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M O'Donnell

Publications and source records attributed to M O'Donnell.

At least 37 records · Page 2Linked to original sources
PubMed2001

Crystal structure of the processivity clamp loader gamma (gamma) complex of E. coli DNA polymerase III.

The gamma complex, an AAA+ ATPase, is the bacterial homolog of eukaryotic replication factor C (RFC) that loads the sliding clamp (beta, homologous to PCNA) onto DNA. The 2.7/3.0 A crystal structure of gamma complex reveals a pentameric arrangement of subunits, with stoichiometry delta':gamma(3):delta. The C-terminal domains of the subunits form a circular collar that supports an asymmetric arrangement of the N-terminal ATP binding domains of the gamma motor and the structurally related domains of the delta' stator and the delta wrench. The structure suggests a mechanism by which the gamma complex switches between a closed state, in which the beta-interacting element of delta is hidden by delta', and an open form similar to the crystal structure, in which delta is free to bind to beta.

Binding Sites↗
PubMed2001

Mechanism of processivity clamp opening by the delta subunit wrench of the clamp loader complex of E. coli DNA polymerase III.

The dimeric ring-shaped sliding clamp of E. coli DNA polymerase III (beta subunit, homolog of eukaryotic PCNA) is loaded onto DNA by the clamp loader gamma complex (homolog of eukaryotic Replication Factor C, RFC). The delta subunit of the gamma complex binds to the beta ring and opens it. The crystal structure of a beta:delta complex shows that delta, which is structurally related to the delta' and gamma subunits of the gamma complex, is a molecular wrench that induces or traps a conformational change in beta such that one of its dimer interfaces is destabilized. Structural comparisons and molecular dynamics simulations suggest a spring-loaded mechanism in which the beta ring opens spontaneously once a dimer interface is perturbed by the delta wrench.

Amino Acid Sequence↗
PubMed2001

Correlation of vascular endothelial growth factor expression with fibroblast growth factor-8 expression and clinico-pathologic parameters in human prostate cancer.

Vascular endothelial growth factor (VEGF) mediates neo-angiogenesis during tumour progression and is known to cooperate with the fibroblast growth factor (FGF) system to facilitate angiogenesis in a synergistic manner. In view of this, we have investigated VEGF expression in 67 cases of prostate cancer previously characterized for fibroblast growth factor-8 (FGF-8) expression. Cytoplasmic VEGF staining was detected in malignant cells in 45 out of 67 cases. Cytoplasmic staining was found in adjacent stromal cells in 32 cases, being particularly strong around nests of invasive tumour. Positive VEGF immunoreactivity in benign glands was restricted to basal epithelium. A significant association was observed between tumour VEGF and FGF-8 expression (P = 0.004). We identified increased VEGF immunoreactivity in both malignant epithelium and adjacent stroma and both were found to be significantly associated with high tumour stage (P = 0.0047 and P = 0.0002, respectively). VEGF expression also correlated with increased serum PSA levels (P = 0.01). Among positively stained tumours, VEGF expression showed a significant association with Gleason score (P = 0.04). Cases showing positive VEGF immunoreactivity in the stroma had a significantly reduced survival rate compared to those with negative staining (P = 0.037). Cases with tumours expressing both FGF-8 in the malignant epithelium and VEGF in the adjacent stroma had a significantly worse survival rate than those with tumours negative for both, or only expressing one of the two growth factors (P = 0.029). Cox multivariate regression analysis of survival demonstrated that stromal VEGF and tumour stage were the most significant independent predictors of survival. In conclusion, we report for the first time a correlation of both tumour and stromal VEGF expression in prostate cancer with clinical parameters as well as its correlation to FGF-8 expression.

Aged↗
PubMed2001

Interaction of the beta sliding clamp with MutS, ligase, and DNA polymerase I.

The beta and proliferating cell nuclear antigen (PCNA) sliding clamps were first identified as components of their respective replicases, and thus were assigned a role in chromosome replication. Further studies have shown that the eukaryotic clamp, PCNA, interacts with several other proteins that are involved in excision repair, mismatch repair, cellular regulation, and DNA processing, indicating a much wider role than replication alone. Indeed, the Escherichia coli beta clamp is known to function with DNA polymerases II and V, indicating that beta also interacts with more than just the chromosomal replicase, DNA polymerase III. This report demonstrates three previously undetected protein-protein interactions with the beta clamp. Thus, beta interacts with MutS, DNA ligase, and DNA polymerase I. Given the diverse use of these proteins in repair and other DNA transactions, this expanded list of beta interactive proteins suggests that the prokaryotic beta ring participates in a wide variety of reactions beyond its role in chromosomal replication.

