Flip-flopping fractional flux quanta.

The d-wave pairing symmetry in high-critical temperature superconductors makes it possible to realize superconducting rings with built-in pi phase shifts. Such rings have a twofold degenerate ground state that is characterized by the spontaneous generation of fractional magnetic flux quanta with either up or down polarity. We have incorporated pi phase-biased superconducting rings in a logic circuit, a flip-flop, in which the fractional flux polarity is controllably toggled by applying single flux quantum pulses at the input channel. The integration of p rings into conventional rapid single flux quantum logic as natural two-state devices should alleviate the need for bias current lines, improve device symmetry, and enhance the operation margins.

Chromosome cohesion and segregation in mitosis and meiosis.

The faithful segregation of the genetic material into daughter cells during cell division is crucial for the production of healthy progeny. Sister chromatid cohesion and separation are fundamental to this process. Progress has been made in our molecular understanding of cohesion and mechanisms for the dissolution of cohesion have been uncovered.

Orchestrating anaphase and mitotic exit: separase cleavage and localization of Slk19.

Anaphase in budding yeast is triggered by cleavage of the central subunit, Scc1, of the chromosomal cohesin complex by the protease separase. Here we show that separase also cleaves the kinetochore-associated protein Slk19 at anaphase onset. Separase activity is also required for the proper localization of a stable Slk19 cleavage product to the spindle midzone in anaphase. The cleavage and localization of Slk19 are necessary to stabilize the anaphase spindle, and we show that a stable spindle is a prerequisite for timely exit from mitosis. This demonstrates the cleavage of targets other than cohesin by separase in the orchestration of high-fidelity anaphase.

Splitting the chromosome: cutting the ties that bind sister chromatids.

In eukaryotic cells, replicated DNA molecules remain physically connected from their synthesis in S phase until they are separated during anaphase. This phenomenon, called sister chromatid cohesion, is essential for the temporal separation of DNA replication and mitosis and for the equal separation of the duplicated genome. Recent work has identified a number of chromosomal proteins required for cohesion. In this review we discuss how these proteins may connect sister chromatids and how they are removed from chromosomes to allow sister chromatid separation at the onset of anaphase.

Doppler microembolic signals in patients with two different types of bileaflet valves.

OBJECTIVES: This study was performed to evaluate the prevalence and counts of Doppler microembolic signals in patients with St Jude Medical valves (St Jude Medical, Inc, St Paul, Minn) and patients with ATS valves (ATS Medical, Inc, Minneapolis, Minn) and their relation to clinical parameters. METHODS: A total of 179 outpatients of the department of cardiothoracic surgery were examined. They included 98 men and 81 women, aged 61 +/- 11 years, with ATS (n = 91) or St Jude Medical (n = 88) valves in the aortic (n = 110), mitral (n = 39), or both positions (n = 30). Neurologic examination was followed by transcranial Doppler monitoring for microembolic signals. Monitoring was performed bilaterally over the middle cerebral arteries for 1 hour per session. RESULTS: Microembolic signal counts and prevalence were significantly higher in patients with St Jude Medical as compared with ATS valves. Valve type and presence of diabetes mellitus were the only predictors of microembolic signal prevalence on multivariate analysis. No influence of microembolic signals on cerebral embolic complications was established. Additionally, patients with a postoperative history of cerebral embolic complications did not have a higher number of microembolic signals than remaining patients. Interobserver variability was satisfactory. CONCLUSIONS: Patients with St Jude Medical valves were shown to have significantly higher microembolic signal counts than patients with ATS valves. However, our results suggest that microembolic signal counts cannot be used to predict cerebral embolic complications. Their relation to neuropsychologic deficits remains to be evaluated.

Secured cutting: controlling separase at the metaphase to anaphase transition.

