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The clinical outcome of total knee arthroplasty (TKA) remains suboptimal in some patients. One of the factors that might hinder improved functionality may be postoperative limb length discrepancy due to increase in limb length of the operative limb. The objective of this cross-sectional prospective study was to examine the extent to which limb length change occurs after TKA and to compare the change in limb length to the degree of valgus or varus joint position preoperatively. The role of body mass index and Kellgren-Lawrence grade in limb length change was also assessed. The data of 137 TKAs were analyzed and separated into categories to compare change in limb length pre- versus postoperatively. In all, 59.1% of patients experienced an increase in limb length with an average increase of 0.438 cm, but overall, there was no statistically significant difference in limb length pre- versus postoperatively ( P value 0.598). Similar trends were seen within all other groups. It is the conclusion of this study that limb lengthening after TKA does not frequently occur to a statistically significant extent, regardless of preoperative joint state.

Although DNA DSBs are known to be important in producing the damaging effects of ionizing radiation in cells, bistranded clustered DNA damages-two or more oxidized bases, abasic sites or strand breaks on opposing DNA strands within a few helical turns-are postulated to be difficult to repair and thus to be critical radiation-induced lesions. Gamma rays can induce clustered damages in DNA in solution, and high-energy iron ions produce DSBs and oxidized pyrimidine clusters in human cells, but it was not known whether sparsely ionizing radiation can produce clustered damages in mammalian cells. We show here that X rays induce abasic clusters, oxidized pyrimidine clusters, and oxidized purine clusters in DNA in human cells. Non-DSB clustered damages comprise about 70% of the complex lesions produced in cells. The relative levels of specific cluster classes depend on the environment of the DNA.

Measuring DNA damage in higher plants is important in assessing the impaets of environmental conditions, e.g., increased UV resulting from ozone depletion, and in testing the relationship of productivity to DNA damage and repair. Sunlight exposure of plants produces UV induced DNA damages measurable by treating DNA with damage-specific enzymes and dispersion of DNA molecules in denaturing media. Such DNA must be enzyme-digestible, with few single strand breaks. DNA isolation must preclude repair, providing a snapshot of DNA damage. We developed a method for isolating DNA from several crop plants, both monocols and dicots-alfalfa (Medicago sativa L.), pea (Pisam sativam L.), rice (Oryza sativa L.), soybean [Glycine max (L.) Merr.], sorghum [Sorghum bicolor (L.) Moench], and spinach (Spinacia oleraceae L.). This method is simple, readily deals with multiple samples, and avoids organic solvents. We show that pyrimidine dimers can readily be quantified in DNA prepared by this method. This method should also be useful for other experiments requiring high molecular length, enzymatically digestible plant DNA.

Repair of cyclobutyl pyrimidine dimers (CPDs) in DNA is essential in most organisms to prevent biological damage by ultraviolet (UV) light. In higher plants tested thus far, UV-sensitive strains had higher initial damage levels or deficient repair of nondimer DNA lesions but normal CPD repair. This suggested that CPDs might not be important for biological lesions. The photosynthetic apparatus has also been proposed as a critical target. We have analyzed CPD induction and repair in the UV-sensitive rice (Oryza sativa L.) cultivar Norin 1 and its close relative UV-resistant Sasanishiki using alkaline agarose gel electrophoresis. Norin 1 is deficient in cyclobutyl pyrimidine dimer photoreactivation and excision; thus, UV sensitivity correlates with deficient dimer repair.

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland, Anal. Biochem. 210, 378-388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish-line imaging provided higher resolving power. The ratio of the resolving power of finish-line images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish-line images exceeded that of snapshot images by about 50%.

The relative UV sensitivities of alfalfa seedlings grown outdoors versus plants grown in a growth chamber under UV-filtered cool white fluorescent bulbs have been determined using three criteria: (1) level of endogenous DNA damage as sites for the UV endonuclease from Micrococcus luteus. (2) susceptibility to pyrimidine dimer induction by a UV challenge exposure and (3) ability to repair UV-induced damage. We find that outdoor-grown plants contain approximately equal frequencies of endogenous DNA damages, are less susceptible to dimer induction by a challenge exposure of broad-spectrum UV and photorepair dimers more rapidly than plants grown in an environmental chamber under cool white fluorescent lamps plus a filter that removes most UV radiation. These data suggest that plants grown in a natural environment would be less sensitive to UVB-induced damage than would be predicted on the basis of studies on plants grown under minimum UV.

