Sucrose-induced spindle elongation in mitotic PtK-1 cells.

Brief treatment of mitotic metaphase and anaphase PtK-1 cells with tissue culture medium containing 0.5 M sucrose resulted in spindle elongation without chromosome motion. Spindle birefringence also changed from a uniform appearance to one of highly birefringent bundles. Electron microscopic analysis indicated these birefringent bundles were composed of tightly packed arrays of spindle microtubules. No kinetochores could be seen following a 10 min sucrose treatment. Upon removal of sucrose, metaphase spindles returned to pretreatment lengths and the normal birefringence pattern returned. Reduction in spindle length could be temporally coupled with the reappearance of kinetochores and the reassociation of microtubules with these structures. In contrast to treated and released metaphase cells, anaphase spindles did not return to pretreatment lengths. Replacement of sucrose with medium showed the resumption of chromosome-to-pole motion within 2 min of sucrose removal. Chromosome motion could be correlated with the reappearance of kinetochores and kinetochore microtubules. These data have led us to postulate the existence of two microtubule continuums in the spindle and to discuss their roles in spindle organization and chromosome motion.

Role of non-kinetochore microtubules in spindle elongation in mitotic PtK1 cells.

PtK1 metaphase cells were treated with varying concentrations of nocodazole to reduce spindle microtubule number and spindle length. The range of concentrations employed reduced spindle length from approximately 47% to 82% of the original pole-pole distance. Electron microscopy of cells treated with the lowest concentration of nocodazole employed (0.01 microgram/ml) showed a small decrease in the number of non-kinetochore microtubules (nkMTs), particularly evident in the astral region, with no significant effect on kinetochore microtubule number. Metaphase cells treated with 1 microgram/ml nocodazole for 2 min demonstrated a reduction in spindle length and loss of most non-kinetochore microtubules with little effect on the number and arrangement of the kinetochore class of microtubules. Following nocodazole treatment, the cells were perfused with 0.5 M sucrose dissolved in tissue culture medium, a treatment which has previously been shown to induce spindle elongation in metaphase cells. In cells where nocodazole effected a large decrease in non-kinetochore microtubule number with a concomitant decrease in spindle length, sucrose treatment had a reduced effect in inducing spindle elongation. In cells treated with lower concentrations of nocodazole, where numerous non-kinetochore microtubules remained, sucrose had a greater effect in inducing spindle elongation. These data suggest that the non-kinetochore population of microtubules is responsible for the extent of sucrose-induced spindle elongation. An explanation of these data is provided which suggests that the role of non-kinetochore microtubules is to trap energy in the developing spindle, such that it can be used to separate spindle poles during anaphase B.

Structural implications of kinetochore function in sucrose-treated PtK1 cells.

Treatment of PtK1 cells during metaphase with solutions containing hyperosmotic concentrations of sucrose resulted in an alteration of kinetochore structure and function in a concentration-dependent manner. This alteration in kinetochore morphology was shown to be rapidly reversible upon removal of the sucrose-containing tissue culture medium. A 10-min treatment with both 0.2 M and 0.4 M sucrose resulted in a concentration-dependent aggregation of spindle fibers into bundles, loss of trilaminar kinetochore morphology as judged by electron microscopy, and induction of anaphase B-like spindle elongation as previously described. Electron microscopy showed that a 10-min treatment of metaphase cells with hyperosmotic concentrations of sucrose changed the trilaminar kinetochore structure to one of a single lamina, with an amorphous, lightly staining material distally associated with it. Sucrose-induced bundles of microtubules could usually be seen embedded or tangentially associated with this material. Rate and extent of spindle elongation in sucrose-treated metaphase cells were greater in the higher concentrations of sucrose employed. The degree of microtubule bundling was also concentration dependent, with reduced bundling occurring at lower sucrose concentrations. Within 2 min after sucrose removal kinetochores returned to a bi- or trilaminar morphology with reduction in the amount of amorphous material. Reformation of the kinetochore trilaminar structure resembled that of the normal maturation process which occurs from prophase through anaphase. These rapid changes in kinetochore morphology following release from sucrose treatment were temporally associated with restoration of spindle function and suggested that kinetochore integrity was necessary for the expression of spindle forces responsible for spindle shortening. These forces are probably generated or transduced by the continuum formed between the two spindle poles, the kinetochore microtubules, and the sister chromatids.

Prognostic importance of scintigraphic left ventricular cavity dilation during intravenous dipyridamole technetium-99m sestamibi myocardial tomographic imaging in predicting coronary events.

