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D L Mitchell

Publications and source records attributed to D L Mitchell.

At least 19 recordsLinked to original sources
PubMed2001

Identification of a non-dividing subpopulation of mouse and human epidermal cells exhibiting high levels of persistent ultraviolet photodamage.

The distribution and persistence of cyclobutane pyrimidine dimers were investigated in mouse skin after chronic and acute exposures to ultraviolet-B radiation. We found that DNA damage accumulated in response to chronic irradiation and persisted in a unique set of epidermal cells located at the basal layer. Treatment with a tumor promoter caused the heavily damaged epidermal cells to divide and p53-immunopositive clusters to form within 24 h suggesting that these cells may be progenitors of the mutant p53 clusters associated with actinic keratoses and squamous cell carcinomas. In contrast to low fluence chronic irradiation, daily treatment with a higher fluence of ultraviolet-B produced extensive hyperplasia and considerably reduced penetration of photodamage. Exposure of chronically irradiated skin to an acute "sunburn dose" of ultraviolet-B also produced significant epidermal hyperplasia and resulted in complete loss of heavily damaged basal cells within 4 d postirradiation. The occurrence and distribution of cyclobutane dimers in human skin correlated well with putative sunlight exposure and resembled that observed in ultraviolet-B-irradiated mice. Heavily damaged basal cells were observed at various sites, including those receiving sporadic sunlight exposure, suggesting that these cells may play an important role in carcinoma formation in humans.

Animals↗
PubMed2001

Effects of chronic exposure to ultraviolet B radiation on DNA repair in the dermis and epidermis of the hairless mouse.

It has previously been shown that chronic exposure to low fluences of ultraviolet B radiation reduced DNA repair capacity in mouse skin. In this study we now extend this to examine the concentration dependence and tissue dependence of this phenomenon. We found that (6-4) photoproducts were repaired considerably faster than cyclobutane dimers and that the kinetics for photoproduct removal were comparable in the dermis and epidermis. Chronic ultraviolet B irradiation significantly reduced the initial rate and extent of DNA repair. After low daily doses of ultraviolet B (6-4) photoproduct repair was most affected and after high daily doses the repair of both cyclobutane and (6-4) dimers was reduced. Whereas cyclobutane dimer repair was most affected in the dermis, reduced (6-4) photoproduct repair was observed in both tissues. The deleterious effects of chronic ultraviolet exposure were sustained for a considerable time after the chronic treatment ended.

Animals↗
PubMed2001

Might within the madness: solution-focused therapy and thought-disordered clients.

Nurses working with thought-disordered clients in inpatient psychiatric settings may find that much of their role is defined by the administration and monitoring of antipsychotic medications. Therefore, a challenge for these nurses can be to find other nursing interventions for these clients that are effective, efficient, and clearly and uniquely within the scope of nursing. In response to this challenge, this article presents the use of solution-focused therapy (SFT) to help thought-disordered clients better cope with some of their negative experiences and symptomatology. The article provides an overview of SFT, with a focus on how these techniques might be used on an inpatient psychiatry setting with clients experiencing thought disorders. The authors include three case studies demonstrating the use of SFT with clients experiencing thought disorders, and conclude with some of the lessons they have learned using SFT techniques with these kinds of clients in inpatient psychiatric settings.

Adult↗
PubMed2001

Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter.

The relationship between purified transcription factor p50 binding and ultraviolet light-induced DNA damage formation in the NF-kappa B promoter element was investigated. The effect of bound transcription factor on cyclobutane dimer formation was quantified using Maxam-Gilbert analysis of irradiated substrate digested with T4 phage endonuclease V. Two methods were employed for cleaving (6-4) photoproducts. Sites of (6-4) photoproducts cleaved by piperidine showed a general suppression in the presence of bound p50 protein similar to that observed for cyclobutane dimers. In contrast to piperidine, digestion with ultraviolet damage endonuclease (UVDE) from Saccharomyces pombe subsequent to cyclobutane dimer reversal by photolyase displayed a broader spectrum of damaged sites. Whereas some of these sites were suppressed by bound p50 protein, some remained unaffected and one site showed increased (6-4) photoproduct induction. These data illustrate the advantage of UVDE over piperidine for studying (6-4) photoproducts at the sequence level and suggest that this approach may be useful for footprinting transcription factor binding in other promoters.

