The impact of socioeconomic and demographic factors on the utilization of smoking cessation medications in patients hospitalized with cardiovascular disease in Nova Scotia, Canada.

OBJECTIVE: To determine whether any demographic or socioeconomic factors affect the use of smoking cessation medications in patients hospitalized with heart disease. METHOD: Data were obtained from the Improving Cardiovascular Outcomes in Nova Scotia (ICONS) Canada database, which includes a registry of all hospitalized patients with a diagnosis of ischaemic heart disease, congestive heart failure, or atrial fibrillation since October 1997. Patients agreeing to provide follow-up were sent an enrollment survey to determine demographic and socioeconomic factors including household income, educational background and private drug insurance plans. RESULTS: Between 15 October 1997 and 31 December 2000, 5442 patients who were current smokers and 270 patients using a smoking cessation medication were admitted to hospital registered in the ICONS database. An enrollment survey was completed by 1071 current smokers and 77 patients using a smoking cessation agent. CONCLUSION: Higher education level, presence of private drug insurance plans, and less difficulty paying for basic needs were associated with higher use of smoking cessation medications.

Keratoconus, myopia, and personality.

PURPOSE: To determine whether there is an association between keratoconus and personality attributes including obsessionality traits. METHODS: We reviewed all charts in the regional contact lens clinic, identifying patients who had attended from January 1997 to January 2000 and had a diagnosis of either keratoconus or myopia of at least 6 diopters. This yielded 289 keratoconics and 149 myopes who were contacted by mail and invited to complete two standardized personality questionnaires (Maudsley Obsessive-Compulsive Inventory and the revised Eysenck Personality Questionnaire). On receipt of consent, questionnaires and an explanatory letter were sent to potential participants. RESULTS: Completed replies from 118 keratoconic and 75 myopic controls were suitable for analysis after exclusion of patients who returned incomplete data or were deemed unreliable by scoring highly on the lie scale. The only finding between the two groups was that myopes scored higher than keratoconics on the psychoticism scale (p < 0.05). This was a small effect and became insignificant when the Bonferroni procedure was applied. CONCLUSION: This study indicated that there is little evidence to suggest that keratoconics differ significantly in personality from a group of moderate to high myopes who also depend on contact lens correction for distance vision. Although myopes showed marginally higher levels of psychoticism than did keratoconics, analysis of the range of personality traits assessed indicates that the differences between the two groups is not significant. The authors could not substantiate the clinical notion of the keratoconic personality.

Gun culture and symbolism among U.K. and U.S. women.

The authors explored attitudes of young women in the United Kingdom (n = 108) and the United States (n = 91) toward (a) the possession and use of guns through the Attitude to Guns Scale (N. R. Branscombe, J. A. Weir, & P. Crosby, 1992) and (b) guns' perceived functional and symbolic significance through the Symbolic Nature of Guns Scale (C. A. Cooke & J. E. Puddifoot, 1997). There were significant differences in beliefs concerning the right to own a gun and the protective effect of guns but not in the perceived contribution of guns to crime. Although neither group strongly equated guns symbolically with power or control, the U.S. women were more likely to perceive guns as expressions of freedom or independence, and the U.K. women were more likely to view guns as expressions of danger and violence. The findings were contextualized by comparison with samples of male control participants of similar ages.

Transition from caspase-dependent to caspase-independent mechanisms at the onset of apoptotic execution.

We have compared cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway. Extracts from morphologically normal "committed stage" cells induce apoptotic morphology and DNA cleavage in substrate nuclei but require ongoing caspase activity to do so. In contrast, extracts from frankly apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. Further characterization of the "execution phase" extracts revealed the presence of an ICAD/DFF45 (inhibitor of caspase-activated DNase/DNA fragmentation factor)- inhibitable nuclease resembling CAD, plus another activity that was required for the apoptotic chromatin condensation. Despite the presence of active caspases, committed stage extracts lacked these downstream activities, suggesting that the caspases and downstream factors are segregated from one another in vivo during the latent phase. These observations not only indicate that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves, but they also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to active phase of apoptosis.

The hBUB1 and hBUBR1 kinases sequentially assemble onto kinetochores during prophase with hBUBR1 concentrating at the kinetochore plates in mitosis.