Left ventricular (LV) cavity dilation during stress myocardial perfusion imaging has been associated with multivessel disease, and may be an independent prognostic marker in addition to perfusion defects. The present study examines the predictive value for future cardiac events of transient or fixed LV dilation during dipyridamole technetium-99m (Tc-99m) sestamibi single-photon emission computed tomography (SPECT) imaging. The study included 512 consecutive patients who underwent SPECT imaging with Tc-99m sestamibi after dipyridamole infusion. Transient LV dilation was seen in 70 patients (14%) and 74 had fixed cavity dilation (14%); cavity size was normal in 368 patients (72%). Each perfusion scan was classified as normal or abnormal, and if abnormal, defects were categorized as transient or fixed, and as small, medium, or large (depending upon the number of abnormal vascular territories). Events during a mean follow-up of 12.8 +/- 6.8 months were tabulated by direct review of hospital charts and death certificates. The cardiac event rate (cardiac death or nonfatal infarction) was 1.9% in patients with normal cavity size, 11.4% with transient LV dilation, and 13.5% with fixed LV dilation (p < 0.01). Compared with patients with normal cavity size, those with transient LV dilation were more likely to sustain a myocardial infarction (p < 0.01) and those with fixed dilation more frequently suffered cardiac death (p < 0.01) and hospitalization for heart failure (p < 0.01). The group with the highest risk had both a large perfusion defect and cavity dilation. By Cox proportional hazard regression analysis, both transient and fixed LV dilation were strong independent predictors of cardiac events. Transient or fixed LV dilation are commonly seen during dipyridamole Tc-99m sestamibi SPECT imaging (14% incidence for each) and are useful predictors of cardiac events.

Interpretive reproducibility of stress Tc-99m sestamibi tomographic myocardial perfusion imaging.

BACKGROUND: Observer variability has been shown with interpretation of planar thallium-201 images. The interpretive reproducibility of technetium-99m sestamibi tomographic imaging is unknown. This study evaluated the interpretive reproducibility of interpretable Tc-99m sestamibi tomographic images among nuclear cardiologists with a wide range of training and experience. METHODS: Three experienced readers (EX) and 3 less-experienced readers (LEX) interpreted 138 exercise and rest Tc-99m sestamibi tomographic images (101 were abnormal in patients with coronary artery disease [CAD], 37 were normal in patients with <5% likelihood of CAD) twice in random sequence without clinical data. Images of good to excellent quality were randomly selected from a database at 2 nuclear cardiology laboratories. Intraobserver and interobserver agreement for global, left anterior descending (LAD) territory, non-LAD first (normal/abnormal) and second (normal/fixed/reversible) order, and defect extent (normal/single-vessel CAD/multi-vessel CAD) were assessed with percent agreement and Cohen's kappa (kappa) statistic. RESULTS: With regard to intraobserver agreement, first and second order ranged from 87% to 94% and 80% to 90% for global, 82% to 96% and 78% to 95% for LAD, and 88% to 91% and 80% to 90% for non-LAD, respectively. Defect extent ranged from 75% to 90%. There were no differences between EX and LEX for global and non-LAD first and second order, LAD first order, and defect extent. LAD second order was 93% for EX compared with 88% (P = .015) for LEX. With regard to interobserver agreement, first and second order ranged from 73% to 89% and 64% to 85% for global, 73% to 93% and 69% to 91% for LAD, and 76% to 88% and 68% to 84% for non-LAD, respectively. Defect extent ranged from 61% to 82%. Global first and second order ranged from 85% to 87% and 78% to 82% for EX compared with 73% to 84% and 64% to 79% for LEX. LAD first and second order ranged from 89% to 91% and 88% to 89% for EX compared with 73% to 91% and 69% to 70% for LEX. Non-LAD first and second order ranged from 82% to 86% and 76% to 77% for EX compared with 76% to 86% and 68% to 81% for LEX. Defect extent ranged from 69% to 75% for EX compared with 59% to 77% for LEX. CONCLUSIONS: There is moderate to excellent interpretive reproducibility with stress Tc-99m sestamibi SPECT imaging among nuclear cardiologists with a wide range of training and experience.

Effectiveness of nuclear cardiology training guidelines: a comparison of trainees with experienced readers.

BACKGROUND: To evaluate the effectiveness of published nuclear cardiology training guidelines, the diagnostic accuracy of image interpretation by nuclear cardiology trainees was compared with that of experienced nuclear cardiologists. METHODS AND RESULTS: The accuracy of three experienced nuclear cardiologists and three trainees with level II experience following Society of Nuclear Medicine/American College of Cardiology/American Society of Nuclear Cardiology guidelines in the interpretation of 114 exercise 99mTc-labeled sestamibi single-photon emission computed tomographic imaging studies was evaluated. Studies were selected randomly and included patients with less than 5% likelihood of coronary artery disease, as well as patients with angiographically demonstrated single and multivessel disease. Studies were interpreted by each reader without knowledge of clinical or exercise data. Each reader classified perfusion as normal or abnormal. Accuracy was assessed according to sensitivity, normalcy rate, and predictive accuracy. In addition, the ability of experienced readers and trainees to identify abnormal perfusion in patients with multivessel disease was compared. Trainees had high accuracy, comparable to experienced readers for sensitivity, normalcy rate, and predictive accuracy, as well as the ability to identify abnormal perfusion in patients with multivessel disease. In all categories, experienced interpretors demonstrated a trend toward greater accuracy with less observer variability than did trainees. CONCLUSION: Structured training in nuclear cardiology following Society of Nuclear Medicine/American College of Cardiology/American Society of Nuclear Cardiology guidelines during clinical cardiology fellowship is effective, and trainees possess the skills to interpret myocardial perfusion images accurately. Interpretive skills can be expected to improve further with experience.