We tested the ability of melatonin (N-acetyl-5 methoxytryptamine), a highly effective radical scavenger and human hormone, to protect DNA in solution and in human cells against induction of complex DNA clusters and biological damage induced by low or high linear energy transfer radiation (100 kVp X-rays, 970 MeV/nucleon Fe ions).Plasmid DNA in solution was treated with increasing concentrations of melatonin (0.0-3.5 mM) and were irradiated with X-rays. Human cells (28SC monocytes) were also irradiated with X-rays and Fe ions with and without 2 mM melatonin. Agarose plugs containing genomic DNA were subjected to Contour Clamped Homogeneous Electrophoretic Field (CHEF) followed by imaging and clustered DNA damages were measured by using Number Average length analysis. Transformation experiments on human primary fibroblast cells using soft agar colony assay were carried out which were irradiated with Fe ions with or without 2 mM melatonin.In plasmid DNA in solution, melatonin reduced the induction of single- and double-strand breaks. Pretreatment of human 28SC cells for 24 h before irradiation with 2 mM melatonin reduced the level of X-ray induced double-strand breaks by ∼50%, of abasic clustered damages about 40%, and of Fe ion-induced double-strand breaks (41% reduction) and abasic clusters (34% reduction). It decreased transformation to soft agar growth of human primary cells by a factor of 10, but reduced killing by Fe ions only by 20-40%.Melatonin's effective reduction of radiation-induced critical DNA damages, cell killing, and striking decrease of transformation suggest that it is an excellent candidate as a countermeasure against radiation exposure, including radiation exposure to astronaut crews in space travel.

Outer surface protein A (OspA) is a major antigen of Borrelia burgdorferi, the etiological agent of Lyme disease. A recombinant form of OspA (OspA-257) from B. burgdorferi, strain B31, contains 257 amino acids and a single tryptophan residue at position 216 (Trp-216). Mapping studies indicate that Trp-216 is involved in the epitope for the agglutinating monoclonal antibody 105.5. However, the fluorescence emission maximum of the native protein is 330 nm, indicating that Trp-216 is not solvent-exposed. Primary structure analysis suggests an alpha-helical conformation for residues approx. 204-217, which, if located on the protein surface, would allow Trp-216 to be buried, while leaving hydrophilic residues on the opposite side of the helix exposed. This helix would place Lys-212 within approx. 6 A of Trp-216; the presence of such a positively-charged residue can, in principle, be ascertained from fluorescence quenching studies. Stern-Volmer plots confirm that Trp-216 is indeed buried in the native protein, but is readily accessible to the small polar quencher, acrylamide. Furthermore, the dominant component of the fluorescence emission shows only weak dynamic quenching by the positively-charged quencher, Cs+, while the minor component undergoes static quenching by I-, indicating the proximity of a positively-charged residue. These data are consistent with the existence of an alpha-helix from residues 204-217 in the predicted orientation at the protein surface, hence indicating the structure of the antigenic determinant.

The etiological agent of Lyme disease is the tick-borne spirochete, Borrelia burgdorferi. A major antigen of B. burgdorferi is a 31 kDa lipoprotein called outer surface protein A (OspA). Recently, a truncated form of OspA (lacking 17 amino acids at the N-terminus) was cloned, expressed and purified in large quantities (Dunn, J.J., Lade, B.A. and Barbour, A.G. (1990) Protein Expression and Purification 1, 159-168). The truncated protein (OspA-257) is water-soluble, retains the ability to bind antibodies from the sera of Lyme disease patients and may prove useful in development of a vaccine against Lyme disease. We have used far UV circular dichroism (CD) and fluorescence spectroscopy to characterize the secondary structure of and to study conformational changes in OspA-257. CD spectra from 260 to 178 nm predict five classes of secondary structure: alpha-helix (11%), anti-parallel beta-sheet (32%), parallel beta-sheet (10%), beta-turns (18%) and aperiodic structures (including 'random coil') (30%). Analysis of the primary sequence of OspA yielded the most likely sites for alpha-helical regions (residues 100-107, 121-134, 253-273) and for antigenic determinants (Lys-46, Asp-82, Lys-231). CD spectra of the native protein show little change from pH 3 to 11. Thermal denaturation curves, indicate that 'salt bridges' play a role in stabilizing the native protein. Both thermal and chemical denaturations that eliminate all secondary structure as judged by CD or fluorescence are reversible. Denaturation by guanidine-HCl (gdn-HCl) appears to be a cooperative, two-state transition, as indicated by a sudden change in the CD spectrum at approximately 0.75 M gdn-HCl, and an isodichroic point at 208 nm in all CD spectra measured from 0.0-1.75 M gdn-HCl.

