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Biomedical subjects

M O'Donnell

Publications and source records attributed to M O'Donnell.

At least 55 records · Page 3Linked to original sources
PubMed2001

Modified weighting method for forward-looking ring-annular arrays.

Side-looking Intravascular Ultrasound Systems (IVUS) systems provide high resolution cross-sectional images of vascular structure. However, blood velocity estimation is hampered by the orthogonality of the primary blood flow direction to the imaging plane, and do not image in front of the catheter. A forward-looking aperture would improve the ability to guide interventions and allow Doppler processing for improved blood speed estimates; however, catheter-based systems delivered over a guide wire limit forward-looking array geometry to annular variations. Unfortunately, the imaging characteristics of annular arrays are inferior to a full aperture disk. A modified weighting method for a practical synthetic phased, ring-annular array is presented that achieves a transmit-receive aperture and on-axis performance comparable to a full aperture disk array.

Blood Flow Velocity↗
PubMed2001

Compounding for a forward-viewing ring-annular ultrasound array system.

The 3-D data set from a forward-viewing ring-annular array opens up opportunities for compounding in both axial and lateral directions. To explore this potential, we performed a simulation study analyzing 3-D ultrasound data from various phantoms. For a vessel phantom, our results indicate compounding could boost contrast to noise ratio (CNR) (up to a factor of 4 in our results) without significant compromise in in-plane image resolution. For an isotropic cyst phantom, axial and lateral compounding performs similarly. Such improvement should be helpful for device guidance with IVUS (Intravascular Ultrasound) in interventional procedures.

Arterial Occlusive Diseases↗
PubMed2001

Educating patients with type 2 diabetes.

The patient is the self-manager of type 2 diabetes. The role of the health care professional is to provide the knowledge, skills, and behavior change support to empower the patient to do so. Recent governmental, financial, and clinical factors influence how health care professionals perform this role. Such factors coupled with a growing body of research evidence are shaping the way diabetes self-management education is provided.

Attitude to Health↗
PubMed2001

Discussing wisdom teeth with patients: a critique of information materials.

OBJECTIVE: To assess the quality of information leaflets about third molar extraction. DESIGN: Quality assessment of information leaflets about third molar extraction. We focused particularly on the extent to which they reflect current research evidence. SETTING: All fifteen oral and maxillofacial surgery departments in Scotland were asked to provide copies of existing patient information materials on third molar surgery. RESULTS: Six different leaflets were assessed. Most were apparently intended for people who were about to undergo and/or had just undergone the procedure rather than for people facing decisions about extraction. The leaflets appeared to reflect varying policies about third molar extraction and some of the information provided did not reflect current research evidence. CONCLUSIONS: There is currently a lack of good quality information materials available to people who are considering third molar extraction. It is likely that there are also problems with the quality of other patient information leaflets. Health care professionals need to review the quality of patient information leaflets to ensure that the information provided reflects current research evidence.

Humans↗
PubMed2000

The delta subunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli.

In Escherichia coli, the circular beta sliding clamp facilitates processive DNA replication by tethering the polymerase to primer-template DNA. When synthesis is complete, polymerase dissociates from beta and DNA and cycles to a new start site, a primed template loaded with beta. DNA polymerase cycles frequently during lagging strand replication while synthesizing 1-2-kilobase Okazaki fragments. The clamps left behind remain stable on DNA (t(12) approximately 115 min) and must be removed rapidly for reuse at numerous primed sites on the lagging strand. Here we show that delta, a single subunit of DNA polymerase III holoenzyme, opens beta and slips it off DNA (k(unloading) = 0.011 s(-)(1)) at a rate similar to that of the multisubunit gamma complex clamp loader by itself (0.015 s(-)(1)) or within polymerase (pol) III* (0.0065 s(-)(1)). Moreover, unlike gamma complex and pol III*, delta does not require ATP to catalyze clamp unloading. Quantitation of gamma complex subunits (gamma, delta, delta', chi, psi) in E. coli cells reveals an excess of delta, free from gamma complex and pol III*. Since pol III* and gamma complex occur in much lower quantities and perform several DNA metabolic functions in replication and repair, the delta subunit probably aids beta clamp recycling during DNA replication.

