Raman spectroscopy of uracil DNA glycosylase-DNA complexes: insights into DNA damage recognition and catalysis

Using off-resonance Raman spectroscopy, we have examined each complex along the catalytic pathway of the DNA repair enzyme uracil DNA glycosylase (UDG). The binding of undamaged DNA to UDG results in decreased intensity of the DNA Raman bands, which can be attributed to an increased level of base stacking, with little perturbation in the vibrational modes of the DNA backbone. A specific complex between UDG and duplex DNA containing 2'-beta-fluorodeoxyuridine shows similar increases in the level of DNA base stacking, but also a substrate-directed conformational change in UDG that is not observed with undamaged DNA, consistent with an induced-fit mechanism for damage site recognition. The similar increases in the level of DNA base stacking for the nonspecific and specific complexes suggest a common enzyme-induced distortion in the DNA, potentially DNA bending. The difference spectrum of the extrahelical uracil base in the substrate-analogue complexes reveals only a small electron density reorganization in the uracil ring for the ground state complex, but large 34 cm(-)(1) downshifts in the carbonyl normal modes. Thus, UDG activates the uracil ring in the ground state mainly through H bonds to its C=O groups, without destroying its quasi-aromaticity. This result is at variance with the conclusion from a recent crystal structure, in which the UDG active site significantly distorts the flipped-out pseudouridine analogue such that a change in hybridization at C1 occurs [Parikh, S. S., et al. (2000) Proc. Natl. Acad. Sci. USA 97, 5083]. The Raman vibrational signature of the bound uracil product differs significantly from that of free uracil at neutral pH, and indicates that the uracil is anionic. This is consistent with recent NMR results, which established that the enzyme stabilizes the uracil anion leaving group by 3.4 pK(a) units compared to aqueous solution, contributing significantly to catalysis. These observations are generally not apparent from the high-resolution crystal structures of UDG and its complexes with DNA; thus, Raman spectroscopy can provide unique and valuable insights into the nature of enzyme-DNA interactions.     

Structural characterization of the entire 1.3S subunit of transcarboxylase from Propionibacterium shermanii.

Transcarboxylase (TC) from Propionibacterium shermanii, a biotin-dependent enzyme, catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. Within the multisubunit enzyme complex, the 1.3S subunit functions as the carboxyl group carrier. The 1.3S is a 123-amino acid polypeptide (12.6 kDa), to which biotin is covalently attached at Lys 89. We have expressed 1.3S in Escherichia coli with uniform 15N labeling. The backbone structure and dynamics of the protein have been characterized in aqueous solution by three-dimensional heteronuclear nuclear magnetic resonance (NMR) spectroscopy. The secondary structure elements in the protein were identified based on NOE information, secondary chemical shifts, homonuclear 3J(HNHalpha) coupling constants, and amide proton exchange data. The protein contains a predominantly disordered N-terminal half, while the C-terminal half is folded into a compact domain comprising eight beta-strands connected by short loops and turns. The topology of the C-terminal domain is consistent with the fold found in both carboxyl carrier and lipoyl domains, to which this domain has approximately 26-30% sequence similarity.     

Fish and Phytoplankton Exhibit Contrasting Temporal Species Abundance Patterns in a Dynamic North Temperate Lake

Temporal patterns of species abundance, although less well-studied than spatial patterns, provide valuable insight to the processes governing community assembly. We compared temporal abundance distributions of two communities, phytoplankton and fish, in a north temperate lake. We used both 17 years of observed relative abundance data as well as resampled data from Monte Carlo simulations to account for the possible effects of non-detection of rare species. Similar to what has been found in other communities, phytoplankton and fish species that appeared more frequently were generally more abundant than rare species. However, neither community exhibited two distinct groups of “core” (common occurrence and high abundance) and “occasional” (rare occurrence and low abundance) species. Both observed and resampled data show that the phytoplankton community was dominated by occasional species appearing in only one year that exhibited large variation in their abundances, while the fish community was dominated by core species occurring in all 17 years at high abundances. We hypothesize that the life-history traits that enable phytoplankton to persist in highly dynamic environments may result in communities dominated by occasional species capable of reaching high abundances when conditions allow. Conversely, longer turnover times and broad environmental tolerances of fish may result in communities dominated by core species structured primarily by competitive interactions.     

Effects of Hormone Therapy on Cognition and Mood in Recently Postmenopausal Women: Findings from the Randomized,Controlled KEEPS–Cognitive and Affective Study

Background

Menopausal hormone therapy (MHT) reportedly increases the risk of cognitive decline in women over age 65 y. It is unknown whether similar risks exist for recently postmenopausal women, and whether MHT affects mood in younger women. The ancillary Cognitive and Affective Study (KEEPS-Cog) of the Kronos Early Estrogen Prevention Study (KEEPS) examined the effects of up to 4 y of MHT on cognition and mood in recently postmenopausal women.

