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Linear dichroism provides information on the orientation of chromophores part of, or bound to, an orientable molecule such as DNA. For molecular alignment induced by hydrodynamic shear, the principal axes orthogonal to the direction of alignment are not equivalent. Thus, the magnitude of the flow-induced change in absorption for light polarized parallel to the direction of flow can be more than a factor of two greater than the corresponding change for light polarized perpendicular to both that direction and the shear axis. The ratio of the two flow-induced changes in absorption, the dichroic increment ratio, is characterized using the orthogonal orientation model, which assumes that each absorbing unit is aligned parallel to one of the principal axes of the apparatus. The absorption of the alienable molecules is characterized by components parallel and perpendicular to the orientable axis of the molecule. The dichroic increment ratio indicates that for the alignment of DNA in rectangular flow cells, average alignment is not uniaxial, but for higher shear, as produced in a Couette cell, it can be. The results from the simple model are identical to tensor models for typical experimental configurations. Approaches for measuring the dichroic increment ratio with modern dichrometers are discussed.

A method for obtaining fluorescence polarization data from an instrument designed to measure circular and linear dichroism is compared with a previously reported approach. The new method places a polarizer between the sample and a detector mounted perpendicular to the direction of the incident beam and results in determination of the fluorescence polarization ratio, whereas the previous method does not use a polarizer and yields the fluorescence anisotropy. A similar analysis with the detector located axially with the excitation beam demonstrates that there is no frequency modulated signal due to fluorescence polarization in the absence of a polarizer.

A single-beam spectrometer equipped with a photoelastic modulator can be configured to measure a number of different parameters useful in characterizing chemical and biochemical materials including natural and magnetic circular dichroism, linear dichroism, natural and magnetic fluorescence-detected circular dichroism, and fluorescence polarization anisotropy as well as total absorption and fluorescence. The derivations of the mathematical expressions used to extract these parameters from ultraviolet, visible, and near-infrared light-induced electronic signals in a dichrometer assume that the dichroic signals are sufficiently small that certain mathematical approximations will not introduce significant errors. This article quantifies errors resulting from these assumptions as a function of the magnitude of the dichroic signals. In the case of linear dichroism, improper modulator programming can result in errors greater than those resulting from the assumption of small signal size, whereas for fluorescence polarization anisotropy, improper modulator phase alone gives incorrect results. Modulator phase can also impact the values of total absorbance recorded simultaneously with linear dichroism and total fluorescence. Chirality 24:706–717, 2012. © 2012 Wiley Periodicals, Inc.†

We have investigated the absorption, the fluorescence and phosphorescence emission and the fluorescence lifetimes of adenine in low-temperature argon and nitrogen matrices at 15 K. Compared to other environments the absorption spectrum shows higher intensity at the shortest wavelengths, and a weak apparent absorption peak is observed at 280 nm. The resolved fluorescence excitation spectrum has five peaks at positions corresponding to those observed in the absorption spectrum. The position of the fluorescence maximum depends on the excitation wavelength. Excitation below 220 nm displays a fluorescence maximum at 305 nm, while for excitations at higher wavelengths the maximum occurs at 335 nm. The results suggest that multiple-emission excited electronic states are populated in low-temperature gas matrices. Excitation at 265 nm produces a phosphorescence spectrum with a well-resolved vibrational structure and a maximum at 415 nm. The fluorescence decays corresponding to excitation at increasing energy of each resolved band could be fit with a double exponential, with the shorter and longer lifetimes ranging from 1.7 to 3.3 ns and from 12 to 23 ns, respectively. Only for the excitation at 180 nm one exponential is required, with the calculated lifetimes of 3.3 ns. The presented results provide an experimental evidence of the existence of multiple site-selected excited electronic states, and may help elucidate the possible deexcitation pathways of adenine. The additional application of synchrotron radiation proved to result in a significant enhancement of the resolution and spectral range of the phenomena under investigation.

Numerous studies have explored the relationship between female beauty and positive effects for the woman, product, or ad; however, none has explored women's emotional responses to different beauty types. This study investigated college women's emotional responses to Solomon, Ashmore, and Longo's six beauty types. The survey results revealed that the original six beauty types were not supported. Instead, they combined into two independent dimensions: Sexual/Sensual (SS) and Classic Beauty/Cute/Girl-Next-Door (CCG). After testing emotional reactions to High CCG/Low SS, High SS/Low CCG, and Equal CCG/SS models, models with higher degrees of CCG produced significantly greater pleasure, arousal, and dominance.

