Reversal of a mutator activity by a nearby fidelity-neutral substitution in the RB69 DNA polymerase binding pocket

Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.     

Growth hormone deficiency and replacement in patients with treated Cushing's Disease, prolactinomas and non-functioning pituitary adenomas: effects on body composition, glucose metabolism, lipid status and bone mineral density

BACKGROUND/AIMS: This study was designed to determine whether previous Cushing's disease (CD) or prolactinoma (PRL) could exert adverse effects additional to those of growth hormone (GH) deficiency as a consequence of variable degrees of prior hypogonadism or hypercatabolism. We report the effects of 5 years GH treatment in 124 GH deficiency adults; 42 patients with non-functioning pituitary adenomas (NFPA), 43 with treated PRL and 39 with treated CD. METHODS: Fasting plasma glucose, HbA(1c), lipoprotein profile, anthropometry and bone mineral density (BMD) were measured at baseline, 6 months and annually up to 5 years. RESULTS: Mean body mass index remained unchanged in the PRL group and tended to increase in the NFPA group. In contrast, body mass index decreased in the CD group. Decreases in waist and waist/hip ratio were seen in all groups at 6 months. Decreases in total cholesterol and low-density lipoprotein cholesterol were seen in all groups and remained sustained at 5 years. Plasma glucose and HbA(1c) increased at 6 months. Subsequently, plasma glucose returned to baseline values at 5 years; in contrast, HbA(1c )remained unchanged at the end of the study. Baseline lumbar spine and hip BMD were lower in the PRL and CD groups than in the NFPA group, decreased over 1 year in all groups and subsequently increased by 2 years in NFPA with a subsequent increase in lumbar spine BMD in PRL and CD groups delayed to 3-5 years. CONCLUSIONS: Baseline characteristics and response to GH replacement are qualitatively similar in NFPA, PRL and CD patients. Because improvements in BMD occur later in PRL and CD patients, an extended trial of GH therapy may be indicated in those patients who were commenced on GH therapy as an additional treatment for reduced BMD.     

Multiple Exposures to Chlorhexidine and Xylitol: Adhesion and Biofilm Formation by Streptococcus mutans

Growing evidence from clinical studies suggests that mothers using xylitol gums or lozenges have decreased levels of Streptococcus mutans (SM) and do not transmit these cariogenic bacteria as readily to their children. To begin to determine mechanisms for these clinical findings and to explore potential synergism of antimicrobial combinations, we studied the effect of multiple exposures of chlorhexidine (CHX) combined with copper gluconate (CG) or zinc gluconate (ZG) followed by xylitol (XYL) on the ability of SM to adhere and form biofilms. Cell suspensions of SM were exposed two times to CHX; CG; CHX plus CG; ZG; and CHX plus ZG, and then four times to XYL. Control cells were exposed six times to water or XYL or received no treatment. For biofilm assessment, glass slides were inoculated with treated cells, and numbers of bacteria were enumerated after 48 hours of incubation. To assess the ability of SM to adhere, microtiter plate wells coated with primary S. sanguinis biofilms grown in sucrose were inoculated with treated SM, and adhesion was determined. Cells exposed to CHX–XYL combinations exhibited significant but transient inhibition of growth. The multiple-exposure regimen groups showed significant decreases in the ability of SM to form biofilms (P < 0.05). However, the CHX–XYL group exhibited a much greater effect than the other treatment groups (P < 0.001). Adhesion studies revealed that none of the multiple-exposure regimens had a significant effect on adhesion of SM to primary biofilms of S. sanguinis. We concluded that significant inhibition of SM growth and subsequent inability to grow as biofilms in the presence of sucrose occurs after a staggered exposure regimen to CHX initially and then to XYL. This may help explain the clinical data showing the decreased levels of SM in mothers treated with CHX and XYL.     

Phenylalanine 90 and 93 are localized within the phenol binding site of human UDP-glucuronosyltransferase 1A10 as determined by photoaffinity labeling, mass spectrometry, and site-directed mutagenesis

4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.     

Elimination of electrocardiogram contamination from electromyogram signals: An evaluation of currently used removal techniques.