The final irreversible step in the duplication and distribution of genomes to daughter cells takes place at the metaphase to anaphase transition. At this point aligned sister chromatid pairs split and separate. During metaphase, cohesion between sister chromatids is maintained by the chromosomal multi-subunit cohesin complex. Here, I review recent findings as to how anaphase is initiated by proteolytic cleavage of the Scc1 subunit of cohesin. Scc1 is cleaved by a site-specific protease that is conserved in all eukaryotes, and is now called 'separase'. As a result of this cleavage, the cohesin complex is destroyed, allowing the spindle to pull sister chromatids into opposite halves of the cell. Because of the final and irreversible nature of Scc1 cleavage, this reaction is tightly controlled. Several independent mechanisms seem to impose regulation on Scc1 cleavage, acting on both the activity of separase and the susceptibility of the substrate.

Phosphorylation of the cohesin subunit Scc1 by Polo/Cdc5 kinase regulates sister chromatid separation in yeast.

At the onset of anaphase, a caspase-related protease (separase) destroys the link between sister chromatids by cleaving the cohesin subunit Scc1. During most of the cell cycle, separase is kept inactive by binding to an inhibitory protein called securin. Separase activation requires proteolysis of securin, which is mediated by an ubiquitin protein ligase called the anaphase-promoting complex. Cells regulate anaphase entry by delaying securin ubiquitination until all chromosomes have attached to the mitotic spindle. Though no longer regulated by this mitotic surveillance mechanism, sister separation remains tightly cell cycle regulated in yeast mutants lacking securin. We show here that the Polo/Cdc5 kinase phosphorylates serine residues adjacent to Scc1 cleavage sites and strongly enhances their cleavage. Phosphorylation of separase recognition sites may be highly conserved and regulates sister chromatid separation independently of securin.

Chromosome condensation: packaging the genome.

The packaging of centimetre long DNA molecules into compact metaphase chromosomes is essential for genome segregation in anaphase. The chromosomal condensin complex plays a crucial part in this packaging, and important new insight into condensin action in vitro and in vivo has recently been gained.

Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability.

Cohesion between sister chromatids is established during DNA replication and depends on a protein complex called cohesin. At the metaphase-anaphase transition in the yeast Saccharomyces cerevisiae, the ESP1-encoded protease separin cleaves SCC1, a subunit of cohesin with a relative molecular mass of 63,000 (Mr 63K). The resulting 33K carboxy-terminal fragment of SCC1 bears an amino-terminal arginine-a destabilizing residue in the N-end rule. Here we show that the SCC1 fragment is short-lived (t1/2 approximately 2 min), being degraded by the ubiquitin/proteasome-dependent N-end rule pathway. Overexpression of a long-lived derivative of the SCC1 fragment is lethal. In ubr1Delta cells, which lack the N-end rule pathway, we found a highly increased frequency of chromosome loss. The bulk of increased chromosome loss in ubr1Delta cells is caused by metabolic stabilization of the ESP1-produced SCC1 fragment. This fragment is the first physiological substrate of the N-end rule pathway that is targeted through its N-terminal residue. A number of yeast proteins bear putative cleavage sites for the ESP1 separin, suggesting other physiological substrates and functions of the N-end rule pathway.

Characterization of fission yeast cohesin: essential anaphase proteolysis of Rad21 phosphorylated in the S phase.

Cohesin complex acts in the formation and maintenance of sister chromatid cohesion during and after S phase. Budding yeast Scc1p/Mcd1p, an essential subunit, is cleaved and dissociates from chromosomes in anaphase, leading to sister chromatid separation. Most cohesin in higher eukaryotes, in contrast, is dissociated from chromosomes well before anaphase. The universal role of cohesin during anaphase thus remains to be determined. We report here initial characterization of four putative cohesin subunits, Psm1, Psm3, Rad21, and Psc3, in fission yeast. They are essential for sister chromatid cohesion. Immunoprecipitation demonstrates stable complex formation of Rad21 with Psm1 and Psm3 but not with Psc3. Chromatin immunoprecipitation shows that cohesin subunits are enriched in broad centromere regions and that the level of centromere-associated Rad21 did not change from metaphase to anaphase, very different from budding yeast. In contrast, Rad21 containing similar cleavage sites to those of Scc1p/Mcd1p is cleaved specifically in anaphase. This cleavage is essential, although the amount of cleaved product is very small (<5%). Mis4, another sister chromatid cohesion protein, plays an essential role for loading Rad21 on chromatin. A simple model is presented to explain the specific behavior of fission yeast cohesin and why only a tiny fraction of Rad21 is sufficient to be cleaved for normal anaphase.