Predicting the effects of polychromatic light on biological systems is a central goal of environmental photobiology. If the dose-response function for a process is a linear function of the light incident on a system at each wavelength within the spectrum, the effect of a polychromatic spectrum is obtained by integrating the product of the cross section for the reaction at each wavelength and the spectral irradiance at that wavelength over both wavelength and time. This procedure cannot be used, however, if the dose-response functions for an effect are not linear functions of photon dose. Although many photochemical reactions are linear within the biologically relevant range of doses, many biological end points are not. I describe procedures for calculating the effects of polychromatic irradiations on systems that exhibit certain classes of dose-response functions, including power law responses typical of mutation induction and exponential dose-responses typical of cell survival. I also present an approach to predict the effects of polychromatic spectra on systems in which the ultraviolet components form pyrimidine dimers, and the longer-wavelength ultraviolet and visible components remove them by photoreactivation, thus generating complex dose-response functions for these coupled light-driven reactions.

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.

The treatment of knee osteoarthritis and the preparation for total knee arthroplasty require repetitive imaging to guide preoperative planning and operative technique. Full-length standing anteroposterior images are the gold standard in assessing the alignment of the limb via the measurement of the mechanical axis of the knee. The anatomical axis can be obtained from a more limited image of the knee, and as such is less expensive and exposes the patient to less ionizing radiation. The objective of this cross-sectional prospective study was to examine the extent to which the anatomical axis measured on a fixed-flexed posteroanterior (Rosenberg view) radiograph correlates with the mechanical axis. The data of 209 total knee arthroplasty radiographs were analyzed to compare the preoperative correlation between the mechanical and anatomical axis. The anatomical axis correlated with the mechanical axis when it was measured from both the standing full-length anteroposterior radiograph and from a fixed-flexed posteroanterior radiograph. Using an angle of offset found from linear regression, these correlations become closer. Body mass index and Kellgren-Lawrence grade were not found to have a significant effect. It is the conclusion of this study that the anatomical axis, as measured from a limited knee radiography, may serve as a plausible estimate of the mechanical axis when done with a neutral angle of offset, and that offset angle depends on gender and the imaging technique used to determine the anatomical axis. [ Orthopedics. 2015; 38(12):e1115–e1120.]

The probability of survival of cells or viruses exposed to various forms of radiation is expressed as a function of the probability that a given cell will receive a certain number of lethal damages, the average probability that each such damage is repairable, and an upper bound on the repair capacity of each cell. All lethal damages are presumed induced as a linear function of dose. The probability of survival is found to be the product of a single exponential, which reflects inactivation by unrepairable lethal damages and dominates at low doses, and an Euler gamma function, which reflects inactivation due to repairable damages formed in excess of the upper bound on repair capacity. Computational procedures obtain stochastic parameters from published survival data for the inactivation of bacterial, yeast and mammalian cells exposed to ionizing or ultraviolet radiation, including split-dose experiments. The survival of cells exposed to photodynamic therapy is analysed assuming that lethal damages cannot be repaired, but more than one may be required for inactivation.

Background/Aims: Photoreactivation (PR) of cyclobutyl pyrimidine dimers (CPD) in human skin remains controversial. Recently Whitmore et al. (1) reported negative results of experiments using two photorepair light (PRL) sources on UV-irradiated skin of volunteers. However, their PRL sources induced substantial levels of dimers in skin, suggesting that the additional dimers formed could have obscured PR. We met a similar problem of dimer induction by a PRL source. We designed and validated a PRL source of sufficient intensity to catalyse PR, but that did not induce CPD, and used it to measure photorepair in human skin. Methods and Results: Using a solar simulator filtered with three types of UV-filters, we found significant dimer formation in skin, quantified by number average length analysis using electrophoretic gels of isolated skin DNA. To prevent scattered UV from reaching the skin, we interposed shields between the filters and skin, and showed that the UV-filtered/shielded solar simulator system did not induce damage in isolated DNA or in human skin. We exposed skin of seven healthy human volunteers to 302 nm radiation, then to the improved PRL source (control skin areas were kept in the dark for measurement of excision repair). Conclusions: Using a high intensity PRL source that did not induce dimers in skin, we found that three of seven subjects carried out rapid photorepair of dimers; two carried out moderate or slow dimer photorepair, and three did not show detectable photorepair. Excision repair was similarly variable in these volunteers. Subjects with slower excision repair showed rapid photorepair, whereas those with rapid excision generally showed little or no photoreactivation.

