Cell cycle effects and concomitant p53 expression in hairless murine skin after longwave UVA (365 nm) irradiation: a comparison with UVB irradiation

Ultraviolet A (UVA, 315-400 nm) radiation is known to be a complete carcinogen, but in contrast to UVB (280-315 nm) radiation, much of the cell damage is oxygen dependent (mediated through reactive oxygen species), and the dominant premutational DNA lesion(s) remains to be identified. To investigate further the basic differences in UVA and UVB carcinogenesis, we compared in vivo cellular responses, viz. cell cycle progression and transient p53 expression in the epidermis, after UVA1 (340-400 nm) exposure with those after broadband UVB exposure of hairless mice. Using flow cytometry we found a temporary suppression of bromodeoxyuridine (BrdU) uptake in S-phase cells both after UVB and UVA1 irradiation, which only in the case of UVB is followed by an increase to well over control levels. With equally erythemogenic doses (1-2 MED), the modulation of BrdU uptake was more profound after UVB than after UVA1 irradiation. Also, a marked transient increase in the percentage of S-phase cells occurred both after UVB and after UVA1 irradiation, but this increase evolved more rapidly after UVA1 irradiation. Further, p53 expression increased both after UVB and UVA1 irradiations, with peak expression already occurring from 12 to 24 h after UVA1 exposure and around 24 h after UVB exposure. Overall, UVA1 radiation appears to have less of an impact on the cell cycle than UVB radiation, as measured by the magnitude and duration of changes in DNA synthesis and cells in S phase. These differences are likely to reflect basic differences between UVB and UVA1 in genotoxicity and carcinogenic action.     

Cosmetic results of breast conserving therapy for breast carcinoma. Treatment results from the Heidelberg Radiation Clinic in the years 1984 to 1992]

We examined the effect of an antioxidant and protein kinase inhibitors on prostaglandin E2 (PGE2) release from Balb/c 3T3 mouse fibroblast cells induced by quinolone phototoxicity. Simultaneous administration of sparfloxacin (SPFX) or lomefloxacin (LFLX) at 12.5 to 100 microM and ultraviolet-A (UVA) irradiation for 10 min markedly elevated PGE2 concentration in the incubation medium, whereas levofloxacin (LVFX) at concentrations up to 100 microM and UVA irradiation did not increase PGE2 concentration. Pretreatment with 100 microM pyrrolidine dithiocarbamate (PDTC), an antioxidant, or 1 microM calphostin C, a selective inhibitor of protein kinase C (PKC), completely inhibited the elevation of PGE2 in the 24-h incubation medium; pretreatment with 10 microM H7, a cyclic nucleotide-dependent protein kinase, and PKC or 1 microM herbimycin A, a tyrosine kinase inhibitor, inhibited the PGE2 elevation by 29 to 39%. Conversely, 25 nM staurosporine significantly augmented the PGE2 elevation by quinolones plus UVA. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) were not detected in the incubation medium of 3T3 cells after quinolone plus UVA, corresponding to the lack of effect of antibodies against IL-1alpha, IL-1beta, and TNFalpha on PGE2 release from 3T3 cells. These results suggest that PGE2 production in 3T3 cells by quinolone phototoxicity is modulated by reactive oxygen species, PKC, and tyrosine kinase, but not by IL-1 or TNFalpha.     

Differential regulation of P53 and Bcl-2 expression by ultraviolet A and B

The induction of apoptosis by ultraviolet (UV) radiation and other DNA damaging agents plays a critical role in monitoring the accumulation of genetic damage and the suppression of tumor development. We hypothesize that UVA and UVB induce apoptosis by modulating balances between p53 and/or bcl-2 genes. Using MCF-7 cells that express both wild-type P53 and Bcl-2 proteins, we demonstrated that UVA and UVB induced apoptosis through regulating expression of apoptosis promoting or inhibiting genes. UVA induced immediate apoptosis and downregulated bcl-2 expression. Bcl-2 expression was reduced by approximately 40% at 4 h post-150 kJ UVA irradiation per m2 with a maximum downregulation (over 70%) at 24 h. The dose-response studies revealed that significant reduction of bcl-2 expression was observed at UVA doses ranging from 50 to 200 kJ per m2; however, p53 levels were not affected by UVA. In contrast, UVB exhibited a entirely different action than UVA in that UVB substantially induced p53 expression, but had no effect on bcl-2 expression. The induction of P53 by UVB was dose and time dependent with the maximum expression at 24 h post-2 and post-4 kJ UVB irradiation per m2. Down-regulation of bcl-2 and fragmentation of DNA induced by UVA occurred earlier (approximately at 4 h) than upregulation of p53 and DNA fragmentation by UVB (12-24 h). These results suggest that UVA and UVB cause cell damage through different mechanisms and that the balances between the expression of p53 and bcl-2 may play an important role in regulating the apoptosis induced by UV irradiation.     

