Abnormal epicardial coronary resistance in patients with diffuse atherosclerosis but "Normal" coronary angiography.

BACKGROUND: Coronary arteries without focal stenosis at angiography are generally considered non-flow-limiting. However, atherosclerosis is a diffuse process that often remains invisible at angiography. Accordingly, we hypothesized that in patients with coronary artery disease, nonstenotic coronary arteries induce a decrease in pressure along their length due to diffuse coronary atherosclerosis. METHODS AND RESULTS: Coronary pressure and fractional flow reserve (FFR), as indices of coronary conductance, were obtained from 37 arteries in 10 individuals without atherosclerosis (group I) and from 106 nonstenotic arteries in 62 patients with arteriographic stenoses in another coronary artery (group II). In group I, the pressure gradient between aorta and distal coronary artery was minimal at rest (1+/-1 mm Hg) and during maximal hyperemia (3+/-3 mm Hg). Corresponding values were significantly larger in group II (5+/-4 mm Hg and 10+/-8 mm Hg, respectively; both P<0.001). The FFR was near unity (0.97+/-0.02; range, 0.92 to 1) in group I, indicating no resistance to flow in truly normal coronary arteries, but it was significantly lower (0.89+/-0.08; range, 0.69 to 1) in group II, indicating a higher resistance to flow. In 57% of arteries in group II, FFR was lower than the lowest value in group I. In 8% of arteries in group II, FFR was <0.75, the threshold for inducible ischemia. CONCLUSION: Diffuse coronary atherosclerosis without focal stenosis at angiography causes a graded, continuous pressure fall along arterial length. This resistance to flow contributes to myocardial ischemia and has consequences for decision-making during percutaneous coronary interventions.

Conserved Wat1/Pop3 WD-repeat protein of fission yeast secures genome stability through microtubule integrity and may be involved in mRNA maturation.

Accurate chromosome segregation is dependent upon the integrity of mitotic spindles, which pull each pair of sister chromatids towards opposite poles. In this study, we have characterised fission yeast pop3-5235, a diploidising mutant that is impaired in genome stability. Pop3 is the same as Wat1, a conserved protein containing 7 WD repeats. Pop3/Wat1 has also been isolated from a two-hybrid screen as a binding partner to Prp2, the large subunit of the essential splicing factor U2AF. In wat1 mutants, the cellular amount of alpha-tubulin is decreased to very low levels, which results in compromised microtubules and spindles, consequently leading to unequal chromosome separation. Further analysis shows that, in spite of the binding between Wat1 and Prp2, Wat1 may not be involved directly in splicing reactions per se. Instead, we find that Wat1 is required for the maintenance of alpha-tubulin mRNA levels; moreover, transcript levels of genes other than the alpha-tubulin gene are also equally decreased in this mutant. Wild-type Wat1, but not the mutant protein, forms a large complex in the cell with several other proteins, suggesting that Wat1 functions as a structural linker in the complex. The results suggest that Wat1 plays a role in mRNA maturation as a coupling protein between splicing and synthesis and/or stabilisation.

The distribution of substance P and neuropeptide Y in four songbird species: a comparison of food-storing and non-storing birds.

The distributions of the neuropeptides substance P (SP) and neuropeptide Y (NPY) were investigated in four songbird species that differ in their food-storing behavior. The food-storing black-capped chickadee (Parus atricapillus) was compared to the non-storing blue tit (Parus caeruleus) and great tit (Parus major) within the avian family Paridae, as well as to the non-storing dark-eyed junco (Junco hyemalis). All four species showed a similar distribution of SP throughout the brain with the exception of two areas, the hippocampal complex (including hippocampus (Hp) and parahippocampus (APH)) and the Wulst (including the hyperstriatum accessorium (HA)). SP-like immunoreactivity was found in cells of the Hp in juncos, but not in the three parid species. Two areas within the APH and HA showed SP-like immunoreactivity in all four species. The more medial of these (designated SPm) is a distinctive field of fibers and terminals found throughout the APH and extending into the HA. A positive relationship between SPm and Hp volume was found for all four species with the chickadee having a significantly larger SPm area relative to telencephalon than the other species. The distribution of SP in this region may be related to differences in food-storing behavior. In contrast to substance P, NPY distribution throughout the brain was similar in all four species. Further, NPY-immunoreactive cells were found in the Hp of all four species and no species differences in the number of NPY cells was observed.

