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Biomedical subjects

S P Bell

Publications and source records attributed to S P Bell.

At least 19 recordsLinked to original sources
PubMed2000

Regulation of origin recognition complex conformation and ATPase activity: differential effects of single-stranded and double-stranded DNA binding.

The Saccharomyces cerevisiae origin recognition complex (ORC) is bound to origins of DNA replication throughout the cell cycle and directs the assembly of higher-order protein-DNA complexes during G(1). To examine the fate of ORC when origin DNA is unwound during replication initiation, we determined the effect of single-stranded DNA (ssDNA) on ORC. We show that ORC can bind ssDNA and that ORC bound to ssDNA is distinct from that bound to double-stranded origin DNA. ssDNA stimulated ORC ATPase activity, whereas double-stranded origin DNA inhibited the same activity. Electron microscopy studies revealed two alternative conformations of ORC: an extended conformation stabilized by origin DNA and a bent conformation stabilized by ssDNA. Therefore, ORC appears to exist in two distinct states with respect to its conformation and ATPase activity. Interestingly, the effect of ssDNA on these properties of ORC is correlated with ssDNA length. Since double-stranded origin DNA and ssDNA differentially stabilize these two forms of ORC, we propose that origin unwinding triggers a transition between these alternative states.

Adenosine Triphosphatases↗
PubMed2000

Alterations in the determinants of diastolic suction during pacing tachycardia.

In cardiomyocytes, generation of restoring forces (RFs) responsible for elastic recoil involves deformation of the sarcomeric protein titin in conjunction with shortening below slack length. At the left ventricular (LV) level, recoil and filling by suction require contraction to an end-systolic volume (ESV) below equilibrium volume (Veq) as well as large-scale deformations, for example, torsion or twist. Little is known about RFs and suction in the failing ventricle. We undertook a comparison of determinants of suction in open-chest dogs previously subjected to 2 weeks of pacing tachycardia (PT) and controls. To assess the ability of the LV to contract below Veq, we used a servomotor to clamp left atrial pressure and produce nonfilling diastoles, allowing measurement of fully relaxed pressure at varying volumes. We quantified twist with sonomicrometry. We also assessed transmural ratios of N2B to N2BA titin isoforms and total titin to myosin heavy chain (MHC) protein. In PT, the LV did not contract below Veq, even with marked reduction of volume (end-diastolic pressure [EDP], 1 to 2 mm Hg), whereas in controls ESV was less than Veq when EDP was less than approximately 5 mm Hg. In PT, both systolic twist and diastolic untwisting rate were reduced, and there was exaggerated transmural variation in titin isoform and titin-to-MHC ratios, consistent with the more extensible N2BA being present in larger amounts in the subendocardium. Thus, in PT, determinants of suction at the level of the LV are markedly impaired. The altered transmural titin isoform gradient is consistent with a decrease in RFs and may contribute to these findings.

Animals↗
PubMed2000

ATPase switches controlling DNA replication initiation.

Proteins that bind and hydrolyze ATP are frequently involved in the early steps of DNA replication. Recent studies of Saccharomyces cerevisiae suggest that two members of the AAA+ ATPase family--the origin recognition complex and Cdc6p--have separable roles for ATP binding and ATP hydrolysis during eukaryotic DNA replication. Intriguingly, the proposed regulation of these eukaryotic replication proteins by ATP has functional similarities to the ATP-dependent control of the DnaA and DnaC initiation factors from Escherichia coli. Comparison of the ATP regulation of these factors suggests that ATP binding and hydrolysis acts as a molecular switch that couples key events during initiation of replication. This switch results in a significant change in protein function.

Adenosine Triphosphatases↗
PubMed1999

Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element.

In the yeast Saccharomyces cerevisiae, sequence-specific DNA binding by the origin recognition complex (ORC) is responsible for selecting origins of DNA replication. In metazoans, origin selection is poorly understood and it is unknown whether specific DNA binding by metazoan ORC controls replication. To address this problem, we used in vivo and in vitro approaches to demonstrate that Drosophila ORC (DmORC) binds to replication elements that direct repeated initiation of replication to amplify the Drosophila chorion gene loci in the follicle cells of egg chambers. Using immunolocalization, we observe that ACE3, a 440-bp chorion element that contains information sufficient to drive amplification, directs DmORC localization in follicle cells. Similarly, in vivo cross-linking and chromatin immunoprecipitation assays demonstrate association of DmORC with both ACE3 and two other amplification control elements, AER-d and ACE1. To demonstrate that the in vivo localization of DmORC is related to its DNA-binding properties, we find that purified DmORC binds to ACE3 and AER-d in vitro, and like its S. cerevisiae counterpart, this binding is dependent on ATP. Our findings suggest that sequence-specific DNA binding by ORC regulates initiation of metazoan DNA replication. Furthermore, adaptation of this experimental approach will allow for the identification of additional metazoan ORC DNA-binding sites and potentially origins of replication.