Base Sequence↗
PubMed2001

Attenuation of DNA damage in the dermis and epidermis of the albino hairless mouse by chronic exposure to ultraviolet-A and -B radiation.

Mammalian skin is vulnerable to the photocarcinogenic and photoaging effects of solar UV radiation and defends itself using a variety of photoprotective responses including epidermal thickening, tanning and the induction of repair and antiradical systems. We treated Skh-1 albino hairless mice for 60 days with ultraviolet-A (UVA) or ultraviolet-B (UVB) radiation and measured the frequency of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts induced by a single acute sunburn dose of UVB at different stages of the chronic treatment. We found that both UVA and UVB exposure produced a photoprotective response in the dermis and epidermis and that the degree of photoproduct attenuation was dependent on dose, wavelength and the type of damage induced. Although epidermal thickening was important, our data suggest that UV protective compounds other than melanin may be involved in mitigating the damaging effects of sunlight in the skin.

Animals↗
PubMed2001

Repeated exposures of human skin equivalent to low doses of ultraviolet-B radiation lead to changes in cellular functions and accumulation of cyclobutane pyrimidine dimers.

Chronic exposure to sunlight may induce skin damage such as photoaging and photocarcinogenesis. These harmful effects are mostly caused by ultraviolet-B (UVB) rays. Yet, less is known about the contribution of low UVB doses to skin damage. The aim of this study was to determine the tissue changes induced by repeated exposure to a suberythemal dose of UVB radiation. Human keratinocytes in monolayer cultures and in skin equivalent were irradiated daily with 8 mJ/cm2 of UVB. Then structural, ultrastructural, and biochemical alterations were evaluated. The results show that exposure to UVB led to a generalized destabilization of the epidermis structure. In irradiated skin equivalents, keratinocytes displayed differentiated morphology and a reduced capacity to proliferate. Ultrastructural analysis revealed, not only unusual aggregation of intermediate filaments, but also disorganized desmosomes and larger mitochondria in basal cells. UVB irradiation also induced the secretion of metalloproteinase-9, which may be responsible for degradation of type IV collagen at the basement membrane. DNA damage analysis showed that both single and repeated exposure to UVB led to formation of (6-4) photoproducts and cyclobutane pyrimidine dimers. Although the (6-4) photoproducts were repaired within 24 h after irradiation, cyclobutane pyrimidine dimers accumulated over the course of the experiment. These studies demonstrate that, even at a suberythemal dose, repeated exposure to UVB causes significant functional and molecular damage to keratinocytes, which might eventually predispose to skin cancer.

Adult↗
PubMed2000

Repair of UV damage in plants by nucleotide excision repair: Arabidopsis UVH1 DNA repair gene is a homolog of Saccharomyces cerevisiae Rad1.

To analyze plant mechanisms for resistance to UV radiation, mutants of Arabidopsis that are hypersensitive to UV radiation (designated uvh and uvr) have been isolated. UVR2 and UVR3 products were previously identified as photolyases that remove UV-induced pyrimidine dimers in the presence of visible light. Plants also remove dimers in the absence of light by an as yet unidentified dark repair mechanism and uvh1 mutants are defective in this mechanism. The UVH1 locus was mapped to chromosome 5 and the position of the UVH1 gene was further delineated by Agrobacterium-mediated transformation of the uvh1-1 mutant with cosmids from this location. Cosmid NC23 complemented the UV hypersensitive phenotype and restored dimer removal in the uvh1-1 mutant. The cosmid encodes a protein similar to the S. cerevisiae RAD1 and human XPF products, components of an endonuclease that excises dimers by nucleotide excision repair (NER). The uvh1-1 mutation creates a G to A transition in intron 5 of this gene, resulting in a new 3' splice site and introducing an in-frame termination codon. These results provide evidence that the Arabidopsis UVH1/AtRAD1 product is a subunit of a repair endonuclease. The previous discovery in Lilium longiflorum of a homolog of human ERCC1 protein that comprises the second subunit of the repair endonuclease provides additional evidence for the existence of the repair endonuclease in plants. The UVH1 gene is strongly expressed in flower tissue and also in other tissues, suggesting that the repair endonuclease is widely utilized for repair of DNA damage in plant tissues.