Imaging of the endothelial dysfunction in coronary atherosclerosis.

Coronary endothelial dysfunction is characterised by coronary vasoconstrictive responses to endothelium-dependent vasodilators. It is associated with coronary artery disease (CAD) and is considered an early phase of coronary atherosclerosis. Patients with CAD benefit from vigorous risk factor interventions and medical treatment, with a marked decrease in coronary events and an improvement in survival that are not reported following revascularisation procedures. Therefore, early detection of anatomical and functional changes in the coronary vasculature due to atherosclerosis provides the basis for integrated pharmacological, dietary and lifestyle modifications to prevent cardiovascular events and revascularisation procedures. The question arises as to whether these alterations in regional myocardial tone can be detected by any of the current non-invasive methods. Several methods are reviewed. We consider that intracoronary ultrasonography is the most accurate method, but non-invasive positron emission tomography and magnetic resonance imaging technology is of growing importance for identifying endothelial dysfunction of early coronary atherosclerosis.

A precise, three-dimensional atlas of myocardial perfusion correlated with coronary arteriographic anatomy.

To map precise myocardial perfusion anatomy, we correlated detailed coronary arteriographic anatomy for every coronary artery and all secondary branches in the heart that had flow-limiting stenosis with corresponding specific, circumscribed, myocardial perfusion defects by positron emission tomography. Eight hundred ninety-five patients with abnormal coronary arteriograms showing any visible coronary artery narrowing of greater than 10% diameter stenosis underwent positron emission tomography perfusion imaging at rest and after dipyridamole stress; the data obtained were processed automatically into 3-dimensional topographic displays of relative radionuclide uptake in anterior, septal, left lateral, and inferior quadrant views, without attenuation artifacts, depth-dependent resolution, or spatial distortion of polar displays. The selection criterion for detailed anatomic analysis was the presence of a discrete, localized, moderate to severe, dipyridamole-induced perfusion defect, defined by automated algorithms as 1 quadrant view outside 2 SDs of healthy control subjects with which a specific stenotic coronary artery and/or its secondary branches could be correlated unequivocally on the coronary arteriogram for mapping precise perfusion anatomy, not for determining sensitivity or specificity. Because the anatomy of myocardial perfusion is inherently not statistical data, the results are presented as a summary atlas and series of individual cases that illustrate myocardial perfusion anatomy. Because the patterns of myocardial perfusion anatomy were derived from a large number of subjects, the atlas provides generalized information, not previously published, that correlates detailed arteriographic anatomy with perfusion anatomy including secondary diagonal, marginal, and posterior descending branches of the coronary arteries.

Study of cyclin proteolysis in anaphase-promoting complex (APC) mutant cells reveals the requirement for APC function in the final steps of the fission yeast septation initiation network.

Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombe mutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APC cut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cut mutants and the normal reorganization of septation initiation network components during anaphase.

Coordination between fission yeast glucan formation and growth requires a sphingolipase activity.

css1 mutants display a novel defect in Schizosaccharomyces pombe cell wall formation. The mutant cells are temperature-sensitive and accumulate large deposits of material that stain with calcofluor and aniline blue in their periplasmic space. Biochemical analyses of this material indicate that it consists of alpha- and beta-glucans in the same ratio as found in cell walls of wild-type S. pombe. Strikingly, the glucan deposits in css1 mutant cells do not affect their overall morphology. The cells remain rod shaped, and the thickness of their walls is unaltered. Css1p is an essential protein related to mammalian neutral sphingomyelinase and is responsible for the inositolphosphosphingolipid-phospholipase C activity observed in S. pombe membranes. Furthermore, expression of css1(+) can compensate for loss of ISC1, the enzyme responsible for this activity in Saccharomyces cerevisiae membranes. Css1p localizes to the entire plasma membrane and secretory pathway; a C-terminal fragment of Css1p, predicted to encode a single membrane-spanning segment, is sufficient to direct membrane localization of the heterologous protein, GFP. Our results predict the existence of an enzyme(s) or process(es) essential for the coordination of S. pombe cell wall formation and division that is, in turn, regulated by a sphingolipid metabolite.