Adenosine Triphosphatases↗
PubMed2001

Simple bedside additive tool for prediction of in-hospital mortality after percutaneous coronary interventions.

BACKGROUND: Risk-adjustment models for percutaneous coronary intervention (PCI) mortality have been recently reported, but application in bedside prediction of prognosis for individual patients remains untested. METHODS AND RESULTS: Between July 1, 1997 and September 30, 1999, 10 796 consecutive procedures were performed in a consortium of 8 hospitals. Predictors of in-hospital mortality were identified by use of multivariate logistic regression analysis. The final model was validated by use of the bootstrap technique. Additional validation was performed on an independent data set of 5863 consecutive procedures performed between October 1, 1999, and August 30, 2000. An additive risk-prediction score was developed by rounding coefficients of the logistic regression model to the closest half-integer, and a visual bedside tool for the prediction of individual patient prognosis was developed. In this patient population, the in-hospital mortality rate was 1.6%. Multivariate regression analysis identified acute myocardial infarction, cardiogenic shock, history of cardiac arrest, renal insufficiency, low ejection fraction, peripheral vascular disease, lesion characteristics, female sex, and advanced age as independent predictors of death. The model had excellent discrimination (area under the receiver operating characteristic curve, 0.90) and was accurate for prediction of mortality among different subgroups. Near-perfect correlation existed between calculated scores and observed mortality, with higher scores associated with higher mortality. CONCLUSIONS: Accurate predictions of individual patient risk of mortality associated with PCI can be achieved with a simple bedside tool. These predictions could be used during discussions of prognosis before and after PCI.

Age Factors↗
PubMed2001

Development of a novel helicase assay using electrochemiluminescence.

DNA helicases are ubiquitous enzymes involved in DNA replication, recombination, and repair. These enzymes use the energy of ATP to unwind duplex DNA. A common approach to measure DNA helicase activity utilizes electrophoresis to visualize product formation in a gel. The present study develops a rapid helicase assay based on electrochemiluminescence. The assay is adaptable to rapid high-throughput screening for chemical inhibitors. The assay is applied to Escherichia coli DnaB. Three other technologies have also been applied to DnaB and are compared to results of the electrochemiluminescence-based assay.

Base Sequence↗
PubMed2001

Mechanism of beta clamp opening by the delta subunit of Escherichia coli DNA polymerase III holoenzyme.

The beta sliding clamp encircles the primer-template and tethers DNA polymerase III holoenzyme to DNA for processive replication of the Escherichia coli genome. The clamp is formed via hydrophobic and ionic interactions between two semicircular beta monomers. This report demonstrates that the beta dimer is a stable closed ring and is not monomerized when the gamma complex clamp loader (gamma(3)delta(1)delta(1)chi(1)psi(1)) assembles the beta ring around DNA. delta is the subunit of the gamma complex that binds beta and opens the ring; it also does not appear to monomerize beta. Point mutations were introduced at the beta dimer interface to test its structural integrity and gain insight into its interaction with delta. Mutation of two residues at the dimer interface of beta, I272A/L273A, yields a stable beta monomer. We find that delta binds the beta monomer mutant at least 50-fold tighter than the beta dimer. These findings suggest that when delta interacts with the beta clamp, it binds one beta subunit with high affinity and utilizes some of that binding energy to perform work on the dimeric clamp, probably cracking one dimer interface open.

Adenosine Triphosphate↗
PubMed2001

A model for SOS-lesion-targeted mutations in Escherichia coli.