The kinetochore binds an evolutionarily conserved set of checkpoint proteins that function to monitor whether chromosomes have aligned properly at the spindle equator. Human cells contain two related protein kinases, hBUB1 and hBUBR1, that appear to have evolved from a single ancestral BUB1 gene. We generated hBUB1- and hBUBR1-specific antibodies so that the localization patterns of these kinases could be directly compared. In the human U2OS osteosarcoma cell line, hBUB1 first appeared at kinetochores during early prophase before all kinetochores were occupied by hBUBR1 or CENP-F. Both proteins remained at kinetochores throughout mitosis but their staining intensity was reduced from anaphase onward. Kinetochores of unaligned chromosomes exhibited stronger hBUB1 and hBUBR1 staining. Immunoelectron microscopy showed that hBUBR1 appeared to be concentrated in the outer kinetochore plate and in some instances the inner plate as well. When chromosome spreads were examined by light microscopy, hBUB1 and hBUBR1 were coincident with CENP-E. This suggests that both kinases are concentrated near the surface of the kinetochore where they can monitor kinetochore-microtubule interactions.

Localization of CENP-E in the fibrous corona and outer plate of mammalian kinetochores from prometaphase through anaphase.

We have conducted a detailed ultrastructural analysis of the distribution of the kinesin-related centromere protein CENP-E during mitosis in cultured human, rat kangaroo and Indian muntjac cells. Using an affinity-purified polyclonal antibody and detection by 0.8 nm colloidal gold particles, CENP-E was localized primarily to the fibrous corona of the kinetochore in prometaphase and metaphase cells. Some labeling of the kinetochore outer plate was also observed. The distribution of fibrous corona-associated CENP-E did not change dramatically following the attachment of microtubules to the kinetochore. Thus, the normal disappearance of this kinetochore substructure in conventional electron micrographs of mitotic chromosomes with attached kinetochores is not due to the corona becoming stretched along the spindle microtubules as has been suggested. Examination of cells undergoing anaphase chromatid movement revealed the presence of CENP-E still associated with the outer surface of the kinetochore plate. At the same time, the majority of detectable CENP-E in these cells was associated with the bundles of antiparallel microtubules in the central spindle. CENP-E in this region of the cell is apparently associated with the stem body matrix material. The simultaneous localization of CENP-E on centromeres and the central spindle during anaphase was confirmed by both wide-field microscopy of human cells and conventional fluorescence microscopy of rat kangaroo cells. Together, the observations reported here are consistent with models in which CENP-E has a role in promoting the poleward migration of sister chromatids during anaphase A.

Immunolocalization of CENP-A suggests a distinct nucleosome structure at the inner kinetochore plate of active centromeres.

The trilaminar kinetochore directs the segregation of chromosomes in mitosis and meiosis. Despite its importance, the molecular architecture of this structure remains poorly understood [1]. The best known component of the kinetochore plates is CENP-C, a protein that is required for kinetochore assembly [2], but whose molecular role in kinetochore structure and function is unknown. Here we have raised for the first time monospecific antisera to CENP-A [3], a 17 kD centromere-specific histone variant that is 62% identical to the carboxy-terminal domain of histone H3 [4,5] and that resembles the yeast centromeric component CSE4 [6]. We have found by simultaneous immunofluorescence with centromere antigens of known ultrastructural location that CENP-A is concentrated in the region of the inner kinetochore plate at active centromeres. Because CENP-A was previously shown to co-purify with nucleosomes [7], our data suggest a specific nucleosomal substructure for the kinetochore. In human cells, these kinetochore-specific nucleosomes are enriched in alpha-satellite DNA [8]. However, the association of CENP-A with neocentromeres lacking detectable alpha-satellite DNA, and the lack of CENP-A association with alpha-satellite-rich inactive centromeres of dicentric chromosomes together suggest that CENP-A association with kinetochores is unlikely to be determined solely by DNA sequence recognition. We speculate that CENP-A binding could be a consequence of epigenetic tagging of mammalian centromeres.

Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.