Quantitation of the conversion of nonradioactive supercoiled DNA to its open circular or linear forms on ethidium-stained electrophoretic gels has been difficult because of differential binding of ethidium to supercoiled DNA vs other forms under different conditions and the nonlinear response of photographic film. We have developed methods for adding a linear DNA as an internal fluorescence standard to “normalize” the quantity of DNA loaded into each lane of a gel. Inclusion of a linear normalizing DNA in samples before partitioning for individual supercoil cleavage reactions allows the quantitation of the resultant species, is technically easy, and does not require quantitative application of the sample to the gel. If the presence of a normalizing DNA during supercoil cleavage is undesirable, the addition of a normalizing plasmid to each sample after supercoil cleavage (but before electrophoresis) or the quantitative application of samples containing test DNA alone to the gel gives similar data, but with increased variability. We use the normalizing DNA method in cleavage by a physical agent (ionizing radiation) and in a more complex situation, by a protein-based, light-dependent synthetic endonuclease. We show how the fraction of intact supercoiled DNA can be calculated from measurement of the cleaved and normalizing species only. The method also can be used in reactions involving the depletion of one DNA species, whether supercoiled or not, such as protein-DNA interactions as detected by gel retardation assays.

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.

Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

Double strand breaks in DNA can be quantified down to very low frequencies (a few per Gigabase pair) in nanogram quantities of nonradioactive, genomic DNA by dispersing the DNAs on electrophoretic gels, digitizing them by quantitative electronic imaging, and calculating the DNA lengths by number average length analysis. No specific distribution of damages is required for number average length analysis. To test the validity of this approach, we used DNA populations of known absolute lengths and break frequencies as experimental DNAs and calculated the number average lengths and double strand break levels. Experimental DNAs and length standards were dispersed using pulsed field electrophoretic modes (unidirectional pulsed field, contour clamped homogeneous field, or transverse alternating field) appropriate for their size range, stained with ethidium, destained, and a quantitative electronic image obtained. A dispersion curve was constructed from the migration-mobility relationships of the length standard DNAs, and the number average lengths of the experimental DNAs were calculated. The calculated DNA lengths agreed well with the actual lengths. Furthermore, the double strand break frequencies calculated through number average length analysis of DNAs dispersed by these pulsed field gel modes and digitized by quantitative electronic imaging were in excellent agreement with the actual values for populations of DNA over the size range of approximately 4 kbp to approximately 3 Mbp. The use of this approach in quantifying DNA damages is illustrated for double strand breaks and damage clusters (e.g., OxyPurine clusters recognized by Escherichia coli Fpg protein) induced in T7 DNA by ionizing radiation.

Quantifying DNA lesions provides a powerful way to assess the level of endogenous damage or the damage level induced by radiation, chemical or other agents, as well as the ability of cells to repair such damages. Quantitative gel electrophoresis of experimental DNAs along with DNA length standards, imaging the resulting dispersed DNA and calculating the population average length allows accurate measurement of lesion frequencies. Number average length analysis provides high sensitivity and does not require any specific distribution of lesions within the DNA molecules. These methods are readily applicable to strand breaks and ultraviolet radiation induced pyrimidine dimers, but can also be used-with appropriate modifications-for ionizing radiation-induced lesions such as oxidized bases and abasic sites.

The number of bases that can be read in a single run by a DNA sequencing instrument that detects fluorophore labeled DNA arriving at a ``finish-line`` located a fixed distance from the starting wells is influenced by numerous parameters. Strategies for improving the length-of-read of a DNA sequencer can be based on quantitative models of the separation of DNA by gel electrophoresis. The dispersion function of the electrophoretic system--the relationship between molecular contour length and time of arrival at the detector--is useful in characterizing the performance of a DNA sequencer. We adapted analytical representations of dispersion functions, originally developed for snapshot imaging of DNA gels, (samples electrophoresed for constant time), to finish-line imaging, and demonstrated that a logistic-type function with non-integral exponent is required to describe the experimental data. We use this dispersion function to determine the resolution length and resolving power of a LI-COR DNA sequencing system and a custom built capillary gel electrophoresis system, and discuss the factors that presently limit the number of bases that can be determined reliably in a single sequencing run.