Amino Acid Sequence↗
PubMed2000

Sterilization of reusable implant components: a pilot study.

STATEMENT OF PROBLEM: The placement and restoration of dental implants require the use of numerous reusable instruments and components. The adequate sterilization of reusable instruments and components is essential to prevent cross contamination between patients. Sterilization usually is accomplished with single-use sterilization envelopes. A reusable sterilization vehicle would reduce costs as well as the waste generated in patient care. PURPOSE: This study was designed to determine the efficacy of a 10-cc Pyrex test tube as a sterilization vehicle for reusable dental implant instruments and components. MATERIAL AND METHODS: In this study, a reusable dental implant component was placed in a Pyrex test tube, along with a biologic test strip. A control biologic test strip was kept for each test tube. The test tube was closed with a cotton roll folded in half and placed in the opening. Twenty test tubes were prepared. five sets of 4 test tubes were placed in an autoclave in different locations with varying orientations. The autoclave completed a standard sterilization cycle. RESULTS: The biological monitoring service indicated that the biologic test strips in 100% of the test tubes were sterile, whereas the control strips were 100% nonsterile. CONCLUSION: A Pyrex test tube sealed with a cotton roll can serve as a sterilization vehicle for reusable dental implant instruments and components.

Bacillus subtilis↗
PubMed2000

The Arabidopsis UVH1 gene is a homolog of the yeast repair endonuclease RAD1.

Ultraviolet radiation induces DNA damage products, largely in the form of pyrimidine dimers, that are both toxic and mutagenic. In most organisms, including Arabidopsis, these lesions are repaired both through a dimer-specific photoreactivation mechanism and through a less efficient light-independent mechanism. Several mutants defective in this "dark repair" pathway have been previously described. The mechanism of this repair has not been elucidated, but is thought to be homologous to the nucleotide excision repair mechanisms found in other eukaryotes. Here we report the complementation of the Arabidopsis uvh1 dark repair mutant with the Arabidopsis homolog of the yeast nucleotide excision repair gene RAD1, which encodes one of the subunits of the 5'-repair endonuclease. The uvh1-2 mutant allele carries a glycine-->aspartate amino acid change that has been previously identified to produce a null allele of RAD1 in yeast. Although Arabidopsis homologs of genes involved in nucleotide excision repair are readily identified by searching the genomic database, it has not been established that these homologs are actually required for dark repair in plants. The complementation of the Arabidopsis uvh1 mutation with the Arabidopsis RAD1 homolog clearly demonstrates that the mechanism of nucleotide excision repair is conserved among the plant, animal, and fungal kingdoms.

Amino Acid Sequence↗
PubMed2000

Effects of cytosine methylation on pyrimidine dimer formation in DNA.

The relative induction of cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4)pyrimidone photoproducts ([6-4]PD) was quantified in the duplex homopolymers polydeoxyadenosine:polydeoxythymidine, polydeoxyguanosine:polydeoxycytidine and polydeoxyguanosine:polydeoxy-5-methylcytidine irradiated with UVC or UVB radiation. Cytosine methylation significantly increased the yield of cytosine (6-4)PD after irradiation with UVC light and of cytosine CPD and (6-4)PD after irradiation with UVB light. The data suggest that CPD and (6-4)PD are preferentially induced at 5-methylcytosine bases in DNA of cells exposed to sunlight and comprise a major component of the mutation spectrum leading to the initiation of sunlight-induced skin cancer.

Cytosine↗
PubMed2000

Induction, distribution and repair of UV photodamage in the platyfish, Xiphophorus signum.