Animals↗
PubMed1999

Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication.

Chromosomes are replicated in characteristic, temporal patterns during S phase. We have compared the timing of association of replication proteins at early- and late-replicating origins of replication. Minichromosome maintenance proteins assemble simultaneously at early- and late-replicating origins. In contrast, Cdc45p association with late origins is delayed relative to early origins. DNA polymerase alpha association is similarly delayed at late origins and requires Cdc45p function. Activation of the S phase checkpoint inhibits association of Cdc45p with late-firing origins. These studies suggest that Cdc45p is poised to serve as a key regulatory target for both the temporal and checkpoint-mediated regulation of replication origins.

Carrier Proteins↗
PubMed1999

ORC localization in Drosophila follicle cells and the effects of mutations in dE2F and dDP.

We isolated mutations in Drosophila E2F and DP that affect chorion gene amplification and ORC2 localization in the follicle cells. In the follicle cells of the ovary, the ORC2 protein is localized throughout the follicle cell nuclei when they are undergoing polyploid genomic replication, and its levels appear constant in both S and G phases. In contrast, when genomic replication ceases and specific regions amplify, ORC2 is present solely at the amplifying loci. Mutations in the DNA-binding domains of dE2F or dDP reduce amplification, and in these mutants specific localization of ORC2 to amplification loci is lost. Interestingly, a dE2F mutant predicted to lack the carboxy-terminal transcriptional activation and RB-binding domain does not abolish ORC2 localization and shows premature chorion amplification. The effect of the mutations in the heterodimer subunits suggests that E2F controls not only the onset of S phase but also origin activity within S phase.

Animals↗
PubMed1998

Left ventricular restoring forces: modulation by heart rate and contractility.

We used a servomotor system in open-chest dogs to rapidly clamp left atrial pressure below left ventricular (LV) diastolic pressure in order to produce nonfilling diastoles during which the LV fully relaxed at its end-systolic volume (ESV). Restoring forces (RFs) generated during contraction which result in LV filling by suction were considered to be present when the fully relaxed pressure (FRP) was negative. We characterized RFs in terms of the fully relaxed pressure-volume relation (FRPV relation, FRP plotted vs ESV), which has negative and positive portions and an equilibrium volume (FRP = 0 mmHg). A negative FRP is ordinarily present over the lower half of the physiologic filling range. Increased contractility (systemic dobutamine) shifts the FRPV relation downward, indicating greater RFs at any ESV. Intracoronary dobutamine administered via the left anterior descending coronary artery has the same effect. Acute increases in heart rate from about 100 to 150 beats/min did not alter the FRPV relation. In contrast, chronic tachycardia heart failure resulted in marked depression of the ability to generate RFs, even at very low volumes. Thus, RFs normally contribute to LV filling. They are augmented by acute increases in global and anterior wall contractility but not heart rate, within the range specified above. Chronic tachycardia heart failure markedly attenuated RFs. The latter may constitute a previously unappreciated mechanism of diastolic dysfunction in heart failure.

Animals↗
PubMed1998

Mechanoenergetic studies in isolated mouse hearts.

We tested the feasibility of an isolated, balloon-in-ventricle, isovolumically contracting, crystalloid-perfused mouse heart preparation (n = 10) for studies of cardiac mechanoenergetics using the end-systolic pressure-volume relation (ESPVR) and myocardial oxygen consumption (VO2)-pressure-volume area (PVA) framework employed in larger species. The intraventricular balloon method was shown to be accurate for measurement of left ventricular volume, especially at relatively higher volumes. The ESPVR demonstrated contractility-dependent curvilinearity. Average slope of the ESPVR was 1,299 +/- 369 (SD) mmHg.g.ml-1, with a volume intercept of 0.018 +/- 0.006 ml. The VO2-PVA relation was well fitted by a straight line, with average slope and VO2 intercept of 3.57 +/- 1.31 x 10(-5) ml O2.mmHg-1.ml-1 and 0.92 +/- 0.21 x 10(-3) ml O2.beat-1.g-1, respectively. Decreasing perfusate Ca2+ concentration resulted in a decrease in the slope of the ESPVR, a decrease in the VO2 intercept of the VO2-PVA relation, but no significant change in its slope. Hearts from hypothyroid (n = 8) mice demonstrated similar mechanoenergetic changes. We conclude that delineation of the ESPVR and the VO2-PVA relation is feasible in the mouse heart. Our method should allow an assessment of cardiac mechanoenergetics as sophisticated as that previously possible only in larger hearts.

Animals↗
PubMed1998

Effects of dobutamine on left ventricular restoring forces.