Ultraviolet radiation in sunlight damages DNA in plants, but little is understood about the types, lesion capacity, and coordination of repair pathways. We challenged intact alfalfa seedlings with UV doses that induced different initial levels of cyclobutyl pyrimidine dimers and measured repair by excision and photoreactivation. By using alkaline gel electrophoresis of nonradioactive DNAs treated with a cyclobutyl pyrimidine dimer-specific UV endonuclease, we quantitated ethidium-stained DNA by electronic imaging and calculated lesion frequencies from the number average molecular lengths. At low initial dimer frequencies (less than ~30 dimers per million bases), the seedlings used only photoreactivation to repair dimers; excision repair was not significant. At higher damage levels, both excision and photorepair contributed significantly. This strategy would allow plants with low damage levels to use error-free repair requiring only an external light energy source, whereas seedlings subjected to higher damage frequencies could call on additional repair processes requiring cellular energy. Characterization of repair in plants thus requires an investigation of a range of conditions, including the level of initial damage.

Quantitation of UV-induced DNA damages in nanogram quantities of non-radioactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.

Steady-state and time-resolved fluorescence techniques were used to monitor pH-induced conformational changes in spinach ferredoxin. An increase was seen in the wave-length maximum of tryptophan-73 (Trp-73) emission, from 325 nm below pH 6.0 to 342 nm above pH 7.0, indicating significantly diminished hydrophobicity, at pH 7.0, in the environment of the indole ring. Raising the solution pH from 6.0 to 7.6 also decreased the binding of the detergent Brij-96, showing that the ferredoxin molecule as a whole became more hydrophilic at higher pH. Nonionic (acrylamide) and ionic (I- and Cs+) quenchers were used to probe the tryptophan environment. Trp-73 is partially shielded from I-, presumably by negatively charged residues, as predicted from the amino acid sequence and three-dimensional structure of plant-type ferredoxins. Ionic strength and pH effects on tryptophan fluorescence lifetimes follow a pattern common to single-tryptophan proteins: the emission decays can be fit to a biexponential model in which the lifetime of the excited state increases with increasing pH. The indication of a pH-induced conformational change in the range pH 6.0 to 7.6 is discussed with reference to the physiological association of ferredoxin with ferredoxin:NADP+ oxidoreductase and the rise in chloroplast stromal pH in the light.

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.

Skin, the principal barrier between human internal organs and the external environment, is assaulted daily with physical and chemical carcinogens, promoters, and modifiers of biological responses to such agents. DNA is the principal target for most carcinogens, and DNA in skin is particularly at risk. It is subject to damage not only from ingested compounds and their metabolic products, but also from externally applied or encountered chemicals, as well as from physical carcinogens such as sunlight and cosmetic or medical sources of ultraviolet radiation. Three major factors determine the balance between damage to DNA of skin and the biological consequences of that damage: (1) the frequencies and types of lesions, (2) the ability of the individual to repair a lesion, and (3) the strategy that skin employs to deal with the different spectra of lesions inflicted under varying environmental conditions. Thus, cellular responses to DNA damage, including repair of DNA lesions, are critical factors in determining the final level of damage and its consequences.

We describe a method based on gel electrophoresis for the quantitation of strand breaks in DNA and demonstrate its application to the measurement of single- and double-strand breaks formed by gamma-rays for DNA irradiated in vitro. For single-strand breaks, our data span the dose range from 0.1 to 1 Gy, while for double-strand breaks doses were from 3 to 15 Gy. In agreement with results obtained using other techniques, we find that the dose response function for single-strand breaks is linear while the dose response function for double-strand breaks is curved, indicating that it is the sum of both linear and quadratic components. We discuss factors that determine the sensitivity of the method and indicate approaches to make possible the quantitation of strand breaks in the DNA of cells irradiated with sublethal doses.