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.

The repair-dependent model of cell radiation survival is extended to include radiation-induced transformations. The probability of transformation is presumed to scale with the number of potentially lethal damages that are repaired in a surviving cell or the interactions of such damages. The theory predicts that at doses corresponding to high survival, the transformation frequency is the sum of simple polynomial functions of dose; linear, quadratic, etc, essentially as described in widely used linear-quadratic expressions. At high doses, corresponding to low survival, the ratio of transformed to surviving cells asymptotically approaches an upper limit. The low dose fundamental- and high dose plateau domains are separated by a downwardly concave transition region. Published transformation data for mammalian cells show the high-dose plateaus predicted by the repair-dependent model for both ultraviolet and ionizing radiation. For the neoplastic transformation experiments that were analyzed, the data can be fit with only the repair-dependent quadratic function. At low doses, the transformation frequency is strictly quadratic, but becomes sigmodial over a wider range of doses. Inclusion of data from the transition region in a traditional linear-quadratic analysis of neoplastic transformation frequency data can exaggerate the magnitude of, or create the appearance of, a linear component. Quantitative analysis of survival and transformation data shows good agreement for ultraviolet radiation; the shapes of the transformation components can be predicted from survival data. For ionizing radiations, both neutrons and x-rays, survival data overestimate the transforming ability for low to moderate doses. The presumed cause of this difference is that, unlike UV photons, a single x-ray or neutron may generate more than one lethal damage in a cell, so the distribution of such damages in the population is not accurately described by Poisson statistics. However, the complete sigmodial dose-response data for neoplastic transformations can be fit using the repair-dependent functions with all parameters determined only from transformation frequency data.

Clustered DNA damages--two or more lesions (oxidised bases. abasic sites, or strand breaks) within a few DNA helical turns on opposing strands--are induced in DNA in solution and in vivo in human cells by ionising radiation. They have been postulated to be difficult to repair, and thus of potentially high biological significance. Since the total of clustered damages produced by ionising radiation is at about 3 to 4 times higher levels than double-strand breaks and are apparently absent in unirradiated cells, levels of clustered damages present immediately alter radiation exposure could serve as sensitive dosemeters of radiation exposure. Since some clusters may not be repairable and may accumulate in cells, they might also be useful as integrating dosemeters of biological effects of radiation damage.

DNA damages that can be converted to single- or double strand breaks can be quantified by separating DNA by gel electrophoresis and obtaining a quantitative image of the resulting distribution of DNA in the gel. We review the theory of this method and discuss its implementation, including the charge-coupled device (CCD) camera systems we developed to acquire images of fluorophore labeled 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.

We have assembled a system using a personal computer workstation equipped with standard office software, an audio system, speech recognition software and an inexpensive radio-based wireless microphone that permits laboratory workers to enter or modify data while performing other work. Speech recognition permits users to enter data while their hands are holding equipment or they are otherwise unable to operate a keyboard. The wireless microphone allows unencumbered movement around the laboratory without a “tether” that might interfere with equipment or experimental procedures. To evaluate the potential of voice data entry in a laboratory environment, we developed a prototype relational database that records the disposal of radionuclides and/or hazardous chemicals. Current regulations in our laboratory require that each such item being discarded must be inventoried and documents must be prepared that summarize the contents of each container used for disposal. Using voice commands, the user enters items into the database as each is discarded. Subsequently, the program prepares the required documentation.

Recently, I was asked to introduce a session on spectroscopy at the Biophysics and Synchrotron Radiation 98 meeting. I wanted to recognize the “hot new areas” in synchrotron-based spectroscopy, and the rapidly expanding research in the infrared came immediately to mind. As it happens, however, I am acquainted with several of the scientists responsible for the development of infrared research using synchrotron radiation sources, and thus knew that while “hot” is certainly appropriate, “new” is not quite correct. Other speakers at the same conference emphasized the recent, rapid growth in determinations of macromolecu-lar structures by X-ray crystallography using synchrotron radiation, but from personal contacts with leaders in this field I knew that this was also a long and arduous process. These observations led me to consider the evolution of new research areas within the synchrotron radiation community.