GA strategy for variable selection in QSAR studies: GA-based PLS analysis of calcium channel antagonists

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.     

Immunotherapy of head and neck cancer

Understanding the mechanisms responsible for photodamage to the skin is most important for dermatology. 3-D cultures have been used as tools to mimic the in vivo situation for several years. We irradiated such a system containing human dermal fibroblasts cultured in collagen gels, a well-known model considered to be a dermal equivalent, which reproduces the interaction between cells and the surrounding extracellular matrix. The effects of solar irradiation (315-800 nm) on the steady-state levels of the mRNAs of extracellular matrix components (type I and III collagens) and their degrading enzymes (interstitial collagenase, MMP-1 and stromelysin 1, MMP-3) were measured. Exposure to low levels of solar radiation (0-10 J cm-2 in the UVA, i.e. suberythemal UVA doses) caused a transient decrease in type I procollagen mRNA, an increase in MMP-mRNA, and no change in type III procollagen mRNA steady-state levels. These results describe the early changes in the connective tissue of the skin following exposure to low-level solar stimulation, and may help explain the long-term changes in photodamaged skin.     

Adhesion of leukocytes to dermal endothelial cells is induced after single-dose, but reduced after repeated doses of UVA

Approximately 20-50% of ultraviolet A (UVA) irradiation delivered to the skin surface may reach the human dermal microvascular endothelial cells (HDMEC) that play a pivotal role in cellular inflammatory tissue; however, the pathophysiologic role of HDMEC in UVA-induced skin changes is largely unknown. Based on previous in vivo and in vitro studies revealing UVA-induced expression of endothelial adhesion molecules, we studied isolated HDMEC under various conditions in order to further delineate the impact of UVA on these cells. The expression of cell adhesion molecules was determined by flow cytometry and the resulting changes of stable adhesion of leukocytes to endothelial cells were quantitated for granulocytes, lymphocytes, and monocytes using a newly developed multicellular adhesion assay. Additionally, antibody blocking experiments were performed to delineate the role of individual cell adhesion molecules in UVA-induced leukocyte adherence. High-dose polychromatic UVA (25 J per cm2, maximal emission at 375 nm) induced intercellular adhesion molecule-1 and E-selectin with different kinetics but correlating the adhesion of leukocyte subsets. This effect subsided, however, in the course of 3-6 daily applied UVA doses. Moreover, pro-inflammatory cytokine challenge by tumor necrosis factor-alpha and interleukin-1-alpha resulted in significantly weaker induction of intercellular adhesion molecule-1 and E-selectin in repeatedly UVA-exposed HDMEC. Differential quantitation of peripheral blood derived granulocytes, lymphocytes, and monocytes revealed reduced adhesion particularly of lymphocytes followed by monocytes and granulocytes compared with leukocyte adhesion to nonirradiated but cytokine-stimulated HDMEC. It is concluded that UVA substantially influences endothelial cell adhesion molecules expression and thus directly interferes with leukocyte adhesion to endothelial cells. Divergent UVA-induced effects in this respect can be attributed to the mode of UV exposure as well as to the condition of endothelial cells prior to UVA exposure.     

Culture of chondrocytes in alginate and collagen carrier gels

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.     