The genus Xiphophorus is an important model for investigating the etiology and genetics of sunlight-induced melanoma as well as other cancers. We used immunological techniques to determine the induction, distribution and repair of cyclobutane pyrimidine dimers (CPD) and pyrimidine(6-4)pyrimidone dimers ([6-4]PD) in different tissues of Xiphophorus signum exposed to ultraviolet-B light. We found that the (6-4)PD was induced at 5 to 10-fold lower frequency than the CPD and that scalation provided considerable photoprotection against both photoproducts. Photoenzymatic repair (PER) was very efficient in X. signum with most of the lesions removed within 20 min; PER of CPD occurred at about twice the rate of (6-4)PD. Nucleotide excision repair (NER) was much less efficient than PER and the rates of CPD and (6-4)PD removal were comparable. PER was more efficient in the caudal fin compared to the lateral epidermis; the opposite was true for NER. Although the initial rate of CPD excision was five-fold faster in the lateral epidermis compared to the caudal fin a considerable amount of residual damage remained in both tissues. The diverse photochemical and photobiological responses observed in X. signum suggest that heritable traits governing deoxyribonucleic acid damage induction and repair may be involved in the susceptibility of other Xiphophorus species to melanomagenesis.

Animals↗
PubMed2000

The dose dependence of cyclobutane dimer induction and repair in UVB-irradiated human keratinocytes.

UVB and UVA components of the solar spectrum or from artificial UV-sources might be important etiological factors for the induction and development of skin cancer. In particular, deficiencies in the capacity to repair UV-induced DNA-lesions have been linked to this phenomenon. However, until now only limited data are available on the biological and physical parameters governing repair capacity. We have, therefore, developed a flowcytometric assay using fluorescence-labeled monoclonal antibodies to study the dose-dependence of induction and repair of UVB-induced cyclobutane pyrimidine dimers in a spontaneously immortalized keratinocytic cell line (HaCaT). Our results show that the kinetics of recognition and incision of UVB-induced DNA lesions slows down by a factor of about 3 in a dose range of 100-800 J m-2. Furthermore, a thorough analysis of repair kinetics indicates that this reduction in repair capacity might not be dependent on saturation of enzymatic repair capacity (Michaelis-Menten) but may be caused by a UV-induced impairment of enzymes involved in DNA repair. Because this effect is evident in vitro at doses comparable to the minimal erythemal dose in vivo, our results might have significant impact on risk assessment for UV-induced carcinogenesis.

Cell Line↗
PubMed2000

Common fluorescent sunlamps are an inappropriate substitute for sunlight.

Fluorescent sunlamps are commonly employed as convenient sources in photobiology experiments. The ability of Kodacel to filter photobiologically irrelevant UVC wavelengths has been described. Yet there still remains a major unaddressed issue--the over representation of UVB in the output. The shortest terrestrial solar wavelengths reaching the surface are approximately 295 nm with the 295-320 nm range comprising approximately 4% of the solar UV irradiance. In Kodacel-filtered sunlamps, 47% of the UV output falls in this range. Consequently, in studies designed to understand skin photobiology after solar exposure, the use of these unfiltered sunlamps may result in misleading, or even incorrect conclusions. To demonstrate the importance of using an accurate representation of the UV portion of sunlight, the ability of different ultraviolet radiation (UVR) sources to induce the expression of a reporter gene was assayed. Unfiltered fluorescent sunlamps (FS lamps) induce optimal chloramphenicol acetyltransferase (CAT) activity at apparently low doses (10-20 J/cm2). Filtering the FS lamps with Kodacel raised the delivered dose for optimal CAT activity to 50-60 mJ/cm2. With the more solar-like UVA-340 lamps somewhat lower levels of CAT activities were induced even though the apparent delivered doses were significantly greater than for either the FS or Kodacel-filtered sunlamp (KFS lamps). When DNA from parallel-treated cells was analyzed for photoproduct formation by a radioimmuneassay, it was shown that the induction of CAT activity correlated with the level of induced photoproduct formation regardless of the source employed.

Animals↗
PubMed2000

The efficacy of a broad-spectrum sunscreen to protect engineered human skin from tissue and DNA damage induced by solar ultraviolet exposure.