Restoring forces, which are generated when the left ventricle contracts below its equilibrium volume (Veq), are responsible for diastolic suction. Their magnitude is inversely related to end-systolic volume (ESV). In previous studies in which the mitral valve was replaced with a prosthesis, increased contractility was shown to augment restoring forces independently of ESV. In the present study, we quantified restoring forces in the presence of an intact mitral valve in open-chest dogs (n = 6) as the fully relaxed pressure (FRP) after completion of left ventricular pressure (LVP) fall during nonfilling diastoles produced by a servomotor system that clamped left atrial pressure below LVP. A negative FRP indicated a restoring force was present. We related FRP to ESV during control, intravenous, and left anterior descending coronary artery (intracoronary) administration of dobutamine. With intravenous dobutamine, we observed an approximately parallel downward and rightward shift of the FRP-ESV relation, indicating increased restoring forces at any ESV less than Veq. The downward shift averaged -2.6 +/- 1.6 (SD) mmHg at the control Veq. A similar shift occurred with intracoronary dobutamine. In additional experiments (n = 2), we found that over a common range of ESV dobutamine slightly increased wall thickness (<10%) during nonfilling diastoles, consistent with an increase in coronary blood volume. We conclude that dobutamine increases restoring forces independently of changes in ESV in conjunction with an increase in Veq. This effect may partly be related to increased coronary blood volume.

Animals↗
PubMed1997

Decrease in forces responsible for diastolic suction during acute coronary occlusion.

BACKGROUND: The production of left ventricular (LV) restoring forces generated during contraction, which are responsible for diastolic suction, is dependent on end-systolic volume (ESV) and systolic transmural and 3D deformation. We tested the hypothesis that acute coronary occlusion would result in loss of forces that cause suction. METHODS AND RESULTS: Ten open-chest dogs were subjected to a 10-minute acute coronary occlusion (proximal left anterior descending coronary artery). A servomotor connected to the left atrium (LA) was used to rapidly clamp LA pressure during systole below the level of the succeeding LV diastolic pressure, resulting in nonfilling diastoles during which the LV fully relaxed at its ESV. LA clamps at multiple ESVs (conductance catheter) allowed delineation of positive and negative portions of the fully relaxed LV pressure-volume relation (FRPVR). A negative fully relaxed pressure (FRP) indicates the presence of restoring forces. After 10 minutes of acute coronary occlusion, there was an upward shift of the FRPVR. Thus, for example, at matched ESVs before and during coronary occlusion, FRP was -1.1+/-1.1 (+/-SD) mm Hg before versus 0.2+/-1.2 mm Hg after 10 minutes of coronary occlusion (P<.05). CONCLUSIONS: Acute coronary occlusion results in a rapid decrease in forces responsible for suction. This phenomenon is independent of the level of ESV and may contribute to ischemic diastolic dysfunction.

Animals↗
PubMed1997

Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase.

In S. cerevisiae, the chromatin structure of DNA replication origins changes as cells become competent for DNA replication, suggesting that G1 phase-specific association of replication factors with origin DNA regulates entry into S phase. We demonstrate that ORC, Cdc45p, and MCM proteins are components of prereplication complexes (pre-RC). The MCM-origin association is dependent upon ORC and Cdc6p. During S phase, MCM proteins and Cdc45p dissociate from origin DNA and associate with nonorigin DNA with similar kinetics as DNA Polymerase epsilon, which is present at DNA replication forks. Our results identify protein components of the pre-RC and a novel replication complex appearing at the G1/S transition (the RC), and suggest that after initiation MCM proteins and Cdc45p move with eukaryotic replication forks.

Carrier Proteins↗
PubMed1997

Coordinate binding of ATP and origin DNA regulates the ATPase activity of the origin recognition complex.

The Origin Recognition Complex (ORC) is a six-protein assembly that specifies the sites of DNA replication initiation in S. cerevisiae. Origin recognition by ORC requires ATP. Here, we demonstrate that two subunits, Orc1p and Orc5p, bind ATP and that Orc1p also hydrolyzes ATP. ATP binding and hydrolysis by Orc1p are both regulated by origin DNA in a sequence-specific manner. ATP binding to Orc1p, but not ATP hydrolysis, is responsible for the ATP dependence of the ORC-origin interaction, indicating that ATP is a cofactor that locks ORC on origin DNA. These data demonstrate that occupancy of the Orc1p ATP-binding site has a profound effect on ORC function and that ATP hydrolysis by Orc1p has the potential to drive transitions between different functional states of ORC.

Adenosine Triphosphatases↗
PubMed1997

A new butadiene derivative, T-686, inhibits plasminogen activator inhibitor type-1 production in vitro by cultured human vascular endothelial cells and development of atherosclerotic lesions in vivo in rabbits.