Summary and recommendations for future research

A novel triblock copolymer of epsilon-caprolactone (CL) and ethylene oxide (E), CL6E90CL6, intended for use in implantable drug-delivery systems, has been subjected to gamma irradiation, in the solid state and in aqueous solution, under different controlled environmental conditions, to assess its stability to a radiation sterilization process. When copolymer matrices were irradiated with doses of irradiation up to 72 kGy in the presence of oxygen, negligible changes were observed in the molar mass, molecular mobility (assessed by pulsed nuclear magnetic resonance spectroscopy) and thermal properties. However, irradiation of matrices in the absence of oxygen (anoxia) induced the formation of cross-links, as indicated by a reduction in the molecular mobility of the copolymer, but without affecting its molar mass and thermal properties. Gamma irradiation of aqueous solutions of CL6E90CL6 in the presence of oxygen induced random polymer chain scission, as evidenced by a reduction in the molar mass, and the formation of a distribution of copolymer chain lengths in solution. Nuclear magnetic resonance relaxation studies showed that irradiation of solutions of CL6E90CL6 at concentrations greater than 4% w/v under anoxic conditions with doses of 54 kGy produced polymer gels with a network structure. These differences in the effects of gamma irradiation on the physicochemical properties of CL6E90CL6 might be germane to the method selected for sterilization of the polymer before its use in implantable drug-delivery systems.     

Continuous infusion of 5-fluorouracil plus low-dose cisplatin in tumor-bearing mice

We previously reported that the topical application of ascorbic acid 2-O-alpha-glucoside (AA-2G) suppressed the cutaneous inflammation by ultraviolet irradiation in human and guinea pigs (Miyai et al., Nishinihon J. Dermatol., 58, 439-443 (1996)). In this paper, the effect of AA-2G on the lethal damage induced by ultraviolet B (UVB) was studied using a human keratinocyte cell line, SCC, established from squamous cell carcinoma. The photoprotective effect of AA-2G on cytotoxicity of UVB in SCC cells was dose dependent (0.125-1 mM) and more effective than that of ascorbic acid (AsA) at 1 mM. This protection was completely abolished in the presence of an alpha-glucosidase inhibitor, castanospermine, indicating that release of AsA from this derivative was essential for reduction of the actinic injury. AA-2G significantly suppressed cytotoxicities of hydrogen peroxide and superoxide anion produced by xanthine and xanthine oxidase. AA-2G exhibited a preventive effect against the cytotoxicity produced by tert-butylhydroperoxide, an inducer of lipid peroxidation, in the presence of alpha-tocopherol, but not in the absence of alpha-tocopherol. Cytotoxicity of UVB was also effectively reduced by the combination of AA-2G and alpha-tocopherol. In addition, AA-2G reduced UVB-promoted formation of lipid peroxide and accumulation of lipofuscin, which is known to be a complex of cellular proteins and metabolites of lipid peroxide. These data suggest that AA-2G prevents the acute inflammation induced by UVB irradiation partly through scavenging reactive oxygen species and potentiating the antioxidative activity of alpha-tocopherol.     

To see but not to read; the magnocellular theory of dyslexia

Ultraviolet (UV) radiation induces cytokine release from cultured keratinocytes as well as from epidermis in vivo. The purpose of this study was to determine whether differentiation of cultured keratinocytes into stratified epithelium decreases the effects of UVA and UVB radiation on cytokine release. Interleukin-1 (IL-1) alpha, IL-1 beta and tumor necrosis factor (TNF)-alpha release from human keratinocytes and reconstituted human epidermis was measured after exposure to UVA or UVB radiation. Release of IL-1 alpha, IL-1 beta, and TNF-alpha was induced by both UVA and UVB radiation from both keratinocytes and reconstituted epidermis. Release of these cytokines was correlated with cytotoxicity. Keratinocyte cultures were far more sensitive to UVB radiation than reconstituted epidermis, in terms of both cytotoxicity and cytokine release. In contrast, epidermal stratification/differentiation had much less effect on the sensitivity to UVA radiation. We conclude that epidermal stratification and the formation of a stratum corneum provide protection against UVB radiation but have limited barrier effect against UVA radiation.     