New high-flux synchrotron radiation circular dichroism (SRCD) beamlines are providing important information for structural biology, but can potentially cause denaturation of the protein samples under investigation. This effect has been studied at the new CD1 dedicated SRCD beamline at ISA in Denmark, where radiation-induced thermal damage effects were observed, depending not only on the radiation flux but also on the focal spot size of the light. Comparisons with similar studies at other SRCD facilities worldwide has lead to the estimation of a flux density threshold under which SRCD beamlines should be operated when samples are to be exposed to low-wavelength vacuum ultraviolet radiation for extended periods of time.

Modern scientific instruments can produce huge quantities of data, usually in digital form. However, data acquisition is only one of three important functions. To be useful, data must also be stored and analyzed. Fortunately, the same computer-based technologies that facilitate the generation of large data-sets provide tools to accomplish these tasks. We describe a data system based on computers connected to a network, developed for this purpose.

We report a fluorescence analyzer that records simultaneously the temporal profiles for both orthogonal linear polarizations for all wavelengths in a fluorescence emission spectrum. The Analyzer combines a resistive-anode single-photon-counting photomultiplier, imaging spectrograph, Wollaston polarizer, multiparameter analyzer with histograming memory, and standard timing electronics. The spectrograph disperses the fluorescence spectrum across the photocathode of the photomultiplier, and the Wollaston polarizer separates the spectra of the two polarizations in opposite directions from the center of the photocathode perpendicular to the direction of spectral dispersion. The locations at which each photon reaches the photocathode is determined by the ratios of the charges read from the four corners of the resistive anode. One of the two address coordinates that determine where in histogramming memory each photon is recorded is obtained by measuring the time of arrival of the photon at the detector relative ...

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.

— We have determined the capacity of soybean seedlings to repair DNA damage by UV doses that do not produce apparent injury in the plants. They remove cyclobutane pyrimidine dimers by both excision and photoreactiva-tion. The rates and relative contributions of these repair processes were determined as a function of initial level of cyclobutyl pyrimidine dimers. Photoreactivation was detected in seedlings at all initial dimer levels. Although excision was not observed at the lowest dimer frequencies, at higher initial dimer levels it was quite effective in dimer removal. The rates of repair in soybean were substantially higher than in alfalfa seedlings at the same DNA damage levels.

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.

The pulsed nature or ‘‘time structure’’ of synchrotron radiation from electron storage rings is used to measure the kinetics of the decay of electronically excited states and is particularly useful because the wavelength of excitation can be chosen at will. However, changes in the length of the pulses of radiation from a storage ring resulting from the gradual decrease of current circulating in the ring during the course of a ‘‘fill’’ limit the duration of data collection, and hence photometric sensitivity. A fourth‐harmonic cavity that was recently added to the vacuum ultraviolet (VUV) storage ring at the National Synchrotron Light Source slows the loss of current during a fill, and thus increases the total number of photons produced. When operated in a passive (unpowered) mode, however, the fourth‐harmonic cavity increases both the average width of the photon pulses and the changes in width that occur during a fill, thus reducing the usefulness of the VUV ring in timing experiments. We demonstrate that ...

A single-beam spectrometer equipped with a photoelastic modulator can be configured to measure a number of different parameters useful in characterizing chemical and biochemical materials including natural and magnetic circular dichroism, linear dichroism, natural and magnetic fluorescence-detected circular dichroism, and fluorescence polarization anisotropy as well as total absorption and fluorescence. The derivations of the mathematical expressions used to extract these parameters from ultraviolet, visible, and near-infrared light-induced electronic signals in a dichrometer assume that the dichroic signals are sufficiently small that certain mathematical approximations will not introduce significant errors. This article quantifies errors resulting from these assumptions as a function of the magnitude of the dichroic signals. In the case of linear dichroism, improper modulator programming can result in errors greater than those resulting from the assumption of small signal size, whereas for fluorescence polarization anisotropy, improper modulator phase alone gives incorrect results. Modulator phase can also impact the values of total absorbance recorded simultaneously with linear dichroism and total fluorescence.

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.

