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Biomedical subjects

J C Sutherland

Publications and source records attributed to J C Sutherland.

At least 37 records · Page 2Linked to original sources
PubMed1992

Quantitation of pyrimidine dimers in DNA from UVB-irradiated alfalfa (Medicago sativa L.) seedlings.

Depletion of stratospheric ozone will increase the solar ultraviolet radiation in the range from 290-320 nm (UVB) that reaches the surface of the earth, placing an increased UV burden on exposed organisms. One consequence of increased UVB may be decreased productivity of crop plants. A principal lesion caused by UV in DNA is the cyclobutyl pyrimidine dimer. We have adapted a method for measuring these dimers in nanogram quantities of non-radioactive DNA for use in UV-irradiated plants. We find that biologically relevant doses of broad band UVB radiation induce easily detectable frequencies of pyrimidine dimers in the DNA of irradiated alfalfa sprout leaves and that the dose response for dimer formation is linear up to doses of at least 690 J m-2. We also find easily measurable frequencies of dimers in the leaves of seedlings grown in glass filtered sunlight but not exposed to additional UVB, suggesting that significant numbers of dimers are formed in plants exposed to normal sunlight.

DNA↗
PubMed1991

Vacuum UV CD spectra of homopolymer duplexes and triplexes containing A.T or A.U base pairs.

Vacuum UV circular dichroism (CD) spectra were measured down to 174 nm for five homopolymers, five duplexes, and four triplexes containing adenine, uracil, and thymine. Near 190 nm, the CD bands of poly[d(A)] and poly[r(A)] were larger than the CD bands of the polypyrimidines, poly[d(T)], poly[d(U)], and poly[r(U)]. Little change was observed in the 190 nm region upon formation of the duplexes (poly[d(A).d(T)], poly[d(A).d(U)], poly[r(A).d(T)], poly[r(A).d(U)], and poly[r(A).r(U)]) or upon formation of two of the triplexes (poly[d(T).d(A).d(T)] and poly[d(U).d(A).d(U)]). This showed that the purine strand had the same or a similar structure in these duplexes and triplexes as when free in solution. Both A.U and A.T base pairing induced positive bands at 177 and 202 nm. For three triplexes containing poly[d(A)], the formation of a triplex from a duplex and a free pyrimidine strand induced a negative band centered between 210 and 215 nm. The induction of a band between 210 and 215 nm indicated that these triplexes had aspects of the A conformation.

Base Composition↗
PubMed1991

Effects of electrocautery on midline laparotomy wound infection.

This study compared the healing of midline fascial incisions made with either scalpel or electrocautery and inoculated with Escherichia coli in 57 Sprague-Dawley rats. At 7 days, tensile strength was significantly less when incisions were made with electrocautery than with a scalpel. Additionally, would strength was inversely related to the concentration of the inoculum of E coli. The use of electrocautery was also associated with more frequent bacteremia at 48 hours and higher mortality at 7 days. Our results suggest that the technique used to incise the abdominal fascia influences subsequent wound healing, particularly in contaminated wounds.

Animals↗
PubMed1991

Quantitative gel electrophoresis of DNA: resolution of overlapping bands of restriction endonuclease digests.

Accurate quantitation of the distribution of DNA digested to completion by a restriction endonuclease and separated in an electrophoretic gel is greatly facilitated by electronic imaging systems employing charge-coupled device detectors. Such quantitation permits the extraction of more accurate values of the lengths of both the parent molecular species and the fragments produced by a restriction endonuclease. We describe a method that determines the number of components in bands containing two or more daughter fragments that are of similar length and hence not resolved by gel electrophoresis. We also report a constrained deconvolution procedure based on the equal number of daughter molecules in a terminal digestion by a restriction endonuclease that resolves the distribution of each of the overlapping species in a composite band on a gel and hence increases the accuracy of the lengths assigned to each.

Calibration↗
PubMed1991

Do pyrimidine dimer yields correlate with erythema induction in human skin irradiated in situ with ultraviolet light (275-365 nm)?

Ultraviolet radiation produces erythema in human skin, and damages the DNA of living cells in skin. Previous work showed that broad-band UV-B (290-320 nm) radiation produced higher levels of cyclobutyl pyrimidine dimers in DNA of individuals with high UV-B sensitivity (low minimal erythema dose) than in subjects of low UV-B sensitivity [Freeman et al. (1986) J. Invest. Dermatol., 86, 34-36]. We examined the relationship between erythema induction and dimer yields in DNA of human skin irradiated in situ with narrow band radiation spanning the wavelength range 275-365 nm. We find that, in general, higher dimer yields are produced per incident photon in volunteers with higher susceptibility to erythema induced by radiation of the same wavelength.