Blotting, Western↗
PubMed2000

Reproductive factors, subfertility, and risk of neural tube defects: a case-control study based on the Oxford Record Linkage Study Register.

Periconceptual exposure to subfertility treatments is increasingly common, raising concerns about the possibility of malformations in the offspring. The authors conducted a case-control study to determine whether subfertility or its treatment was associated with increased risk of neural tube defects (NTDs). Cases were 694 women diagnosed with an NTD-affected pregnancy in Oxfordshire or West Berkshire, England, between 1970 and 1987. Cases were individually matched on maternal year of birth and year of index pregnancy to controls randomly selected from a computerized database. Data on demographic, reproductive, and obstetric factors were abstracted from patient hospital records. Overall, the period prevalences of subfertility and of subfertility treatment were 7% and 3%, respectively, No evidence was found that the risk of NTD-affected pregnancies was increased by either subfertility (odds ratio (OR) = 1.2, 95% confidence interval (CI): 0.7, 2.1) or its treatment (OR = 0.9, 95% CI: 0.4, 2.0). After adjustment, NTD-affected pregnancies were associated with female offspring (OR = 2.3, 95% CI: 1.8, 3.1), multiple birth (OR = 4.8, 95% CI: 1.2, 18.8), and higher numbers of pregnancies (p for trend = 0.005). The findings from this large, population-based study were wholly consistent with those from smaller studies that found no increased risk of NTD associated with exposure to fertility treatments but reported associations with various pregnancy outcomes.

Adult↗
PubMed2000

Pathophysiologic role of selectins and their ligands in ischemia reperfusion injury.

Research findings are unveiling the potential role of leukocytes and leukocyte adhesion molecules such as selectins in ischemia-reperfusion injury (IRI). "Anti-adhesion" therapy using selectin blocking agents may represent a new approach to treatment of the many diverse clinical disorders in which ischemia-reperfusion occurs, including transplantation, reperfusion after thrombotic events and shock. In this paper we review the pathophysiology of IRI, the different types of selectins and selectin ligands, the clinical implications of selectin blockade in different organs with IRI, and new insights into mechanism of action.

Animals↗
PubMed2000

The DNA replication machine of a gram-positive organism.

This report outlines the protein requirements and subunit organization of the DNA replication apparatus of Streptococcus pyogenes, a Gram-positive organism. Five proteins coordinate their actions to achieve rapid and processive DNA synthesis. These proteins are: the PolC DNA polymerase, tau, delta, delta', and beta. S. pyogenes dnaX encodes only the full-length tau, unlike the Escherichia coli system in which dnaX encodes two proteins, tau and gamma. The S. pyogenes tau binds PolC, but the interaction is not as firm as the corresponding interaction in E. coli, underlying the inability to purify a PolC holoenzyme from Gram-positive cells. The tau also binds the delta and delta' subunits to form a taudeltadelta' "clamp loader." PolC can assemble with taudeltadelta' to form a PolC.taudeltadelta' complex. After PolC.taudeltadelta' clamps beta to a primed site, it extends DNA 700 nucleotides/second in a highly processive fashion. Gram-positive cells contain a second DNA polymerase, encoded by dnaE, that has homology to the E. coli alpha subunit of E. coli DNA polymerase III. We show here that the S. pyogenes DnaE polymerase also functions with the beta clamp.

Amino Acid Sequence↗
PubMed2000

Molecular mechanism and energetics of clamp assembly in Escherichia coli. The role of ATP hydrolysis when gamma complex loads beta on DNA.

Escherichia coli DNA polymerase III holoenzyme is a multisubunit composite containing the beta sliding clamp and clamp loading gamma complex. The gamma complex requires ATP to load beta onto DNA. A two-color fluorescence spectroscopic approach was utilized to study this system, wherein both assembly (red fluorescence; X-rhodamine labeled DNA anisotropy assay) and ATP hydrolysis (green fluorescence; phosphate binding protein assay) were simultaneously measured with millisecond timing resolution. The two temporally correlated stopped-flow signals revealed that a preassembled beta. gamma complex composite rapidly binds primer/template DNA in an ATP hydrolysis independent step. Once bound, two molecules of ATP are rapidly hydrolyzed (approximately 34 s(-1)). Following hydrolysis, gamma complex dissociates from the DNA ( approximately 22 s(-1)). Once dissociated, the next cycle of loading is severely compromised, resulting in steady-state ATP hydrolysis rates with a maximum of only approximately 3 s(-1). Two single-site beta dimer interface mutants were examined which had impaired steady-state rates of ATP hydrolysis. The pre-steady-state correlated kinetics of these mutants revealed a pattern essentially identical to wild type. The anisotropy data showed that these mutants decrease the steady-state rates of ATP hydrolysis by causing a buildup of "stuck" binary-ternary complexes on the primer/template DNA.