Resolving power is a quantitative measure of the ability of an electrophoretic system to separate DNA (and other) molecules of similar size. It is a dimensionless quantity, and hence facilitates comparison of the performance of electrophoretic systems that operate very differently. Resolving power can be determined as a function of molecular length from experimental data consisting of a series of completely resolved bands on a gel or blot; closely spaced bands are not required. We discuss factors such as the mass of DNA in a particular band and the spatial resolution of the system used to image the distribution of DNA on a gel or blot that, while not an intrinsic part of the electrophoretic system, may influence the observed resolving power. We derive an empirical global dispersion function that applies both to images of gels obtained after a fixed time of electrophoresis of all the samples and to images obtained as each species reaches a detector located at a fixed distance from the starting well. We use this dispersion function to show that the improvement in resolving power produced by extending the time or distance of electrophoresis in a static, uniform electric field asymptotically approaches a limiting value that is a function of the length of the DNA. When plotted as a function of molecular length, this limiting value defines an envelope that characterizes the intrinsic limits of performance of a particular electrophoretic system (e.g., electric field strength, gel type and concentration, buffer, temperature). Comparing the resolving power of static field agarose gel electrophoresis as routinely practiced for separating DNA molecules from 10(3) to 10(5) bp long with other electrophoretic schemes suggests that significant improvements should be achievable.

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.

The organization of pigment-protein complexes into large chiral macrodomains was investigated in wild-type and chlorophyll b-less mutant thylakoid membranes of barley. The variations in the anomalous circular dichroism bands and in the angular-dependence of circular intensity differential scattering showed that in wild-type chloroplasts, the formation of macrodomains was governed by interactions of the light-harvesting chlorophyll alb complexes (LHCII). Two external factors could be identified which regulate the parameters of the anomalous circular dichroism signal: (i) electrostatic screening by divalent cations under conditions that favor membrane stacking and (ii) the osmotic pressure of the medium, which is suggested to affect the lateral interactions between complexes and influence the packing-density of particles. These two factors governed preferentially the negative and the positive anomalous circular dichroism signals, respectively. In the chlorina f-2 mutant thylakoid membranes, deficient in most chlorophyll b binding proteins, the formation of macrodomains which gave rise to the anomalous circular dichroism signals was still regulated by these same external factors. However, in the absence of major LHCII polypeptides the formation of macrodomains was apparently mediated by other complexes having weaker interaction capabilities. As a consequence, the size of the macrodomains under comparable conditions appeared smaller in the mutant than in the wild-type thylakoid membranes. Circular dichroism is a valuable probe for examining the long-range interactions between pigment-protein complexes which participate in the formation and stabilization of membrane ultrastruc-ture. A functional role of macrodomains in long-range energy migration processes is proposed.

— 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.

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.

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.

The vacuum UV CD spectra of GpC, GpC, 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-straded 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 specturm 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. A · U base pairs induced two positive bands at about 177 and 201 nm, whereas G · C base pair induced a large positive band at 186 nm and negative bands at about 175 and 202 nm.

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 of the fact that poly[d(A) · d(T)] exists in a noncanonical B conformation.

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.

A new fluorescence spectrometer has been assembled at the U9B beamline of the National Synchrotron Light Source to allow simultaneous multiwavelength and time‐resolved fluorescence detection, as well as spatial imaging of the sample fluorescence. The spectrometer employs monochromatized, tunable UV and visible excitation light from a synchrotron bending magnet and an imaging spectrograph equipped with a single‐photon sensitive emission detector. The detector is comprised of microchannel plates in series, with a resistive anode for encoding the position of the photon‐derived current. The centroid position of the photon‐induced electron cascade is derived in a position analyzer from the four signals measured at the corners of the resistive anode. Spectral information is obtained by dispersing the fluorescence spectrum across one dimension of the detector photocathode. Timing information is obtained by monitoring the voltage divider circuit at the last MCP detector. The signal from the MCP is used as a ‘‘start’’ signal to perform a time‐correlated single photon counting experiment. The analog signal representing the position, and hence wavelength, is digitized concomitantly with the start/stop time difference and stored in the two‐dimensional histogramming memory of a multiparameter analyzer.

Assessing DNA damage induction, repair and consequences of such damages requires measurement of specific DNA lesions by methods that are independent of biological responses to such lesions. Lesions affecting one DNA strand (altered bases, abasic sites, single strand breaks (SSB)) as well as damages affecting both strands (clustered damages, double strand breaks) can be quantified by direct measurement of DNA using gel electrophoresis, gel imaging and number average length analysis. Damage frequencies as low as a few sites per gigabase pair (10(9)bp) can be quantified by this approach in about 50ng of non-radioactive DNA, and single molecule methods may allow such measurements in DNA from single cells. This review presents the theoretical basis, biochemical requirements and practical aspects of this approach, and shows examples of their applications in identification and quantitation of complex clustered damages.

