DNA Damage Levels Determine Cyclobutyl Pyrimidine Dimer Repair Mechanisms in Alfalfa Seedlings.

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.

Action spectra for ultraviolet light-induced transformation of human cells to anchorage-independent growth.

We have determined action spectra for transformation of human embryonic skin and muscle fibroblasts to anchorage-independent growth. Tests under our experimental conditions indicate that reciprocity holds for photon rate and exposure time and that all the dose-effect curves in the wavelength range of 248 to 297 nm are smaller. These data can be used to construct action spectra with a maximum at about 265 nm, which do not implicate moieties other than nucleic acids as absorbers in the transformation process.

Evidence for an alpha-helical epitope on outer surface protein A from the Lyme disease spirochete, Borrelia burgdorferi: an application of steady-state and time-resolved fluorescence quenching techniques.

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.

Structural analysis of an outer surface protein from the Lyme disease spirochete, Borrelia burgdorferi, using circular dichroism and fluorescence spectroscopy.

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.

Ultraviolet B-Sensitive Rice Cultivar Deficient in Cyclobutyl Pyrimidine Dimer Repair.

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.

A randomized trial of high dose cyclophosphamide, vincristine, and prednisone plus or minus doxorubicin (CVP versus CAVP) with long-term follow-up in advanced non-Hodgkin's lymphoma.

Ninety-three stage III and IV patients with non-Hodgkin's lymphoma were randomized to either high dose CVP (cyclophosphamide 1500 mg/m2 i.v. day 1, vincristine 1.4 mg/m2 day 1, and prednisone 40 mg/m2 orally days 1-10) or high dose CAVP (cyclophosphamide 1000 mg/m2 i.v. day 1, doxorubicin 45 mg/m2 i.v. day 1, vincristine and prednisone as above). Overall, the complete response (CR) rates were similar (CVP 51%, CAVP 51%). Patients with the International Working Formulation diffuse large cell lymphoma had significantly higher CR with CAVP. No difference in CR duration was detected between the two regimens. CRs were durable with 68% of diffuse and 86% of diffuse large cell complete responders alive and disease free at 7 years. Survival was similar with both regimens except for patients with diffuse large cell lymphoma who survived longer with CAVP. Both regimens were equitoxic with neutropenia less than 1.0 x 10(9)/liter in 36% of courses, infections in 15% of courses, and fatal infections in three patients. These intermittent high dose cyclophosphamide equitoxic regimens produced durable responses. However, the doxorubicin-containing regimen is superior in diffuse large cell lymphoma.

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

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

Radiation-related pericardial effusions in patients with Hodgkin's disease.

Pericardial effusions following radiotherapy for Hodgkins Disease have previously been described as infrequent and related to the total dose of radiation received. Analysis of all chest x-rays on 81 patients who received upper-mantle radiotherapy for Hodgkins Disease at the Baltimore Cancer Research Center between 1968 and 1972 disclosed an incidence of pericardial effusions of 30.9% (25 of 81), with 13.6% (11 of 81) requiring limitation of activity (5) or pericardiectomy (6). Clinical presentation of radiation-related percardial effusions was subtle, with signs and symptoms a late finding if they occurred. Radiotherapy data was reviewed and no difference in total dose (rads) or time-dose relationships (rets) was found between the groups who did or did not develop effusions. Analysis of multiple pre-treatment clinical and pathological characteristics disclosed four parameters that were felt to be related to the development of pericardial effusions; elevated ESR, normal absolute lymphocyte count, initial presence of extensive mediastinal adenopathy and the addition of adjuvant chemotherapy. The presence of increasing combinations of these pretreatment 'risk factors' led to an increasing likelihood of developing a radiation-related pericardial effusion such that six of seven patients with all four 'risk factors' developed a pericardial effusion. Nine of 13 clinically significant effusions were associated with the addition of adjuvant chemotherapy. Possible pathogenetic mechanisms that include factors other than radiation dosage and the clinical management of radiation-related pericardial effusions are discussed.

Torulopsis glabrata infections in patients with cancer. Increasing incidence and relationship to colonization.

Torulopsis glabrata, an opportunistic pathogen, was found to be the etiologic agent of infections in patients with cancer. This observation prompted a retrospective review to determine the incidence and underlying factors of infection with this organism. This study showed that T. glabrata had been cultured frequently and that the incidence of infection has been progressively increasing. During a 48-month period (9/70-8/74), T. glabrata was cultured from routine surveillance and diagnostic cultures in 167 patients, 27 of whom had either presumed or documented infection. Review of clinical and necropsy records implicated T. glabrata infections as a contributory factor in the death of 14 of the 27 patients. Etiologic diagnosis of infection was established antemortem in only three patients. Pulmonary isolation in pure growth occurred in 24 of the 27 patients. Seventeen of 19 infected patients who had prior routine surveillance cultures were colonized prior to infection. Infection occurred in the setting of far advanced malignancy or leukopenia and followed the use of systemic, broad spectrum antibiotics. T. glabrata is a frequently overlooked opportunistic pathogen which, in the proper setting, appears to be producing increasing numbers of infections.

Laboratory voice data entry system.

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.

Circular dichroism user facility at the National Synchrotron Light Source: estimation of protein secondary structure.