Adenosine Triphosphate↗
PubMed2000

Cross-utilization of the beta sliding clamp by replicative polymerases of evolutionary divergent organisms.

Chromosomal replicases are multiprotein machines comprised of a DNA polymerase, a sliding clamp, and a clamp loader. This study examines replicase components for their ability to be switched between Gram-positive and Gram-negative organisms. These two cell types diverged over 1 billion years ago, and their sequences have diverged widely. Yet the Escherichia coli beta clamp binds directly to Staphylococcus aureus PolC and makes it highly processive, confirming and extending earlier results (Low, R. L., Rashbaum, S. A. , and Cozzarelli, N. R. (1976) J. Biol. Chem. 251, 1311-1325). We have also examined the S. aureus beta clamp. The results show that it functions with S. aureus PolC, but not with E. coli polymerase III core. PolC is a rather potent polymerase by itself and can extend a primer with an intrinsic speed of 80-120 nucleotides per s. Both E. coli beta and S. aureus beta converted PolC to a highly processive polymerase, but surprisingly, beta also increased the intrinsic rate of DNA synthesis to 240-580 nucleotides per s. This finding expands the scope of beta function beyond a simple mechanical tether for processivity to include that of an effector that increases the intrinsic rate of nucleotide incorporation by the polymerase.

Amino Acid Sequence↗
PubMed2000

A TOPRIM domain in the crystal structure of the catalytic core of Escherichia coli primase confirms a structural link to DNA topoisomerases.

Primases synthesize short RNA strands on single-stranded DNA templates, thereby generating the hybrid duplexes required for the initiation of synthesis by DNA polymerases. We present the crystal structure of the catalytic unit of a primase enzyme, that of a approximately 320 residue fragment of Escherichia coli primase, determined at 2.9 A resolution. Central to the catalytic unit is a TOPRIM domain that is strikingly similar in its structure to that of corresponding domains in DNA topoisomerases, but is unrelated to the catalytic centers of other DNA or RNA polymerases. The catalytic domain of primase is crescent-shaped, and the concave face of the crescent is predicted to accommodate about 10 base-pairs of RNA-DNA duplex in a loose interaction, thereby limiting processivity.

Amino Acid Sequence↗
PubMed2000

Fidelity of eucaryotic DNA polymerase delta holoenzyme from Schizosaccharomyces pombe.

The fidelity of Schizosaccharomyces pombe DNA polymerase delta was measured in the presence or absence of its processivity subunits, proliferating cell nuclear antigen (PCNA) sliding clamp and replication factor C (RFC) clamp-loading complex, using a synthetic 30-mer primer/100-mer template. Synthesis by pol delta alone was distributive. Processive synthesis occurred in the presence of PCNA, RFC, and Escherichia coli single strand DNA-binding protein (SSB) and required the presence of ATP. "Passive" self-loading of PCNA onto DNA takes place in the absence of RFC, in an ATP-independent reaction, which was strongly inhibited by SSB. The nucleotide substitution error rate for pol delta holoenzyme (HE) (pol delta + PCNA + RFC) was 4.6 x 10(-4) for T.G mispairs, 5.3 x 10(-5) for G.G mispairs, and 4.5 x 10(-6) for A.G mispairs. The T.G misincorporation frequency for pol delta without the accessory proteins was unchanged. The fidelity of pol delta HE was between 1 and 2 orders of magnitude lower than that measured for the E. coli pol III HE at the same template position. This relatively low fidelity was caused by inefficient proofreading by the S. pombe polymerase-associated proofreading exonuclease. The S. pombe 3'-exonuclease activity was also extremely inefficient in excising primer-3'-terminal mismatches in the absence of dNTP substrates and in hydrolyzing single-stranded DNA. A comparison of pol delta HE with E. coli pol IIIalpha HE (lacking the proofreading exonuclease subunit) showed that both holoenzymes exhibit similar error rates for each mispair.