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.

Predicting the effects of polychromatic light on biological systems is a central goal of environmental photobiology. If the dose-response function for a process is a linear function of the light incident on a system at each wavelength within the spectrum, the effect of a polychromatic spectrum is obtained by integrating the product of the cross section for the reaction at each wavelength and the spectral irradiance at that wavelength over both wavelength and time. This procedure cannot be used, however, if the dose-response functions for an effect are not linear functions of photon dose. Although many photochemical reactions are linear within the biologically relevant range of doses, many biological end points are not. I describe procedures for calculating the effects of polychromatic irradiations on systems that exhibit certain classes of dose-response functions, including power law responses typical of mutation induction and exponential dose-responses typical of cell survival. I also present an approach to predict the effects of polychromatic spectra on systems in which the ultraviolet components form pyrimidine dimers, and the longer-wavelength ultraviolet and visible components remove them by photoreactivation, thus generating complex dose-response functions for these coupled light-driven reactions.

Circular dichroism and fluorescence polarization anisotropy are important tools for characterizing biomolecular systems. Both are used extensively in kinetic experiments involving stopped- or continuous flow systems as well as titrations and steady-state spectroscopy. This paper presents the theory for determining circular dichroism and fluorescence polarization anisotropy simultaneously, thus insuring the two parameters are recorded under exactly the same conditions and at exactly the same time in kinetic experiments. The approach to measuring circular dichroism is that used in almost all conventional dichrographs. Two arrangements for measuring fluorescence polarization anisotropy are described. One uses a single fluorescence detector and signal processing with a lock-in amplifier that is similar to the measurement of circular dichroism. The second approach uses classic ''T'' format detection optics, and thus can be used with conventional photon-counting detection electronics. Simple extensions permit the simultaneous measurement of the absorption and excitation intensity corrected fluorescence intensity.

Circular dichroism (CD) spectroscopy is a well-established technique in structural biology. The use of synchrotron radiation as an intense light source for these measurements extends the applications possible using lab-based instruments. In recent years, there has been a major growth in synchrotron radiation circular dichroism (SRCD) beamlines worldwide, including ones at the NSLS, ISA, SRS, HiSOR, BSRF, NSRRC, SOLEIL, Diamond, TERAS, BESSYII, and ANKA synchrotrons. Through the coordinated efforts of beamline scientists and users at these sites, important proof-of-principle studies have been done enabling the method to be developed for novel and productive studies on biological systems. This paper describes the characteristics of SRCD beamlines and some of the new types of applications that have been undertaken using these beamlines.

DEPLETION of stratospheric ozone will increase the intensity of solar mid-ultraviolet (280–320 nm) radiation reaching the biosphere1. Predictions of increases in biologically effective ultraviolet radiation require knowledge of both the solar spectral intensity and the wavelength-dependent sensitivity (action spectrum) for damaging the biological target2. A generalized action spectrum for plant damage encompassing wavelengths from 280 to 313 nm3–5 has been widely used to predict the consequences of ozone depletion. Calculations6 based on this spectrum and new satellite measurements of atmospheric ozone suggest that plants will be among those organisms most severely affected. Here we report an absolute action spectrum for cyclobutyl pyrimidine dimer induction in DNA in intact alfalfa seedlings, which reveals damage by wavelengths as long as 365 nm. Calculations based on this new action spectrum predict significantly smaller increases in biologically effective ultraviolet radiation resulting from ozone depletion, particularly at high latitudes, than calculations based on either the generalized plant action spectrum or the action spectrum for damaging unshielded DNA.

Measurements of circular dichroism (CD) in the UV and vacuum UV have used photoelastic modulators (PEMs) for high sensitivity (to about 10{sup -6}). While a simple technique for wavelength calibration of the PEMs has been used with good results, several features of these calibration curves have not been understood. The authors have calibrated a calcium fluoride PEM and a lithium fluoride PEM using the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory as a light source. These experiments showed calibration graphs that are linear bit do not pass through the graph origin. A second ''multiple pass'' experiment with laser light of a single wavelength, performed on the calcium fluoride PEM, demonstrates the linearity of the PEM electronics. This implies that the calibration behavior results from intrinsic physical properties of the PEM optical element material. An algorithm for generating calibration curves for calcium fluoride and lithium fluoride PEMs has been developed. The calibration curves for circular dichroism measurement for the two PEMs investigated in this study are given as examples.