Ran is a small GTPase that is required for protein import, mRNA export, and the maintenance of nuclear structures. To gain a better understanding of Ran's role in the nucleus, we have sought to use Xenopus egg extracts for the purification and characterization of proteins from egg extracts bound with a high affinity to a glutathione-S-transferase-Ran fusion protein (GST-Ran). We found that GST-Ran associates specifically with at least 10 extract proteins. We determined the identifies of six Ran-interacting proteins (Rips), and found that they include RanBP2/Nup358, Nup153, Importin beta, hsc70, RCC1, and RanBP1. On the basis of peptide sequence, a seventh Rip (p88) seems to be similar but not identical to Fug1/RanGAP1, the mammalian Ran-GTPase-activating protein. Gel filtration analysis of endogenous extract proteins suggests that Importin beta acts as a primary GTP-Ran effector. Both Ran and Importin beta are coimmunoprecipitated by anti-p340RanBP2 antibodies in the presence of nonhydrolyzable GTP analogues, suggesting that Ran-Importin beta complexes interact with p340RanBP2. Two other Rips, p18 and p88, are coprecipitated with p340RanBP2 in a nucleotide-independent manner. Analysis of the Ran-GTPase pathway in Xenopus extracts allows the examination of interactions between Ran-associated proteins under conditions that resemble in vivo conditions more closely than in assays with purified components, and it thereby allows additional insights into the molecular mechanism of nuclear transport.

CENP-C is required for maintaining proper kinetochore size and for a timely transition to anaphase.

The human autoantigen CENP-C has been demonstrated by immunoelectron microscopy to be a component of the inner kinetochore plate. Here we have used antibodies raised against various portions of CENP-C to probe its function in mitosis. We show that nuclear microinjection of anti-CENP-C antibodies during interphase causes a transient arrest at the following metaphase. Injection of the same antibodies after the initiation of prophase, however, does not disrupt mitosis. Correspondingly, indirect immunofluorescence using affinity-purified human anti-CENP-C antibodies reveals that levels of CENP-C staining are reduced at centromeres in cells that were injected during interphase, but appear unaffected in cells which were injected during mitosis. Thus, we suggest that the injected antibodies cause metaphase arrest by reducing the amount of CENP-C at centromeres. Examination of kinetochores in metaphase-arrested cells by electron microscopy reveals that the number of trilaminar structures is reduced. More surprisingly, the few remaining kinetochores in these cells retain a normal trilaminar morphology but are significantly reduced in diameter. In cells arrested for extended periods, these small kinetochores become disrupted and apparently no longer bind microtubules. These observations are consistent with an involvement of CENP-C in kinetochore assembly, and suggest that CENP-C plays a critical role in both establishing and/or maintaining proper kinetochore size and stabilizing microtubule attachments. These findings also support the idea that proper assembly of kinetochores may be monitored by the cell cycle checkpoint preceding the transition to anaphase.

Nuclear events of apoptosis in vitro in cell-free mitotic extracts: a model system for analysis of the active phase of apoptosis.

We have developed a cell-free system that induces the morphological transformations characteristic of apoptosis in isolated nuclei. The system uses extracts prepared from mitotic chicken hepatoma cells following a sequential S phase/M phase synchronization. When nuclei are added to these extracts, the chromatin becomes highly condensed into spherical domains that ultimately extrude through the nuclear envelope, forming apoptotic bodies. The process is highly synchronous, and the structural changes are completed within 60 min. Coincident with these morphological changes, the nuclear DNA is cleaved into a nucleosomal ladder. Both processes are inhibited by Zn2+, an inhibitor of apoptosis in intact cells. Nuclear lamina disassembly accompanies these structural changes in added nuclei, and we show that lamina disassembly is a characteristic feature of apoptosis in intact cells of mouse, human and chicken. This system may provide a powerful means of dissecting the biochemical mechanisms underlying the final stages of apoptosis.

Mapping DNA within the mammalian kinetochore.

The location of the cis-acting DNA sequences that direct the assembly of the mammalian kinetochore is not known. A variety of circumstantial evidence, however, has led to the widespread belief that they are present throughout the kinetochore including the kinetochore outer plate. To investigate this question directly, we have used two independent methods to localize DNA in and around the mammalian kinetochore. Both methods fail to reveal DNA in the outer kinetochore plate, finding instead that the outer-most detectable DNA in the centromere is located in the inner kinetochore plate. Our results imply that the outer kinetochore plate is primarily a proteinaceous structure. It is thus unlikely that fibers observed in the outer plate correspond to chromatin, as previously assumed. Our observations suggest that current models of kinetochore structure may need to be reconsidered.