The UmuD'2C protein complex (Escherichia coli pol V) is a low-fidelity DNA polymerase (pol) that copies damaged DNA in the presence of RecA, single-stranded-DNA binding protein (SSB) and the beta,gamma-processivity complex of E. coli pol III (ref. 4). Here we propose a model to explain SOS-lesion-targeted mutagenesis, assigning specific biochemical functions for each protein during translesion synthesis. (SOS lesion-targeted mutagenesis occurs when pol V is induced as part of the SOS response to DNA damage and incorrectly incorporates nucleotides opposite template lesions.) Pol V plus SSB catalyses RecA filament disassembly in the 3' to 5' direction on the template, ahead of the polymerase, in a reaction that does not involve ATP hydrolysis. Concurrent ATP-hydrolysis-driven filament disassembly in the 5' to 3' direction results in a bidirectional stripping of RecA from the template strand. The bidirectional collapse of the RecA filament restricts DNA synthesis by pol V to template sites that are proximal to the lesion, thereby minimizing the occurrence of untargeted mutations at undamaged template sites.

Adenosine Triphosphate↗
PubMed2001

The ring-type polymerase sliding clamp family.

Ring-type polymerases consist of a DNA polymerase, a ring-shaped sliding clamp protein and a clamp-loading complex. Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and the beta clamp in prokaryotes. Both PCNA and beta form a ring around DNA, which is made up of two subunits of three domains each in beta but three subunits of two domains each in PCNA. Despite this difference and a lack of detectable sequence homology, the structures of the two rings are very similar. The sliding clamp slides along DNA and tethers the polymerase to the DNA, enabling rapid and processive DNA replication.

Animals↗
PubMed2001

Health status of children with moderate to severe cerebral palsy.

The aim of the study was to evaluate the health of children with cerebral palsy (CP) using a global assessment of quality of life, condition-specific measures, and assessments of health care use. A multicenter population-based cross-sectional survey of 235 children, aged 2 to 18 years, with moderate to severe impairment, was carried out using Gross Motor Function Classification System (GMFCS) levels III (n = 56), IV (n = 55), and V (n = 122). This study group scored significantly below the mean on the Child Health Questionnaire (CHQ) for Pain, General Health, Physical Functioning, and Impact on Parents. These children used more medications than children without CP from a national sample. Fifty-nine children used feeding tubes. Children in GMFCS level V who used a feeding tube had the lowest estimate of mental age, required the most health care resources, used the most medications, had the most respiratory problems, and had the lowest Global Health scores. Children with the most severe motor disability who have feeding tubes are an especially frail group who require numerous health-related resources and treatments. Also, there is a relationship among measures of health status such as the CHQ, functional abilities, use of resources, and mental age, but each appears to measure different aspects of health and well-being and should be used in combination to reflect children's overall health status.

Activities of Daily Living↗
PubMed2001

A prospective, single-blind, randomized controlled trial of antiseptic cream for recurrent epistaxis in childhood.

Epistaxis is common in children. Trials show antiseptic cream is as effective as cautery, but it is not known whether either is better than no treatment. We wished to know the efficacy of cream in children with recurrent epistaxis. The design was a single-blind, prospective, randomized controlled trial set in the Otolaryngology clinic in a children's hospital. The participants were 103 children referred by their general practitioner for recurrent epistaxis. Excluded were those with suspected tumours, bleeding disorders or allergies to constituents of the cream. Referral letters were randomized to treatment and no treatment groups. Treatment was antiseptic cream to the nose twice daily for 4 weeks, which was prescribed by the general practitioner before clinic attendance. All children were given an appointment for 8 weeks after randomization. The main outcome measures were the proportion of children in each group with no epistaxis in the 4 weeks preceding clinic review. Complete data were available for 88 (85%) of the children. Of the treatment group, 26/47 (55%) had no epistaxis in the 4 weeks before the clinic appointment. Of the controls, 12/41 (29%) had no epistaxis over the 4 weeks. This is a relative risk reduction of 47% for persistent bleeding (95% CI 9-69%) and an absolute risk reduction of 26% (95% CI 12-40%), giving a number needed to treat of 3.8 (95% CI 2.5-8.5). We conclude that antiseptic cream is an effective treatment for recurrent epistaxis in children.

Administration, Topical↗
PubMed2001

A comparative study: conventional preparation and ThinPrep 2000 in respiratory cytology.

