Late-Life Body Mass Index, Rapid Weight Loss, Apolipoprotein E ε4 and the Risk of Cognitive Decline and Incident Dementia.

OBJECTIVES: To examine the effect of late-life body mass index (BMI) and rapid weight loss on incident mild cognitive impairment (MCI) and Alzheimer's disease (AD). DESIGN: Prospective longitudinal cohort study. SETTING: National Alzheimer's Coordinating Center (NACC) Uniform Data Set, including 34 past and current National Institute on Aging-funded AD Centers across the United States. PARTICIPANTS: 6940 older adults (n=5061 normal cognition [NC]; n=1879 MCI). MEASUREMENTS: BMI (kg/m2) and modified Framingham Stroke Risk Profile (FSRP) score (sex, age, systolic blood pressure, anti-hypertension medication, diabetes mellitus, cigarette smoking, prevalent cardiovascular disease, atrial fibrillation) were assessed at baseline. Cognition and weight were assessed annually. RESULTS: Multivariable binary logistic regression, adjusting for age, sex, race, education, length of follow-up, and modified FSRP related late-life BMI to risk of diagnostic conversion from NC to MCI or AD and from MCI to AD. Secondary analyses related late-life BMI to diagnostic conversion in the presence of rapid weight loss (>5% decrease in 12 months) and apolipoprotein E (APOE) ε4. During a mean 3.8-year follow-up period, 12% of NC participants converted to MCI or AD and 49% of MCI participants converted to AD. Higher baseline BMI was associated with a reduced probability of diagnostic conversion, such that for each one-unit increase in baseline BMI there was a reduction in diagnostic conversion for both NC (OR=0.977, 95%CI 0.958-0.996, p=0.015) and MCI participants (OR=0.962, 95%CI 0.942-0.983, p<0.001). The protective effect of higher baseline BMI did not persist in the setting of rapid weight loss but did persist when adjusting for APOE ε4. CONCLUSIONS: Higher late-life BMI is associated with a lower risk of incident MCI and AD but is not protective in the presence of rapid weight loss.

Interactions between two catalytically distinct MCM subgroups are essential for coordinated ATP hydrolysis and DNA replication.

The six MCM (minichromosome maintenance) proteins are essential DNA replication factors that each contain a putative ATP binding motif and together form a heterohexameric complex. We show that these motifs are required for viability in vivo and coordinated ATP hydrolysis in vitro. Mutational analysis discriminates between two functionally distinct MCM protein subgroups: Mcm4p, 6p, and 7p contribute canonical ATP binding motifs essential for catalysis, whereas the related motifs in Mcm2p, 3p, and 5p serve a regulatory function. Reconstitution experiments indicate that specific functional interactions between these two subgroups are required for robust ATP hydrolysis. Our observations show parallels between the MCM complex and the F1-ATPase, and we discuss how ATP hydrolysis by the MCM complex might be coupled to DNA strand separation.

Genome-wide distribution of ORC and MCM proteins in S. cerevisiae: high-resolution mapping of replication origins.

DNA replication origins are fundamental to chromosome organization and duplication, but understanding of these elements is limited because only a small fraction of these sites have been identified in eukaryotic genomes. Origin Recognition Complex (ORC) and minichromosome maintenance (MCM) proteins form prereplicative complexes at origins of replication. Using these proteins as molecular landmarks for origins, we identified ORC- and MCM-bound sites throughout the yeast genome. Four hundred twenty-nine sites in the yeast genome were predicted to contain replication origins, and approximately 80% of the loci identified on chromosome X demonstrated origin function. A substantial fraction of the predicted origins are associated with repetitive DNA sequences, including subtelomeric elements (X and Y') and transposable element-associated sequences (long terminal repeats). These findings identify the global set of yeast replication origins and open avenues of investigation into the role(s) ORC and MCM proteins play in chromosomal architecture and dynamics.

Activation of PKC decreases myocardial O2 consumption and increases contractile efficiency in rats.