CENP-C, an autoantigen in scleroderma, is a component of the human inner kinetochore plate.

We have isolated and characterized a set of overlapping cDNA clones that encode the human centromere autoantigen centromere protein C (CENP-C). The identity of these clones has been established using several criteria. First, they were shown to encode a polypeptide that migrates at the expected position for CENP-C on SDS-polyacrylamide gel electrophoresis. Second, we have demonstrated that this polypeptide shares at least two epitopes with human CENP-C. Polyclonal antibodies were raised to fusion proteins encoded by nonoverlapping regions of the cDNA clones. These antibodies were shown to recognize a protein at a position appropriate for CENP-C on immunoblots of human chromosomal proteins. In addition, we used indirect immunofluorescence to demonstrate that these antibodies recognize centromeres of HeLa chromosomes in the expected pattern for CENP-C. Localization of CENP-C by immunoelectron microscopy reveals that this protein is a component of the inner kinetochore plate.

Compartmentalization within the nucleus: discovery of a novel subnuclear region.

Antibodies to a set of structurally related autoantigens (p23-25) bind to a previously uncharacterized, large structural domain in the nucleus of a variety of human cell types. This subnuclear domain is visible by phase contrast alone as a region of decreased density after several different fixation protocols. The morphology of this region changes dramatically during the cell cycle and we have given it the name PIKA (for polymorphic interphase karyosomal association) based on preliminary evidence that the PIKA proteins may be associated with chromatin. The function of the PIKA is not yet known, but our immunolocalization data indicate that it is unlikely to be associated with regions of ongoing DNA replication, heterogeneous nuclear RNA storage, or mRNA processing. The discovery of the PIKA provides evidence supporting an emerging model of nuclear structure. It now appears that the nucleus is organized into distinct domains which include not only the nucleolus, but also previously unidentified regions such as the PIKAs. Furthermore, structural rearrangements undergone by the nucleolus and the PIKAs may be indicative of a broad tendency for nuclear organization to change in a cell cycle-specific fashion.

Analysis of the distribution of the INCENPs throughout mitosis reveals the existence of a pathway of structural changes in the chromosomes during metaphase and early events in cleavage furrow formation.

The INCENPs are two polypeptides of 135 x 10(3) and 150 x 10(3) Mr that enter mitosis as tightly bound chromosomal proteins, but subsequently leave the chromosomes altogether and become associated with the central spindle and cell cortex at the contractile ring. In the experiments reported here we have used confocal microscopy and immunoelectron microscopy to provide a detailed picture of the intracellular location of these proteins during mitosis. The experiments have not only revealed a number of new details concerning the properties of the INCENPs in mitosis, but have revealed a number of novel aspects of the mitotic process itself. The first of these is the existence of a sequential pathway of structural changes in the chromosomes that occurs during metaphase. This pathway is revealed by the existence of four distinct INCENP staining patterns in mitotic cells. In 'early' and 'early/mid' metaphase, the INCENPs gradually become concentrated at the centromeres, forming a ring at the center of the metaphase plate. During 'mid/late' metaphase they exit from the chromosomes, so that by late metaphase they are found solely in streaks that traverse the plate parallel to the spindle axis. The streaks probably correspond to INCENPs closely associated with microtubule bundles, perhaps as part of the stem body material. Examination of transverse optical sections of the spindle interzone during early anaphase reveals an unexpectedly high degree of order. The INCENP antigens are localized on fibers that are organized into a hollow ring 8 microns in diameter and approximately 4 microns beneath the cell cortex. Measurement of cellular dimensions in the confocal microscope reveals that the maximum diameter of early anaphase cells lies across the spindle equator, so that when the cleavage furrow forms, it does so around the maximum circumference of the cell. During anaphase, a subpopulation of the INCENP antigen becomes localized to the cortex where the furrow will subsequently form. This occurs prior to any other evidence of furrowing. Thus, binding of the INCENPs to this region may represent an early step in furrow formation. Together, these results suggest that the INCENPs may represent a new class of 'chromosomal passenger' proteins that are carried to the spindle equator by the chromosomes and subsequently perform a cytoskeletal role following their release from the chromosomes at the metaphase:anaphase transition.