Fission yeast Clp1p phosphatase regulates G2/M transition and coordination of cytokinesis with cell cycle progression.

BACKGROUND: In Saccharomyces cerevisiae the mitotic-exit network (MEN) functions in anaphase to promote the release of the Cdc14p phosphatase from the nucleolus. This release causes mitotic exit via inactivation of the cyclin-dependent kinase (Cdk). Cdc14p-like proteins are highly conserved; however, it is unclear if these proteins regulate mitotic exit as in S. cerevisiae. In Schizosaccharomyces pombe a signaling pathway homologous to the MEN and termed the septation initiation network (SIN) is required not for mitotic exit, but for initiation of cytokinesis and for a cytokinesis checkpoint that inhibits further cell cycle progression until cytokinesis is complete. RESULTS: We have identified the S. pombe Cdc14p homolog, Clp1p, and show that it is not required for mitotic exit but rather functions together with the SIN in coordinating cytokinesis with the nuclear-division cycle. As cells enter mitosis, Clp1p relocalizes from the nucleolus to the spindle and site of cell division. Clp1p exit from the nucleolus does not depend on the SIN, but the SIN is required for keeping Clp1p out of the nucleolus until completion of cytokinesis. Clp1p, in turn, may promote the activation of the SIN by antagonizing Cdk activity until cytokinesis is complete and thus ensuring that cytokinesis is completed prior to the initiation of the next cell cycle. In addition to its roles in anaphase, Clp1p regulates the G2/M transition since cells deleted for clp1 enter mitosis precociously and cells overexpressing Clp1p delay mitotic entry. Unlike Cdc14p, Clp1p appears to antagonize Cdk activity by preventing dephosphorylation of Cdc2p on tyrosine. CONCLUSIONS: S. pombe Clp1p affects cell cycle progression in a markedly different manner than its S. cerevisiae homolog, Cdc14p. This finding raises the possibility that related phosphatases in animal cells will prove to have important roles in coordinating the onset of cytokinesis with the events of mitosis.

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe.

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.

High prevalence of myocardial perfusion abnormalities on positron emission tomography in asymptomatic persons with a parent or sibling with coronary artery disease.

BACKGROUND: We hypothesized that asymptomatic persons with a parent or sibling with coronary artery disease (CAD) have myocardial perfusion defects on positron emission tomography (PET) as markers of early CAD. METHODS AND RESULTS: After medical and family histories were recorded, 90 subjects underwent rest-dipyridamole cardiac PET perfusion imaging, including 18 index cases (a subject with CAD documented by PET and arteriography), 32 asymptomatic adults without known CAD who had a parent or sibling with CAD among these index cases, 30 asymptomatic subjects with comparable coronary risk factors without CAD or a family history of CAD, and 10 volunteer control subjects with no risk factors and no family history. PET perfusion images were quantified with automated software for size of abnormalities as percent of the cardiac image outside 95% CIs of normal controls and for severity as the lowest quadrant average relative activity. Of asymptomatic subjects with a parent or sibling with CAD (first-degree relatives), 50% had dipyridamole-induced myocardial perfusion defects that involved >/=5% of the cardiac image outside normal 95% CIs with or without other risk factors. The size of perfusion defects was larger in first-degree relatives than in control subjects (11+/-13% versus 1+/-1%, P:=0.02) and larger than in asymptomatic subjects with comparable risk factors but no family history of CAD (11+/-13% versus 5+/-6%, P:=0.02). CONCLUSIONS: This study documents the presence of quantitative, statistically significant, dipyridamole-induced myocardial perfusion abnormalities on PET in 50% of asymptomatic persons with a parent or sibling with CAD, independent of other risk factors, indicating preclinical coronary atherosclerosis.