Sunscreens are known to protect against sunlight-induced erythema and sunburn, but their efficiency at protecting against skin cancer is still a matter of debate. Specifically, the capacity of sunscreens to prevent or reduce tissue and DNA damage has not been thoroughly investigated. The present study was undertaken to assess the ability of a chemical broad-spectrum sunscreen to protect human skin against tissue and DNA damage after solar UV radiation. Engineered human skin was generated and either treated or not with a broad-spectrum SPF 30 sunscreen and exposed to increasing doses of simulated sunlight (SSL). Immediately after irradiation, histological, immunohistochemical, and molecular quantitative analyses were performed. The unprotected irradiated engineered human skin showed significant epidermal disorganization accompanied by a complete absence of laminin deposition. The sunscreen prevented SSL-induced epidermal damage at low doses and allowed laminin deposition at almost all SSL doses tested. The frequencies of cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts, and photooxidative lesions measured by alkaline gel electrophoresis and radioimmunoassay were significantly reduced by the sunscreen. Thus, tissue and DNA damage may provide excellent quantitative end points for assessing the photoprotective efficacy of sunscreens.

Adolescent↗
PubMed1999

Effect of oxidative DNA damage in promoter elements on transcription factor binding.

Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters.

Base Sequence↗
PubMed1999

Effects of chronic low-dose ultraviolet B radiation on DNA damage and repair in mouse skin.

Chronic exposure to sunlight causes skin cancer in humans, yet little is known about how habitual exposure to low doses of ultraviolet B radiation (UVB) affects DNA damage in the skin. We treated Skh-1 hairless mice with daily doses of suberythemal UVB for 40 days and analyzed the amount and distribution of DNA photodamage using RIAs and immunofluorescence micrography. We found that DNA damage accumulated in mouse skin as a result of chronic irradiation and that this damage persisted in the dermis and epidermis for several weeks after the chronic treatment was terminated. Although the persistent damage was evenly distributed throughout the dermis, it remained in the epidermis as a small number of heavily damaged cells at the dermal-epidermal boundary. Rates of DNA damage induction and repair were determined at different times over the course of chronic treatment in response to a higher challenge dose of UVB light. The amount of damage induced by the challenge dose increased in response to chronic exposure, and excision repair of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone dimers was significantly reduced. The sensitization of mouse epidermal DNA to photoproduct induction, the reduction in excision repair, and the accumulation of nonrepairable DNA damage in the dermis and epidermis suggest that chronic low-dose exposure to sunlight may significantly enhance the predisposition of mammalian skin to sunlight-induced carcinogenesis.

Animals↗
PubMed1999

DNA repair and survival in human lens epithelial cells with extended lifespan.

PURPOSE: Ultraviolet-B radiation (290-320 nm) produces cataracts in animals and has been associated with human cataract formation in several epidemiological studies. UVB radiation decreases the long-term cell survival and changes the pattern of protein synthesis in cultured lens epithelial cells. However, the relationship between DNA photoproduct formation and long term cell survival in human lens epithelial cells is not known. In the present work, we used human lens epithelial cells with extended lifespan (HLE B-3 cells) to examine the kinetics of DNA repair and cell survival after UVB exposure. METHODS: Cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts were analyzed by radioimmunoassay. Long-term survival of the cells was determined by measuring their ability form colonies when plated at low density. RESULTS: HLE B-3 cells were repair competent after UVB (302 nm) exposure. Excision repair of the (6-4) photoproduct was more efficient than that of the cyclobutane dimer. Ninety five percent of the (6-4) photoproducts were repaired 24 h after 400 J/m2 UVB exposure, whereas 50% of the cyclobutane dimers were repaired during this time. When cells were split for the clonogenic assay immediately after irradiation, only 10% of the cells formed colonies following 7 days of culture in the serum-containing medium. When cells were split for the clonogenic assay after a 48 hour incubation in serum-containing medium, the colony-forming ability of the irradiated cells increased to 60% following culture in a serum-containing medium. CONCLUSIONS: These results indicate a close correlation between the repair of cyclobutane dimers and the increase in the long-term survival of the cells as measured by their colony-forming ability. The extended lifespan human lens epithelial cells HLE B-3 may be a useful model to investigate the mechanism and regulation of UVB-induced DNA repair in human lens cells.

Cell Survival↗