Trunk electromyographic signals (EMG) are often contaminated with heart muscle electrical activity (ECG) due to the proximity of the collection sites to the heart and the volume conduction characteristics of the ECG through the torso. Few studies have quantified ECG removal techniques relative to an uncontaminated EMG signal (gold standard or criterion measure), or made direct comparisons between different methods for a given set of data. Understanding the impacts of both untreated contaminated EMG and ECG elimination techniques on the amplitude and frequency parameters is vital given the widespread use of EMG. The purpose of this study was to evaluate four groups of current and commonly used techniques for the removal of ECG contamination from EMG signals. ECG recordings at two intensity levels (rest and 50% maximum predicted heart rate) were superimposed on 11 uncontaminated biceps brachii EMG signals (rest, 7 isometric and 3 isoinertial levels). The 23 removal methods used were high pass digital filtering (finite impulse response (FIR) using a Hamming window, and fourth-order Butterworth (BW) filter) at five cutoff frequencies (20, 30, 40, 50, and 60 Hz), template techniques (template subtraction and an amplitude gating template), combinations of the subtraction template and high pass digital filtering, and a frequency subtraction/signal reconstruction method. For muscle activation levels between 10% and 25% of maximum voluntary contraction, the template subtraction and BW filter with a 30 Hz cutoff were the two best methods for maximal ECG removal with minimal EMG distortion. The BW filter with a 30 Hz cutoff provided the optimal balance between ease of implementation, time investment, and performance across all contractions and heart rate levels for the EMG levels evaluated in this study.     

Gas exchange responses of Chesapeake Bay tidal marsh species under field and laboratory conditions

Summary Laboratory and field gas exchange measurements were made on C₃ (Scirpus olneyi Gray) and C₄ (Spartina patens (Ait.) Mahl., Distichlis spicata (L.) Green) species from an irregularly flooded tidal marsh on the Chesapeake Bay. Laboratory measurements were made on plants grown from root stocks that were transplanted to a greenhouse and grown under high light and high nutrient conditions. The two C₄ species were similar in their laboratory gas exchange characteristics: both had higher net carbon exchange rates, higher mesophyll conductances, higher photosynthetic temperature optima and lower leaf conductances than the C₃ species. The laboratory photosynthetic water use efficiency of the C₄ species was approximately three times that of the C₃ species.Field gas exchange responses of the above species were measured in situ a Chesapeake Bay tidal marsh. Despite differences in biological potential measured in the laboratory, all three species had similar in situ carbon exchange rates on a leaf area basis. On a dry weight basis, leaves of the two C₄ species had about 1.4 times higher light saturated CO₂ assimilation rates than the C₃ species. Light saturation of CO₂ exchange occurred at photosynthetic photon flux densities of 80 n Einstein cm^-2s^-1, compared with 160 n Einstein cm ^-2s^-1 in the laboratory grown plants. Spartina patens and Scirpus olneyi had similar daily CO₂ assimilation rates, but the daily transpiration rate of the C₃ species was almost twice that of the C₄ species. Spartina patens showed greater seasonal decrease in photosynthesis than Distichlis spicata and Scirpus olneyi. The two C₄ grass species maintained higher mesophyll conductances and photosynthetic water use efficiencies than the C₄ sedge.     

Coordinated regulation of caveolin-1 and Rab11a in apical recycling compartments of polarized epithelial cells

Recent studies have identified caveolin-1, a protein best known for its functions in caveolae, in apical endocytic recycling compartments in polarized epithelial cells. However, very little is known about the regulation of caveolin-1 in the endocytic recycling pathway. To address this question, in the current study we compared the relationship between compartments enriched in sub-apical caveolin-1 and Rab11a, a well-defined marker of apical recycling endosomes, using polarized MDCK cells as a model. We show that caveolin-1-containing vesicles define a compartment that partially overlaps with Rab11a, and that the distribution of subapical caveolin-1 and Rab11a shows a similar dependence on microtubule disruption. Mutants of the Rab11a effector, Rab11-FIP2 also altered the localization of caveolin-1. These findings indicate that caveolin-1 is coordinately regulated with Rab11a within the apical recycling system of polarized epithelial cells, suggesting that the two proteins are components of the same pathway.     

COSA-1 reveals robust homeostasis and separable licensing and reinforcement steps governing meiotic crossovers

Crossovers (COs) between homologous chromosomes ensure their faithful segregation during meiosis. We identify C.?elegans COSA-1, a cyclin-related protein conserved in metazoa, as a key component required to convert meiotic double-strand breaks (DSBs) into COs. During late meiotic prophase, COSA-1 localizes to foci that correspond to the single CO site on each homolog pair and indicate sites of eventual concentration of other conserved CO proteins. Chromosomes gain and lose competence to load CO proteins during meiotic progression, with competence to load COSA-1 requiring prior licensing. Our data further suggest a self-reinforcing mechanism maintaining CO designation. Modeling of a nonlinear dose-response relationship between IR-induced DSBs and COSA-1 foci reveals efficient conversion of DSBs into COs when DSBs are limiting and a robust capacity to limit cytologically differentiated CO sites when DSBs are in excess. COSA-1 foci serve as a unique live cell readout for investigating CO formation and CO interference.