Methods and Findings

KEEPS, a randomized, double-blinded, placebo-controlled clinical trial, was conducted at nine US academic centers. Of the 727 women enrolled in KEEPS, 693 (95.3%) participated in the ancillary KEEPS-Cog, with 220 women randomized to receive 4 y of 0.45 mg/d oral conjugated equine estrogens (o-CEE) plus 200 mg/d micronized progesterone (m-P) for the first 12 d of each month, 211 women randomized to receive 50 μg/d transdermal estradiol (t-E2) plus 200 mg/d m-P for the first 12 d of each month, and 262 women randomized to receive placebo pills and patches. Primary outcomes included the Modified Mini-Mental State examination; four cognitive factors: verbal learning/memory, auditory attention/working memory, visual attention/executive function, and speeded language/mental flexibility; and a mood measure, the Profile of Mood States (POMS). MHT effects were analyzed using linear mixed-effects (LME) models, which make full use of all available data from each participant, including those with missing data. Data from those with and without full data were compared to assess for potential biases resulting from missing observations. For statistically significant results, we calculated effect sizes (ESs) to evaluate the magnitude of changes.On average, participants were 52.6 y old, and 1.4 y past their last menstrual period. By month 48, 169 (24.4%) and 158 (22.8%) of the 693 women who consented for ancillary KEEPS-Cog were lost to follow-up for cognitive assessment (3MS and cognitive factors) and mood evaluations (POMS), respectively. However, because LME models make full use all available data, including data from women with missing data, 95.5% of participants were included in the final analysis (n = 662 in cognitive analyses, and n = 661 in mood analyses). To be included in analyses, women must have provided baseline data, and data from at least one post-baseline visit. The mean length of follow-up was 2.85 y (standard deviation [SD] = 0.49) for cognitive outcomes and 2.76 (SD = 0.57) for mood outcomes. No treatment-related benefits were found on cognitive outcomes. For mood, model estimates indicated that women treated with o-CEE showed improvements in depression and anxiety symptoms over the 48 mo of treatment, compared to women on placebo. The model estimate for the depression subscale was −5.36 × 10⁻² (95% CI, −8.27 × 10⁻² to −2.44 × 10^−2; ES = 0.49, p < 0.001) and for the anxiety subscale was −3.01 × 10⁻² (95% CI, −5.09 × 10⁻² to −9.34 × 10⁻³; ES = 0.26, p < 0.001). Mood outcomes for women randomized to t-E2 were similar to those for women on placebo. Importantly, the KEEPS-Cog results cannot be extrapolated to treatment longer than 4 y.

Conclusions

The KEEPS-Cog findings suggest that for recently postmenopausal women, MHT did not alter cognition as hypothesized. However, beneficial mood effects with small to medium ESs were noted with 4 y of o-CEE, but not with 4 y of t-E2. The generalizability of these findings is limited to recently postmenopausal women with low cardiovascular risk profiles.

Trial Registration

ClinicalTrials.gov NCT00154180 and NCT00623311     

A Case Control Study Reveals that Polyomaviruria Is Significantly Associated with Interstitial Cystitis and Vesical Ulceration

Objectives

To investigate whether polyomaviruses contribute to interstitial cystitis pathogenesis.

Subjects and Methods

A prospective study was performed with 50 interstitial cystitis cases compared with 50 age-matched, disease-free controls for the frequency of polyomaviruria. Associations between polyomaviruria and disease characteristics were analysed in cases. Polyomavirus in urine and bladder tissue was detected with species (JC virus vs. BK virus) specific, real-time PCR.

Results

Case patients were reflective of interstitial cystitis epidemiology with age range from 26–88 years (median 58) and female predominance (41/50 F). There was a significant increase in the frequency of polyomavirus shedding between cases and controls (p<0.02). Polyomavirus shedding, in particular BK viruria, was associated with vesical ulceration, a marker of disease severity, among interstitial cystitis cases after adjustment for age and sex (OR 6.8, 95% CI 1.89–24.4). There was a significant association among cases between the presence of BK viruria and response to intravesical Clorpactin therapy (OR 4.50, 95% CI 1.17–17.4).

Conclusion

The presence of polyomaviruria was found to be associated with the ulcerative form of interstitial cystitis. Clorpactin, which has anti-DNA virus activity, was more likely to improve symptoms in the presence of BK viruria. These data from this pilot study suggest associations between polyomaviruria and interstitial cystitis warranting further investigation.     

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions.