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Biomedical subjects

M O'Donnell

Publications and source records attributed to M O'Donnell.

At least 19 recordsLinked to original sources
PubMed1995

Structural and functional similarities of prokaryotic and eukaryotic DNA polymerase sliding clamps.

The remarkable processivity of cellular replicative DNA polymerases derive their tight grip to DNA from a ring-shaped protein that encircles DNA and tethers the polymerase to the chromosome. The crystal structures of prototypical 'sliding clamps' of prokaryotes (beta subunit) and eukaryotes (PCNA) are ring shaped proteins for encircling DNA. Although beta is a dimer and PCNA is a trimer, their structures are nearly superimposable. Even though they are not hexamers, the sliding clamps have a pseudo 6-fold symmetry resulting from three globular domains comprising each beta monomer and two domains comprising each PCNA monomer. These domains have the same chain fold and are nearly identical in three-dimensions. The amino acid sequences of 11 beta and 13 PCNA proteins from different organisms have been aligned and studied to gain further insight into the relation between the structure and function of these sliding clamps. Furthermore, a putative embryonic form of PCNA is the size of beta and thus may encircle DNA as a dimer like the prokaryotic clamps.

Amino Acid Sequence↗
PubMed1995

The outcome of matched unrelated donor bone marrow transplantation in patients with hematologic malignancies using molecular typing for donor selection and graft-versus-host disease prophylaxis regimen of cyclosporine, methotrexate, and prednisone.

Graft-versus-host disease (GVHD) is a major obstacle to successful bone marrow transplantation (BMT) from matched unrelated donor (MUD). Currently available HLA-A, -B, and -DR serologic testing may not be sensitive enough to detect clinically relevant donor/recipient (D/R) nonidentity. Better HLA matching of D/R pairs using molecular typing for class II antigens in combination with intensive GVHD prophylaxis may potentially reduce the incidence of GVHD and lead to an improved outcome of MUD transplantation. Between July 1991 and August 1993, thirty consecutive patients with hematologic malignancies underwent MUD transplantation from donors who were identical for HLA -A, -B, and -DR by serologic typing. Twenty-five D/R pairs were matched for DRB and DQB by molecular typing (restriction fragment-length polymorphism and sequence-specific oligonucleotide probe hybridization analyses), whereas five were allele mismatched at either DRB or DQB. All patients also received GVHD prophylaxis with the combination of cyclosporine (CSA), methotrexate (MTX), and prednisone (PSE). The median age was 35 years (range, 15 to 50). The diagnoses were: chronic myelogenous leukemia (CML) in chronic phase (CP) (16), CML in more than CP (3), acute leukemia in more than first complete remission (CR) (8), acute leukemia in first CR (1), and advanced high-grade lymphoma (2). The preparative regimen consisted of 1,320 cGy fractionated total body irradiation (FTBI) and 60 mg/kg cyclophosphamide (CY) daily for 2 days in 17 good-risk patients (CML/CP and acute leukemia first CR); and 1,320 cGy FTBI in combination with 60 mg/kg etoposide and 20 to 60 mg/kg CY in 13 patients with advanced leukemia and lymphoma. All patients received CSA, PSE, and MTX on days 1, 3, 6 for GVHD prophylaxis, and 10 patients also received day +11 MTX. All patients engrafted except one who died early of regimen-related toxicity. The incidence of grade III or IV acute GVHD was 24% (95% confidence interval [CI], 10% to 44%) and that of extensive chronic GVHD was 65% (95% CI, 43% to 84%). At a median follow-up of 13.6 months, 57% of the patients are alive in remission with a median Karnofsky performance status of 90%. The cumulative probability of 2-year disease-free survival for all patients was 53% (95%) CI, 33% to 71%); for good-risk patients, 71% (95% CI, 46% to 87%) and for the poor-risk group, 34% (95% CI, 13% to 64%).(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1995

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. I. Organization of the clamp loader.