Adult↗
PubMed1990

The vacuum UV CD bands of repeating DNA sequences are dependent on sequence and conformation.

CD spectra were obtained for eight synthetic double-stranded DNA polymers down to at least 175 nm in the vacuum uv. Three sets of sequence isomers were studied: (a) poly[d(A-C).d(G-T)] and poly[d(A-G).d(C-T)], (b) poly[d(A-C-C).d(G-G-T)] and poly[d(A-C-G).d(C-G-T)], and (c) poly[d(A).d(T)], poly[d(A-T).d(A-T)], poly[d(A-A-T).d(A-T-T)], and poly[d(A-A-T-T).d(A-A-T-T)]. There were significant differences in the CD spectra at short wavelengths among each set of sequence isomers. The (G.C)-containing sequences had the largest vacuum uv bands, which were positive and in the wavelength range of 180-191 nm. There were no large negative bands at longer wavelengths, consistent with the polymers all being in right-handed conformations. Among the set of sequences containing only A.T base pairs, poly[d(A).d(T)] had the largest vacuum uv CD band, which was at 190 nm. This CD band was not present in the spectra of the other (A.T)-rich polymers and was absent from two first-neighbor estimations of the poly[d(A).d(T)] spectrum obtained from the other three sequences. We concluded that the sequence dependence of the vacuum uv spectra of the (A.T)-rich polymers was due in part to the fact that poly[d(A).d(T)] exists in a noncanonical B conformation.

Base Composition↗
PubMed1990

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1990

Does exposure of human skin in situ to 385 or 405 nm UV induce pyrimidine dimers in DNA?

A previous report [Freeman et al. (1986) Photochem. Photobiol. 43S, 93S] indicated that irradiation of human skin in situ with 385 or 405 nm radiation produced detectable levels of pyrimidine dimers in DNA. Since these wavelengths are absorbed poorly by DNA, these results suggested that DNA damage was sensitized by other absorbing molecules present in skin. Examination of two experimental aspects of the previous work indicates that (1) the static gel electrophoresis method for DNA dispersion used in lesion determination gave accurate values of the levels of induced dimers, and (2) the DNA damage apparently induced by 385 nm was actually induced by shorter wavelength UV present in the 20 nm bandpass beam of the monochromator. The current results indicate that monochromatic 385 and 405 nm radiation are ineffective in dimer production in human skin in situ.

Adult↗
PubMed1989

Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ with ultraviolet light.

The UV components of sunlight are believed to be a major cause of human skin cancer, and DNA is thought to be the principal molecular target. Alterations of the intensity and wavelength distribution of solar UV radiation reaching the surface of the earth, for example by depletion of stratospheric ozone, will change the effectiveness of solar radiation in damaging DNA in human skin. Evaluation of the magnitude of such effects requires knowledge of the altered sunlight spectrum and of the action spectrum for damaging DNA in human skin. We have determined an action spectrum for the frequency of pyrimidine dimer formation induced in the DNA of human skin per unit dose of UV incident on the skin surface. The peak of this action spectrum is near 300 nm and decreases rapidly at both longer and shorter wavelengths. The decrease in our action spectrum for wavelengths less than 300 nm is attributed to the absorption of the upper layers of the skin, an in situ effect that is inherently included in our measurements. Convolution of the dimer action spectrum with the solar spectra corresponding to a solar angle of 40 degrees under current levels of stratospheric ozone (0.32-cm O3 layer) and those for 50% ozone depletion (0.16-cm O3 layer), indicate about a 2.5-fold increase in dimer formation. If the action spectrum for DNA damage that results in skin cancer resembles that for dimer induction in skin, our results, combined with epidemiological data, suggest that a 50% decrease in stratospheric ozone would increase the incidence of nonmelanoma skin cancers among white males in Seattle, Washington, by 7.5- to 8-fold, to a higher incidence than is presently seen in the corresponding population of Albuquerque, New Mexico.

Adult↗
PubMed1989

Excited state properties of the N-(deoxyguanosin-8-yl)-2-aminofluorene adducts.

