Killing of Escherichia coli K-12 by near-ultraviolet radiation in the presence of hydrogen peroxide: Role of double-strand DNA breaks in absence of recombinational repair

Near-ultraviolet (NUV) radiation killing of Escherichia coli K-12 can be enhanced by a sub-lethal concentration of hydrogen peroxide. This can be divided into a “RecA-dependent” and “RecA-independent” synergistic killing action. Stationary phase wild-type and 8 closely related repair-deficient mutants were examined for their NUV sensitivities in the presence and absence of H₂O₂. All exhibited the “RecA-independent” synergism; i.e., H₂O₂ enhanced NUV lethality when RecA repair was not operating. The “RecA-independent” synergism did not result from destruction of repair enzymes. Very few DNA—protein crosslinks could be detected following NUV plus H₂O₂ treatment. However, double-strand (DS) DNA breaks were produced, apparently by conversion of closely spaced single-strand (SS) breaks on opposite strands. The correlation between DS-break formation and lethality in wild-type and a polA mutant indicates that the RecA-independent synergistic killing results from the conversion of SS into lethal DS breaks.     

Rational Design and Biophysical Characterization of Thioredoxin-Based Aptamers: Insights into Peptide Grafting

Peptide aptamers are simple structures, often made up of a single-variable peptide loop constrained within a constant scaffold protein. Aptamers were rationally designed by inserting peptides into a solvent-exposed loop on thioredoxin (Trx). They were designed to interact with the proteins elongation initiation factor 4E (eIF4E) and mouse double minute 2 (MDM2) and were then validated by competitive fluorescence anisotropy experiments. The constructed aptamers interacted with eIF4E and MDM2 with apparent K_d values of 1.25 ± 0.06 μM and 0.09 ± 0.01 μM, respectively, as determined by isothermal titration calorimetry (ITC). The MDM2 aptamer (SuperTIP) interacted ∼ 2-fold more tightly with MDM2 than the free linear peptide (12.1 peptide), while the eIF4E aptamer elongation initiation factor 4GI-SG interacted ∼ 5-fold less strongly than the free linear peptide (elongation initiation factor 4GI). These differences in binding with respect to each aptamer's free peptide reveal that there are more factors involved than just constraining a peptide in a scaffold that lead to tighter binding. ITC studies of aptamer interactions reveal an enthalpic component more favorable than that for the free linear peptides, as well as a larger unfavorable entropic component. These results indicated that stapling of the free peptide in the scaffold increases the favorable enthalpy of the interaction with the target protein. Thermostability studies also revealed that peptide insertion significantly destabilized the Trx scaffold by ∼ 27 °C. It is this destabilization that leads to an increase in the flexibility of the Trx scaffold, which presumably is lost upon the aptamer's interaction with the target protein and is the cause of the increase in unfavorable entropy in the ITC studies. The precise origin of the enthalpic effect was further studied using molecular dynamics for the MDM2-SuperTIP system, which revealed that there were also favorable electrostatic interactions between the Trx scaffold and the MDM2 protein itself, as well as with the inserted peptide. This work reveals that any increase in the binding affinity of an aptamer over a free peptide is dependent on the increase in the favorable enthalpy of binding, which is ideally caused by stapling of the peptide or by additional interactions between the aptamer protein and its target. These need to be sufficient to compensate for the destabilization of the scaffold by peptide insertion. These observations will be useful in future aptamer designs.     

Dissecting the Alternatively Folded State of the Antibody Fab Fragment

Intact antibodies and antigen binding fragments (Fab) have been previously shown to form an alternatively folded state (AFS) at low pH. This state consists primarily of secondary structure interactions, with reduced tertiary structure content. The AFS can be distinguished from the molten globule state by the formation of nonnative structure and, in particular, its high stability. In this study, the isolated domains of the MAK33 (murine monoclonal antibody of the subtype κ/IgG1) Fab fragment were investigated under conditions that have been reported to induce the AFS. Surprising differences in the ability of individual domains to form the AFS were observed, despite the similarities in their native structures. All Fab domains were able to adopt the AFS, but only for V_H (variable domain of the heavy chain) could a significant amount of tertiary structure be detected and different conditions were needed to induce the AFS. V_H, the least stable of the domains under physiological conditions, was the most stable in the AFS, yet all domains showed significant stability against thermal and chemical unfolding in their AFS. Formation of the AFS was found to generally proceed via the unfolded state, with similar rates for most of the domains. Taken together, our data reveal striking differences in the biophysical properties of the AFS of individual antibody domains that reflect the variation possible for domains of highly homologous native structures. Furthermore, they allow individual domain contributions to be dissected from specific oligomer effects in the AFS of the antibody Fab fragment.     