The gamma complex of DNA polymerase III holoenzyme, the replicase of Escherichia coli, couples ATP hydrolysis to the loading of beta sliding clamps onto primed DNA. The beta sliding clamp tethers the holoenzyme replicase to DNA for rapid and processive synthesis. In this report, the gamma complex has been constituted from its five different subunits. Size measurements and subunit stoichiometry studies show a composition of gamma 2 delta 1 delta' 1 1 chi 1 psi 1. Strong intersubunit contacts have been identified by gel filtration, and weaker contacts were identified by surface plasmon resonance measurements. An analogous tau complex has also been constituted and characterized; it is nearly as active as the gamma complex in clamp loading activity, but as shown in the fourth report of this series, it is at a disadvantage in binding the delta, delta', chi, and psi subunits when core is present (Xiao, H., Naktinis, V., and O'Donnell, M. (1995) J. Biol. Chem. 270, 13378-13383). The single copy subunits within the gamma complex provide the basis for the structural asymmetry inherent within DNA polymerase III holoenzyme.

Chromatography, High Pressure Liquid↗
PubMed1995

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. III. Interface between two polymerases and the clamp loader.

The nine-subunit DNA polymerase (Pol) III* coupled to its beta sliding clamp is a rapid and highly processive replicating machine. The multiple subunits are needed for the complicated task of duplicating the Escherichia coli chromosome. In this report, Pol III* was constituted from individual pure proteins, and its structure was studied. Constitution of the Pol III* particle requires an ordered addition of the subunits, and the final structure contains 14 polypeptides in the ratio alpha 2 epsilon 2 theta 2 tau 2 gamma 2 delta 1 delta' 1 chi 1 psi 1. The structure can be summarized as being composed of two core polymerases (alpha epsilon theta) held together by a dimer of tau and one gamma complex clamp loader (gamma 2 delta 1 delta' 1 chi 1 psi 1) for loading beta onto DNA. At the center of the structure, the related tau and gamma subunits form a heterotetramer upon which the two core polymerases and clamp loader proteins assemble. The single copy nature of the delta, delta', chi, and psi subunits confers a structural asymmetry with respect to the two polymerases, presumably for the different functions of replicating the leading and lagging strands.

Chromosomes, Bacterial↗
PubMed1995

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. IV. ATP-binding site mutants identify the clamp loader.

The gamma complex (gamma delta delta' chi psi) and tau complex (tau delta delta' chi psi) clamp loaders require ATP hydrolysis to load beta sliding clamps onto DNA. The beta sliding clamp tethers the polymerase (Pol) III* replicase to DNA for processive synthesis. Pol III* contains both gamma and tau, but only one each of the delta, delta', chi, and psi subunits. Hence, there is ambiguity with respect to which clamp loader, the gamma or tau complex, exists in the Pol III* replicase structure. In this study, ATP-binding site mutants of gamma and tau have been prepared, and these mutants, when assembled into either the gamma or tau complex, are inactive in clamp loading. These mutants have been used as a tool to determine the identity of the clamp loader in Pol III*. The nine-subunit Pol III* has been assembled using either mutant gamma or tau in place of wild-type gamma or tau. The results show that mutation of gamma inactivates Pol III* activity, but mutation of tau does not, indicating that the gamma complex (and not the tau complex) is the clamp loader of Pol III*. The tau subunit carries the task of dimerizing the core polymerase, and it is this association of tau with core that appears to direct the single copy subunits away from tau and onto gamma.

Adenosine Triphosphate↗
PubMed1995

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. V. Four different polymerase-clamp complexes on DNA.