Automated identification of Doppler microembolic signals: comparison of two techniques.

An alternative technique for identification of Doppler microemboli signals (MES), based on intensity measurements in the vessel and in an arbitrary sample volume was recently reported. We evaluated the applicability of this approach as stand alone system, and compared it to the standard bigate method (TCD 8, version 8T). Bilateral TCD monitoring was performed in 11 patients with prosthetic heart valves and 15 patients during elective cardiac surgery, using three sample volumes (29 mm, 50 mm and 55 mm). All data was saved on digital audio tapes and evaluated by two experienced observers. Only signals unanimously identified as MES or artifacts by both observers were evaluated. A total of 6189 MES and 11,241 artifacts were further analysed. Sensitivity and specificity of the bigate approach and the technique utilising the arbitrary sample volume were 90.7%, 91.3% and 88%, 91.9% respectively. Simultaneous monitoring over three sample volumes and combination of the two detection algorithms could potentially provide an adequate stand-alone system for MES detection.

Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast.

In eukaryotic cells, replicated DNA strands remain physically connected until their segregation to opposite poles of the cell during anaphase. This "sister chromatid cohesion" is essential for the alignment of chromosomes on the mitotic spindle during metaphase. Cohesion depends on the multisubunit cohesin complex, which possibly forms the physical bridges connecting sisters. Proteolytic cleavage of cohesin's Sccl subunit at the metaphase to anaphase transition is essential for sister chromatid separation and depends on a conserved protein called separin. We show here that separin is a cysteine protease related to caspases that alone can cleave Sccl in vitro. Cleavage of Sccl in metaphase arrested cells is sufficient to trigger the separation of sister chromatids and their segregation to opposite cell poles.

Disjunction of homologous chromosomes in meiosis I depends on proteolytic cleavage of the meiotic cohesin Rec8 by separin.

It has been proposed but never proven that cohesion between sister chromatids distal to chiasmata is responsible for holding homologous chromosomes together while spindles attempt to pull them toward opposite poles during metaphase of meiosis I. Meanwhile, the mechanism by which disjunction of homologs is triggered at the onset of anaphase I has remained a complete mystery. In yeast, cohesion between sister chromatid arms during meiosis depends on a meiosis-specific cohesin subunit called Rec8, whose mitotic equivalent, Sccl, is cleaved at the metaphase to anaphase transition by an endopeptidase called separin. We show here that cleavage of Rec8 by separin at one of two different sites is necessary for the resolution of chiasmata and the disjunction of homologous chromosomes during meiosis.

Chromosome cohesion: a polymerase for chromosome bridges.

How do cells ensure that sister chromatids produced during DNA replication stay connected with each other until their separation in anaphase? New insight is provided by the discovery of DNA polymerase kappa, which has been found to be required for building the connections between sister chromatids.

Splitting the chromosome: cutting the ties that bind sister chromatids.

In eukaryotic cells, sister DNA molecules remain physically connected from their production at S phase until their separation during anaphase. This cohesion is essential for the separation of sister chromatids to opposite poles of the cell at mitosis. It also permits chromosome segregation to take place long after duplication has been completed. Recent work has identified a multisubunit complex called cohesin that is essential for connecting sisters. Proteolytic cleavage of one of cohesin's subunits may trigger sister separation at the onset of anaphase.

Differentiation between true microembolic signals and artefacts using an arbitrary sample volume.