Timing is everything: regulation of mitotic exit and cytokinesis by the MEN and SIN.

Proper completion of mitosis requires careful coordination of numerous cellular events. It is crucial, for example, that cells do not initiate spindle disassembly and cytokinesis until chromosomes have been properly segregated. Cells have developed numerous safeguards or checkpoints to delay exit from mitosis and initiation of the next cell cycle in response to defects in late mitosis. In this review, we discuss recent work on two homologous signaling pathways in budding and fission yeast, termed the mitotic exit network (MEN) and septation initiation network (SIN), respectively, that are essential for coordinating completion of mitosis and cytokinesis with other mitotic events.

Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe.

An oligonucleotide labeling system was developed that can produce radiolabeled hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase. Yet the system is as rapid and simple as kinase labeling. The reaction uses the Klenow fragment of E. coli DNA polymerase to add alpha-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity. The reaction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 of the tf2d gene, a TATA-box binding transcription factor. Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.

Isolation of an essential Schizosaccharomyces pombe gene, prp31(+), that links splicing and meiosis.

We carried out a screen for mutants that arrest prior to premeiotic S phase. One of the strains we isolated contains a temperature-sensitive allele mutation in the fission yeast prp31(+) gene. The prp31-E1 mutant is defective in vegetative cell growth and in meiotic progression. It is synthetically lethal with prp6 and displays a pre-mRNA splicing defect at the restrictive temperature. We cloned the wild-type gene by complementation of the temperature-sensitive mutant phenotype. Prp31p is closely related to human and budding yeast PRP31 homologs and is likely to function as a general splicing factor in both vegetative growth and sexual differentiation.

Sid4p is required to localize components of the septation initiation pathway to the spindle pole body in fission yeast.

A mutation in the Schizosaccharomyces pombe sid4(+) (septation initiation defective) gene was isolated in a screen for mutants defective in cytokinesis. We have cloned sid4(+) and have found that sid4(+) encodes a previously unknown 76.4-kDa protein that localizes to the spindle pole body (SPB) throughout the cell cycle. Sid4p is required for SPB localization of key regulators of septation initiation, including the GTPase Spg1p, the protein kinase Cdc7p, and the GTPase-activating protein Byr4p. An N-terminally truncated Sid4p mutant does not localize to SPBs and when overproduced acts as a dominant-negative mutant by titrating endogenous Sid4p and Spg1p from the SPB. Conversely, the Sid4p N-terminal 153 amino acids are sufficient for SPB localization. Biochemical studies demonstrate that Sid4p interacts with itself, and yeast two-hybrid analysis shows that its self-interaction domain lies within the C-terminal half of the protein. Our data indicate that Sid4p SPB localization is a prerequisite for the execution of the Spg1p signaling cascade.

The role of the sid1p kinase and cdc14p in regulating the onset of cytokinesis in fission yeast.

Coordination of mitosis and cytokinesis is crucial for ensuring proper chromosome segregation and genomic stability. In Schizosaccharomyces pombe, the sid genes (cdc7, cdc11, cdc14, spg1, sid1, sid2 and sid4) define a signaling pathway that regulates septation and cytokinesis. Here we describe the characterization of a novel protein kinase, Sid1p. Sid1p localizes asymmetrically to one spindle pole body (SPB) in anaphase. Sid1p localization is maintained during medial ring constriction and septum synthesis and disappears prior to cell separation. Additionally, we found that Cdc14p is in a complex with Sid1p. Epistasis analysis places Sid1p-Cdc14p downstream of Spg1p-Cdc7p but upstream of Sid2p. Finally, we show that cyclin proteolysis during mitosis is unaffected by inactivating the sid pathway; in fact, loss of Cdc2-cyclin activity promotes Sid1p-Cdc14p association with the SPB, possibly providing a mechanism that couples cytokinesis with mitotic exit.