Luminescence of the near‐ultraviolet blue‐emitting Ba9Al2Si6O24:Ce3+ phosphor

A near‐ultraviolet (NUV) blue‐emitting phosphor Ba₉Al₂Si₆O₂₄:Ce³⁺ (BAS:Ce³⁺) was synthesized using a high‐temperature solid‐state reaction. BAS:Ce³⁺ had an excitation band peak at about 328 nm and showed a blue emission band. The NUV‐blue emission band had a peak at about 386 nm with a band width of about 60 nm, attributed to the 5d–4f transition of Ce³⁺. Fluorescent decay showed an exponential model with a lifetime of 27.2 nsec. At 150°C, the luminescence intensity decreased to 68.7% compared with the intensity at room temperature.     

Optimising sporulation and virulence in Drechslera avenacea

Studies were conducted on agar media to optimise sporulation of Drechslera avenacea, a fungal pathogen being evaluated as a biological control agent for Avena species (wild oats). Conidium production was affected by nutrition, pH, temperature and light conditions. Of the agar media tested, Czapek Dox agar (CZA) and half-strength oatmeal agar (½OMA) were the only media where sporulation occurred at all temperatures tested under a 12-h light:12-h dark photoperiod (L/D). The optimum temperature for conidium production was 20°C on ½OMA, whereas there was no optimum temperature on CZA. Under a 12-h near-ultraviolet (NUV):12-h dark photoperiod (NUV/D), similar numbers of conidia were produced on CZA at 6.66, 14.56, and 22.78 W m^?2, whereas on ½OMA conidium production was the highest at 14.56 W m^?2. When NUV/D and L/D conditions were compared, similar numbers of conidia where produced on CZA, whereas ½OMA conidium production was superior under the NUV/D photoperiod. Considerable variation in sporulation and degree of virulence of D. avenacea was detected among isolates from different geographic areas. The most virulent conidia were obtained on ½OMA at 20°C incubated under continuous illumination NUV light. Therefore, the most suitable conditions for conidium production of D. avenacea were growth for 1 week on ½OMA at 20°C under continuous NUV at an intensity of 14.56 W m^?2. Under these conditions, 1.1×10⁵ conidia mL^?1 were produced which is the highest sporulation yet reported for any Drechslera spp., which are traditionally poor sporulators.     

Effects of Temperature on the Pattern of Anthocyanin Accumulation in Seedlings of Polygonum cuspidatum

Polygonum cuspidatum seedling. Anthocyanin accumulated first in the lower part of hypocotyls and then the site of accumulation gradually extended toward the upper part of hypocotyls when seedlings were irradiated with white light (WL) at 25 C. Etiolated seedlings accumulated anthocyanin only in the upper parts (hook and cotyledons) when the seedlings were irradiated with WL at 5 C. De-etiolated seedlings that had been pre-irradiated with WL for 1 day at 25 C accumulated anthocyanin both in upper and lower parts of the seedlings when the seedlings were irradiated with WL at 5 C. Spectral sensitivity was dependent on the temperature during irradiation. Red light (R), blue light (B), and near ultra-violet light (NUV) induced the accumulation of anthocyanin at 5 C but only NUV was effective in inducing the accumulation of anthocyanin at 25 C. Dichlorophenyl dimethylurea (DCMU) inhibited WL-induced anthocyanin accumulation but did not NUV-induced anthocyanin accumulation at 25 C. However, sucrose promoted NUV action at 25 C, indicating that photosynthesis can promote NUV-induced anthocyanin accumulation. Distribution of phytochrome in etiolated seedlings, that was examined by spectrophotometry, was similar to the distribution of anthocyanin at 5 C. Furthermore, phytochrome remained after 48 hr irradiation with WL at 5 C although phytochrome was rapidly degraded at 25 C. Received 12 July 1999/ Accepted in revised form 24 December 1999     

Psoralen plus near ultraviolet light inactivation of cultured Chinese hamster cells and its relation to DNA cross-links

The survival curve of Chinese hamster cells exposed to near ultraviolet (NUV) light in the presence of 4,5′,8-trimethylpsoralen exhibits a pronounced shoulder, i.e. cells accumulate sublethal damage before survival decreases exponentially. During incubation between fractionated exposures, the cells are able to recover their capacity to accumulate sublethal damage, although at a slower rate than after X-irradiation. Synchronized cells demonstrate a pronounced age-response variation, similar to that of X-irradiated cells, in which maximum resistance occurs in the second half of the S phase. During a second NUV exposure without psoralen, following a first treatment consisting of psoralen-plus-NUV, only DNA cross-links are produced. Using this procedure a correlation was found between cell killing and the production of DNA cross-links.     

The random (non-specific) forward mutational response of gene loci in Aspergillus conidia after photosensitisation to near ultraviolet light (365 nm) by 8-methoxypsoralen

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Evidence is presented that a treatment with 8-methoxypsoralen plus near UV light generally produces a random (non-specific) mutational effect on different gene loci in Aspergillus conidia.