Base Sequence↗
PubMed2000

Human CD4(+) T lymphocytes consistently respond to the latent Epstein-Barr virus nuclear antigen EBNA1.

The Epstein-Barr virus (EBV)-encoded nuclear antigen EBNA1 is critical for the persistence of the viral episome in replicating EBV-transformed human B cells. Therefore, all EBV-induced tumors express this foreign antigen. However, EBNA1 is invisible to CD8(+) cytotoxic T lymphocytes because its Gly/Ala repeat domain prevents proteasome-dependent processing for presentation on major histocompatibility complex (MHC) class I. We now describe that CD4(+) T cells from healthy adults are primed to EBNA1. In fact, among latent EBV antigens that stimulate CD4(+) T cells, EBNA1 is preferentially recognized. We present evidence that the CD4(+) response may provide a protective role, including interferon gamma secretion and direct cytolysis after encounter of transformed B lymphocyte cell lines (B-LCLs). Dendritic cells (DCs) process EBNA1 from purified protein and from MHC class II-mismatched, EBNA1-expressing cells including B-LCLs. In contrast, B-LCLs and Burkitt's lymphoma lines likely present EBNA1 after endogenous processing, as their capacity to cross-present from exogenous sources is weak or undetectable. By limiting dilution, there is a tight correlation between the capacity of CD4(+) T cell lines to recognize autologous B-LCL-expressing EBNA1 and DCs that have captured EBNA1. Therefore, CD4(+) T cells can respond to the EBNA1 protein that is crucial for EBV persistence. We suggest that this immune response is initiated in vivo by DCs that present EBV-infected B cells, and that EBNA1-specific CD4(+) T cell immunity be enhanced to prevent and treat EBV-associated malignancies.

Adult↗
PubMed2000

Roles of E. coli DNA polymerases IV and V in lesion-targeted and untargeted SOS mutagenesis.

The expression of the Escherichia coli DNA polymerases pol V (UmuD'2C complex) and pol IV (DinB) increases in response to DNA damage. The induction of pol V is accompanied by a substantial increase in mutations targeted at DNA template lesions in a process called SOS-induced error-prone repair. Here we show that the common DNA template lesions, TT (6-4) photoproducts, TT cis-syn photodimers and abasic sites, are efficiently bypassed within 30 seconds by pol V in the presence of activated RecA protein (RecA*), single-stranded binding protein (SSB) and pol III's processivity beta,gamma-complex. There is no detectable bypass by either pol IV or pol III on this time scale. A mutagenic 'signature' for pol V is its incorporation of guanine opposite the 3'-thymine of a TT (6-4) photoproduct, in agreement with mutational spectra. In contrast, pol III and pol IV incorporate adenine almost exclusively. When copying undamaged DNA, pol V exhibits low fidelity with error rates of around 10(-3) to 10(-4), with pol IV being 5- to 10-fold more accurate. The effects of RecA protein on pol V, and beta,gamma-complex on pol IV, cause a 15,000- and 3,000-fold increase in DNA synthesis efficiency, respectively. However, both polymerases exhibit low processivity, adding 6 to 8 nucleotides before dissociating. Lesion bypass by pol V does not require beta,gamma-complex in the presence of non-hydrolysable ATPgammaS, indicating that an intact RecA filament may be required for translesion synthesis.

DNA Damage↗
PubMed2000

DNA structure requirements for the Escherichia coli gamma complex clamp loader and DNA polymerase III holoenzyme.