Several different subassemblies of DNA polymerase III holoenzyme can be purified from Escherichia coli. Toward the goal of understanding the functional significance of these subassemblies, we have used the gamma complex clamp loader and the beta ring to assemble each different polymerase onto DNA. Through use of radioactive labeled proteins, the subunit structure of each resulting processive polymerase has been determined. Use of DNA polymerase III core, the gamma complex, and beta results in a core-beta complex on DNA; the gamma complex is not incorporated into the structure. The addition of tau to the assembly reaction to form either core1-tau 2 or core2-tau 2 results in a more efficient polymerase and more stabile association of core-tau beta on DNA, although the gamma complex still does not remain on DNA. The gamma complex clamp loader was retained on DNA with the other subunits only if it was first assembled into the polymerase (Pol) III* structure. The clamp loader within Pol III* appeared to be capable of loading two beta clamps onto DNA for both core polymerases within Pol III*, consistent with the hypothesis that one replicase can simultaneously replicate both strands of a duplex chromosome. These findings extend those of an earlier study showing that distinctive polymerases can be assembled depending on the presence or absence of tau (Maki, S., and Kornberg, A. (1988) J. Biol. Chem. 263, 6561-6569). The significance of these distinct polymerases in separate paths of DNA metabolism is discussed.

Chromosomes, Bacterial↗
PubMed1995

Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp.

The Escherichia coli replicase, DNA polymerase III holoenzyme, derives its processivity from the beta subunit sliding clamp that encircles DNA and tethers the replicase to the template. The beta dimer is assembled around DNA by the gamma complex clamp loader in an ATP-dependent reaction. In this report, the essential contact between the clamp loader and beta is identified as mediated through the delta subunit of the gamma complex. The delta subunit appears to contact the face of the beta dimer ring that contains the two C termini. Surprisingly, ATP is required for the gamma complex to bind beta, but not for delta to bind beta. This indicates that delta is buried in the gamma complex and suggests a role for ATP in exposing delta for interaction with beta. A protease protection assay has been developed to specifically probe the delta subunit within the gamma complex. The results of the assay are consistent with an ATP-induced conformational change in the gamma complex that alters the state of the delta subunit within it. The implication of these key features to the clamp loading mechanism of the gamma complex is discussed.

Adenosine Triphosphate↗
PubMed1995

Elasticity imaging for early detection of renal pathology.

Early detection of renal pathology may be possible with elasticity imaging. This hypothesis was experimentally tested by quantitatively imaging internal mechanical strain due to surface deformations in an in vitro animal model of nephritis. Preliminary data support the hypothesis that kidney elasticity changes with renal damage and concomitant scarring before problems are detectable by traditional diagnostic techniques such as laboratory measurements of renal function.

Animals↗
PubMed1995

Quantitative determination of functional thiol groups on intact cell surfaces by resonance Raman spectroscopy.

The sensitivity and selectivity of resonance Raman spectroscopy, combined with electronic spectroscopy, has been used to develop a method to quantify the membrane thiol population in situ in viable erythrocytes. This technique is based on the thiol-disulfide reaction of Ellman's reagent (5,5'-dithio-bis-2-nitrobenzoic acid). It has the advantage that continuous monitoring of lysis is simple and a correction can be made for any interference resulting from lysis. In addition, the extent of reaction can be expressed as a ratio of the reagent signal, providing an internal calibrant.

Calibration↗
PubMed1995

Intracytoplasmic lumina and mucinous inclusions in ovarian carcinomas.

Intracytoplasmic mucinous inclusions and lumina have been previously described in non-glandular neoplasms such as urothelial carcinoma. We describe their presence in 93% of non-mucinous ovarian carcinomas. They were found in abundance in all 25 cases (100%) of clear cell carcinoma, in 48 of 50 cases (96%) of serous carcinoma and 20 of 25 cases (80%) of endometrioid carcinoma. The degree of the differentiation of the tumour did not influence the number of inclusions or lumina observed. These results suggest that the presence of intracytoplasmic lumina and mucinous inclusions is more widespread than hitherto appreciated. Their presence in an otherwise poorly differentiated metastatic carcinoma might, at the very last, prompt one to consider the ovary as a possible primary site. In addition, an abundance of intracytoplasmic mucinous inclusion and lumina with microcyst formation, in an otherwise poorly differentiated malignant primary ovarian epithelial tumour, might suggest the possibility of a clear cell carcinoma.

Adenocarcinoma, Clear Cell↗
PubMed1995

DNA polymerase III holoenzyme: structure and function of a chromosomal replicating machine.

