside the nuclei. After illumination, sites of DNA repair were labeled by means of a monoclonal antibody against proliferating cell nuclear antigen (PCNA) bound in the nuclei. The amount of bound PCNA in non-S-phase cells was proportional to the light dose. The bound PCNA was homogeneously distributed in the nuclei 0.5 h after photodynamic treatment (PDT) with TSPP. In contrast, for cells given PDT with 3-THPP, the periphery of the nuclei was selectively labeled, indicating that the initial DNA damage was localized close to the sensitizer at the nuclear membrane.
Assuntos
Reparo do DNA/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Linfócitos B/efeitos dos fármacos , Células Cultivadas , Humanos , Luz , Proteínas Nucleares/análise , Antígeno Nuclear de Célula em ProliferaçãoRESUMO
DNA strand breaks, measured by alkaline elution, and hypoxanthine guanine phosphoribosyltransferase (HPRT) mutation were studied in V79 cells after photochemical treatment (PCT) or exposure to X-rays. Cells were incubated with the photosensitizers Photofrin II (PII) and three closely related porphyrins tetra-(3-hydroxyphenyl) porphyrin (3THPP), meso-tetra-(4-sulfonatophenyl) porphine (TPPS4) and meso-tetra-(N-methyl-4-pyridyl) porphine (TMPyPH2). These dyes are assumed to act on cellular targets mainly via singlet oxygen when excited by light. While the hydrophilic TPPS4 and TMPyPH2 did not photoinduce mutants to any significant extent, both lipophilic dyes, 3THPP and PII, were significantly mutagenic when excited by light. On the other hand, TPPS4 was the most efficient sensitizer of alkali-labile DNA strand breaks, while TMPyPH2 did not induce any significant amount of either type of DNA damage. Surprisingly, no correlation between the two parameters was found for PCT, either after exposures inactivating 50% of the cells or after exposures inactivating 90% of them. The lack of correlation between the yields of DNA strand breaks and of mutants could not be explained by differences in the intracellular localization pattern of the dyes.
Assuntos
DNA/efeitos da radiação , Hipoxantina Fosforribosiltransferase/genética , Luz , Animais , Células Cultivadas , Cricetinae , DNA/efeitos dos fármacos , Dano ao DNA , Reparo do DNA , Mutação , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/farmacologia , Raios XRESUMO
DNA and free nucleosides were photosensitized with the DNA-binding dyes methylene blue (MB) and meso-tetra(4-N-methyl-pyridyl) porphyrin (p-TMPyP) and the non-binding meso-tetra (4-sulphonatophenyl) porphyrin (TSPP). After light exposure DNA was enzymatically digested to nucleosides. Only the guanine residues were photodegraded. By measuring optical absorption, at least 20 photoproducts were detected. Singlet oxygen (1O2) was involved in induction of all these products since D2O enhanced their yields from 4 to 10 times. The photoproducts were the same for all sensitizers. However, several photoproducts were found only with DNA or only with free 2'-deoxyguanosine. Four of 20 photoproducts were induced both in DNA and free 2'-deoxy-guanosine. The yield of the photoproduct 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) relative to the degree of 2'-deoxy-guanosine degradation depended on which sensitizer was used and on whether nucleosides or DNA was exposed. Apparently, DNA structure affected the types of as well as the yields of photo-products induced by 1O2.
Assuntos
DNA/química , Guanina/química , Nucleosídeos/química , Oxigênio/química , Desoxiguanosina/química , Radicais Livres , Azul de Metileno , Nucleosídeos/isolamento & purificação , Fotoquímica , Porfirinas , Oxigênio SingleteRESUMO
DNA strand breaks and hypoxanthine guanine phosphoribosyl transferase (HPRT) mutants were measured in parallel in photochemically treated (PCT) cells and compared at the same level of cell survival. Chinese hamster fibroblasts (V79 cells) were either incubated with the lipophilic dyes tetra(3-hydroxyphenyl)porphyrin (3THPP) and Photofrin II (PII), the anionic dye meso-tetra(4-sulfonatophenyl)porphine (TPPS4) or the cationic dye meso-tetra(N-methyl-4-pyridyl)porphine (p-TMPyPH2) before light exposure. In the cells, the lipophilic dyes were localized in membranes, including the nuclear membrane, while the hydrophilic dyes were taken up primarily into spots in the cytoplasm. In addition, the hydrophilic TPPS4 was distributed homogeneously throughout the whole cytoplasm and nucleoplasm. According to the HPRT mutation test, the mutagenicity of light doses survived by 10% of the cells was a factor of six higher in the presence of 3THPP than of PII, whereas for X-rays it was a factor of three higher than for PCT with 3THPP. Light exposure in the presence of the hydrophilic dyes TPPS4 and p-TMPyPH2 was not significantly mutagenic. There was no correlation between the induced rates of HPRT mutants and of DNA strand breaks. Thus, TPPS4 was the most efficient sensitizer with regard to DNA strand breaks when compared at the same level of cell survival, followed by 3THPP, PII and p-TMPyPH2. Hence, the rate of DNA strand breaks cannot be used to predict the mutagenicity of PCT.