The spectroscopic characteristics of adducts derived from the covalent binding of the carcinogen 2-aminofluorene to the C8 position of deoxyguanosine [N-(deoxyguanosin-8-yl)-2-amino-fluorene, dGuo-C8-AF], and from an adduct of similar structure formed with the synthetic polynucleotide poly(dG-dC).poly(dG-dC), were investigated. At 77 K both adducts are characterized by well-defined and rather narrow fluorescence emission spectra with maxima at 370 and 390 nm characteristic of the aromatic, monomolecular 2-aminofluorene (AF) residue. In contrast, at room temperature, the fluorescence is characterized by a broad, structureless emission band with a maximum at 460 nm in aqueous mixtures, shifting to 415 nm in solvents of lower polarity (100% propanol); the maxima are located at intermediate wavelengths in solutions of different propanol/water compositions, and this emission is attributed to an excited state complex (exciplex). The fluorescence quantum yield decreases when either the solvent polarity or the temperature are increased, varying from 5.4% (100% propanol) to 0.04-0.05% (100% H2O). The fluorescence decay profiles of dGuo-C8-AF adducts (measured at the National Synchrotron Light Source facility at the Brookhaven National Laboratory) can be roughly, but not exactly, modeled in terms of two exponential decay components in the range of about 0.3-1.0 ns with the propanol concentration greater than 60%; at lower propanol concentrations, a single short lifetime is observed and in 100% water solutions its value is 0.08 ns. The shorter lifetime, favored in solvent mixtures of higher polarities, is attributed to an exciplex with significant charge-transfer character.(ABSTRACT TRUNCATED AT 250 WORDS)

2-Acetylaminofluorene↗
PubMed1987

Unidirectional pulsed-field electrophoresis of single- and double-stranded DNA in agarose gels: analytical expressions relating mobility and molecular length and their application in the measurement of strand breaks.

Unidirectional pulsed-field electrophoresis improves the separation of single-stranded DNA molecules longer than 20 kilobases (kb) in alkaline agarose gels compared to static-field electrophoresis. The greatest improvement in separation is for molecules longer than 100 kb. The improved resolution of long molecules with unidirectional pulsed-field electrophoresis makes possible the measurement of lower frequencies of single-strand breaks. The analytical function that relates the length and mobility of single-stranded DNA electrophoresed with a static field also applies to unidirectional pulsed field separations. Thus, the computer programs used to measure single-strand breaks are applicable to both undirectional pulsed- and static-field separations. Unidirectional pulsed-field electrophoresis also improves the separation of double-stranded DNA in neutral agarose gels. The function relating molecular length and mobility for double-stranded DNA separated by unidirectional pulsed-field electrophoresis is a superset of the function for single-stranded DNA. The coefficients of this function can be determined by iterative procedures.

DNA↗
PubMed1987

Electronic imaging system for direct and rapid quantitation of fluorescence from electrophoretic gels: application to ethidium bromide-stained DNA.

We have built an electronic imaging system based on a modified charge-coupled-device television camera that directly quantitates the distribution of fluorescence from electrophoretic gels, chromatograms, and other stationary sources. Exposure times can exceed 1 min. Unlike the photographic system that it replaces, the response of the camera is directly proportional to the intensity of incident fluorescence, and image data are digitized and stored in computer memory ready for analysis immediately upon completion of an exposure. We describe procedures for the display, normalization, and archival storage of image data and programs that use images of ethidium bromide-stained DNA in alkaline agarose gels to quantitate single-strand breaks in DNA.

DNA↗
PubMed1987

Production of pyrimidine dimers in DNA of human skin exposed in situ to UVA radiation.

Cyclobutyl pyrimidine dimers, measured as sites recognized by the dimer-specific ultraviolet (UV) endonuclease from Micrococcus luteus, were produced in DNA of human skin exposed in situ to UVA (320-400 nm) radiation. The dimer yields produced by a broadband UVA source, by broadband UVA filtered to remove all light of wavelength less than 340 nm, and by narrow band radiation centered at 365 nm were similar, indicating that UVA radiation, and not stray shorter wavelength radiation, was responsible for dimer production. The identity of the UVA-induced DNA lesions was confirmed as pyrimidine dimers by photoreactivation of approximately 100% of the endonuclease-sensitive sites in vitro with the 40,000 dalton Escherichia coli photoreactivating enzyme.

Adult↗