Effect of the prenatal maternal environment on the control of breathing during non-rapid-eye-movement sleep in the developing lamb

The specific recognition of DNA modifications by repair endonucleases was used to characterize damage induced by 3-carbethoxypsoralen (3-CPs) plus UvA in M13mp8 replicative form I (RF-I) DNA. Under the conditions used, 3-CPs plus UVA generates DNA base modifications which are recognized by the UvrABC complex and the Fpg protein of E. coli. The rate of formation of UvrABC sensitive sites is 3-4-fold higher than that of Fpg sensitive sites. In addition a small number of sites of base loss (sensitive to Nfo protein) were observed. M13mp8 RF-I DNA treated with 3-CPs plus UVA was tested for transfection efficiency in E. coli mutants defective in either Fpg protein and/or UvrABC complex. The survival of 3-CPs plus UVA damaged M13mp8 RF-I DNA was significantly reduced when transfected into uvrA mutants compared to that in the wild-type strain. On the other hand, the survival of 3-CPs plus UVA damaged RF-I DNA was not altered in fpg-1 mutants. These results show that nucleotide excision repair mediated by the UvrABC complex is the major repair pathway involved in the elimination of lethal lesions induced in DNA by 3-CPs plus UVA. Our data suggest that in vitro exposure of M13mp8 RF-I DNA to 3-CPs plus UVA produces predominantly thymine photoaddition and to a lesser extent guanine photooxidation partially due to singlet oxygen generated during photoreaction. The photoaddition products are primarly responsible for the observed lethal effect.     

Molecular and cellular effects of ultraviolet light-induced genotoxicity

Exposure to the solar ultraviolet spectrum that penetrates the Earth's stratosphere (UVA and UVB) causes cellular DNA damage within skin cells. This damage is elicited directly through absorption of energy (UVB), and indirectly through intermediates such as sensitizer radicals and reactive oxygen species (UVA). DNA damage is detected as strand breaks or as base lesions, the most common lesions being 8-hydroxydeoxyguanosine (8OHdG) from UVA exposure and cyclobutane pyrimidine dimers from UVB exposure. The presence of these products in the genome may cause misreading and misreplication. Cells are protected by free radical scavengers that remove potentially mutagenic radical intermediates. In addition, the glutathione-S-transferase family can catalyze the removal of epoxides and peroxides. An extensive repair capacity exists for removing (1) strand breaks, (2) small base modifications (8OHdG), and (3) bulky lesions (cyclobutane pyrimidine dimers). UV also stimulates the cell to produce early response genes that activate a cascade of signaling molecules (e.g., protein kinases) and protective enzymes (e.g., haem oxygenase). The cell cycle is restricted via p53-dependent and -independent pathways to facilitate repair processes prior to replication and division. Failure to rescue the cell from replication block will ultimately lead to cell death, and apoptosis may be induced. The implications for UV-induced genotoxicity in disease are considered.     

Identification of a basophil leukocyte interleukin-3-regulated protein that is identical to IgE-dependent histamine-releasing factor

This study aimed to identify basophil leukocyte proteins associated with interleukin (IL)-3 and/or anti-IgE activation by two-dimensional (2-D) gel electrophoresis. We noticed one particular protein showing increased synthesis after recombinant human (rh)IL-3 and, to a lesser extent, anti-IgE stimulation. The protein was also present in the culture medium in increased amounts after rhIL-3 stimulation. On the basis of comigration with proteins in published 2-D gel electrophoresis databases and immunoblotting with a specific monoclonal antibody, we identified this protein as translationally controlled tumor protein (TCTP), also known as p23 or IgE-dependent histamine-releasing factor. The antibody was shown to be specific for TCTP/IgE-dependent histamine-releasing factor by blotting on 2-D gels of proteins from human lymphocytes and the human basophilic cell line KU812, followed by N-terminal amino-acid sequencing of the bound protein. Densitometric analysis of the gels showed that the synthesis of IgE-dependent histamine-releasing factor in human basophil leukocytes was dose dependent upon rhIL-3 stimulation with an optimum of 100 ng/ml. The level of the protein in the medium was also highest at an optimal rhIL-3 concentration of 100 ng/ml. Supernatants from cultured basophils were able to stimulate histamine release from other basophils. This histamine release was decreased by precipitation of TCTP/IgE-dependent histamine-releasing factor from these supernatants.     