The ultraviolet circular dichroism of a protein can be used to estimate the net fraction of its amino acids in different classes of secondary structure. Recent advances in the accuracy of such calculations have resulted from improved computational techniques, as well as extension of the spectral region analyzed to wavelengths less than 180 nm, a wavelength range beyond the limit of most laboratory-based circular dichroism spectrometers. We describe a spectrometer that uses UV radiation from the National Synchrotron Light Source at the Brookhaven National Laboratory to record circular dichroism spectra of proteins (and other biologically important molecules) in aqueous solution over the optimum wavelength range required for calculation of secondary structures. This instrument is available for use by scientists from academic, commercial and research institutions.

Acute renal failure and renal tubular squamous metaplasia following treatment with streptozotocin.

Nephrotoxicity, in the form of transient proteinuria, azotemia, abnormalities of tubular function, and acute renal failure, is the major toxic condition following administration of streptozotocin. The renal morphologic and ultrastructural abnormalities associated with streptozotocin remain poorly defined. We describe a patient with metastatic islet cell tumor of the pancreas who was treated with 16 weekly courses of 1 g/m2 of streptozotocin without marked change in renal function. Following a six-week hiatus without change in renal function, a single course of 1 g/m2 of streptozotocin was administered and resulted in acute renal failure. Light microscopic examination of the kidneys showed irregularly dilated renal tubules lined by low cuboid epithelium. The cells were pleomorphic and showed some mitoses. Nuclei were irregular and variably hyperchromatic. Electron microscopic examination disclosed large aggregates of fine microfilaments in the proximal convoluted tubules and collecting ducts. Microfilament aggregates were both free in the cytoplasm and membrane bound. Microfilaments were proved to be tonofilaments by the demonstration of keratin within the epithelium, using the immunoperoxidase method. These data suggest that squamous metaplasia may be an important part of streptozotocin renal toxicity, and the suggestion is made that they may be an antecedent of neoplastic change.

Action spectra for ultraviolet photosensitized killing of mammalian cells by misonidazole and para-nitroacetophenone.

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.

Organ selective angiotensin antagonists: sarcosyl1-cysteinyl(S-methyl)8-angiotensin I.

An angiotensin antagonist, Sarcosyl1-Cysteinyl(S-Methyl)8-angiotensin I [Sar1-Cys(Me)8-ANG I] was synthesized and its pharmacological properties evaluated in vivo (rat blood pressure assay) and in vitro (rabbit aortic strips, guinea-pig ileum and rat uterus assays). It was found to be an extremely potent angiotensin II (ANG II) antagonist in the rat pressor assay (dose ratio for ANG II of 1300 during infusion of 5.0 micrograms/kg/min Sar1-Cys(Me)8-ANG I) and a moderately effective antagonist in guinea-pig ileum (pA2 congruent to 8.2). Moderate antagonism was also seen in the rabbit aortic strip preparation (pA2 congruent to 8.1) while the analog was inactive in the rat uterus assay. In each of the preparations where antagonist activity was observed there was evidence of non-competitive antagonism. Most striking was the inability of extremely high doses ( up to 125 micrograms ANG II/kg) of ANG II to overcome the Sar1-Cys(Me)8-ANG I blockade. In both the rat pressor and guinea-pig ileum assays the Sar1-Cys(Me)8-ANG I antagonism is completely abolished in the presence of the converting enzyme inhibitor SQ14225 (Captopril-Squibb). Organ selectivity of this analog is discussed in terms of the inherent activity of the active principle (i.e. the Sar1-Cys(Me)8-angiotensin II [Sar1-Cys(Me)8-ANG II] released by the action of converting enzyme) and the availability of converting enzyme in each bioassay.

Synthesis and pharmacology of a prohormone angiotensin antagonist.

A prohormone angiotensin antagonist, Sarcosyl1-Alanyl8-angiotensin I (Sar1-Ala8-A-I) was synthesized and its pharmacological properties were evaluated in three biological systems. It was found to be a good inhibitor in vivo in the rat blood pressure assay, somewhat less active in guinea pig ileum and a relatively weak antagonist in rat uterus. In vivo the inhibitory effect was greatly attenuated by the presence of the converting enzyme inhibitor BPP5alpha.

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

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

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

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

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

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

Effects of electrocautery on midline laparotomy wound infection.

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

Novel device for quantitatively collecting small volumes of urine from laboratory rats.

A reusable urinary collection device suitable for quantitative collection of uncontaminated urinary samples from rats without using a metabolism cage has been designed. The device can be attached to the pelvic skin within 5 min with minimum handling and discomfort to the unanesthetized rat using an adhesive. The suitability of this device was investigated by collecting and analyzing urine over a 48-h period following the intraperitoneal injection of [14C]inulin. A two-way crossover study was done with samples from one of the two experiments being collected while the animal was housed in a commercially available metabolism cage equipped to separate urine and feces. The percent of the dose excreted, food and water consumption, and the urinary output were not statistically different for rats housed in the metabolism cages or with the collection device attached. Histopathological examination of the rats revealed no pathology after a 24-h period except minor skin inflammation which occurred both in controls and animals to which the device was attached. These studies demonstrate the comparability of the new urine collection device to a commercially available metabolism cage in the quantitative collection of small (0-7 mL) urine samples with less contamination of the samples from the environment.