The effect of protein kinase C (PKC) activation on cardiac mechanoenergetics is not fully understood. To address this issue, we determined the effects of the PKC activator phorbol 12-myristate 13-acetate (PMA) on isolated rat hearts. Hearts were exposed to PMA with or without pretreatment with the PKC inhibitor chelerythrine. Contractile efficiency was assessed as the reciprocal of the slope of the linear myocardial O2 consumption (VO2) pressure-volume area (PVA) relation. PMA decreased contractility (Emax; -30 +/- 8%; P < 0.05) and increased coronary perfusion pressure (+58 +/- 11%; P < 0.01) without altering left ventricular end-diastolic pressure. Concomitantly, PMA decreased PVA-independent VO2 [nonmechanical energy expenditure for excitation-contraction (E-C) coupling and basal metabolism] by 28 +/- 8% (P < 0.05) and markedly increased contractile efficiency (+41 +/- 8%; P < 0.05) in a manner independent of the coronary vascular resistance. Basal metabolism was not affected by PMA. Chelerythrine abolished the PMA-induced vasoconstriction, negative inotropy, decreased PVA-independent VO2, and increased contractile efficiency. We conclude that PKC-mediated phosphorylation of regulatory proteins reduces VO2 via effects on both the contractile machinery and the E-C coupling.

ATP bound to the origin recognition complex is important for preRC formation.

The origin recognition complex (ORC) binds origins of replication and directs the assembly of a higher order protein complex at these sites. ORC binds and hydrolyzes ATP in vitro. ATP binding to the largest subunit of ORC, Orc1p, stimulates specific binding to origin DNA; however, the function of ATP hydrolysis by ORC is unknown. To address the role of ATP hydrolysis, we have generated mutants within Orc1p that are dominant lethal. At physiological ATP concentrations, these mutants are defective for ATP hydrolysis but not ATP binding in the absence of DNA. These mutants inhibit formation of the prereplicative complex when overexpressed. The dominant lethal phenotype of these mutant ORC complexes is suppressed by simultaneous overexpression of wild-type, but not mutant, Cdc6p. Our findings suggest that these hydrolysis-defective mutants inhibit growth by titrating Cdc6p away from the origin. Based on these observations, we propose that Cdc6p specifically recognizes the ATP-bound state of Orc1p and that ATP hydrolysis is coupled to preRC disassembly.

Nucleosomes positioned by ORC facilitate the initiation of DNA replication.

The packaging of eukaryotic DNA into nucleosomes is a critical regulator of nuclear events. To address the interplay between chromatin and replication initiation, we have assessed the determinants and function of the nucleosomal configuration of S. cerevisiae replication origins. Using in vitro and in vivo assays, we demonstrate that the yeast initiator, the origin recognition complex (ORC), is required to maintain the nucleosomal configuration adjacent to origins. Disruption of the ORC-directed nucleosomal arrangement at an origin interferes with initiation of replication, but does not alter the association of ORC with the origin. Instead, the nucleosomes positioned by ORC are important for prereplicative complex formation. These findings suggest that origin-proximal nucleosomes facilitate replication initiation, and that local chromatin structure affects origin function.

Delta opioid receptor stimulation mimics ischemic preconditioning in human heart muscle.