In order to compare and contrast conventional preparation (CP) with ThinPrep 2000 (TP) in respiratory cytology, 207 samples were divided equally and processed by the two different preparation methods, generating three CP and one TP slide per sample. No lesion identified by CP was missed by TP and there were no significant differences between TP and CP in the diagnostic categories. However, two cases of squamous cell carcinoma were detected on TP which had been classified as unsatisfactory and moderate squamous dyskaryosis, respectively, on CP. ThinPrep was found to be superior to CP in many respects as it provided standardized preparations in a greater proportion of cases and problems such as cell overlapping and background debris were markedly reduced. In several instances the diagnostic accuracy in CP was compromised by smears that were either too thick, too thin, or too scanty. Cell preservation was also better on TP when compared with CP, facilitating more accurate diagnosis and significantly reducing the primary screening and reporting time, especially of sputum samples. A major advantage of TP methodology is the fact that it facilitates optimal use of skilled cytotechnologists and streamlines the workflow in the laboratory.

Carcinoma↗
PubMed2001

Tandem-cycle high-dose melphalan and cisplatin with peripheral blood progenitor cell support in patients with breast cancer and other malignancies.

We evaluated the feasibility of tandem-cycle high-dose chemotherapy (HDCT) with cisplatin, melphalan, and peripheral blood progenitor cells (PBPCs). Fifty patients with high-risk primary (n = 17) or stage IV breast cancer (n = 29) or other malignancies (n = 4) received 2 cycles of intravenous melphalan, 20 to 151.8 mg/m2, and cisplatin, 200 mg/m2, followed by granulocyte-macrophage colony-stimulating factor (GM-CSF) or G-CSF. Starting at 40 mg/m2 of melphalan, patients also received PBPCs. Delayed platelet recovery defined the maximum tolerated dose (MTD) for melphalan at 101.2 mg/m2 per cycle. There were no treatment-related deaths. Cycle 2 was delivered at a median of 1.7 months after cycle 1; 72% of patients treated at the MTD received both cycles. Cycle 2 was omitted when patients refused it or had disease progression or toxicities, primarily prolonged thrombocytopenia. Complete response rates in stage IV breast cancer patients increased from 28% pre-HDCT to 55% after cycle 2. At a median follow-up of 4.6 years (range, 1.5-8.1 years), 11 of 29 patients with stage IV breast carcinoma were alive with 5-year projected progression-free and overall survival rates of 19% (95% confidence interval [CI], 7%-41%) and 39% (95% CI, 20%-62%), respectively. Five-year projected progression-free and overall survival rates for patients with stage IV breast cancer in complete response following HDCT versus all others were 35% (95% CI, 15%-70%) versus 0% (P = .01) and 61% (95% CI, 35%-91%) versus 10% (95% CI, 2%-60%) (P = .003; log-rank test), respectively. Estrogen-receptor positivity was predictive of reduced risk of progression (relative risk [RR], 0.25; 95% CI, 0.10-0.65; P = .003) and death (RR, 0.27; 95% CI, 0.10-0.72; P = .009) after adjusting for response status. Five-year projected relapse-free and overall survival rates were 71% (95% CI, 43%-96%) and 82% (95% CI, 56%-100%), respectively, for the 17 patients with high-risk primary breast cancer. Tandem-cycle high-dose melphalan and cisplatin with PBPCs is feasible. Preliminary data suggest significant activity in selected patients with stage IV responding breast carcinoma.

Adult↗
PubMed2001

The Zarit Burden Interview: a new short version and screening version.

PURPOSE: The purpose of the study was to develop a short and a screening version of the Zarit Burden Interview (ZBI) that would be suitable across diagnostic groups of cognitively impaired older adults, and that could be used for cross-sectional, longitudinal, and intervention studies. DESIGN AND METHODS: We used data from 413 caregivers of cognitively impaired older adults referred to a memory clinic. We collected information on caregiver burden with the 22-item ZBI, and information about dependence in activities of daily living (ADLs) and the frequency of problem behaviors among care recipients. We used factor analysis and item-total correlations to reduce the number of items while taking into consideration diagnosis and change scores. RESULTS: We produced a 12-item version (short) and a 4-item version (screening) of the ZBI. Correlations between the short and the full version ranged from 0.92 to 0.97, and from 0.83 to 0.93 for the screening version. Correlations between the three versions and ADL and problem behaviors were similar. We further investigated the behavior of the short version with a two-way analysis of variance and found that it produced identical results to the full version. IMPLICATIONS: The short and screening versions of the ZBI produced results comparable to those of the full version. Reducing the number of items did not affect the properties of the ZBI, and it may lead to easier administration of the instrument.

Activities of Daily Living↗
PubMed2001

Quantitative blood speed imaging with intravascular ultrasound.