We have developed an alkaline agarose gel method for quantitating single strand breaks in nanogram quantities of nonradioactive DNA. After electrophoresis together with molecular length standards, the DNA is neutralized, stained with ethidium bromide, photographed, and the density profiles recorded with a computer controlled scanner. The median lengths, number average molecular lengths, and length average molecular lengths of the DNAs can be computed by using the mobilities of the molecular length standards. The frequency of single strand breaks can then be determined by comparison of the corresponding average molecular lengths of DNAs treated and not treated with single strand break-inducing agents (radiation, chemicals, or lesion-specific endonuclease). Single strand break yields (induced at pyrimidine dimer sites in uv-irradiated human fibroblasts DNA by the dimer-specific endonuclease from Micrococcus luteus) from our method agree with values obtained for the same DNAs from alkaline sucrose gradient analysis. The method has been used to determine pyrimidine dimer yields in DNA from biopsies of human skin irradiated in situ. It will be especially useful in determining the frequency of single strand breaks (or lesions convertible to single strand breaks by specific cleaving reagents or enzymes) in small quantities of DNA from cells or tissues not amenable to radioactive labeling.

Isolated photoreactivating enzyme (PRE) from Escherichia coli exhibits some optical density at wavelengths greater than 300 nm. After correcting for the effects of light scattering, however, we find no true absorption in the spectral region that is required for enzymatic activity (320-450 nm). At shorter wavelengths, there is an absorption maximum near 260 nm that is due primarily to an RNA cofactor. Heating to 60/sup 0/ C and subsequently cooling to 4/sup 0/ C released the RNA cofactor from association with apoprotein and result in hyperchromicity. Circular dichroism indicates that the RNA associated with native enzyme is partially double stranded. At low ionic strength (..mu.. = 0.01), heating to 15/sup 0/ C or protease treatment at 4/sup 0/ C results in irreversible loss of part of the double strandedness. We show that the difference spectrum at 4/sup 0/ C between the absorption spectra of native enzyme and heat-treated enzyme can be fit by a superposition of reference spectra for denaturation of A-U and G-C base pairs derived from model polynucleotides. The coefficients of the linear combination of reference spectra were used to calculate the fraction of A-U and G-C base pairs. We find that both A-U and G-C base pairsmore » are present in equal concentrations and that about 20% are in a double-standard conformation in the native enzyme.« less

Anti-peripheral-nerve myelin antibodies (anti-PNM Ab) can be detected in the serum of all patients with acute-phase Guillain-Barre syndrome (GBS) thus far tested. Correlation of the titer of this antibody with the clinical course would help to establish a role for the humoral immune system in the pathophysiology of GBS. In this study, anti-PNM Ab levels were measured in serial serum samples of 7 patients with GBS with an assay that detects antibodies bound to peripheral nerve myelin antigens by fixation of the first component of complement. Although the titers of anti-PNM Ab detected in these patients varied between 0 and 256 U/ml, the antibody titer was always highest on admission (35 to 256 U/ml) and rapidly declined during a one-to-three-week period. Disappearance of antibodies or very low levels of them correlated with cessation of progression and considerable clinical improvement as documented by increased pulmonary vital capacity and muscular strength. Low but measurable antibody titers (5 to 12 U/ml) were frequently found up to four months following the acute neurological deficit. The close temporal relationship between anti-PNM Ab titer and the clinical course in GBS suggests that antibody most likely participates through complement activation in peripheral nerve demyelination.

— Non-dividing human cells degenerate and eventually detach from a culture vessel surface when exposed to UV light. Action spectra for this kind of cell inactivation were determined using eight monochromatic wavelengths from 240 to 313 nm and both a normal DNA excision-repair-proficient strain and a repair-deficient Xeroderma pigmentosum (XP12BE) strain. The action spectra for both strains have similar shapes with a broad peak between 254 and 280 nm followed by a steep decline at wavelengths greater than 280 nm. The relative action spectra are similar to those for inactivation of reproductive capacity and pyrimidine dimer formation in rodent cells suggesting that the critical target and critical damage for inactivation of non-dividing human cells is DNA and damage to DNA, respectively. Normal repair-proficient cells are 5–7 times more resistant at all wavelengths, based on a comparison of Do values, than repair-deficient XP12BE cells, supporting the conclusion that the inactivating damage at all wavelengths is to DNA.

The ocular pathologic findings in an immunosuppressed patient who died of disseminated histoplasmosis are described. Histoplasma capsulatum was found in large numbers within the endothelial cells of the choroid, in one area each of the ciliary body and trabecular meshwork. There was a minimal inflammatory response. No granulomas were noted. Previously reported cases of ocular histoplasmosis in immunosuppressed patients are similar in that, with one exception, granulomatous inflammation was not demonstrated. Thirteen eyes in which histoplasma organisms have been found in the eye are reviewed.

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.