Assuntos
Dano ao DNA/efeitos dos fármacos , Hipoxantina Fosforribosiltransferase/genética , Radiossensibilizantes/toxicidade , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Cricetinae , Cricetulus , Éter de Diematoporfirina/toxicidade , Luz , Mutagênese , Porfirinas/toxicidade , Raios XRESUMO
We have exploited properties of photosensitizers to study an aspect of the packing of chromatin in the cell nucleus. The fluorescent photosensitizers mesotetra(3-hydroxyphenyl) porphyrin and Photofrin II were both localized in the nuclear membrane and other membrane structures, but could not be found inside the nuclei. Light exposure of cells at 1 degrees C in the presence of the sensitizers induced DNA double-strand breaks. The length distributions of DNA fragments were determined by pulsed field gel electrophoresis. Because DNA damage is produced mainly via singlet oxygen diffusing less than 0.1 microns from the sensitizer, DNA double-strand breaks were supposedly produced within this distance of the nuclear membrane. Consistent with this, with prolonged illumination and with increasing concentrations of sensitizer the distribution of DNA fragment lengths reached a plateau level. In contrast, with the hydrophilic, intranuclear sensitizer meso-tetra(4-sulphonatophenyl)porphyrin, no such plateau level was found. The plateau distributions of DNA fragment lengths of different cell types had the same general shape with average fragment lengths ranging from 174 to 194 kilobasepairs. Particular genes, c-myc, fos and p53, were found on broad distributions of photocleaved fragment lengths. The results indicate that on each side of the genes the locus of the chromatin fibre situated close to the nuclear membrane, varied randomly.
Assuntos
DNA/efeitos dos fármacos , Luz , Oligodesoxirribonucleotídeos/isolamento & purificação , Porfirinas/farmacologia , Radiossensibilizantes/farmacologia , Linhagem Celular , DNA/isolamento & purificação , Sondas de DNA , Eletroforese em Gel de Ágar , Genes fos , Genes myc , Genes p53 , Humanos , Microscopia de Fluorescência , Peso MolecularRESUMO
Intracellular properties of three photosensitizers relevant to photodynamic cancer therapy were compared using cultured human NHIK 3025 cells. When taken up in the cells, the hydrophilic photosensitizer aluminum phthalocyanine tetra sulfonate required about 10 times more quanta of light absorbed per cell to kill 90% of the cells than did the hydrophobic dyes Photofrin II and tetra(3-hydroxyphenyl)porphyrin. In spite of this, the phthalocyanine molecule was the most efficient dye per quantum of excitation light, since the extinction coefficient of the phthalocyanine is more than 10 times higher than that of the two hydrophobic photosensitizers at therapeutic wavelengths. The two hydrophobic dyes had significantly higher fluorescence quantum yields when taken up by cells than when bound to human plasma or human serum albumin, whereas the opposite was true for the hydrophilic phthalocyanine dye--suggesting intracellular aggregation. Finally, the potential genetic toxicities of the drugs in the form of DNA strand breaks were compared. The aluminum phthalocyanine tetra sulfonate photosensitized more DNA strand breaks than did Photofrin II and tetra(3-hydroxyphenyl)porphyrin when compared at the same level of cell survival.
Assuntos
Dano ao DNA/efeitos dos fármacos , Fotoquimioterapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Éter de Diematoporfirina , Hematoporfirinas/farmacologia , Indóis/farmacologia , Compostos Organometálicos/farmacologia , Porfirinas/farmacologia , Espectrometria de FluorescênciaRESUMO
The first aim of this study was to devise a meaningful, objective, and reproducible method of measuring premolar rotation on study casts. A new method of measurement was devised, using standardized arch forms as the reference line superimposed on the study casts. The method error of the difference in two determinations was calculated as +/- 2.95 degrees. Using this method, premolar rotations were measured on study casts at 15 years of 203 children enrolled in the Nittedal Growth Study. Histograms of the frequency distribution for each premolar were drawn and the mean rotations compared. Correlation coefficients were then calculated to determine if any intra-quadrant, intra-arch, or inter-arch relationships existed. Significant correlations were found between contra-lateral teeth in the same arch (P less than 0.001) and to a lesser degree between premolars in the same quadrant. Significant inter-arch correlation was found between left upper and lower second premolars (P less than 0.001). Surprisingly, some cross-arch relationships were found between the upper left second premolar and lower right second premolar (P less than 0.001), and between the upper right second premolar and lower left second premolar (P less than 0.01). It is suggested that aetiological factors in premolar rotation are genetic and, to a lesser extent, local factors. The significant correlation found between the upper and lower left second premolars suggests that occlusal factors may have a role to play.