Previously, we presented a method of real-time arterial color flow imaging using an intravascular ultrasound (IVUS) imaging system, where real-time RF A-scans were processed with an FIR (finite-impulse response) filter bank to estimate relative blood speed. Although qualitative flow measurements are clinically valuable, realizing the full potential of blood flow imaging requires quantitative flow speed and volume measurements in real time. Unfortunately, the rate of RF echo-to-echo decorrelation is not directly related to scatterer speed in a side-looking IVUS system because the elevational extent of the imaging slice varies with range. Consequently, flow imaging methods using any type of decorrelation processing to estimate blood speed without accounting for spatial variation of the radiation pattern will have estimation errors that prohibit accurate comparison of speed estimates from different depths. The FIR filter bank approach measures the rate of change of the ultrasound signal by estimating the slow-time spectrum of RF echoes. A filter bank of M bandpass filters is applied in parallel to estimate M components of the slow-time DFT (discrete Fourier transform). The relationship between the slow-time spectrum, aperture diffraction pattern, and scatterer speed is derived for a simplified target. Because the ultimate goal of this work is to make quantitative speed measurements, we present a method to map slow time spectral characteristics to a quantitative estimate. Results of the speed estimator are shown for a simulated circumferential catheter array insonifying blood moving uniformly past the array (i.e., plug flow) and blood moving with a parabolic profile (i.e., laminar flow).

Animals↗
PubMed2001

Strain rate imaging using two-dimensional speckle tracking.

Strain rate images (SRI) of the beating heart have been proposed to identify non-contracting regions of myocardium. Initial attempts used spatial derivatives of tissue velocity (Doppler) signals. Here, an alternate method is proposed based on two-dimensional phase-sensitive speckle tracking applied to very high frame rate, real-time images. This processing can produce high resolution maps of the time derivative of the strain magnitude (i.e., square root of the strain intensity). Such images complement traditional tissue velocity images (TVI), providing a more complete description of cardiac mechanics. To test the proposed approach, SRI were both simulated and measured on a thick-walled, cylindrical, tissue-equivalent phantom modeling cardiac deformations. Real-time ultrasound images were captured during periodic phantom deformation, where the period was matched to the data capture rate of a commercial scanner mimicking high frame rate imaging of the heart. Simulation results show that SRI with spatial resolution between 1 and 2 mm are possible with an array system operating at 5 MHz. Moreover, these images are virtually free of angle-dependent artifacts present in TVI and simple strain rate maps derived from these images. Measured results clearly show that phantom regions of low deformation, which are difficult to identify on tissue velocity-derived SRI, are readily apparent with SRI generated from two-dimensional phase-sensitive speckle tracking.

Biomedical Engineering↗
PubMed2001

A new polymorphism for the RI22H mutation in hereditary pancreatitis.

BACKGROUND AND AIMS: Hereditary pancreatitis (HP) is a rare form of recurrent acute and chronic pancreatitis. Mutations in the cationic trypsinogen (protease serine 1, PRSS1) gene have been identified as causing HP. The R122H (previously known as R117H) mutation is the commonest and can be detected by a single and rapid polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) based technique using the AflIII enzyme. This test however may give a false negative result in the presence of a neutral polymorphism within the enzyme recognition site. The frequency of this event was examined by sequencing studies in patients with HP and in healthy controls. METHODS: Of 60 families identified by the UK and Ireland consortium of EUROPAC (European Registry for Hereditary Pancreatitis and Familial Pancreatic Cancer), 51 were screened for R122H, N29I, and A16V mutations using standard techniques, and by sequencing of all five exons of cationic trypsinogen. RESULTS: Twelve families had the N29I mutation, one family had A16V and, on standard testing, 15 families had the R122H mutation. An additional family with the R122H mutation was found on direct sequencing. The false negative result was due to a neutral polymorphism C-->T at the third base of the codon, not affecting the amino acid coded for, destroying the AflIII restriction site. This polymorphism was not observed in 50 DNA specimens (100 chromosomes) from controls nor from 50 individuals from PRSS1 mutation negative HP families. A novel mutation specific PCR was developed to avoid this pitfall. CONCLUSIONS: One of the 16 families with HP and an R122H mutation contained a polymorphism affecting the AflIII restriction site. Adoption of an alternative R122H assay is important for genetic studies in individuals with apparent HP.

Acute Disease↗