Photohaptenic properties of fluoroquinolones

Although quinolone antibacterial agents have both phototoxicity and photoallergenicity, the latter's potency has been poorly investigated compared with the former's. Some of the photoallergic chemicals serve as photohaptens, which lead to T-cell-mediated immune reactions after photobinding to protein by UVA radiation. We examined the photohaptenic potential of fluoroquinolones, including lomefloxacin (LFLX), ciplofloxacin, norfloxacin, ofloxacin, levofloxacin, fleroxacin, enoxacin and sparfloxacin (SPFX). The absorption spectra of the quinolones were altered by UVA irradiation, with an exception of SPFX that seems to be photostable toward UVA. Bovine serum albumin and murine epidermal cells were coupled with these fluoroquinolones other than SPFX by exposure to UVA. Subcutaneous inoculation of fluoroquinolone-photomodified epidermal cells induced and elicited a delayed-type hypersensitivity reaction in mice. However, epidermal cells incubated with LFLX without UVA exposure also induced and elicited a significant hypersensitivity reaction to a lesser degree than LFLX-photomodified epidermal cells. Furthermore, there was cross-reactivity between LFLX-photomodified epidermal cells and simply LFLX-incubated cells. This suggests that cells can be weakly modified with LFLX even in the dark and that UVA irradiation promotes this modification. Our study demonstrated that fluoroquinolones have photohaptenic properties to which their photoallergenicity is probably ascribed.     

Photoprotective actions of natural and synthetic melanins

Melanins are thought to be important modulators of photochemistry in skin. Eumelanin, a black-brown pigment, is believed to protect against UV-induced photodamage, whereas pheomelanin, a red-yellow pigment, is believed to possess photosensitizing properties. To investigate the hypothesized dichotomy of melanins as both photoprotectants and photosensitizers, we examined the effects of melanins on UV-induced liposomal lipid peroxidation. Sepia melanin, a representative eumelanin, and both red hair pheomelanin and synthetic pheomelanin were employed in these studies. Both eumelanin and pheomelanin inhibited UVA/B- and UVA-induced liposomal lipid peroxidation in a concentration-dependent manner as measured by inhibition of conjugated diene formation. No change in protective properties of the melanins was observed in the presence of saturating levels of O2 during UVA irradiation. Pheomelanin irradiated with UVA/B or UVA induced superoxide-catalyzed reduction of nitroblue tetrazolium, whereas eumelanin did not. Melanins are known to bind various metals, and we examined the effect of iron on the photoproperties of melanins. Eumelanin complexed with Fe(III) did not inhibit UVA/B-induced lipid peroxidation, whereas pheomelanin complexed with Fe(III) stimulated UVA/B-induced lipid peroxidation. Thus, complexation with iron reversed the antioxidant effect of eumelanin and converted pheomelanin into a prooxidant. Analysis of lipid peroxidation products indicated that the oxidation was mediated by free radicals rather than by singlet oxygen. These data indicate that both eumelanin and pheomelanin exert antioxidant effects against UV-induced lipid peroxidation but that the prooxidant activities of pheomelanin result from pheomelanin-metal complexation.     

X-ray-activated UVA long persistent luminescence from defective fluoride elpasolites

Long persistent phosphors have received significant attention owing to their attractive photophysical properties.Here,we report a new long persistent phosphor exhibiting strong ultraviolet A(UVA)afterglow.The phosphors were synthesized by a solid-state reaction method.We find that the obtained phosphors demonstrate super long UVA-afterglow emissions after irradiation by X-ray source,and the afterglow can last more than 50 h.A wide range of experimental characterizations indicate that the Tb~(3+)doped fluoride elpasolite phosphors are defective and some fluoride ions are replaced by oxygen ions,which creates electron traps with suitable trap depths.Our results establish that Tb3+can act as optical emitters in wide-bandgap hosts that can result in the UVA afterglow.This work enriches the bank of UV long persistent phosphors,and may stimulate more efforts for the design and synthesis of this kind of optical materials.     

Proteins induced in Escherichia coli by benzoic acid

Proteins induced by benzoic acid in Escherichia coli were observed on two-dimensional electrophoretic gels (2-D gels). Cultures were grown in glucose-rich medium in the presence or absence of 20 mM benzoate at an external pH of 6.5, where the pH gradient (deltapH) is large and benzoate accumulates, and at an external pH of 8.0, where deltapH is inverted and little benzoate is taken up. Radiolabeled proteins were separated on 2-D gels and were identified on the basis of the index of VanBogelen and Neidhardt. In the absence of benzoic acid, little difference was seen between pH 6.5 and pH 8.0; this confirms that the mechanisms of protein homeostasis in this range are constitutive, including the transition between positive and inverted deltapH. Addition of benzoate at pH 6.5 increased the expression of 33 proteins. Twelve of the benzoate-induced proteins were induced at pH 8.0 as well, and nine of these matched proteins induced by the uncoupler dinitrophenol. Eighteen proteins were induced by benzoate only at pH 6.5, not at pH 8.0, and were not induced by dinitrophenol. One may be the iron and pH regulator Fur, which regulates acid tolerance in Salmonella spp. The other 13 proteins had not been identified previously. The proteins induced by benzoate only at a low pH may reflect responses to internal acidification or to accumulation of benzoate.     