DNA polymerase III holoenzyme contains two DNA polymerases embedded in a particle with 9 other subunits. This multisubunit DNA polymerase is the Eschericia coli chromosomal replicase, and it has several special features that distinguish it as a replicating machine. For example, one of its subunits is a circular protein that slides along DNA while clamping the rest of the machinery to the template. Other subunits act together as a matchmaker to assemble the ring onto DNA. Overall, E. coli DNA polymerase III holoenzyme is very similar in both structure and function to the chromosomal replicases of eukaryotes, from yeast all the way up to humans. This review summarizes our present knowledge about the function of the 10 subunits of this replicating machine and how they coordinate their actions for smooth duplication of chromosomes.

Adenosine Triphosphate↗
PubMed1995

Experimental studies on an efficient catheter array imaging system.

A new synthetic aperture system for intraluminal imaging has been tested using a 32 element, 20 MHz circular array wrapped around the surface of a catheter appropriate for coronary artery applications. This system is based on an optimal reconstruction method that has been extended to reduce grating lobes using a slight modification to classic synthetic aperture data acquisition. Optimal reconstruction filters have been derived for two different operating modes based on this new data acquisition strategy. Imaging results on a wire target phantom show that spatial resolution is a simple linear function of depth, reaching a minimum 6 dB beam width of approximately 2.2 wavelengths. Sidelobe levels are inherently high for this system because of the small number of firings used to synthesize an aperture. Optimal reconstruction filters, however, can reduce these sidelobes to at least -20 dB in all cases. Finally, images of an excised segment of porcine femoral artery demonstrate the overall performance of the system as an intraluminal imager.

Animals↗
PubMed1995

Lipids and lipoprotein(a) as risk factors for vascular disease in patients on renal replacement therapy.

A large cohort of patients on renal replacement therapy were screened for the presence of symptomatic arterial disease affecting the coronary, cerebral or peripheral circulations. Ninety-two of 325 patients were found to have vascular disease. Those with vascular disease had significantly higher median lipoprotein(a) [Lp(a)] levels than those without (38.4 vs 14.2 mg/dl, P < 0.001), with a preponderance of Lp(a) levels greater than 30 mg/dl (58% vs 25% P < 0.001). Apolipoprotein(a) [apo(a)] isoform distribution was similar between the groups, but those with vascular disease had higher Lp(a) levels in the S2, S3/S4 and S4 isoform types. Comparison of 76 matched pairs of patients confirmed elevated Lp(a) levels in those with vascular disease. These patients also had significantly higher total cholesterol (6.66 vs 6.02 mmol/l) and low-density lipoprotein cholesterol (4.49 vs 3.86 mmol/l). Only Lp(a) was independently associated with vascular disease (P = 0.02). Elevated Lp(a) levels are significantly associated with the presence of vascular disease in patients on renal replacement therapy and may constitute another risk factor for the development of such disease in these patients.

Adult↗
PubMed1994

Use of local neural tube defect registers to interpret national trends.

To conduct a number of studies into the prevalence of neural tube defects (NTD) in the area covered by the Oxford Record Linkage Study (ORLS), multiple sources were used to build a local register of cases occurring in Oxfordshire and West Berkshire between 1968-1990. One source of potential cases--namely, termination and congenital malformation monitoring data available for the locality from the Office of Population Censuses and Surveys (OPCS) data--were kept separate. Comparison of the local cases recorded by OPCS and those known to the register from 1974-1990, using the method of capture-recapture, suggested that national data are only about two thirds complete, but that this underreporting is likely to be reasonably consistent from year to year. OPCS data can therefore be used to study NTD trends if not absolute risks. The local register seemed, by the same yardstick, to be very complete and is being used in a variety of studies of the occurrence of NTD. Survival to one year in this area, over the period 1968-1990, has only improved in the recent past, if at all. Most NTD pregnancies now end in termination rather than birth, and there has been a true decline in the occurrence of NTDs, and likewise the different subtypes.

Abortion, Legal↗