Pressure-derived fractional flow reserve to assess serial epicardial stenoses: theoretical basis and animal validation.

Background-Fractional flow reserve (FFR) is an index of stenosis severity validated for isolated stenoses. This study develops the theoretical basis and experimentally validates equations for predicting FFR of sequential stenoses separately. Methods and Results-For 2 stenoses in series, equations were derived to predict FFR (FFR(pred)) of each stenosis separately (ie, as if the other one were removed) from arterial pressure (P(a)), pressure between the 2 stenoses (P(m)), distal coronary pressure (P(d)), and coronary occlusive pressure (P(w)). In 5 dogs with 2 stenoses of varying severity in the left circumflex coronary artery, FFR(pred) was compared with FFR(app) (ratio of the pressure just distal to that just proximal to each stenoses) and to FFR(true) (ratio of the pressures distal to proximal to each stenosis but after removal of the other one) in case of fixed distal and varying proximal stenoses (n=15) and in case of fixed proximal and varying distal stenoses (n=20). The overestimation of FFR(true) by FFR(app) was larger than that of FFR(true) by FFR(pred) (0.070+/-0.007 versus 0.029+/-0.004, P<0.01 for fixed distal stenoses, and 0.114+/-0.01 versus 0.036+/-0. 004, P<0.01 for fixed proximal stenoses). This overestimation of FFR(true) by FFR(app) was larger for fixed proximal than for fixed distal stenoses. Conclusions-The interaction between 2 stenoses is such that FFR of each lesion separately cannot be calculated by the equation for isolated stenoses (P(d)/P(a) during hyperemia) applied to each separately but can be predicted by more complete equations taking into account P(a), P(m), P(d), and P(w).

Frequency and clinical implications of fluid dynamically significant diffuse coronary artery disease manifest as graded, longitudinal, base-to-apex myocardial perfusion abnormalities by noninvasive positron emission tomography.

BACKGROUND: Diffuse coronary atherosclerosis is the substrate for plaque rupture and coronary events. Therefore, in patients with mild arteriographic coronary artery disease without significant segmental dipyridamole-induced myocardial perfusion defects, we tested the hypothesis that fluid dynamically significant diffuse coronary artery narrowing is frequently manifest as a graded, longitudinal, base-to-apex myocardial perfusion abnormality by noninvasive PET. METHODS AND RESULTS: In this study, 1001 patients with documented coronary artery disease by coronary arteriography showing any visible coronary artery narrowing underwent rest-dipyridamole PET perfusion imaging. Quantitative severity of dipyridamole-induced, circumscribed, segmental PET perfusion defects was objectively measured by automated software as the minimum quadrant average relative activity indicating localized flow limiting stenoses. Quantitative severity of the graded, longitudinal, base-to-apex myocardial perfusion gradient indicating fluid dynamic effects of diffuse coronary artery narrowing was objectively measured by automated software as the spatial slope of relative activity along the cardiac longitudinal axis. CONCLUSIONS: In patients with mild arteriographic disease without statistically significant dipyridamole-induced segmental myocardial perfusion defects caused by flow-limiting stenoses compared with normal control subjects, there was a graded, longitudinal, base-to-apex myocardial perfusion gradient significantly different from normal control subjects (P=0. 001) that was also observed for moderate to severe dipyridamole-induced segmental perfusion defects (P=0.0001), indicating diffuse disease underlying segmental perfusion defects; 43% of patients with or without segmental perfusion defects demonstrated graded, longitudinal, base-to-apex perfusion abnormalities beyond +/-2 SD of normal control subjects, indicating diffuse coronary arterial narrowing by noninvasive PET perfusion imaging.

A mutant of Arp2p causes partial disassembly of the Arp2/3 complex and loss of cortical actin function in fission yeast.

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2(+) gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing.

The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M. Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing. Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro. Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo. These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.