OBJECTIVES: The objective of this study was to examine whether the delta (delta) opioid receptor isoform is expressed in the human heart and whether this receptor improves contractile function after hypoxic/reoxygenation injury. BACKGROUND: Delta opioid receptor agonists mimic preconditioning (PC) in rat myocardium, corresponding to known cardiac delta opioid receptor expression in this species. METHODS: The messenger RNA transcript encoding the delta opioid receptor was identified in human atria and ventricles. To evaluate the cardioprotective role of the opioid receptor, human atrial trabeculae from patients undergoing coronary bypass grafting were isolated and superfused with Tyrode's solution. A control group underwent 90 min of simulated ischemia and 120 min of reoxygenation. A second group was preconditioned with 3 min simulated ischemia and 7 min reoxygenation. Additional groups included: superfusion with the delta receptor agonist (DADLE) (10 nM), with the delta receptor antagonist naltrindole (10 nM) and with the mitochondrial K(ATP) channel blocker 5-hydroxydecanoate (5HD) (100 microM) either with or without PC, respectively. A final group was superfused with 5HD before DADLE. The end point used was percentage of developed force after 120 min of reoxygenation. RESULTS: Results, expressed as means +/- SEM, were: control = 32.6 + 3.8%; PC = 50.5% + 1.8*; DADLE = 46.0+/-3.9%*; PC + naltrindole = 25.5+/-3.9%; naltrindole alone = 25.5+/-4.3%; 5HD + PC = 28.9+/-7.4%; 5HD alone = 24.1+/-3.0%; 5HD + DADLE = 26.9+/-4.4% (*p < 0.001 vs. controls). CONCLUSIONS: Human myocardium expresses the delta opioid receptor transcript. Stimulation of this receptor appears to protects human muscle from simulated ischemia, similar to PC, and via opening of the mitochondrial K(ATP) channel.

Genome-wide location and function of DNA binding proteins.

Understanding how DNA binding proteins control global gene expression and chromosomal maintenance requires knowledge of the chromosomal locations at which these proteins function in vivo. We developed a microarray method that reveals the genome-wide location of DNA-bound proteins and used this method to monitor binding of gene-specific transcription activators in yeast. A combination of location and expression profiles was used to identify genes whose expression is directly controlled by Gal4 and Ste12 as cells respond to changes in carbon source and mating pheromone, respectively. The results identify pathways that are coordinately regulated by each of the two activators and reveal previously unknown functions for Gal4 and Ste12. Genome-wide location analysis will facilitate investigation of gene regulatory networks, gene function, and genome maintenance.

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.

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.

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.

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.

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.

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.

Mechanoenergetic alterations during the transition from cardiac hypertrophy to failure in Dahl salt-sensitive rats.

BACKGROUND: The time course and mechanisms of altered mechanoenergetics and depressed cross-bridge cycling in hypertrophied and failing myocardium are uncertain. METHODS AND RESULTS: We studied mechanoenergetics in Dahl salt-sensitive (DS) rats fed high-salt diet (HS) for 6 (HS-6) and 12 (HS-12) weeks to produce compensated hypertrophy and failure. The slope of the end-systolic pressure-volume relation (E'max) was similar in HS-6 and low-salt controls (LS-6), but reduced in HS-12 compared with controls (LS-12). Efficiency [1/slope of oxygen consumption (&f1;O2)-pressure-volume area (PVA) relation] was similar in HS-6 and LS-6 but higher in HS-12 versus LS-12 (59+/-16% versus 44+/-7%, P<0.05). Economy [1/slope of the force-time integral (FTI)-&f1;O2 relation] was similar in HS-6 and LS-6 but higher in HS-12 versus LS-12 (218+/-123 versus 74+/-39x10(3) g. s. mL O2-1. g; P<0.05). Compared with controls, myofibrillar ATPase activity was reduced by 24% in HS-6 and 44% in HS-12. V3 Isomyosin was increased in HS-6 (40+/-12% versus 9+/-8%; P<0.05) and further increased in HS-12 (76+/-10% versus 22+/-18%; P<0.05). Hypothyroid LS-12 rats had 100% V3 isomyosin, yet efficiency, economy, and ATPase values were intermediate between LS-12 and HS-12. HS-12 rats demonstrated increased troponin T3 isoform (17+/-2 versus 23+/-2%, P<0.05). There were no changes in troponin I or tropomyosin isoforms. However, the proportion of phosphorylated troponin T was reduced in HS-12 versus LS-12 hearts (P<.001). CONCLUSIONS: In DS rats, the transition to failure is associated with depressed E'max and increased efficiency and economy. These findings are linked to myofibrillar ATPase activity and suggest that mechanisms other than isomyosin switching are important determinants of ventricular energetics. A troponin T isoform switch is one potential mechanism.

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.

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.

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.

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.