DNA photoreactivating enzyme has been purified from Escherichia coli B and from induced cells of an E. coli strain containing a lysogenic bacteriophage carrying the gene for photoreactivation. The final preparations appear homogeneous by the criteria of presence of one band after electrophoresis on polyacrylamide gels under nondenaturing conditions and of constant specific activity in enzyme fractions eluted from hydroxylapatite. The enzyme has an apparent monomer molecular weight between 40,000 and 46,000 and tends to aggregate in solution. The pH optimum under the present assay conditions is 7.2. The action spectrum for photoreactivation by the purified enzyme has a maximum in the same region and the same spectral shape as the action spectrum obtained for E. coli cells in vivo.

We report the first measurement of the magnetic circular dichroism (MCD) of the basic polypeptide antibiotic netropsin (Nt). The MCD shows that the longest wavelength absorption band of Nt is the sum of more than one component and permits a radically new interpretation of the circular dichroism of the complex which Nt forms with DNA. We conclude that Nt has no major effect on the CD and thus the helical structure of the bases of the DNA to which it is bound. Thus the ability of Nt to inhibit the function of DNA polymerase, RNA polymerase, and the photoreactivating enzyme must be mediated by factors other than a distortion of the helical structure of the bases.

Purified photoreactivating enzyme (PRE) from E. coli has an absorption maximum at 257 nm, but no well defined absorption band for λ > 300 nm, the wavelength region which is required for photorectivation. When PRE is mixed with ultraviolet irradiated DNA (uvDNA), there is an increase in absorption for λ >300 nm. Absorbance changes are not observed if PRE is mixed with unirradiated DNA nor is the increase for λ > 300 nm due to an increase in light scattering. Thus, the long sought “chromophore” appears to be a molecular complex which exists only when E. coli PRE interacts with dimercontaining DNA and leads us to ask what are the moieties of the dimer and the PRE which interact to produce the new absorption band. A preliminary step in identifying the interacting moieties of the dimer is determining whether the PRE approaches the dimer from the major the minor groove of the DNA. If the PRE approaches from the major grove it can interact with the cyclobutyl ring while if it approaches from the minor groove, it might interact with the two carbonyl groups at the 2 positions of the pyrimidine ring which are common features of all photoreactable dimers. Recent data show that the basic peptide antibiotic netropsin, which is known to bind in the minor groove, inhibits binding of the PRE to uvDNA. This result supports the hypothesis (but does not prove)that the carbonyl groups at position 2 of the pyrimidine rings may be key moieties in the interaction between the E. coli PRE and uvDNA.

— DNA molecules that have been exposed to light from a 150 W incandescent spot lamp are nicked by the Micrococcus luteus endonuclease specific for cyclobutyl-type pyrimidine dimers. The production of these enzyme-sensitive sites increases with increasing spot lamp exposure. These sites have been confirmed to be pyrimidine dimers by their property of being photoreversed by an E. coli photoreactivating enzyme. The emission spectrum of the lamp shows detectable output at wavelengths less than 320 nm. These results indicate that the sensitivity of the techniques utilized in this work can be used to detect low levels of contaminating UV radiation.

In photoreactivation, photochemical damage produced in DNA by far ultraviolet radiation (lambda < 320 nm) is repaired in an enzyme-mediated reaction using longer wavelength light (310 < lambda < 450 nm for E. coli). Photoreactivating enzyme (PRE) acts on a single class of photoproducts, cyclobutyl pyrimidine dimers, in an otherwise normal DNA strand at least nine bases long. PRE is one of the few DNA repair enzymes which has been purified to homogeneity in quantities sufficient for physico-chemical studies.

A patient with acute lymphoblastic leukemia and granulocytopenia developed fever and diffuse muscle pain and weakness while under therapy. Blood cultures grew Candida Krusei and autopsy showed diffuse muscle fungal infestation.

V-79 Chinese hamster fibroblasts in monolayer culture were exposed to ultraviolet radiation at 313, 334, 365, 380, and 405 nm in the presence of either misonidazole or para-nitroacetophenone, drugs which act as both photosensitizers and radiosensitizers of cell killing. Survival was measured by a colony-forming assay. The resulting action spectra for cell death photosensitized by the drugs (the reciprocals of the exposures required at each wavelength to reduce cell survival to a given level) closely match their absorption spectra over a range of three orders of magnitude. These results demonstrate that cells can be killed upon excitation of misonidazole or para-nitroacetophenone in the absence of any other types of energy deposition or biomolecular damage.