Plasminogen activator inhibitor-1 (PAI-1), the major physiologic inhibitor of tissue-type plasminogen activator and urokinase, is abundantly expressed in atherosclerotic vascular wall. To determine the role of PAI-1 in vascular wall, we have used a novel inhibitor of PAI-1, (3E, 4E)-3-benzylidene-4-(3,4,5-trimethoxy-benzylidene) -pyrrolidine-2,5-dione (T-686). T-686 was given to human vascular endothelial cells in vitro and to rabbits subjected to high cholesterol diet and mechanical injury in vivo. T-686 attenuated the augmentation of PAI-1 antigen accumulation induced by transforming growth factor beta in conditioned medium from the human umbilical vein endothelial cells. In rabbits with aortic atherosclerosis induced by hypercholesterolemia and implantation of indwelling plastic tubing, oral administration of T-686 (30mg/kg body weight/day) for 8 weeks attenuated the increase in plasma PAI-1 activity induced by vascular injury without decreasing blood triglyceride and cholesterol. This was accompanied by the reduction in aortic PAI-1 mRNA expression and the inhibition of development of atherosclerosis lesions. Thus, T-686 not only decreased PAI-1 synthesis in vascular cells in vitro but also protected against the development of vascular lesions in vivo. This compound may be useful in defining the role of PAI-1 in atherothrombotic states.

Animals↗
PubMed1997

Architecture of the yeast origin recognition complex bound to origins of DNA replication.

In many organisms, the replication of DNA requires the binding of a protein called the initiator to DNA sites referred to as origins of replication. Analyses of multiple initiator proteins bound to their cognate origins have provided important insights into the mechanism by which DNA replication is initiated. To extend this level of analysis to the study of eukaryotic chromosomal replication, we have investigated the architecture of the Saccharomyces cerevisiae origin recognition complex (ORC) bound to yeast origins of replication. Determination of DNA residues important for ORC-origin association indicated that ORC interacts preferentially with one strand of the ARS1 origin of replication. DNA binding assays using ORC complexes lacking one of the six subunits demonstrated that the DNA binding domain of ORC requires the coordinate action of five of the six ORC subunits. Protein-DNA cross-linking studies suggested that recognition of origin sequences is mediated primarily by two different groups of ORC subunits that make sequence-specific contacts with two distinct regions of the DNA. Implications of these findings for ORC function and the mechanism of initiation of eukaryotic DNA replication are discussed.

Animals↗
PubMed1997

Initiation of DNA replication in eukaryotic cells.

The recent identification of proteins that recognize origins of DNA replication and control the initiation of eukaryotic DNA replication has provided critical molecular tools to dissect this process. Dynamic changes in the assembly and disassembly of protein complexes at origins are important for the initiation of DNA replication and occur throughout the cell cycle. Herein, we review the key proteins required for the initiation of DNA replication, their involvement in the protein complex assembly at replication origins, and how the cell cycle machinery regulates this process.

Animals↗
PubMed1997

Cholesterol emboli syndrome--uncommon or unrecognized?

Cholesterol emboli syndrome is a multisystem disorder that can be precipitated by angiographic procedures. We report 5 cases in which the presentation was renal failure. All patients had undergone angiography, but the temporal relation of the procedure to the clinical presentation was highly variable, the interval ranging from one day to four months. With the increase in diagnostic and therapeutic uses of angiography, the cholesterol emboli syndrome is likely to become more frequent and needs to be recognized.

Acute Kidney Injury↗
PubMed1996

Restoring forces assessed with left atrial pressure clamps.

A negative pressure (P) in the fully relaxed left ventricle (LV) indicates the presence of restoring forces generated during contraction. To assess restoring forces in the intact LV under physiological filling conditions, a servomotor system was used in anesthetized open-chest dogs (n = 8) to produce nonfilling diastoles by left atrial pressure (LAP) clamping during systole such that LAP was less than left ventricular pressure (LVP) during the subsequent diastole. Steady-state LV end-diastolic pressure (EDP) was varied by volume infusion from 4.0 +/- 1.5 (+/-SD) to 12.8 +/- 2.1 mmHg. The corresponding fully relaxed LVPs increased from -2.1 +/- 1.9 to 1.1 +/- 3.2 mmHg, P < 0.001. LAP clamping increased the rate of LVP fall by 34 +/- 28% (P < 0.001) during 10 ms after the LVP dropped below the level of the LVP-LAP crossover of the preceding normal beat. During clamped beats, two-dimensional echo revealed substantial downward displacement of the mitral valve (MV) leaflets despite the reversed LA-LV gradient and absence of filling. Thus 1) restoring forces are present at low physiological EDP but absent at high physiological EDP; 2) filling retards the rate of fall of LVP; 3) even in the absence of filling, the process of LV relaxation facilitates MV opening.

Animals↗