Expression of neurofibromatosis 2 protein in human brain tumors: an immunohistochemical study

Examining damage (inactivation) of antioxidant enzymes in the cells and the pattern of recovery after a single UV exposure might be a useful method for analyzing the mechanisms of chronic UV irradiation, because chronic UV irradiation means repeated single exposures. We irradiated human skin fibroblasts with a single exposure to UVA (1, 6 or 12 J/cm2) and examined the time course of changes in antioxidant enzymes over several days. Only catalase activity was inactivated at the end of the irradiation (66% for 6 J/cm2, 53% for 12 J/cm2), recovering by day 5. Superoxide dismutase (SOD) activity decreased on day 3 (63% for 6 J/cm2, 72% for 12 J/cm2), and recovered on day 5, although it was not changed at the end of exposure. The activities of glutathione peroxidase (GSH-Px) and glutathione reductase (GR) were nearly unchanged by irradiation. If repeated UV exposures occur before the inactivated enzyme activities recover, cellular damage will be significant due to the lowered antioxidant defense system. We examined the rates of synthesis and degradation of catalase in response to UVA irradiation. Both synthesis and degradation rates were changed by irradiation. These data indicate that catalase activity was still low on day 2 due to the existence of inactivated catalase produced at the end of UV irradiation, and catalase activities recovered by day 5 due to a significant increase in the synthesis rate. To elucidate the role of bound NADPH in catalase in response to UV irradiation, we measured the NADPH released from catalase after UVA irradiation using bovine liver catalase. UVA irradiation caused a release of NADPH from catalase (25% for 12 J/cm2), but this was not directly related to the inactivation of catalase.     

Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells

Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control (psoSCR30) psoralen-conjugated TFOs in the presence and absence of UVA irradiation. The results demonstrated a 6- to 10-fold induction of supFG1 mutations in the psoAG30-treated cells as compared with psoSCR30-treated or untreated control cells. Interestingly, UVA irradiation had no effect onthe mutation frequencies induced by the psoralen-conjugated TFOs, suggesting a triplex-mediated but photoproduct-independent process of mutagenesis. Sequencing data were consistent with this finding since the expected T.A-->A.T transversions at the predicted psoralen crosslinking site were not detected. However, insertions and deletions were detected within the triplex binding site, indicating a TFO-specific induction of mutagenesis. This result demonstrates the ability of triplex-forming oligonucleotides to influence mutation frequencies at a specific site in a mammalian chromosome.     

First steps from a two-dimensional protein index towards a response-regulation map for Bacillus subtilis

Data on the identification of proteins of Bacillus subtilis on two-dimensional (2-D) gels as well as their regulation are summarized and the identification of 56 protein spots is included. The pattern of proteins synthesized in Bacillus subtilis during exponential growth, during starvation for glucose or phosphate, or after the imposition of stresses like heat shock, salt- and ethanol stress as well as oxidative stress was analyzed. N-terminal sequencing of protein spots allowed the identification of 93 proteins on 2-D gels, which are required for the synthesis of amino acids and nucleotides, the generation of ATP, for glycolyses, the pentose phosphate cycle, the citric acid cycle as well as for adaptation to a variety of stress conditions. A computer-aided analysis of the 2-D gels was used to monitor the synthesis profile of more than 130 protein spots. Proteins performing housekeeping functions during exponential growth displayed a reduced synthesis rate during stress and starvation, whereas spots induced during stress and starvation were classified as specific stress proteins induced by a single stimulus or a group of related stimuli, or as general stress proteins induced by a variety of entirely different stimuli. The analysis of mutants in global regulators was initiated in order to establish a response regulation map for B. subtilis. These investigations demonstrated that the alternative sigma factor sigma B is involved in the regulation of almost all of the general stress proteins and that the phoPR two-component system is required for the induction of a large part but not all of the proteins induced by phosphate starvation.