We evaluated a new discrimination technique between microemboli (MES) and artefact signals. Monitoring was performed over the middle cerebral artery (55 mm) and the brain parenchyma (29 mm). Intensity changes were expressed as percent of change compared to the value measured in the proximal depth. The cut-off value providing the highest sensitivity and specificity in the differentiation was evaluated using 250 MES and 250 artefact signals, and subsequently analysed in the first part of the study. Intensity values derived from the distal depth were subsequently evaluated in 10 patients undergoing cardiac surgery and 45 patients with potential arterial or cardioembolic source. Intensity changes of 87% (84%-90%) and -58% (-71%-(-48%)) were measured in the initial 500 signals for MES and artefact signals, respectively. The best intensity cut-off value was calculated at 27%. This value was subsequently applied to a total of 1858 MES and 1958 artefacts, resulting to sensitivity and specificity of 96% and 98%, respectively. The proposed technique provided adequate results, warranting further evaluation.

Cerebrovascular reactivity is impaired in patients with cardiac failure.

AIMS: We undertook this study to evaluate potential changes in cerebral vasoreactivity in patients with cardiac failure and their consequent dependence upon cardiac functional variables. METHODS AND RESULTS: A total of 50 patients with various degrees of heart failure, 20 age-matched controls and 20 normal controls were examined. Cerebrovascular reactivity was examined with the carbon dioxide technique. Mean flow velocities of both middle cerebral arteries as well as end-tidal carbon dioxide partial pressure were continuously registered. Normal controls were examined on two different occasions, to evaluate the technique's reproducibility. Cerebrovascular reactivity was significantly reduced in all examined patients as compared to controls, and in NYHA IV as compared to NYHA II and III patients. A significant relationship between cerebrovascular reactivity and left ventricular ejection fraction was evident. Reproducibility of the technique was satisfactory. CONCLUSION: Our study provided evidence of significantly reduced cerebrovascular reactivity in patients with cardiac failure, which was significantly related to the NYHA grade and the left ventricular ejection fraction.

Venous microemboli in patients with artificial heart valves.

BACKGROUND: Detection of microemboli signals (MES) in patients with artificial heart valves has been extensively described, but the underlying material remains unclear. We assumed that the detection of MES in the jugular vein of patients with prosthetic valves would clearly argue for gaseous embolic material, since formed emboli are unable to cross through the capillaries. METHODS AND RESULTS: Twenty-five patients with artificial heart valves, 15 patients with asymptomatic carotid artery disease, and 25 normal controls were examined. Monitoring was performed simultaneously over the dominant jugular vein and the ipsilateral middle cerebral artery for 30 min per subject, using 2-MHz transducers of a color duplex scanner for the jugular vein and a pulsed-wave Doppler for the middle cerebral artery. Data were harvested in an eight-channel digital recorder and MES counts evaluated by two separate observers. MES prevalence in the middle cerebral artery was 100, 13 and 0% in patients with artificial heart valves, asymptomatic carotid artery disease, and normal controls, respectively. No MES were detected in the jugular veins of patients with carotid artery disease or in normal controls, while their prevalence was 68% in patients with artificial heart valves. The interobserver agreement was satisfactory. CONCLUSION: Our results suggest that the embolic material of at least a part of MES in patients with artificial heart valves is gaseous.

Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1.

Cohesion between sister chromatids is established during DNA replication and depends on a multiprotein complex called cohesin. Attachment of sister kinetochores to the mitotic spindle during mitosis generates forces that would immediately split sister chromatids were it not opposed by cohesion. Cohesion is essential for the alignment of chromosomes in metaphase but must be abolished for sister separation to start during anaphase. In the budding yeast Saccharomyces cerevisiae, loss of sister-chromatid cohesion depends on a separating protein (separin) called Esp1 and is accompanied by dissociation from the chromosomes of the cohesion subunit Scc1. Here we show that Esp1 causes the dissociation of Scc1 from chromosomes by stimulating its cleavage by proteolysis. A mutant Scc1 is described that is resistant to Esp1-dependent cleavage and which blocks both sister-chromatid separation and the dissociation of Scc1 from chromosomes. The evolutionary conservation of separins indicates that the proteolytic cleavage of cohesion proteins might be a general mechanism for triggering anaphase.