The Escherichia coli chromosomal replicase, DNA polymerase III holoenzyme, is highly processive during DNA synthesis. Underlying high processivity is a ring-shaped protein, the beta clamp, that encircles DNA and slides along it, thereby tethering the enzyme to the template. The beta clamp is assembled onto DNA by the multiprotein gamma complex clamp loader that opens and closes the beta ring around DNA in an ATP-dependent manner. This study examines the DNA structure required for clamp loading action. We found that the gamma complex assembles beta onto supercoiled DNA (replicative form I), but only at very low ionic strength, where regions of unwound DNA may exist in the duplex. Consistent with this, the gamma complex does not assemble beta onto relaxed closed circular DNA even at low ionic strength. Hence, a 3'-end is not required for clamp loading, but a single-stranded DNA (ssDNA)/double-stranded DNA (dsDNA) junction can be utilized as a substrate, a result confirmed using synthetic oligonucleotides that form forked ssDNA/dsDNA junctions on M13 ssDNA. On a flush primed template, the gamma complex exhibits polarity; it acts specifically at the 3'-ssDNA/dsDNA junction to assemble beta onto the DNA. The gamma complex can assemble beta onto a primed site as short as 10 nucleotides, corresponding to the width of the beta ring. However, a protein block placed closer than 14 base pairs (bp) upstream from the primer 3' terminus prevents the clamp loading reaction, indicating that the gamma complex and its associated beta clamp interact with approximately 14-16 bp at a ssDNA/dsDNA junction during the clamp loading operation. A protein block positioned closer than 20-22 bp from the 3' terminus prevents use of the clamp by the polymerase in chain elongation, indicating that the polymerase has an even greater spatial requirement than the gamma complex on the duplex portion of the primed site for function with beta. Interestingly, DNA secondary structure elements placed near the 3' terminus impose similar steric limits on the gamma complex and polymerase action with beta. The possible biological significance of these structural constraints is discussed.

Bacterial Proteins↗
PubMed2000

Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage.

The protein encoded by gene 45 of T4 bacteriophage (gene 45 protein or gp45), is responsible for tethering the catalytic subunit of T4 DNA Polymerase to DNA during high-speed replication. Also referred to as a sliding DNA clamp, gp45 is similar in its function to the processivity factors of bacterial and eukaryotic DNA polymerases, the beta-clamp and PCNA, respectively. Crystallographic analysis has shown that the beta-clamp and PCNA form highly symmetrical ring-shaped structures through which duplex DNA can be threaded. Gp45 shares no sequence similarity with beta-clamp or PCNA, and sequence comparisons have not been able to establish whether it adopts a similar structure. We have determined the crystal structure of gp45 from T4 bacteriophage at 2.4 A resolution, using multiple isomorphous replacement. The protein forms a trimeric ring-shaped assembly with overall dimensions that are similar to those of the bacterial and eukaryotic processivity factors. Each monomer of gp45 contains two domains that are very similar in chain fold to those of beta-clamp and PCNA. Despite an overall negative charge, the inner surface of the ring is in a region of positive electrostatic potential, consistent with a mechanism in which DNA is threaded through the ring.

Amino Acid Sequence↗
PubMed2000

A model for Escherichia coli DNA polymerase III holoenzyme assembly at primer/template ends. DNA triggers a change in binding specificity of the gamma complex clamp loader.

The gamma complex of the Escherichia coli DNA polymerase III holoenzyme assembles the beta sliding clamp onto DNA in an ATP hydrolysis-driven reaction. Interactions between gamma complex and primer/template DNA are investigated using fluorescence depolarization to measure binding of gamma complex to different DNA substrates under steady-state and presteady-state conditions. Surprisingly, gamma complex has a much higher affinity for single-stranded DNA (K(d) in the nM range) than for a primed template (K(d) in the microM range) under steady-state conditions. However, when examined on a millisecond time scale, we find that gamma complex initially binds very rapidly and with high affinity to primer/template DNA but is converted subsequently to a much lower affinity DNA binding state. Presteady-state data reveals an effective dissociation constant of 1.5 nM for the initial binding of gamma complex to DNA and a dissociation constant of 5.7 microM for the low affinity DNA binding state. Experiments using nonhydrolyzable ATPgammaS show that ATP binding converts gamma complex from a low affinity "inactive" to high affinity "active" DNA binding state while ATP hydrolysis has the reverse effect, thus allowing cycling between active and inactive DNA binding forms at steady-state. We propose that a DNA-triggered switch between active and inactive states of gamma complex provides a two-tiered mechanism enabling gamma complex to recognize primed template sites and load beta, while preventing gamma complex from competing with DNA polymerase III core for binding a newly loaded beta.DNA complex.

Adenosine Triphosphate↗